Because of their involvement in regulating transcription, gene promoters contain important *cis*-acting elements (Zhu and Li, 1997). To characterize the possible regulatory mechanisms of *NAP* genes during fruit development, maturation, and softening, we analyzed the promoters of four fruit-specific *NAP* genes. Several motifs associated with responses to phytohormones and environmental factors were detected. These motifs included the ABA-responsive element and coupling element 3, the CGTCA and TGACG motifs associated with responses to methyl jasmonate, and the TCA-element related to responses to salicylic acid (**Table 2**). Exogenous ABA can up-regulate *NAP* expression in *A. thaliana* and rice (Liang et al., 2014; Yang et al., 2014). Zhou et al. (2013) reported that *OsNAP* can regulate leaf senescence by affecting jasmonic acid signaling pathways, and that overexpressing *OsNAP* increases the production of endogenous jasmonic acid in rice. Other studies have demonstrated that hormones, including ABA, jasmonic acid, and salicylic acid, have important regulatory roles during fruit ripening and softening (Creelman and Mullet, 1995; Zhang et al., 2003, 2009a). Therefore, it can be inferred that *PpNAP* genes regulate peach fruit development and softening by influencing specific hormone signal transduction pathways. Moreover, genes containing the MADS-box motif have key roles in flower and fruit development and maturation (Adamczyk and Fernandez, 2009; Smaczniak et al., 2012). We also observed that one to three copies of the MADS-domain site CArG-box were present in the promoters of four *NAP* genes, indicating that *PpNAP* and MADSbox genes may interact to regulate peach fruit development and ripening. However, the specific regulatory mechanisms of *PpNAP* genes that affect peach fruits require further study.

### AUTHOR CONTRIBUTIONS

CZ, MH, HL, and DZ: Design and interpretation of all experiments. FL, JL, and MQ: Performed all plant physiological and biochemical experiments. CZ, FL and LC: Wrote the manuscript.

### ACKNOWLEDGMENTS

We wish to thank Minghui Lu for English language modifying. This research was supported by grants from National Science Foundation of China (Grant No. 31572079) and the Natural Science Foundation of Shaanxi Province, China (Grant No. 2015JM3103).

### REFERENCES

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. 00147

FIGURE S1 | The amplification (A), melt curve (B) and melt park (C) of 18s ribosomal gene in all samples.

FIGURE S2 | Quantitative reverse transcription PCR analysis of PpACO1genes in fruits with different developmental stage. Arrow indicates the time of harvest(121 DAFB).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Li, Li, Qian, Han, Cao, Liu, Zhang and Zhao. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Implication of Abscisic Acid on Ripening and Quality in Sweet Cherries: Differential Effects during Pre- and Post-harvest

Verónica Tijero† , Natalia Teribia† , Paula Muñoz and Sergi Munné-Bosch\*

Department of Plant Biology, Faculty of Biology, University of Barcelona, Barcelona, Spain

### Edited by:

Mario Pezzotti, University of Verona, Italy

#### Reviewed by:

Christoph Martin Geilfus, Christian-Albrechts-Universität zu Kiel, Germany Gianfranco Diretto, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Italy

> \*Correspondence: Sergi Munné-Bosch<EMAIL_ADDRESS>
†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science

Received: 11 February 2016 Accepted: 18 April 2016 Published: 04 May 2016

#### Citation:

Tijero V, Teribia N, Muñoz P and Munné-Bosch S (2016) Implication of Abscisic Acid on Ripening and Quality in Sweet Cherries: Differential Effects during Pre- and Post-harvest. Front. Plant Sci. 7:602. doi: 10.3389/fpls.2016.00602 Sweet cherry, a non-climacteric fruit, is usually cold-stored during post-harvest to prevent over-ripening. The aim of the study was to evaluate the role of abscisic acid (ABA) on fruit growth and ripening of this fruit, considering as well its putative implication in over-ripening and effects on quality. We measured the endogenous concentrations of ABA during the ripening of sweet cherries (Prunus avium L. var. Prime Giant) collected from orchard trees and in cherries exposed to 4◦C and 23◦C during 10 days of postharvest. Furthermore, we examined to what extent endogenous ABA concentrations were related to quality parameters, such as fruit biomass, anthocyanin accumulation and levels of vitamins C and E. Endogenous concentrations of ABA in fruits increased progressively during fruit growth and ripening on the tree, to decrease later during postharvest at 23◦C. Cold treatment, however, increased ABA levels and led to an inhibition of over-ripening. Furthermore, ABA levels positively correlated with anthocyanin and vitamin E levels during pre-harvest, but not during post-harvest. We conclude that ABA plays a major role in sweet cherry development, stimulating its ripening process and positively influencing quality parameters during pre-harvest. The possible influence of ABA preventing over-ripening in cold-stored sweet cherries is also discussed.

Keywords: sweet cherry, ABA, ripening, over-ripening, ascorbate, vitamin E, cold storage

## INTRODUCTION

In recent decades, sweet cherry has become one of the most important non-climacteric fruits worldwide, with an important distribution to international markets from highly productive countries at origin, such as Turkey, United States, Iran, Italy, and Spain, among others (FAO, 2015). However, both its flavor and nutritional quality is strongly dependent on tree growth conditions at pre-harvest, post-harvest treatments and its consumption at an optimum ripening stage (Ashton, 2007). Over-ripening, which leads to a loss of quality during post-harvest, is associated with fruit darkening, softening and a general loss of organoleptic properties (Meheriuk et al., 1995). Cold treatments are generally used to store them properly during post-harvest in order to avoid fruit quality loss, but the physiological and biochemical mechanisms underlying fruit ripening on the tree and over-ripening during post-harvest are still relatively unknown for sweet cherries.

It has been shown that high concentrations of abscisic acid (ABA) are required for ripening in sweet cherries (Luo et al., 2013; Wang et al., 2015). ABA is a sesquiterpenoid hormone, derived from carotenoids, that is implicated in several physiological processes, from seed dormancy to senescence processes, including plant stress responses and the regulation of fruit development (Nambara and Marion-Poll, 2005; Finkelstein, 2013; Leng et al., 2014). ABA has been shown to play a major role in the ripening process of non-climacteric fleshy fruits, such as cherry fruits, modulating color changes (through modulation of anthocyanin biosynthesis) and sugar accumulation (Kumar et al., 2014; Wang et al., 2015). However, nothing is known about the possible role of ABA in the regulation of fruit quality in terms of vitamin C and E accumulation, or to what extent ABA can affect over-ripening processes in sweet cherries.

Among various quality parameters, the content and composition of water- and lipid-soluble vitamins in edible fleshy fruits is of paramount importance for human health (FAO, 2004). Sweet cherries are rich in vitamin C, which is considered one of the most important water-soluble antioxidants, together with anthocyanins, in this fruit (Serrano et al., 2005). Aside from protecting cells from reactive oxygen species, ascorbate is involved in the regulation of growth processes in plants (Veljovic-Jovanovic et al., 2001), and it plays a role, as a cofactor, in the regulation of 9-cis-epoxycarotenoid dioxygenase (NCED), the key limiting step in the biosynthesis of ABA from carotenoids (Conklin and Barth, 2004). Furthermore, ascorbate recycles oxidized tocopherols (vitamin E), when this lipid-soluble antioxidant reacts with lipid peroxyl radicals in its function of inhibiting the propagation of lipid peroxidation in biological membranes (Munné-Bosch and Alegre, 2002). Although vitamin C has received some attention in the ripening of sweet cherries as a component of organic acids (Serrano et al., 2005), nothing is known about the levels of vitamin E, its possible variations with ripening and regulation by phytohormones in non-climacteric fruits. Only in mango, a climacteric fruit, it has been shown that vitamin E biosynthesis may be modulated by ethylene (Singh et al., 2011).

The aim of this study was to get some insights into the role of ABA in the ripening process of sweet cherries, focusing on the endogenous levels of this phytohormone during fruit development in orchard trees and under different conditions of post-harvest. In addition, to better understand the role of ABA in ripening, as well as the loss of quality during fruit storage, we simultaneously analyzed various parameters associated with the ripening process and the fruit quality, such as fruit biomass, anthocyanin accumulation, and levels of antioxidants, including carotenoids, and vitamins C and E.

### MATERIALS AND METHODS

### Experimental Design and Sampling

Three independent, complementary experiments were performed using sweet cherries (Prunus avium L. var. Prime Giant). The first experiment focused on a study of fruit ripening on the tree followed by an over-ripening process at 23◦C, the second one was performed preventing over-ripening at 4◦C, and the third one was performed to test for the tissular location of vitamins in cherry fruits.

For the first experiment, sweet cherries were obtained from trees growing in an exploited orchard at Partida Vall del Sector III (Lleida, NE Spain). Fruits were harvested at various developmental stages on the tree between 23 and 4 days before harvest, and between 3 and 10 days of post-harvest at 23◦C, which led to over-ripening (Supplementary Figure S1). First sampling in orchard trees was performed during 30th April 2015 (23 days before harvest), which corresponds to 34 days after full bloom. For the second experiment, 10 kg from the same cherry cultivar and orchard were brought to the laboratory 3 days after commercial harvest. Fruits without visual defects were chosen for experiments. Then, half of the fruits were kept at 23 ± 2 ◦C in the laboratory, while the other half were subject to 4 ± 1 ◦C in a cold chamber. In both cases, fruits were kept in darkness and samples were taken daily during storage for 1 week.

A third experiment was performed to evaluate possible tissuespecific accumulation of vitamins in sweet cherries. The pit, flesh and skin from fruits collected 23 days pre-harvest or 3 days postharvest were manually separated and immediately immersed in liquid nitrogen for hormone, anthocyanin and vitamins C and E analyses.

All samplings were performed early in the morning (between 9 and 10 a.m. local time) with an average temperature of 10 ± 2 ◦C during pre-harvest and 23 ± 2 ◦C during post-harvest for the first experiment, and with an average temperature of 4 ± 1 ◦C for the second one. Six fruits per tree from eight trees were randomly sampled at each time point during pre-harvest, and six fruits from commercial boxes were randomly sampled daily during postharvest, for each, 23◦C and cold storage. For all experiments, samples were immediately snap frozen in liquid nitrogen and stored at −80◦C until analyses.

### Endogenous Concentrations of Abscisic Acid

Abscisic acid levels were determined by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) as described previously (Müller and Munné-Bosch, 2011). In short, 100 mg per sample were extracted with 200 µL methanol:isopropanol:acetic acid 50:49:1 (v/v/v) using ultrasonication and vortexing (Branson 2510 ultrasonic cleaner, Bransonic, Danbury, CT, USA) for 30 min. Deuterium-labeled ABA was then added, and after centrifugation at 600 g for 15 min at 4◦C, the pellet was re-extracted using the same procedure. Supernatants were pooled and filtered through a 0.22 µm PTFE filter (Waters, Milford, MA, USA) before analyses. ABA levels were analyzed by using UHPLC-ESI-MS/MS as described in Müller and Munné-Bosch (2011). Quantification was made considering recovery rates for each sample by using a deuteriumlabeled internal standard.

### Fruit Quality Parameters

Fruit biomass was estimated by weighing the samples immediately at each sampling time point or after transferring

fpls-07-00602 May 2, 2016 Time: 11:19 # 2

them to the laboratory in bags (with high humidity to avoid desiccation).

Total anthocyanins were determined spectrophotometrically in methanolic extracts as described (Gitelson et al., 2001). In short, 200 mg per sample were extracted with 1 mL methanol using ultrasonication and vortexing. Extracts were centrifuged at 600 g for 10 min at 4◦C and the pellet was re-extracted following the same procedure. Supernatants were pooled and 1% HCl was added. Then, total anthocyanins were measured spectrophotometrically at 530 nm. Total anthocyanins were calculated using the molar extinction coefficient of cyanidin-3 glucoside as a reference, as described (Siegelman and Hendricks, 1958).

Carotenoids levels were estimated by HPLC after extraction with methanol, as described (Munné-Bosch and Alegre, 2000). In short, samples were extracted with methanol, as described for anthocyanins, and separated on a Dupont non-endcapped Zorbax ODS-5 µm column (250 mm long, 4.6 mm i.d.; 20% Carbon, Teknokroma, St. Cugat, Spain) at 30◦C for 38 min at a flow rate of 1 mL min−1 . The solvent mixture for the gradient consisted of (A) acetonitrile:methanol (85:15, v/v) and (B) methanol:ethyl acetate (68:32, v/v). The gradient used was: 0–14 min 100% A, 0% B; 14–16 min decreasing to 0% A, 100% B; 16–28 min 0% A, 100% B; 28–30 min increasing to 100% A, 0% B; and 30–38 min 100% A, 0% B. Detection was carried out at 445 nm and compounds were identified and quantified as described previously (Munné-Bosch and Alegre, 2000).

The analysis of vitamin C was adapted from Takahama and Oniki (1992) and Queval and Noctor (2007). In short, ascorbic acid and its oxidized form, dehydroascorbic acid were extracted with 6% m-phosphoric acid (w/v) and 0.2 mM diethylenetriaminepentaacetic acid, using ultrasonication and vortexing. After centrifugation at 600 g for 10 min at 4◦C, the supernatants were collected and the pellet was re-extracted following the same procedure. Their levels were determined spectrophotometrically at 265 nm, using the ascorbate oxidase assay. The oxidized state of ascorbate was calculated as DHA/(AA + DHA) × 100, where AA is ascorbate and DHA is dehydroascorbate.

The analysis of vitamin E was performed as described (Amaral et al., 2005). In short, 200 mg per sample were extracted with methanol, exactly as described for anthocyanins, and then filtered prior to HPLC analyses. The HPLC equipment consisted of an integrated system with a Jasco PU-2089 Plus pump, a Jasco AS-2055 Plus auto-sampler and a FP-1520 fluorescence detector (Jasco, Tokyo, Japan). All tocopherol and tocotrienol forms were separated on an Inertsil 100A (5 µm, 30 × 250 mm, GL Sciences Inc., Tokyo, Japan) normal-phase column, operating at room temperature. The flow rate was 0.7 mL min−1 and the injection volume was 10 µL. The mobile phase was a mixture of n-hexane and p-dioxane (95.5:4.5, v/v). Detection was carried out at an excitation of 295 nm and emission at 330 nm. Quantification was based on the results obtained from the fluorescence signal and compared to that of a calibration curve made with authentic standards of each compound (Sigma– Aldrich, Steinheim, Germany).

### Statistical Analysis

Data were analyzed by using one-way (first experiment) or twoway (second experiment) factorial analysis of variance (ANOVA). Multiple comparisons tests were carried out by using Bonferroni post-hoc tests. In all cases, differences were considered significant at a probability level of P ≤ 0.05. Furthermore, correlation analyses using the Spearman rank's correlation were made. All statistical analyses were performed using the SPSS 20.0 statistical package.

### RESULTS

### ABA Levels Increase During Ripening on the Tree but Decrease During Over-Ripening

Fruit biomass increased fivefold during ripening on the tree (from 23 days pre-harvest to 3 days post-harvest), to decrease later by 20% due to over-ripening for 1 week (from day 3 to day 10 of post-harvest at 23◦C, **Figure 1**). Anthocyanin levels increased from non-detectable values to 95 µg/g fruit during pre-harvest (between 23 and 4 days preharvesst), to increase even further up to 582 µg/g fruit at 5 days post-harvest. Then, anthocyanin levels remained relatively constant at high levels during over-ripening until the end of the experiment (10 days post-harvest, **Figure 1**).

Abscisic acid levels increased sharply from 26 ng/g fruit at 23 days pre-harvest to 540 ng/g fruit at 11 days pre-harvest, to keep later constant until 4 days pre-harvest (**Figure 2**). Over-ripening at 23◦C led to a depletion of endogenous ABA concentrations in the fruit to attain minimum values of 142 ng/g fruit at 10 days post-harvest. It is noteworthy that ABA increases preceded anthocyanin accumulation during pre-harvest. In contrast, ABA did not change in parallel with anthocyanin accumulation during post-harvest (**Figures 1** and **2**).

Levels of carotenoids decreased sharply during fruit ripening on the tree (**Table 1**). Violaxanthin, an ABA precursor, decreased from 0.58 mg/g FW at 23 days to non-detectable values at 4 days pre-harvest (**Table 1**), which occurred in parallel with increases of ABA levels during fruit ripening on the trees (**Figure 2**). Lutein and zeaxanthin levels also decreased progressively down to nondetectable values during fruit ripening on the tree, while fruits at 4 days pre-harvest still kept 0.17 mg/g FW of β-carotene. The amounts of this antioxidant expressed per fruit unit increased during ripening, attaining maximum levels of 3 mg per fruit unit at 4 days pre-harvest (**Table 1**). Carotenoids were not detected during post-harvest (data not shown).

Total ascorbate levels increased during pre-harvest to decrease later during post-harvest, both when expressed on a fresh weight and a fruit unit basis (**Figure 3**). Interestingly, ascorbate levels showed a biphasic response during post-harvest, with minimum ascorbate levels at 5 and 10 days post-harvest. It is noteworthy that the oxidation state of ascorbate kept constant, both, during pre- and post-harvest, but decreased sharply from around 40% to levels below 20% just after harvest (**Figure 3**).

Vitamin E levels were much lower than those of ascorbate, with maximum levels of 3.5 µg/g fruit being attained at 15 and

11 days pre-harvest and at the end of the experiment (**Figure 4**). Sharp fluctuations in total vitamin E levels were mainly due to those of α-tocopherol, the major vitamin E form present in fruits (Supplementary Figure S1). γ-Tocopherol levels were lower but also more stable than those of α-tocopherol. Vitamin E levels tended to increase during ripening, an effect that was particularly observed for γ-tocopherol (Supplementary Figure S1) and when results where expressed on a fruit unit basis (**Figure 4**). Neither vitamin E levels nor those of α- and γ-tocopherol were altered during post-harvest either when expressed per g fruit or per fruit unit (**Figure 4**; Supplementary Figure S2). β- and δ-tocopherols, and tocotrienols were not detected in cherry fruits.

### Variations in ABA Levels during Cold Storage

Cold storage prevented over-ripening, as observed with the maintenance of visual fruit firmness (Supplementary Figure S2), biomass and anthocyanin levels (**Figure 5**). Cold treatment prevented anthocyanin accumulation, an effect that was already

ABA levels are given both per FW and per fruit unit. Harvest (time 0 in the X axis) corresponds to 57 days after full bloom.

observed at 2 days of cold storage. ABA levels increased in response to cold storage, with an increment at 2 days of treatment (**Figure 6**). Thereafter, ABA levels in cold-stored fruits did not increase further but kept always at higher levels compared to fruits stored at 23◦C. In this case, ABA levels inversely correlated, or simply did not correlate with those of anthocyanins. During over-ripening at 23◦C, ABA levels decreased, while those of anthocyanins increased. When over-ripening was prevented by cold storage, enhanced ABA levels did not lead to changes in anthocyanin accumulation.

Cold storage did not alter total ascorbate levels, but affected its oxidation state. The ascorbate oxidation state increased in response to cold treatment, but differences were small and post hoc analyses did not reveal significant difference at any time point (**Figure 7**). In contrast, ABA levels correlated with vitamin E levels in cold-stored fruits, those of total vitamin E were increasing in parallel with ABA, during the first days of cold treatment (**Figure 8**). The levels of α- and γ-tocopherol were not significantly altered by cold treatment when analyzed separately (Supplementary Figure

Levels of violaxanthin, lutein, zeaxanthin and β-carotene are given at 23, 15, and 4 days pre-harvest, both on a fresh weight (FW) and fruit unit basis. ND, not detected. Different letters indicate significant differences between time points using Bonferroni post hoc tests (ANOVA, P < 0.05).

S3), thus indicating that cold effects on total vitamin E levels (**Figure 8**) were cumulative. It is noteworthy that αand γ-tocopherol followed a completely different tissuespecific accumulation, with γ-tocopherol accumulating, almost exclusively (>99%), in the pit (**Figure 9**). In contrast, α-tocopherol, anthocyanins, ascorbate and ABA were all detected in the pit, flesh and skin during both pre- and post-harvest (**Figure 9**).

### DISCUSSION

Sweet cherry is a non-climacteric fruit, which ripening is known to be promoted by ABA (Setha et al., 2005). Its ethylene concentration is low and has no direct effect in the ripening of sweet cherries (Hartmann, 1992; Kondo and Gemma, 1993), although it may influence anthocyanin accumulation (Kondo and Inoue, 1997). ABA is, however, the phytohormone that plays

significant. ABA levels are given both per FW and per fruit unit.

a major role in the regulation of anthocyanin accumulation and organoleptic sweet cherries properties, such as the ratio of total soluble sugars to total acidity (Kondo and Gemma, 1993; Kondo and Inoue, 1997; Luo et al., 2013). Studies in other non-climacteric fruits, such as grapes, have also shown that ABA not only modulates color development and sugar accumulation, but it may also be implicated in the control of softening during the ripening process (Castellarin et al., 2016). Here, we provide correlative evidence supporting a role for ABA in the regulation of both anthocyanin and vitamin E accumulation during pre-harvest, but not during post-harvest, in sweet cherries "Prime Giant." Furthermore, results suggest that ABA may help prevent over-ripening during post-harvest at 4◦C.

We found that ABA levels strongly and positively correlate with anthocyanin accumulation during ripening of fruits on the tree (**Table 2**), which is in agreement with previous studies (Luo et al., 2013). However, a strong negative correlation was observed between endogenous concentrations of ABA and anthocyanin levels during post-harvest (**Table 2**). Over-ripening

are given both per FW and per fruit unit.

at 23◦C led to progressive decreases in ABA concentrations, while anthocyanin accumulation kept at high levels, thus suggesting an inhibitory role for ABA in over-ripening (**Figures 1** and **2**). Furthermore, ABA levels increased after 2 days of cold storage, while anthocyanin levels kept at lower levels at 4◦C relative to 23◦C (**Figures 5** and **6**), thus suggesting ABA might prevent over-ripening in cold-stored fruits. The role of ABA in over-ripening has been poorly studied to date,

particularly in non-climacteric fruits. However, the application of antitranspirants, such as ABA, in rambutan, a non-climacteric fruit, has been shown to be effective in preventing over-ripening (Siriphollakul et al., 2006), thus supporting further the idea that ABA helps promote ripening in fruits on the tree, but delays over-ripening in detached fruits during post-harvest. This indicates that ABA does not act alone but together with other signaling compounds in the regulation of the ripening

all cases.

Correlation coefficients values are given followed by one or two asterisks when the correlation is significant or highly significant (P ≤ 0.05 and 0.001, respectively). Significant correlations with coefficients above 0.35 are shown in bold. Absence of an asterisk indicates the correlation was not significant. Data was analyzed altogether, and also separately (for each experiment, differentiating also pre- and post-harvest data for Experiment 1).

process in fruits on the tree, an aspect that warrants further investigations.

Aside from its role in the regulation of the ripening process of fruits on the tree, by modulating softening, sugar accumulation and color development (though the modulation of anthocyanin accumulation, Luo et al., 2013; Wang et al., 2015; Castellarin et al., 2016), nothing is known about the possible effects of ABA on vitamin accumulation in sweet cherries or other nonclimacteric fruits. Vitamin C is a water-soluble compound that acts as a cofactor for many iron and copper hydroxylases and dioxygenases involved in key physiological processes in humans, such as in the production of collagen and the synthesis of carnitine (Bender, 2003; Johnston et al., 2007). That is the reason why the frequent intake of foods rich in bioactive compounds, such as vitamin C, is associated with a healthy diet (Arrigoni and De Tullio, 2002). In addition, this compound is considered as one of the most important antioxidants for plant growth and defense (Foyer and Noctor, 2011), which is present in

many plant cell compartments, such as mitochondria, plastids, peroxisomes and the apoplast (Smirnoff, 2000; Foyer, 2001). Moreover, ascorbate is the principal nonenzymatic water-soluble antioxidant that is able to eliminate reactive oxygen species (Cadenas and Packer, 2002). Vitamin C is especially vulnerable to oxidative and enzymatic degradation in raw fruits and vegetables (Redmond et al., 2003). Some studies have reported a loss of vitamin C in many fruits stored under non-optimal conditions after harvest (Munyaka et al., 2010; Neves et al., 2015). Although correlation analyses did not reveal any significant relationship between endogenous concentrations of ABA and vitamin C levels during ripening, over-ripening or cold treatment (**Table 2**), the present study confirmed that this vitamin is present at high amounts in sweet cherries, attaining maximum levels of 3 mg per fruit unit just after harvest, and it was found that its oxidation increases after 7 days of cold storage. Furthermore, ascorbate is known to act as a cofactor of 9-cis-epoxycarotenoid dioxygenase (NCED), the key limiting step in the biosynthesis of ABA from carotenoids, particularly neoxanthin and/or violaxanthin (Conklin and Barth, 2004). In the present study, violaxanthin levels decreased concomitantly with increases of ABA levels during ripening of fruits on the trees, which is consistent with a role for violaxanthin as a precursor of ABA in sweet cherries (Luo et al., 2013).

On the other hand, vitamin E, a lipid-soluble antioxidant in cell membranes, also with health-promoting effects (Booth et al., 2004), is found at high concentrations in some fruits, such as kiwis or avocados (Chun et al., 2006), but it has received little consideration in sweet cherries, mainly due to their low levels in the fruit, at least, compared to other antioxidants, such as anthocyanins or vitamin C. Among vitamin E compounds, both αand β-tocopherol were previously shown to be present in sweet cherries, being α-tocopherol the most abundant with amounts around 1 µg/g fruit (Bastos et al., 2015), which is similar to the amounts obtained in the present study (Supplementary Figure S3). However, we did not detect β- but, instead, γ-tocopherol in sweet cherries, which accumulated particularly in the pit (**Figure 9**). Most importantly, we found a positive correlation between endogenous concentrations of ABA and vitamin E accumulation in sweet cherries, particularly at pre-harvest (**Table 2**). Interestingly, endogenous concentrations of ABA correlated more strongly with γ- than with α-tocopherol levels. Previous studies have shown the presence of an ABAresponsive element (ABRE) in the promoter region of HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD), which encodes for the enzyme responsible of the formation of homogentisate, needed for the biosynthesis of all vitamin E compounds (Chaudhary and Khurana, 2009; Falk and Munné-Bosch, 2010). Therefore, our data supports the contention that ABA is implicated in the biosynthesis of vitamin E compounds in sweet cherries, as it has been shown in leaves of plants exposed to various abiotic stresses (Chaudhary and Khurana, 2009; Munné-Bosch et al., 2009). It is noteworthy that the correlative evidence obtained in the present study supporting a link between ABA and vitamin E biosynthesis was observed in fruits that were ripening on the tree during pre-harvest, but not during post-harvest at 23◦C. Furthermore, enhanced vitamin E levels were preceded by ABA increases during cold storage, thus suggesting ABA may also regulate tocopherol accumulation in response to cold stress in sweet cherries. This may indeed be a defensive response, since both ABA and tocopherols are known to be needed to combat coldinduced reactive oxygen production in plants (El Kayal et al., 2006).

### CONCLUSION

The ABA plays a major role in the control of the ripening process in sweet cherries, particularly stimulating this process during pre-harvest and positively influencing quality parameters, such as the accumulation of anthocyanins and vitamin E. Further research is, however, needed to better understand the mechanisms underlying the regulation of vitamin E biosynthesis by ABA during pre-harvest and cold storage, as well as the inhibitory role of ABA in the over-ripening of sweet cherries, beyond its possible function as an antitranspirant.

### AUTHOR CONTRIBUTIONS

VT and SM-B conceived and designed the experiments with the help of NT. VT, NT, and PM performed the experiments. SM-B wrote the manuscript with the help of VT; all authors contributed to the discussion, revised and approved the final manuscript.

### FUNDING

Research was supported by the Generalitat de Catalunya through the ICREA Academia prize given to SM-B.

### ACKNOWLEDGMENTS

We are very grateful to Maren Müller and Serveis Científicotècnics (University of Barcelona) for their help with ABA analyses. We also thank Josep Maria Gilart for giving us the opportunity to sample the fruits in his orchard.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016.00602

### REFERENCES

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development and in response to abscisic acid and auxin at onset of fruit ripening. Plant Growth Regul. 75, 455–464. doi: 10.1007/s10725-014-0006-x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Tijero, Teribia, Muñoz and Munné-Bosch. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Structural and Functional Analysis of the GRAS Gene Family in Grapevine Indicates a Role of GRAS Proteins in the Control of Development and Stress Responses

### Edited by:

Jérôme Grimplet 1

Richard Sayre, New Mexico Consortium at Los Alamos National Labs, USA

#### Reviewed by:

Vasileios Fotopoulos, Cyprus University of Technology, Cyprus Cordelia Bolle, Ludwig Maximilian University, Germany

> \*Correspondence: Ana M. Fortes<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science

Received: 03 December 2015 Accepted: 07 March 2016 Published: 30 March 2016

#### Citation:

Grimplet J, Agudelo-Romero P, Teixeira RT, Martinez-Zapater JM and Fortes AM (2016) Structural and Functional Analysis of the GRAS Gene Family in Grapevine Indicates a Role of GRAS Proteins in the Control of Development and Stress Responses. Front. Plant Sci. 7:353. doi: 10.3389/fpls.2016.00353 1 Instituto de Ciencias de la Vid y del Vino (Consejo Superior de Investigaciones Científicas-Universidad de La Rioja-Gobierno de La Rioja), Logroño, Spain, 2 Faculdade de Ciências de Lisboa, BioISI, Universidade de Lisboa, Lisboa, Portugal, 3 Instituto de Tecnologia de Química Biológica, Biotecnologia de Células Vegetais, Oeiras, Portugal

\*

, Rita T. Teixeira2

,

, Patricia Agudelo-Romero2

Jose M. Martinez-Zapater 1 and Ana M. Fortes 2, 3

GRAS transcription factors are involved in many processes of plant growth and development (e.g., axillary shoot meristem formation, root radial patterning, nodule morphogenesis, arbuscular development) as well as in plant disease resistance and abiotic stress responses. However, little information is available concerning this gene family in grapevine (Vitis vinifera L.), an economically important woody crop. We performed a model curation of GRAS genes identified in the latest genome annotation leading to the identification of 52 genes. Gene models were improved and three new genes were identified that could be grapevine- or woody-plant specific. Phylogenetic analysis showed that GRAS genes could be classified into 13 groups that mapped on the 19 V. vinifera chromosomes. Five new subfamilies, previously not characterized in other species, were identified. Multiple sequence alignment showed typical GRAS domain in the proteins and new motifs were also described. As observed in other species, both segmental and tandem duplications contributed significantly to the expansion and evolution of the GRAS gene family in grapevine. Expression patterns across a variety of tissues and upon abiotic and biotic conditions revealed possible divergent functions of GRAS genes in grapevine development and stress responses. By comparing the information available for tomato and grapevine GRAS genes, we identified candidate genes that might constitute conserved transcriptional regulators of both climacteric and non-climacteric fruit ripening. Altogether this study provides valuable information and robust candidate genes for future functional analysis aiming at improving the quality of fleshy fruits.

Keywords: abiotic stress, biotic stress, fruit ripening, grapevine, GRAS gene family, transcription factor

## INTRODUCTION

Transcription factors play an important role in the regulation of plant development and disease response. Among them, the plant gene family of GRAS transcription factors was defined based on nuclear localization, DNA binding and transcriptional activation features (Silverstone et al., 1998; Itoh et al., 2002; Morohashi et al., 2003). In addition, in vivo association of specific GRAS proteins with promoter regions of several putative GRAS target genes was confirmed by chromatin immunoprecipitation (Zentella et al., 2007). The name GRAS derives from its first three identified members, namely, gibberellic acid insensitive (GAI), repressor of GA1 (RGA), and scarecrow (SCR; Pysh et al., 1999; Bolle, 2004). Moreover, the Arabidopsis GRAS Protein SCL14 was shown to be essential for the activation of stress-inducible promoters (Fode et al., 2008).

Genome-wide analysis performed in nearly 30 plant species from more than 20 genera revealed that this gene family is widely distributed in the plant kingdom (Tian et al., 2004), reviewed by Hirsch and Oldroyd (2009) and it is likely to have emerged first in bacteria (Zhang et al., 2012). GRAS proteins are typically 400–700 amino acids in length and exhibit considerable sequence homology among each other in their C-terminus, where five conserved motifs, namely LHR I, VHIID, LHR II, PFYRE, and SAW are located (Pysh et al., 1999; Tian et al., 2004). The VHIID domain of a GRAS protein from Brassica napus interacts with a histone deacetylase, supporting the notion that GRAS proteins regulate gene expression at the level of transcription (Gao et al., 2004).

The amino acid sequences of GRAS proteins are highly variable at the N-terminus, which may be responsible for the specificity of their regulatory functions (Tian et al., 2004). For example, a subgroup of GRAS proteins, which function in several plant species as repressors of gibberellin signaling, share in their N-terminal region the amino acid sequence DELLA and are thus referred as DELLA proteins (Silverstone et al., 1998).

The GRAS protein family groups into eight well-known subfamilies: DELLA, HAM, LISCL, PAT1, LAS, SCR, SHR, and SCL3. However, in between 8 and 13 distinct clades can be discriminated in different studies (Huang et al., 2015; Bolle, 2016). Several GRAS genes from plant species such as Arabidopsis, rice, and barley have been functionally characterized, including CIGR (PAT subfamily), GAI, RGL, RGA, and SLN1 (DELLA subfamily), MOC1 (LAS subfamily) as well as other genes from SCR, SHR, LISCL, SCL, and HAM subfamilies (Fu et al., 2002; Stuurman et al., 2002; Day et al., 2004), reviewed by Bolle (2016).  GRAS genes have also been connected with plant disease resistance and abiotic stress response (Mayrose et al., 2006; Ma et al., 2010; Cui, 2012). Furthermore, in the model legume species Medicago truncatula and Lotus japonicus two GRAS proteins were shown to be required for nodule morphogenesis (Kalo et al., 2005; Heckmann et al., 2006). Recently, the GRAS transcription factor RAM1 and the novel GRAS protein RAD1 were reported to be involved in arbuscule development (Xue et al., 2015). The formation of multicomponent GRAS transcription factor complexes with other proteins was suggested to be a prerequisite for elicitation of nodulation or mycorrhization (Oldroyd, 2013). Genes coding for GRAS transcription factors were also identified as targets of miRNAs during tomato fruit development and ripening (Moxon et al., 2008; Karlova et al., 2013).

So far, various in silico genome analyses have predicted the existence of 33, 57 and 48 GRAS genes in the whole genome of Arabidopsis, rice and Chinese cabbage, respectively (Tian et al., 2004; Song et al., 2014). As more species have their complete reference genome sequenced, additional GRAS genes can be identified as it is the case of Vitis vinifera.

Due to its economic relevance, much research in grapevine genomics has been carried out during the last decade. Among these studies, the release of the whole grapevine genome sequence in 2007 represented a breakthrough to promote its molecular genetics analysis (Jaillon et al., 2007). Based on the published sequence data, comprehensive analysis of a given gene family can be performed to uncover its molecular functions, evolution, and gene expression profiles. These analyses can contribute to the understanding of how genes in gene families control traits at a genome-wide level.

Previous comparative analysis with Chinese cabbage genome predicted 43 GRAS transcription factors in V. vinifera (Song et al., 2014). In this work, we update this number to 52, a very similar number of GRAS genes to the 53 recently reported in tomato (Huang et al., 2015). Furthermore, we provide a detailed analysis of the GRAS transcription factors relationships among several plant species through comparative genomics together with the identification, structural analysis, and mapping of the GRAS transcription factors onto the grapevine chromosomes. Finally, expression analyses based on microarray and RNAseq data suggest that GRAS proteins play an important role in grape ripening and in response to abiotic and biotic stresses.

### MATERIALS AND METHODS

### Identification of GRAS Genes

Genes previously identified as encoding GRAS proteins in (Grimplet et al.) were blasted (blastp and tblastn) against the grapevine genome 12x.2 (https://urgi.versailles.inra.fr/ Species/Vitis/Data-Sequences/Genome-sequences), the nonredundant list of genes in (Grimplet et al., 2012) and the COST annotation gene set available at the ORCAE website (http:// bioinformatics.psb.ugent.be/orcae/). Results from different analysis were manually cross-checked to identify new potential loci corresponding to GRAS genes in the grapevine genome. The UGene software (Okonechnikov et al., 2012) was used to design the gene models on the grapevine genome and test their structure.

### Gene Structure Analysis

The potential coding DNA sequences (CDS) were blasted (blastx) against the NCBI public database to compare the structures with other known GRAS genes in other species and the NCBI Refseq predictions of the grapevine genes. When discrepancies were observed, gene models were corrected using the UGene software. Loci bearing genes that were not functional were eliminated from the list. A GFF file with the GRAS genes was designed, uploaded into the IGV software and the RNAseq data available (shoot tips, leaves, flower inflorescences and seed tissues) in the laboratory were used to double-check the exon structure of the genes. Final models were uploaded in the V. vinifera ORCAE database (Sterck et al., 2012; Grimplet et al., 2014).

### Sequence Alignment and Phylogenetic Analysis

Sequence information on previously reported GRAS proteins of Arabidopsis thaliana was retrieved from the Arabidopsis Information Resource (https://www.arabidopsis.org/browse/ genefamily/GRAS.jsp). Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). Multiple sequence alignment was inferred using MUSCLE (Edgar, 2004). The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model (Jones et al., 1992). The bootstrap consensus tree inferred from 100 replicates was taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 30% of bootstrap replicates were collapsed. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The coding data was translated assuming a Standard genetic code table. All positions with less than 95% site coverage were eliminated. The genes were named according to Grimplet et al. (2014) based on the distance homology with Arabidopsis genes.

The alignment file between Arabidopsis and grapevine sequences was uploaded to the Jalview and UGene software for manual adjustment of the alignment and manual motif editing. Motifs identified in Tian et al. (2004) were flagged and labeled for the grapevine genes; additional motifs of high homology were also identified (at least 50% homology within the members of the subfamily on at least 10 amino acids) among grapevine sequences.

### Expression Analysis

Expression data were retrieved from three different microarray platforms (Affymetrix Genchip (16k probesets) GrapeGen (21k probesets), Vitis Nimblegen array (29k probesets), and from our in-house RNAseq projects. Data normalization was performed on all the array of each platform (RMA normalization). After retrieving the values for the probesets corresponding to each gene, the values for the 3 or 4 replicates of the same condition were averaged to obtain a total of 256 conditions (organ, cultivar, treatment, platform). Based on expression data of the grapevine gene expression atlas (Fasoli et al., 2012), a plant ontology ID was attributed to each gene if expression intensity in a tissue was above a defined threshold of absolute log2 value of 8 or absolute value of 256. The same data were used for the co-expression analysis with the whole set of genes available on the Nimblegen platform. Hierarchical clustering with Pearson correlation as metric and average linkage cluster method was performed. Genes considered as having the same profile should present a distance threshold between each other lower than of 0.2.

 Heat maps were performed with the ComplexHeatmap R package (https://github.com/jokergoo/ComplexHeatmap).

### Comparison to Other Plant Species

We performed a sequence comparison using the GRAS genes from 16 plant species (A. thaliana, Brassica rapa, Carica papaya, Eucalyptus grandis, Citrus sinensis, Malus domestica, Prunus persica, Fragaria vesca, Glycine max, M. truncatula, Cucumis melo, Populus trichocarpa, Solanum lycopersicum, Zea mays, Sorghum bicolor, Oryza sativa) retrieved at http://planttfdb.cbi.pku.edu.cn. We identified orthologous genes in genomes from the sixteen species following what was performed in Jaillon et al. (2007). Each pair of predicted gene sets was aligned with the BLASTp algorithm, and alignments with an e-value lower than 1e−20 and sequence homology higher than 40% were retained. If a comparison is above that value, the two genes were considered homologs. Two genes, A from Vitis genome GV and B from genome GX, were considered orthologs one-to-one if B was the best match for gene A in GX and A was the best match for B in GV. A phylogenetic tree was constructed with the GRAS genes from these species with the same parameters as before.

## RESULTS

### Identification and Structural Annotation of the GRAS Genes

Genes that were previously identified as GRAS in the grapevine genome (Grimplet et al., 2012) were used to performed sequence comparison analyses, either against the most up to date gene predictions from CRIBI V1 and V2, the NCBI refseq (on the 12Xv1 of the genome assembly) and the VCOST (on the 12Xv2 of the genome assembly) as well as directly against the reference genome sequence to check whether any potential gene could had been missed by these predictions. In this way, we identified 80 genome regions that shared homology with at least one of the genes.

Gene models were curated using the data collected from gene structure comparisons using different databases as well as the available RNAseq data from our laboratory (Royo et al., 2016) to validate actually expressed exons. This data also allowed evaluating the expression of newly detected genes, not represented in microarray data, by redoing the bioinformatics analysis of original RNAseq data with an updated GFF file. A total of 52 GRAS genes with a functional structure were identified in the grapevine genome (**Table 1**). Data relative to the detection of GRAS genes in previous genome annotations or gene-sets are summarized in **Supplementary Table 1**. Three additional genes were detected compared to the automatic annotation CRIBI V1, one was not seen in the V1, but was known in the annotation from the 8x genome (**Table 1**). The structure of 14 genes CRIBI annotated genes was curated in our work.

Exon/ intron structure is highly conserved amongst GRAS genes in grapevine and most of them presented only one exon which is a common feature of this gene family observed in many plant species (Song et al., 2014; Huang et al., 2015; Lu et al., 2015). Only six genes contained introns (**Table 1**). Five of them contained two exons while VviLISCL7 contained four. No subfamily showed a specific intron/exon structure (**Supplementary Table 1**) while the size of GRAS genes varied greatly, ranging from 294 nucleotides (VviSCL3b) to 2349 nucleotides (VviSCR1). Forty-one genes (79%) had a length longer than 1400 bp.

### Phylogenetic Analysis, Nomenclature, and Motif Analysis

For gene nomenclature, a phylogenetic tree of the GRAS protein coding genes in V. vinifera and Arabidopsis was constructed (**Figure 1**) as recommended by the Super-Nomenclature Committee for Grape Gene Annotation (sNCGGa; Grimplet et al., 2014). This analysis identified the eight subfamilies previously described in other plant species: DELLA, HAM, LISCL, PAT, LS, SCR, SHR, and SCL3. Furthermore, five additional groups were detected that could not been assigned to any of those subfamilies (**Figure 1**). Interestingly, 13 groups were also recently found in tomato (Huang et al., 2015). For individual gene nomenclature, we attributed gene symbols/names using preferentially those previously used when they fit the recommendations of the sNCGGa. If a gene was not described before and had an Arabidopsis ortholog, the corresponding Arabidopsis gene name was used. In addition, to distinguish different subfamily members, names were composed by the subfamily symbol followed by a number or a letter (when the subfamily symbol ended with a number). Among the new detected subfamilies, two showed an Arabidopsis homolog that had not been previously described in a subfamily. These were labeled SCL26 and GRAS8. The 3 remaining new subfamilies were labeled GRASV1, GRASV2 and GRASV3.

Five characteristic conserved motifs were identified in the Cterminus of the GRAS proteins, namely LHRI, VHIID, LHRII, PFYRE, and SAW (summarized by subfamilies in **Figure 2** and detailed in **Supplementary Image 1**). The LHRI motif presented two units (A and B). Leucine repeats found in Unit A were found to be conserved in all GRAS proteins (**Figure 2** and **Supplementary Image 1**) as previously reported (Tian et al.,

subfamilies GRAS8, GRASV1, GRASV2, GRASV3, and SCL26.

#### TABLE 1 | Genome localization of the 52 grapevine GRAS genes.

Bold IDs correspond to genes which CDS structure was curated regarding v1 annotation. Italics indicate the gene is new when compared to v1 annotation. Genes VviSCL3d, VviGRASV3a, and VviGRASV3b correspond to newly detected genes compared to V1. VviSCL3d was already known in the 8x genome. The 14 CRIBI annotated genes with curated structure in this work are in bold.

2004). Unit B contained a putative nuclear localization signal (NLS). The canonical NLS was present in the cluster of DELLA proteins in the phylogenetic tree (**Figure 1**) though it appeared degenerated in VviRGA3 (**Supplementary Image 1**).

The VHIID motif contained three units (A, B, and C). GRAS proteins could be divided into several distinct groups based on conservation of Unit A. Groups such as PAT, DELLA, and HAM presented high conservation of amino acids (VI, IX, and XIII respectively, **Figure 2**). Unit B was extremely conserved and the C unit had a conserved pattern of LRITG (Pysh et al., 1999; Tian et al., 2004). The L was substituted by I or V and in the case of DELLA proteins by F unit.

The LHRII motif embraced units A and B. In Unit A, three regularly spaced leucine heptad repeats (LX6LX6L) could be found followed by several irregularly spaced leucine repeats. In Unit B, many GRAS proteins had a conserved LXXLL pattern (DELLA, SCL3, and LS groups) as previously described (Tian et al., 2004; **Figure 2** and subgroups X, VIII, and XII). The PAT1 and SCR groups presented different conserved patterns (VII and XI).

The PFYRE motif could be divided into three units: P, FY, and RE. On the other hand, the SAW motif was composed of two units, RVER and W-W-W (**Figure 2**). RVER could be noticed in many but not all GRAS proteins. Members in the HAM subfamily lacked the RVER domain in their C-termini as well as some members of the SHR group (**Figure 2** and **Supplementary Image 1**). The W-W-W unit included three subunits: W-G, L-W, and S-W (**Figure 2**).

In the N-terminus several units were found, in accordance with previous reports (Tian et al., 2004). Units I and II of the LISCL group, units III and IV of DELLA proteins, and unit V of SCR group (**Figure 2**). Only one sequence in Arabidopsis

(AtRGL2) and its ortholog in V. vinifera presented domain V in the SCR group. The TVHYNP domain is characteristic of DELLA proteins (unit IV). In two V. vinifera sequences (VviLISCL2 and VviLISCL7) the domains I and/or II of LISCL proteins were missing due to the fact that the N-terminus is too short (**Supplementary Image 1**). The N region was much conserved in LISCL. The N- terminus of SHR proteins was also very short. Furthermore, in HAM subfamily we identified two new motifs named XIV and XV and in PAT subfamily a new motif named XVI (**Figure 2**). The consensus sequences for the new motifs are for XIV: TSVLDTRRSPSPPTSTSTSTL+SS++GGG; and for XV: ++EQS+L+WI+GDV+DPS+G; XVI: RELE+ALLGPDDDD).

Besides these eight known groups, five new additional groups were identified. A new V. vinifera group (formed by four proteins- VviGRAS V1a-Vd) showed similarity with SCR proteins but lacked the SCR motif (**Figures 1**, **2**). This new subfamily was not present in Arabidopsis and was named GRASV1, with V for Vitis. However, this subfamily is apparently only absent in Arabidopsis and Brassica as observed in a comprehensive phylogenetic analysis that includes grapevine and fifteen other plant species (**Supplementary Image 2**).

A subgroup of proteins with much similarity to the SCL group did not present VIII domain including AtGRAS8 and its ortholog in V. vinifera (VviGRAS8). Roman numeric nomenclature for subfamilies as used in Lu et al. (2015) was considered confounding since it was also used to label the motifs, so this subfamily was renamed as VviGRAS8, following the name of the Arabidopsis gene.

Based on the original phylogenetic analysis (**Figure 1**) we detected a third subfamily apparently related to the Arabidopsis gene SCL26 but the broad species analysis (**Supplementary Image 2**) revealed that this subfamily should be split in 3 distinct subfamilies since only two genes were grouped with SCL26 in the species analysis. All these proteins were also phylogenetically related to the HAM subfamily but lacking the XIII domain, a reason why they were not included in the HAM group. Furthermore, we identified GRASV2 and GRASV3 subfamilies within the HAM-like group. Both gene subfamilies had representative genes in other species (**Supplementary Image 2**).

From the alignment of predicted GRAS domain sequences we identified members containing partial GRAS domains with missing motifs (**Supplementary Image 1**). The gene VviSCL3b seemed severely truncated, it presented a premature stop codon lacking the motifs PFYRE and SAW). Interestingly, this gene whose predicted protein has 98 aminoacids is homologous to SlGRAS35 which only contains 85 aminoacids Huang et al., 2015.

As mentioned previously we analyzed the orthologous relationships of GRAS genes in V. vinifera and other species (**Figure 3** and **Supplementary Image 2**). The orthologous relationships were classified into three categories: (i) genes present in grapevine and absent in a given species; (ii) grapevine genes showing a one-to-one relationship with one gene from a given species; (iii) grapevine genes having homologs in a given species, but without no clear putative ortholog (**Figure 3**). When grapevine genes were compared only to Arabidopsis, 18 genes

FIGURE 3 | Grapevine GRAS genes orthology against plant species with sequenced genome. Green: a one-to-one ortholog in the species (ortholog one-to-one = best match in the species that has the grapevine deduced protein as the best match in grapevine.). Gray: the grapevine deduced protein has homology in the species genome but no one-to-one ortholog was detected (the best match do not have the grapevine deduced protein as best match). White: no match in the species.

showed a one-to-one ortholog relationship with an Arabidopsis gene, a value slightly higher to the 15 obtained in the comparative analysis performed between Prunus mume and Arabidopsis (Lu et al., 2015). These genes likely correspond to well-conserved functions between both species. Eleven grapevine genes had homologs in Arabidopsis but no one-to-one relationship could be found. On the other hand, 23 genes do not have homologs in Arabidopsis.

A phylogenetic tree considering several mono and dicotyledonous species together with a sequence comparison were performed to identify genes with widely conserved functions among species (**Figure 3**). Genes that might represent evolutionary conserved functions were VviPAT1, VviSHR1, VviSCR1, and VviSCL26g since orthologs were found in all the species analyzed (**Figure 3**).

GRAS gene family has considerably evolved since the divergence of monocot and eudicot plants as determined by the orthologous relationship of GRAS genes in several species. The phylogenetic analysis of LISCL, HAM, PAT, and SCL groups revealed independent clusters with many members from only monocotyledonous species (**Supplementary Image 2**). On the other hand, E. grandis and P. trichocarpa putative specific subgroups were also noticed. GRAS family expanded significantly in these fast-growing woody tree species. According to Liu and Widmer (2014) there are 106 and 94 GRAS genes in Populus and Eucalyptus, respectively. In V. vinifera no species-specific subgroup was found.

Regarding the new V. vinifera subfamilies, the results indicated that group comprising VviGRASV1a-Vd, existed before the divergence of dicots and monocots and were lost in Arabidopsis and B. rapa (**Figure 3** and **Supplementary Image 2**). However, VviGRASV1c and VviGRASV1d did not appear in monocots.

The genes VviGRASV2a- and VviGRASV3c also presented orthologs in some species but not in Arabidopsis and B. rapa. The gene VviGRASV2a is homologous to two genes from tomato (**Supplementary Image 2**); therefore they may eventually play similar functional roles in fleshy fruits such as grapevine and tomato. Orthologs of VviGRASV2a can be found in many other species whereas for VviGRASV2b no ortholog was detected (**Figure 3**).

Regarding the GRAS8 subfamily, gene VviGRAS8a was included in a large cluster with AtSCL28 and homologous genes in tomato and rice. It has orthologs in several species including tomato but not in rice. VviGRAS8b has homologs in several mono and dicotyledonous species but not in Arabidopsis and B. rapa. Orthologs were not found in Arabidopsis and monocots.

VviSCL26b clustered with AtSCL26 and several other species whereas VviSCL26a did not have homologs/orthologs in Arabidopsis. As expected, since they were never described before in other species, the genes from the new families' shared little homology with genes from Arabidopsis.

### Chomosomal Location of the GRAS Genes

GRAS genes were distributed unevenly among the nineteen chromosomes of the grapevine genome though they were mapped to all the chromosomes (**Figure 4**). The highest number of GRAS genes was found on chr 6 and 13, with 6 and 7 genes respectively. The high number of GRAS sequences in these two chromosomes is mainly due to the presence of repeats of genes belonging to the same group (LISCL). On the other hand, chr 3, 9, 10, 11, 15, 16, and 17 only bore one gene. GRAS genes belonging to the same group were located in chromosomal regions that may represent paralogous segments resulting from ancestral polyploidization events (Jaillon et al., 2007; Velasco et al., 2007). LISCL genes were located in chr 6, 8, and 13 (although most of the LISCL in chr 13 were located just beside the presumed paralogous segment) and PAT genes located in chr 10, 12, and 19.

Concerning LISCL genes, the tandem repetition of almost identical coding sequences (e.g., VviLISCL7 and VviLISCL11) suggests that these duplication events in the grapevine genome are quite recent (Licausi et al., 2010). There is also tandem repetition of genes belonging to different groups such as VviLISCL5 and VviGRASV3c-e as well as VviSCL3a, and VviLISCL1-4). Interestingly, clusters in chr 6 and 13 presented similar sequence string within 4 LISCL genes followed by one SCL3.

Tandem repeats mainly in the LISCL group were also observed in P. mume (Lu et al., 2015).

Interestingly, the new V. vinifera group comprising VviGRAS Va-Vd was distributed in four different chromosomes (1, 14, 17, and 19). Three of them were in paralogous regions in chr 1, 14, and 17.

Therefore, segmental duplication and tandem duplications contributed significantly to the expansion and evolution of the GRAS gene family.

### Expression Analysis of Grapevine GRAS Genes

Three distinct approaches were performed to characterized GRAS genes expression in grapevine. First, we constructed an atlas of expression of the GRAS genes based on the absolute value of gene expression in public data. The results of this study are presented in **Figure 5** that displays the data extracted from the published grapevine gene expression atlas (Fasoli et al., 2012). When a gene was clearly expressed in a given tissue a Plant Ontology (PO) was attributed to the gene and reported in the ORCAE database.

Second, we performed a co-expression analysis based on the same original data using the relative values of expression of all the genes, centered on the average expression. The objective here was to determine expression patterns and to identify genes that were following the same pattern of expression as the GRAS genes and that could be under the same regulatory elements, or under the regulation of the GRAS gene itself. The results are presented in **Table 2** and **Supplementary Table 2**. Nine genes showed a correlation with other genes with a Pearson Correlation Coefficient (PCC) threshold of 0.2. Finding the optimal PCC threshold to retrieve functionally related genes was affected by the method of gene expression database construction and the target gene function (Obayashi and Kinoshita, 2009), but the PCC that was chosen was very stringent.

#### TABLE 2 | Co-expression analysis of GRAS genes.

(Continued)

#### TABLE 2 | Continued

The list of co-expressed genes is complete except for VviGRAS8a and VviSCL26b. Further details are presented in Supplementary Table 2. The list of co-expressed genes are highlighted in bold.

Third, we mined public expression data to identify the behavior of GRAS genes during berry ripening (**Figure 6**) and upon abiotic and biotic stresses (**Figures 7**, **8**) not only in V. vinifera but also in other Vitis species (**Supplementary Table 3**). **Figures 6**–**8** presented the expression values among the experiments where difference in expression of GRAS genes was detected.

Out of the 52 genes analyzed, six were not detected in any analyzed tissue. The rest of the genes mostly showed a general pattern; they were either highly expressed or lightly expressed in all tissues considered. Nevertheless, about one third of the genes showed some tissue-specific expression. Pollen stands out as a different tissue in terms of GRAS genes expression. Differential expression of some GRAS genes among different tissues was previously shown for tomato and Populus (Liu and Widmer, 2014; Huang et al., 2015). Furthermore, differential expression was clearly noticed during grape ripening and stress response.

### PAT Subfamily

Expression studies of VviPAT genes showed that most of them were expressed in all the tissues, including berry, seed, inflorescence, flower and rachis, among others (**Figure 5**). VViPAT6 seemed to be more abundant in reproductive organs (flower, stamen, tendril and berry). VViPAT7 was expressed only in seedling and root. VviPAT genes generally seemed to respond to abiotic stress specifically VviPAT3, VviPAT4, and VviPAT6 were up-regulated after prolonged exposure (**Figure 8**). VViPAT3 and VviPAT4 also seemed to respond to photoperiod and showed a stronger expression under UV light. VviPAT4 was up-regulated in grapevine response to Botrytis cinerea, leaf response to powdery mildew and inflorescence response to Bois Noir suggesting that it could be an important regulator of biotic stress responses (**Figure 8**). VViPAT3, VviPAT4, and VViPAT6 were expressed along grape ripening (**Figure 6**) although differences could be noticed among cultivars and ripening stages (**Supplementary Table 3**). Data on the evolution during ripening confirmed that their expression seems dependent of environmental factors since expression did not seem reproducible over the years in Pinot Noir. However, their expression clearly increased in ripe fruit suggesting that these genes might be related to ripening control.

### SHR Subfamily

Concerning SHR subfamily, VviSHR1, VviSHR2, and VviSHR3 tended to be expressed in all tissues excepted in some floral organs and pollen (**Figure 5**). VviSHR4 and VviSHR5 seemed to

be expressed only in specific vegetative tissues. VviSHR4 showed expression in seedling and VviSHR2 in stem and root. VviSHR3 showed the strongest expression in seedling, root and berry. This gene together with VviSHR5, an ortholog of AtSCL32, was upregulated in berries upon Botrytis cinerea infection (**Figure 8**). VviSHR4 responded positively to Bois Noir attack. VviSHR1 was expressed in several reproductive and vegetative tissues ranging from reproductive tissues (inflorescence and carpel) to root, among others. VviSHR1 presented co-expression with a cluster of 15 genes that included genes involved in cell wall catabolism, defense, and signaling pathways (**Table 2**). During ripening, its expression appeared higher during the earlier stages and seemed to be lower at véraison. In post-harvest berries this gene was also down-regulated.

### LISCL Subfamily

Members of the LISCL subfamily showed distinctive expression patterns. VviLISCL3, VviLISCL5, VviLISCL8, and VviLISCL12 were expressed in all tissues but pollen, while VviLISCL2, VviLISCL7, VviLISCL10 were expressed in almost none tissue (**Figure 5**). Among them, VviLISCL2 expression seemed restricted to older tissues since it was only detected in postharvest fruit, senescent leave and woody stem. The other genes presented a tissues-specific expression. Expression of VviLISCL4 was predominant in male reproductive tissues (stamen and pollen).

VviLISCL3 and VviLISCL12 originated from a duplication event and have high sequence similarity, which resulted in not having a specific probeset for each of them in the GeneChips array. However, their expression seemed to be affected by ripening with the lowest expression around or after véraison and the highest expression in ripe or overripe stages (**Figure 6**). They showed high expression under prolonged abiotic stress and upon virus infection, but distinction between both genes could not be made. Nevertheless, UV light surely affected their expression positively. VviLISCL1 was also over-expressed after 16 days under water deficit and salt stress (**Figure 7**).

Interestingly, VviLISCL7, whose expression was not detected in most tissues, showed slight over-expression upon Botrytis infection (**Figure 8**). Although VviLISCL7 presented a short Nterminal lacking domain I, it might be still functional because it looked expressed in some particular conditions, with motifs II, LHRI, VHIID, LHRII, PFYRE, SAW, and RVER (unit B of

FIGURE 5 | Expression of GRAS genes in grapevine tissues. Gradient color is expressed in RMA-normalized intensity value on the Nimblegen microarray. The value for each tissue corresponds to the condition where the highest expression was reported.

LHRII was also missing). VviLISCL2 also presented a short Nterminal lacking domain I and II; therefore some motifs may not be essential for functionality. VviLISCL11 showed coexpression with a senescence- related gene (**Table 2**) and was over expressed in post-harvest berries.

### DELLA Subfamily

Genes VviRGA3, VviRGA4, and VviRGA5 were expressed in all tissues (**Figure 5**). VviRGA3 and VviRGA5 were up-regulated in the earliest stages of fruit development, at fruit set and might be involved in the transition from inflorescence to flower.

VviRGA3 was also down-regulated under abiotic stresses namely salt, water stress, ABA exposure and high light (**Figure 7**). VviRGA3 co-expressed with an auxin biosynthesis-related coding for gene IAA-amino acid hydrolase (**Table 2**), and might be a key regulator of this enzyme. Moreover, their highest expression was detected in plant tissues commonly responsible for auxin production such as seed and flower. VviRGA5 was up-regulated in berries infected with Botrytis at green stage but its expression severely dropped at véraison so it might participate only in the early response (**Figure 8**).

### SCR Subfamily

The gene VviSCR3 showed peaks of expression in pollen, ripe berries and senescing leaves (**Figure 5**) and co-expressed with a Zinc finger transcription factor (C3HC4 family). Interestingly, VviSCR2, an ortholog of AtSCL23, was down- regulated during ripening in both Trincadeira and Corvina (**Figure 6**). VviSCR1, an ortholog of AtRGL2, was expressed only in some vegetative tissues (seedling, bud and stem) but was slightly up-regulated in green berries upon Botrytis infection and showed a dramatic shift of expression between véraison and medium ripe stage in

SCL3 Subfamily

Corvina. This gene co-expressed with a heat shock transcription factor and an invertase/pectin methylesterase inhibitor (**Table 2**).

Three SCL3 genes (VviSCL3a, VviSCL3b, VviSCL3c) showed similar expression patterns (**Figure 5**). They were predominantly expressed in the stem, seed and berry flesh. Particularly, VviSCL3c might be involved in seed development. The three genes were

### GRAS8 Subfamily

In this subfamily, VviGRAS8a, an ortholog of AtSCL28/GRAS8, exhibited detectable expression in several tissues ranging from inflorescence to tendril and stem (**Figure 5**). VviGRAS8a was down-regulated during grape ripening in Corvina, while no

**108**

differences were observed in Trincadeira (**Figure 6**). In a general manner, VviGRAS8a was more abundant in young tissues (leaf, stem, tendril, rachis, bud) with the only exception of seed. This gene was co-expressed with a large set of genes (79 genes); most of them annotated as genes involved in cell cycle, microtubule organization, nucleotide metabolism or signaling (**Table 2** and **Supplementary Table 2**). This suggests that it might play a role in cell growth and differentiation. It was also over-expressed at ripening and slightly up-regulated upon Botrytis infection in Trincadeira grapes. On the contrary, VviGRAS8b was expressed in older tissues (increased expression during post-harvest stages of ripening, leaf, stem, winter bud). As for VviGRAS8a, the exception was in the seed where no difference between young and old tissues was noticed.

### LAS Subfamily

Genes VviLAS1 and VviLAS2 presented quite a different expression profile with VviLAS1 not being expressed in most tissues (**Figure 5**). VviLAS2 appeared to be more abundant at the beginning of fruit development, with consistency among varieties. VviLAS1 was over expressed in mature berries but not in over-ripe berries (**Figure 6**). VviLAS2 expression also decreased upon Botrytis infection (**Figure 8**) and co-expressed with 11 genes, some of them possibly involved in biotic stress response (**Table 2** and **Supplementary Table 2**).

### GRASV1, GRASV2, GRASV3, and SCL26 Subfamilies

Expression of genes belonging to these new subfamilies was low. For some of them, their possible expression could not be confirmed (VviGRASV1d, VviGRASV3a, VviGRASV3b, although for the latter two we only had RNAseqdata for expression validation). The VviGRASV1 genes shared a similar expression profile during Corvina ripening, peaking at the medium-ripe or ripe stage and showing expression in the first post-harvest stage (**Figure 6**). VviGRASV2 genes also showed this profile. Interestingly, VviGRASV1 and VviGRASV2 genes might also play a role during Botrytis attack (**Figure 8**).

VviGRASV3c was mostly expressed in post-harvest berries. In addition, these 2 subfamilies did not show expression in other tissues, with the exception for VviGRASV3c in root and VviGRASV2a in young inflorescence.

The SCL26 genes showed a reduced expression level in various tissues. Most notably VviSCL26b seemed more abundant in berries at ripe stage (**Figure 6**). VviSCL26b co-expressed with genes involved in the pathogen response and in cell wall metabolism but the function of many of the co-expressed genes was unknown (**Table 2**, **Supplementary Table 2**). The expression profile of these genes was intriguing since little consistency was observed among replicates of the same condition. This inconsistency might be caused by a response to unidentified factors during sampling, which appears in experiments performed by independent laboratories.

### HAM Subfamily

This subfamily is present in all tissues with notable lower values in pollen (**Figure 5**). VviHAM3 was up-regulated during ripening, upon Bois Noir attacks, and in response to drought in the seed and shoot tip (**Figures 6**–**8**). VviHAM1 and VviHAM2 were down-regulated in all the cultivars during ripening; they might play a role in early stages of fruit development.

### DISCUSSION

The availability of sequenced genomes, expression data and associated bioinformatics tools enable the study of the genomic information to predict the putative function of a gene family in developmental processes and in stress response. In general, transcription regulators belonging to the same taxonomic group exhibit common evolutionary origins and specific conserved motifs related to molecular functions, making their genome-wide analysis an effective and practical method to predict unknown protein functions.

We have performed an exhaustive analysis of GRAS genes on the 12x grapevine genome sequence based on the isolation of the complete set of genes identified in PN40024. Chromosome localization, gene structure analyses, phylogenetic analyses with other genome sequenced species and expression analysis allowed to propose an extended characterization of the GRAS gene family in grapevine and to draw hypotheses on the function of newly described genes.

### Expansion of GRAS Family in Grapevine

The grapevine GRAS gene family was greatly expanded by segment/chromosomal duplications as it occurred in other species belonging to different taxonomic groups (Liu and Widmer, 2014; Huang et al., 2015; Lu et al., 2015). Duplicated genes might show functional redundancy and their identification may contribute to decipher gene functions, the evolutionary consequences of gene duplication and their contribution to evolutionary change. Duplicated genes face one of these fates: nonfunctionalization, neofunctionalization (evolving novel functions), or subfunctionalization (partition of gene functions; Prince and Pickett, 2002). The process of non-functionalization can occur when a redundant gene degenerates to a pseudogene or is lost from the genome due to the vagaries of chromosomal remodeling, locus deletion or point mutation (Prince and Pickett, 2002). Likely candidate pseudogenes are some of the outliers in our sequence alignments such as gene VviSCL3b which presents only 294 nucleotides and a premature stop codon and lacks motifs PFYRE and SAW. Interestingly, this gene showed an ortholog only in cabbage (**Figure 3**). However, this gene was found to be expressed suggesting that it could still maintain some functionality. No expression was found for VviSCL3d which may also be a pseudogene that lost its function during the evolution of the gene family.

We have also identified duplicated grapevine genes such as VviLISCL7 and VviLISCL11 whose expression analysis with specific probes might indicate they have evolved into distinct functions. Expression divergence in duplicated GRAS gene was previously detected in several plant species (Wu et al., 2014). Furthermore, no GRAS genes were coexpressed together, reflecting a wide diversity of the functions, or specialization. Unlike other species, tandem duplication events in grapevine seemed mainly restricted to the LISCL subfamily which contained tandem repeated genes with the highest homology. However, other genes from specific subfamilies were in paralogous areas of the genome resulting from polyploidization event (Jaillon et al., 2007). Amongst them, the PAT subfamily had members in chr 10, 12, and 19 (**Figure 4**), GRASV1 in chr 1, 14, and 17, LISCL in chr 6, 8, 10, and GRASV2 in chr 5 and 7 (only two genes). Although V. vinifera has a smaller size genome than S. lycopersicum (487 and 760 Mb, respectively), it contained a similar number of GRAS genes (52 and 53 genes, respectively). In addition, P. mume with a genome size of 280 Mb, almost half the size of the V. vinifera genome, contained 46 GRAS genes, a close number to the 52 V. vinifera genes (Lu et al., 2015). Therefore, the density of GRAS genes varies greatly among plant species (Song et al., 2014; Huang et al., 2015; Lu et al., 2015).

The exon-intron organization analysis showed that 88.46% (46 out of 52) of VviGRAS genes were intronless in grapevine, the highest percentage found so far, though similar to P. mume (82.2%) (Lu et al., 2015). Interestingly, this percentage is much smaller in Populus (54.7%) where the GRAS family greatly expanded (Liu and Widmer, 2014). Horizontal gene transfer of plant GRAS genes that originated from prokaryotic genomes has been proposed (Zhang et al., 2012). This prokaryotic origin followed by extensive duplication events in their evolutionary history might explain the abundance of intronless genes within the GRAS gene family. The grapevine GRAS genes also exhibited a highly variable N-terminal domain, as in other species, indicating the functional versatility of this gene family in grapevine. By contrast, highly conserved C-terminal domains (GRAS domain) were observed in all non-truncated proteins.

### GRAS Family Members are Putatively Involved in Grapevine Development and Defense

### Expression Patterns across a Variety of Tissues Revealed Divergent Functions

GRAS genes showed broad expression patterns across a variety of tissues, as previously observed in Populus and P. mume (Liu and Widmer, 2014; Lu et al., 2015). For example, VviSCR1 was highly expressed in the bud whereas the other VviSCR genes were not detected in this tissue. In Arabidopsis, SCR was located downstream of SHR, and both genes were required for stem cell maintenance of the root meristem to ensure its indeterminate growth (Lee et al., 2008). In V. vinifera, VviSHR3 was the gene from SCR and SHR subfamilies presenting highest expression in the root. Its tomato ortholog (SlGRAS16) also displayed its highest expression in the root comparing to several tissues and organs tested and was also predicted to be involved in root development (Huang et al., 2015).

VviSCR1, ortholog of AtSCR, co-expressed with an invertase/pectin methylesterase inhibitor putatively involved in cell wall organization and biogenesis. VviSHR1 was expressed in several reproductive and vegetative tissues and was co-expressed with a cluster of genes putatively involved in cell wall biogenesis (pectate lyase, endo-1,4-beta-glucanase, glycosyl hydrolase family 10 protein) and signaling mechanisms (leucine-rich repeat protein kinase, receptor protein kinase, wall-associated kinase 4). Previous analysis of a short-root (shr) mutant showed that the AtSHR protein is also involved in root and shoot radial patterning (Helariutta et al., 2000). These transcription factors are likely to play a role in cell wall reorganization and signaling events during cell growth and differentiation in grapevine. SHR and SCR were referred to be expressed in leaves, in young leaf primordia, in developing leaf vascular tissue, and bundle sheet cells (reviewed by Bolle, 2016). Recently, AtSHR, AtSCR, and AtSCL23 were described to control bundle sheath cell fate and function in A. thaliana and this developmental pathway seemed to be evolutionarily conserved (Cui et al., 2014). AtSCR was identified as primarily involved in sugar transport whereas AtSCL23 might play a role in mineral transport. Their expression seemed regulated by SHR protein. Their orthologs in V. vinifera (VviSHR1, VviSCR1, and VviSCR2, respectively) might play similar cellular functions. The tomato genes SlGRAS25 and SlGRAS15 (respective orthologs of VviSHR1 and VviSCR1) in addition to SlGRAS39, ortholog of another SHR gene, VviSHR2, showed high mRNA expression levels in root and stem (Huang et al., 2015), suggesting conserved functions with their homologous gene AtSHR (Cui et al., 2007), and AtSCR (Helariutta et al., 2000) which are involved in root and shoot radial patterning in Arabidopsis. These genes had orthologs in most species (**Figure 3**) indicating that their function might also be conserved in grapevine.

GRAS proteins have also been involved in axillary meristem development. Knock-out Arabidopsis plants for AtLAS/SCL18 are unable to form lateral shoots during vegetative development (Greb et al., 2003). In tomato, mutant plants for the ortholog lateral suppressor (LeLs) were blocked in the initiation of axillary meristems and showed lower number of flowers per inflorescence, absence of petals, reduced fertility, and altered hormone levels (Schumacher et al., 1999). The grapevine ortholog (VviLAS1) was not expressed in most tissues, except for berry pericarp, mature berry and leaf; however the other member of this subfamily, VviLAS2, showed tissue expression that could be more in accordance to the role described for LeLs. The ortholog of VviLAS2 in tomato (GRAS17) is also differentially expressed from mature green stage fruits to breaker stage fruits (Huang et al., 2015).

In grapevine, VviHAM1 is strongly expressed during fruit set and in several tissues such as bud, leaf, and stem.  In Arabidopsis, the GRAS proteins from the HAM branch (SCL6, 22, and 27) are also involved in leaf development (Wang et al., 2010). VviHAM1 may be involved in the regulation of meristematic activity in growing tissues.

Many VviPAT genes showed expression in a wide range of tissues and might be involved in several developmental processes, through the regulation of phytochrome signaling mechanisms, as in Arabidopsis (Bolle, 2004, 2016). PAT genes PAT1, SCL5, SCL21 are positive regulators of phytochrome-A signal transduction while SCL13 is mainly involved in phytochrome-B signal transduction (Bolle et al., 2000; Torres-Galea et al., 2006, 2013). The grapevine PAT subfamily showed the weakest expression in the less photosynthetic tissues (pollen, roots), with the exception of VviPAT7 that displayed an opposite expression profile. VviPAT7 was also one of the few PAT genes with no orthology in other species, except in monocots.

DELLA genes presented a wide range of expression patterns among tissues consistent with their role as negative regulators of gibberellin signal transduction (Peng et al., 1997; Silverstone et al., 1998; Zentella et al., 2007). They interfere with a variety of growth and developmental processes such as stem elongation, flower development, and seed germination (Bolle, 2004). In addition, DELLA proteins integrate not only gibberellin -signaling pathways but also jasmonate, auxin, brassinosteroid, and ethylene pathways, constituting a main signaling hub (Wild et al., 2012; Bolle, 2016). VviRGA5, a one-to-one ortholog of AtRGA/AtGAI, was highly expressed in seed, flower and stem supporting a role in developmental processes.

The rice DLT gene modulates brassinosteroid-related gene expression (Tong et al., 2009). The homologous gene in Arabidopsis is AtSCL28 and in V. vinifera VviGRAS8a. Interestingly, this gene co-expressed with a large set of genes involved in cell cycle, nucleotide metabolism or signaling. In general, the transcripts of this gene were more abundant in young tissues (leaves, stem tendril, rachis, bud) and in inflorescence which is not surprising since brassinosteroids promote growth (reviewed by Fortes et al., 2015). The tomato ortholog SlGRAS41 was suggested to be involved in flowerfruit transition with a potential role in fruit development by modulating brassinosteroid signaling (Huang et al., 2015). A role that is likely to be played by VviGRAS8a in grapevine eventually through an involvement in mechanisms of cell division and differentiation.

As previously mentioned, expression of GRAS genes in pollen tissue differed from other tissues. VviLISCL4 was almost specifically expressed in the stamen and particularly in pollen. Interestingly, a LISCL gene has been shown to be involved in transcriptional regulation during microsporogenesis in the lily anther (Morohashi et al., 2003). Future functional analysis of VviLISCL4 gene during pollen development is required to confirm the importance of this GRAS gene in grapevine reproduction.

Several GRAS genes (VviLISCL2, VviGRASV2b) showed higher expression in senescent tissues (senescent leaves, woody stem, post-harvest berries) than in younger tissues, including ripe/mature tissues. In this way, a wheat LISCL gene, TaSCL14, was identified as promoting senescence in leaves (Chen et al., 2015). VviGRASV2b seemed completely grapevine-specific and its potential involvement in senescence has yet to be clarified.

### GRAS are Likely to Play a Role in Berry Development and Ripening

Several grapevine GRAS genes showed differential expression among berry ripening stages (Fortes et al., 2011; Agudelo-Romero et al., 2013) namely VviLISCL3/12, VviLISCL11, VviPAT3, VviPAT4, VviPAT6, VviSCR3, VviGRAS8b, VviLAS1, VviHAM3, VviSCL26b (up-regulated), VviHAM1, VviHAM2, VviRGA3, VviSHR1, VviLAS2 (down-regulated). Genes VviHAM1, VviHAM2, VviRGA3, VviSHR1, and VviLAS2 seemed to be involved in fruit set and in the early stages of fruit development when there is intense cell division activity and sugar transport. During these stages, the levels of phytohormones such as auxins, cytokins, gibberellins, and jasmonic acid also peaked (reviewed by Fortes et al., 2015), that might be related to the up-regulation of RGA3 since DELLA proteins integrate several phytohormone- signaling pathways (Bolle, 2016). Furthermore, RGA3 co-expressed with a gene coding for IAA-amino acid hydrolase 1 involved in auxin metabolism (auxin activation by conjugation hydrolysis) supporting the role of VviRGA3 in hormonal regulation.

VviLISCL3/VviLISCL12, VviPAT4, VviPAT6, and VviHAM3 were up-regulated at mature stages (ripe, harvest, and postharvest) whereas VviSCR3 was up-regulated in medium ripe and ripe berries and co-expressed with a gene coding for a Zinc finger protein (C3HC4-type ring finger). These transcription factors have been previously described as being modulated during grape ripening (Fortes et al., 2011). VviGRAS8b was overexpressed at post-harvest stages and VviLAS1 and VviSCL26b at medium ripe, ripe and initial post-harvest stage. The gene VviSCL26b co-expressed with genes involved in pathogen response (pathogenesis related protein 1 precursor, heat shock protein 81-2, peroxidase precursor) and cell wall metabolism (endoxylanase, polygalacturonase GH28, cellulase). This could be associated to the activation of genes that are related to biotic stress response as well as cell wall rearrangements taking place during grape ripening (Fortes et al., 2011). VviLISCL11 was over expressed in post-harvest berries and might be linked to the regulation of cell wall degradation processes. In agreement with this hypothesis, it was co-expressed with a senescence related gene.

Altogether, these observations could suggest the relevance of GRAS genes as regulators of the different stages of grape berry development. GRAS transcription factors have been previously associated with the control of tomato fruit ripening (Fujisawa et al., 2012). Authors suggested that SlGRAS38 gene could play a role in fruit ripening due to its ripening-specific expression and direct transcriptional regulation by RIN. In tomato, a typical climacteric fruit, the MADS-box transcription factor RIN is one of the earliest-acting ripening regulators, required for both ethylene-dependent and ethylene- independent pathways. By contrast, VviSH4, the grapevine ortholog of SlGRAS38, did not seem to be involved in grapevine ripening. Since grape is a non-climacteric fruit in which ethylene does not play a central role in the regulation of ripening (reviewed by Fortes et al., 2015), a different transcriptional regulatory pathway of ripening could be expected. Still, common aspects between ripening pathways in both type of fruits can be observed. Grapevine VviPAT3, VviPAT4, and VviPAT6 have expression patterns consistent with their involvement in berry ripening and their tomato orthologs, SlGRAS1, SlGRAS2, and SlGRAS10 (respectively) were differentially expressed from mature green stage fruits to breaker stage fruits (Huang et al., 2015). The same holds true for VviHAM3 and its tomato ortholog SlGRAS8 as well as VviLISCL3 and its ortholog SlGRAS13 (Huang et al., 2015). Therefore, these grapevine GRAS genes (**Figure 9**) could likely be conserved and represent pivotal transcriptional regulators of fruit ripening in both climacteric and non-climacteric species.

levels and red to higher levels. The data is presented considering the tendency of the orthologs across cultivars in grape and tomato.

### Grapevine GRAS Genes are Putatively Involved in Stress and Defense Responses

Several GRAS proteins have been associated with a role in stress signaling (reviewed by Bolle, 2016). Arabidopsis scr and shr loss of function mutants were found to be hypersensitive to abscisic acid (ABA) and to high levels of glucose but were not affected by high salinity or osmotic stress (Cui, 2012). In grapevine VviSHR1 expression seemed to be affected by ABA but not by salt (**Figure 7**). Interestingly, expression of VviSHR1 decreased during grape ripening when glucose levels significantly increased. Moreover, VviSHR1 may be involved in grapevine response against virus whereas VviSCR1 was up-regulated in green berries upon Botrytis infection. In fact, GRAS genes seem to be expressed upon abiotic and biotic factors (reviewed by Bolle, 2016). Furthermore, VviSHR1 co-expressed with genesinvolved in stress response (glutaredoxin family protein, subtilase). A poplar GRAS gene showing the highest identity to Arabidopsis SCL7, conferred salt and drought tolerance to this plant (Ma et al., 2010). The duplicated gene of AtSCL7, AtSCL4, is orthologous of the grapevine VviLAS2 which was down-regulated in response to salt but up-regulated upon UV light and long day exposure. VviLAS2 expression also decreased upon Botrytis and co-expressed with up to 11 genes possibly involved in biotic stress response (epoxide hydrolase 2, DEFENSE NO death 1). VviLAS2 might be a negative regulator of expression of these genes.

Other grapevine GRAS genes were found to show differential stress responses. VviRGA5 was recently shown to be up-regulated in grape berries at initial stage of fungal infection (Agudelo-Romero et al., 2015) and VviRGA3 was down-regulated under abiotic stresses such as salt, water stress, ABA exposure, and high light. Inhibition of growth by DELLA subfamily genes has been proposed as a response to environmental variability (Harberd et al., 2009) so these transcription factors may play an important role in the regulation of abiotic and biotic stress response pathways by regulating growth. Furthermore, DELLA proteins control plant immune responses by modulating the balance of jasmonic acid and salicylic acid signaling (Navarro et al., 2008; Wild et al., 2012), growth regulators which involvement in stress responses is well-known.

The Arabidopsis GRAS protein SCL14 was shown to be essential for the activation of stress-inducible promoters (Fode et al., 2008). The closest grapevine homologs are VviLISCL12 and VviLISCL3 that were also up-regulated after biotic stress. VviLISCL12 was recently shown to be up-regulated upon guazatine treatment, an inhibitor of polyamine catabolism (Agudelo-Romero et al., 2014). In rice, OsGRAS2, the ortholog of AtSCL14 is involved in the regulation of drought stress response (Xu et al., 2015). Other grapevine LISCL genes could likely be involved in abiotic stress response namely VviLISCL1 which was over-expressed after long exposure to water deficit and salt stress (**Figure 7**).

The Brassica oleracea gene BoGRAS, was up-regulated under heat stress (Park et al., 2013) and its grapevine ortholog, VviPAT3, was also over-expressed during biotic stress. The ortholog of VviPAT3 in tomato, SlGRAS1, was also referred to be involved in biotic stress response (Mayrose et al., 2006). Moreover, VviPAT4 might be a good candidate in regulating abiotic and biotic stress responses in grapevine since it was up-regulated under both conditions. In tomato SlGRAS2, the VviPAT4 ortholog, was involved in hormone signaling and abiotic stress response (Huang et al., 2015). VviHAM3 was also up-regulated during ripening, upon Bois Noir attacks, and in response to drought in the seed and shoot tip. Therefore, VviHAM3 exhibited expression patterns that indicate a role in broad stress responses.

Altogether, the expression of several grapevine GRAS genes in response to several stress treatments highlights the wide involvement of this gene family in environmental adaptation, showing diverse responses under different environmental conditions and treatments (Huang et al., 2015). The same results were observed in tomato for the expression of many SlGRAS genes.

### CONCLUSIONS

GRAS transcription factors have been characterized in several species and were proven to be involved in diverse developmental processes and stress responses. However, their involvement in fruit ripening is only now starting to be disclosed. Grape berry development and ripening could be under control of GRAS genes, since the expression of many of them is modulated during this process. The involvement of grapevine GRAS genes in stress responses was also confirmed in this study. Both ripening and stress responses involved genes from new GRAS subfamilies identified in grapevine (GRASV1, GRASV2, GRASV3, SCL26, and GRAS8). Robust candidates for further functional analysis were established and compared with the results of a similar analysis recently performed in tomato, another fleshy fruit. Altogether this data may contribute to the improvement of fruit quality and resilience to biotic and abiotic stresses.

### AUTHOR CONTRIBUTIONS

AF and JG designed the study. JG, PA, RT, and AF analyzed the data. AF wrote the manuscript with valuable input from JG and JM. All the authors revised and approved the manuscript.

### REFERENCES

### ACKNOWLEDGMENTS

Funding was provided by the Portuguese Foundation for Science and Technology (SFRH/BPD/100928/2014, UID/MULTI/04046/2013 and PEst-OE/BIA/UI4046/2014) and is integrated in the COST (European Cooperation in Science and Technology) Action FA1106 "Quality fruit." JG was supported by the Ramon y Cajal program (RYC-2011-07791) and the AGL2014-59171-R project from the Spanish MINECO.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. 00353

Supplementary Image 1 | Structure and subfamily-specific motifs of GRAS proteins. The size varies within the subfamily. Several proteins such as VviLISCL2 and VviLISCL7 present shorter N-terminal sequences. The protein VviSCL3b lacks the motifs PFYRE and SAW.

#### Supplementary Image 2 | Molecular phylogenetic analysis by Maximum Likelihood method between Grapevine and 15 plant species.

Lineage-specific groups can be noticed for Populus and Eucalyptus whereas Arabidopsis putatively lacks specific subgroups.

Supplementary Table 1 | Complete annotation of the grapevine GRAS genes. Alternative Names correspond to previous annotation (8X and 12Xv0). The probesets ID for microarray platform are given for Genechips, Grapegen and Nimblegen. The Nimblegen ID is also the 12Xv1 ID.IEP: evidence code inferred by expression pattern. Positions are given for both the 12X v1 and v2 genome.

Supplementary Table 2 | List of genes co-expressed with GRAS genes. GRAS genes are highlighted in yellow.

Supplementary Table 3 | GRAS genes expression in experiments related to ripening, abiotic stress and biotic stress.

one of which is functionally conserved in a non-legume. Plant Physiol. 142, 1739–1750. doi: 10.1104/pp.106.089508

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Grimplet, Agudelo-Romero, Teixeira, Martinez-Zapater and Fortes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Evolutionary Recycling of Light Signaling Components in Fleshy Fruits: New Insights on the Role of Pigments to Monitor Ripening

Briardo Llorente\*, Lucio D'Andrea and Manuel Rodríguez-Concepción\*

Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Barcelona, Spain

### Edited by:

Antonio Granell, Consejo Superior de Investigaciones Científicas, Spain

### Reviewed by:

Cornelius Barry, Michigan State University, USA Maria Jesus Rodrigo, Instituto de Agroquímica y Tecnología de Alimentos – Consejo Superior de Investigaciones Científicas, Spain

### \*Correspondence:

Briardo Llorente<EMAIL_ADDRESS>Manuel Rodríguez-Concepción<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science

Received: 26 November 2015 Accepted: 19 February 2016 Published: 07 March 2016

### Citation:

Llorente B, D'Andrea L and Rodríguez-Concepción M (2016) Evolutionary Recycling of Light Signaling Components in Fleshy Fruits: New Insights on the Role of Pigments to Monitor Ripening. Front. Plant Sci. 7:263. doi: 10.3389/fpls.2016.00263 Besides an essential source of energy, light provides environmental information to plants. Photosensory pathways are thought to have occurred early in plant evolution, probably at the time of the Archaeplastida ancestor, or perhaps even earlier. Manipulation of individual components of light perception and signaling networks in tomato (Solanum lycopersicum) affects the metabolism of ripening fruit at several levels. Most strikingly, recent experiments have shown that some of the molecular mechanisms originally devoted to sense and respond to environmental light cues have been re-adapted during evolution to provide plants with useful information on fruit ripening progression. In particular, the presence of chlorophylls in green fruit can strongly influence the spectral composition of the light filtered through the fruit pericarp. The concomitant changes in light quality can be perceived and transduced by phytochromes (PHYs) and PHYinteracting factors, respectively, to regulate gene expression and in turn modulate the production of carotenoids, a family of metabolites that are relevant for the final pigmentation of ripe fruits. We raise the hypothesis that the evolutionary recycling of light-signaling components to finely adjust pigmentation to the actual ripening stage of the fruit may have represented a selective advantage for primeval fleshy-fruited plants even before the extinction of dinosaurs.

### Keywords: photosensory pathways, light, fleshy fruits, ripening, evolution

## INTRODUCTION

Light has a dual role in plants as an essential source of energy for driving photosynthesis and, on the other hand, as an environmental cue that modulates many aspects of plant biology such as photomorphogenesis, germination, phototropism, and entrainment of circadian rhythms (Chen et al., 2004; Jiao et al., 2007). The ability to perceive and respond to light changes is mediated by a set of sophisticated photosensory pathways capable of discriminating the quality (spectral composition), intensity (irradiance), duration (including day length), and direction of light (Moglich et al., 2010). In particular, plants perceive light through at least five types of sensory photoreceptors that are distinct from photosynthetic components and detect specific regions of the electromagnetic spectrum. Cryptochromes (CRYs), phototropins, and Zeitlupe family members function in the blue (390–500 nm) and ultraviolet-A (320–390 nm) wavelengths, while the

photoreceptor UVR-8 operates in the ultraviolet-B (280–315 nm) region. Phytochromes (PHYs), which are probably the best studied photoreceptors, function in a dynamic photoequilibrium determined by the red (R, ca. 660 nm) to far-red (FR, ca. 730 nm) ratio in land plants and throughout the visible spectrum (blue, green, orange, red, and far-red) in different algae (Moglich et al., 2010; Rizzini et al., 2011; Rockwell et al., 2014). The photonic information gathered by these photoreceptors is then transduced into changes in gene expression that ultimately promote optimal growth, development, survival and reproduction (Jiao et al., 2007).

Photosensory pathways are thought to have occurred early in plant evolution, probably at the time of the Archaeplastida ancestor (i.e., the last common ancestor of glaucophyte, red algae, green algae and land plants) or perhaps even earlier, before the occurrence of the endosymbiotic event that gave rise to photosynthetic eukaryotes over more than a billion years ago (Duanmu et al., 2014; Mathews, 2014; Fortunato et al., 2015). Through the ages, these mechanisms diverged to play particular roles in different branches of the plant lineage, ranging from presumably acclimative roles in algae (Duanmu et al., 2014; Rockwell et al., 2014) to resource competition functions in land plants (Jiao et al., 2007). In particular, the ability of PHYs to detect changes in the R/FR ratio allows land plants to detect the presence of nearby vegetation that could potentially compete for light. Light filtered or reflected by neighboring leaves (i.e., shade) has a distinctive spectral composition that is characterized by a decreased R/FR ratio due to a preferential absorption of R light by chlorophyll (Casal, 2013). Low R/FR ratios reduce PHY activity, allowing PHY-interacting transcription factors (PIFs) to bind to genomic regulatory elements that tune the expression of numerous genes (Casal, 2013; Leivar and Monte, 2014). Oppositely, high R/FR ratios enhance PHY activity, causing the inactivation of PIF proteins mainly by proteasome-mediated degradation (Bae and Choi, 2008; Leivar and Monte, 2014). Carotenoid biosynthesis represents a rather well characterized example of this regulation. In Arabidopsis thaliana, shade decreases the production of carotenoids in photosynthetic tissues (Roig-Villanova et al., 2007; Bou-Torrent et al., 2015) in part by promoting the accumulation of PIF proteins that repress the expression of the gene encoding phytoene synthase (PSY), the main rate-determining enzyme of the carotenoid pathway (Roig-Villanova et al., 2007; Toledo-Ortiz et al., 2010; Bou-Torrent et al., 2015). De-repression of PSY under sunlight induces carotenoid biosynthesis, which in turn maximizes light harvesting and protects the photosynthetic machinery from harmful oxidative photodamage caused by intense light (Sundstrom, 2008).

Light signals in general and PHYs in particular also modulate the genetic programs associated to fruit development and ripening. Here we will revise current and emerging knowledge on this area based on work carried out in tomato (Solanum lycopersicum), which is the main model system for fleshy fruits, that is, fruits containing a juicy fruit pulp. Further, we will discuss potential selection pressures that might account for the evolutionary recycling of light-signaling components in fleshy fruits.

## FLESHY FRUIT RIPENING: THE CASE OF TOMATO

Fleshy fruits are differentiated floral tissues that evolved 80–90 million years ago (Ma), i.e., relatively recently in the history of plants (Givnish et al., 2005; Eriksson, 2014), as an adaptive characteristic promoting the animal-assisted dissemination of viable seeds (Tiffney, 2004; Seymour et al., 2013; Duan et al., 2014). After seed maturation, fleshy fruits typically undergo a ripening process that involves irreversible changes in organoleptic characteristics such as color, texture, and flavor, all of which result in the production of an appealing food to frugivorous animals. In this manner, the ripening process orchestrates the mutualistic relationship between fleshy-fruited plants and seed-disperser animals (Tiffney, 2004; Seymour et al., 2013; Duan et al., 2014).

Upon fertilization, the development of fleshy fruits such as tomato can be divided into three distinct phases: cell division, cell expansion, and ripening (Gillaspy et al., 1993; Seymour et al., 2013). These different stages are characterized by hormonal, genetic, and metabolic shifts that have been reviewed in great detail elsewhere (Carrari and Fernie, 2006; Klee and Giovannoni, 2011; Seymour et al., 2013; Tohge et al., 2014). Before ripening occurs, tomato fruits have a green appearance due to the presence of chloroplasts that contain the whole photosynthetic machinery. The transition to ripening is characterized by a loss of chlorophylls, cell wall softening, accumulation of sugars, and drastic alterations in the profile of volatiles and pigments. Most distinctly, chlorophyll degradation is accompanied by a conversion of chloroplasts into chromoplasts that progressively accumulate high levels of the health-promoting carotenoids β-carotene (pro-vitamin A) and lycopene (Tomato Genome Consortium, 2012; Fantini et al., 2013; Seymour et al., 2013). These carotenoid pigments give the characteristic orange and red colors to ripe tomatoes. A large number of other fruits (including bananas, oranges, or peppers) also lose chlorophylls and accumulate carotenoids during ripening, resulting in a characteristic pigmentation change (from green to yellow, orange or red) that acts as a visual signal informing animals when the fruit is ripe and healthy (Klee and Giovannoni, 2011).

### THE EFFECT OF LIGHT SIGNALING COMPONENTS ON FRUIT RIPENING

Multiple lines of evidence have exposed the relevance of fruit-localized photosensory pathways as important players in the regulation of fruit ripening and the potential of their manipulation to improve the nutritional quality of tomatoes (Azari et al., 2010). Among many light-signaling mutants displaying altered fruit phenotypes, the tomato high pigment (hp) mutants hp1 and hp2 are two of the best characterized. These mutants owe their name to a deep fruit pigmentation derived from an increment in the number and size of plastids, which in turn result in elevated levels of carotenoids such as lycopene (Yen et al., 1997; Mustilli et al., 1999; Levin

et al., 2003). Detailed characterization of the hp1 and hp2 mutants, which also show increased levels of extraplastidial metabolites such as flavonoids, revealed that the mutated genes encode tomato homologs of the previously described light signal transduction proteins DAMAGED DNA BINDING PROTEIN 1 (DDB1) and DEETIOLATED1 (DET1), respectively (Mustilli et al., 1999; Schroeder et al., 2002; Levin et al., 2003; Liu et al., 2004) (**Figure 1**). Other components that participate in the same light-signaling pathway that HP1 and HP2 have also been shown to impact tomato fruit metabolism. For instance, silencing the tomato E3 ubiquitin-ligase CUL4, which directly interacts with HP1, also produces highly pigmented fruits (Wang et al., 2008). Another example is the E3 ubiquitin-ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), which specifically promotes the degradation of the light-signaling effector ELONGATED HYPOCOTYL 5 (HY5) (Schwechheimer and Deng, 2000) (**Figure 1**). Transgenic plants with downregulated transcripts of COP1 and HY5 produce tomato fruits with increased and reduced levels of carotenoids, respectively (Liu et al., 2004).

Work with photoreceptors (**Figure 1**) has also shed light on the subject. Tomato plants overexpressing the blue light photoreceptor cryptochrome 2 (CRY2) produce fruits with increased levels of flavonoids and carotenoids (Giliberto et al., 2005). PHYs have been found to control different aspects of tomato fruit ripening as well. Activation of fruit-localized PHYs with R light treatments promotes carotenoid biosynthesis, while subsequent PHY inactivation by irradiation with FR light reverts it (Alba et al., 2000; Schofield and Paliyath, 2005). Furthermore, preventing light exposure from the very early stages of fruit set and development results in white fruits completely devoid of pigments (Cheung et al., 1993), a phenotype that resembles that of phyA phyB1 phyB2 PHY triple mutant plants (Weller et al., 2000). In addition to regulating carotenoid levels in tomato fruits, PHYs seem to regulate the timing of phase transition during ripening (Gupta et al., 2014).

### A MECHANISM TO MONITOR RIPENING BASED ON SELF-SHADING AND LIGHT SIGNALING

Although light signaling components have long been known to modulate fruit ripening, another important piece of the puzzle was revealed recently. In tomato, fruit pericarp cells are morphologically similar to leaf palisade cells (Gillaspy et al., 1993). Thus, fruits can be viewed as modified leaves that, besides enclosing the seeds, have suffered a change in organ geometry, namely, a shift from a nearly planate conformation to an expanded three-dimensional anatomy. This anatomy imposes spatial constrains coercing light to pass through successive cell layers, so that the quality of the light that reaches inner sections of the fruit is influenced by the cells of outer pericarp sections (**Figure 2**). Another key difference between tomato leaves and fruits is the cuticle, which is far more pronounced in the fruit. While a potential role of the cuticle in altering the spectral properties of the light that reaches the pericarp cells remains to

be investigated, it is now well established that the occurrence of chlorophyll in fruit chloroplasts significantly reduces the R/FR ratio of the light filtered through the fruit fresh (Alba et al., 2000; Llorente et al., 2015). A reduction in R/FR ratio (also referred to as shade) normally informs plants about the proximity of surrounding vegetation (Casal, 2013). In tomato fruit, however, changes in R/FR ratio can inform of the ripening status. As a consequence of self-shading, it is proposed that a relatively high proportion of PHYs remain inactive in green fruit. This condition stabilizes the tomato PIF1a transcription factor, that binds to a PBE-box located in the promoter of the gene encoding the PSY isoform that controls the metabolic flux to the carotenoid pathway during fruit ripening, PSY1. PIF1a binding directly represses PSY1 expression (**Figure 2**). Chlorophyll breakdown at the onset of ripening reduces the self-shading effect, consequently

promoting PHY activation, degradation of PIF1a, derepression of PSY1, and eventually carotenoid biosynthesis (**Figure 2**). In this manner, the genetically controlled expression of PSY1 (and hence the production of carotenoid pigments) is finetuned to the actual progression of ripening (Llorente et al., 2015).

Translation of molecular insights from tomato to other fleshyfruited plants has indicated that many regulatory networks are conserved across a wide range of species (Seymour et al., 2013). Thus, given the ubiquitous nature of PHYs in land plants and the widespread occurrence of ripening-associated fruit pigmentation changes that typically involve the substitution of an initially chlorophyll-based green color with distinctive non-green (i.e., non-R-absorbing) eye-catching colors, it is possible that similar self-shading regulatory mechanisms might operate in other plant species to inform on the actual stage of ripening (based on the pigment profile of the fruit at every moment) and thus finely coordinate fruit color change. However, the composition of the cuticle or even the anatomy of the most external layer of the pericarp (i.e., the exocarp) might also impact the quality and quantity of light that penetrates the fruit flesh. The self-shading mechanism is expected to be irrelevant in fleshy fruits with a thick skin or exocarp that prevents light to pass through and reach more internal fruit layers.

## FRUIT COLORS AS RIPENING SIGNALS IN AN EVOLUTIONARY CONTEXT

Fleshy fruits are considered to have first appeared in the Late Cretaceous (circa 90 Ma) (Givnish et al., 2005; Eriksson, 2014), at a time when the Earth's vegetation was dense and exuberant, and where most ecological niches were taken over by angiosperms (Lidgard and Crane, 1988; Berendse and Scheffer, 2009). The plentiful surplus of nutritious food gave rise to a huge explosion in the Cretaceous fauna, bringing about the coexistence of numerous herbivorous and omnivorous reptiles (dinosaurs, pterosaurs, lizards), birds and mammals (Lloyd et al., 2008; Prentice et al., 2011; Vullo et al., 2012; Wilson et al., 2012; Jones et al., 2013; Jarvis et al., 2014). With such an abundance of planteating animals, being able to display a change in fruit color when ripe probably represented a valuable trait among early fleshyfruited plants to call the attention of these various potential seed dispersers.

Although deep time co-evolutionary scenarios may be difficult to support, this idea gains plausibility if we consider that the same strategy had been successfully implemented beforehand by gymnosperms, which had already evolved fleshy fruitlike structures by the Early Cretaceous, at least some 20-30 million years before the first fleshy fruits (Yang and Wang, 2013). Several gymnosperms (e.g., Ginkgo biloba, Taxus baccata, and Ephedra distachya) produce fleshy colorful tissues around their seeds and, similar to that occurring in angiosperms, these fruit-like structures undergo a ripening process that also serves as a visual advertisement for animals to eat them and disperse their seeds. Recent evidence supports the hypothesis that the main molecular networks underlying the formation of the fleshy fruit were originally established in gymnosperms (Lovisetto et al., 2012, 2015), thus suggesting that the ripening phenomenon was first selected as an ecological adaptation in gymnosperms and that angiosperms merely exploited it afterwards. If correct, this would imply that Cretaceous plant-eater animals would have already been used to feeding on color-changing fleshy fruit-like tissues by the time that angiosperm fleshy-fruited plants evolved, something that may have facilitated the establishment of the latter.

biosynthesis.

Another relevant fact is that the dominant land animals during the Cretaceous period, the dinosaurs, as well as pterosaurs, lizards, and birds, had highly differentiated color vision, much superior to that of most mammals (Rowe, 2000; Chang et al., 2002; Bowmaker, 2008). Differentiated color vision, or tetrachromacy, is a basal characteristic of land vertebrates derived from the presence of four spectrally distinct retinal cone cells that allow discriminating hues ranging from ultraviolet to red (Bowmaker, 2008; Koschowitz et al., 2014). Turtles, alligators, lizards and birds, are all known to have tetrachromatic color vision, a shared trait inherited from their common reptilian ancestry (Rowe, 2000; Bowmaker, 2008). We have recently come to know that some dinosaurs even sported plumage color patterns and flamboyant cranial crests that may have served for visual display purposes (Li et al., 2010, 2012; Zhang et al., 2010; Bell et al., 2014; Foth et al., 2014; Koschowitz et al., 2014). Altogether, these insights suggest that color cues were likely an important means of signaling among dinosaurs. Although purely speculative at the moment, it is reasonable to assume that there could have also been dinosaurs that, analogously to several birds and reptiles nowadays (Svensson and Wong, 2011), consumed fleshy fruits within their diet as a source of carotenoid pigments used for ornamental coloration. Even though the relevance of, now extinct, Cretaceous megafauna as biological vectors involved in the seed dispersal of primeval fleshyfruited plants remains speculative and controversial (Tiffney, 2004; Butler et al., 2009; Seymour et al., 2013), it is clear that they certainly had fleshy fruit available to eat during the last 25–35 million years of their existence, until the occurrence of the Cretaceous-Paleogene mass extinction event (65 Ma).

Fruit color change meets the criteria of a classical signal, which can be defined as a cue that increases the fitness of the sender (i.e., fleshy-fruited plants) by altering the behavior of the receivers (i.e., seed-disperser animals) (Maynard Smith and Harper, 1995). Importantly, besides visibility conditions and the visual aptitude

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of the receiver, the detectability of a visual signal is determined by its contrast against the background, that is, the conspicuousness of the signal (Schmidt et al., 2004). Ripe fruits displaying a distinct coloration against the foliage leaves are more conspicuous for animals than green fruits and there is no evidence to consider that it was any different to Cretaceous animals. In fact, the invention of fruit fleshiness took place along with expanding tropical forests, suggesting it may have evolved as an advantageous trait related to changes in vegetation from open to more closed environments (Seymour et al., 2013; Eriksson, 2014). In this context, light signaling pathways already established in land plants may have had the chance to evolutionary explore novel phenotypic space in fleshy fruits. Subsequent adaptations under selection in the fruit may have then integrated these pathways as modulatory components of the pigmentation process during ripening. For instance, the self-shading regulation of the tomato fruit carotenoid pathway (Llorente et al., 2015) (**Figure 2**) might have evolved by co-option of components from the preexisting shade-avoidance responses (Mathews, 2006; Casal, 2013). This evolutionary recycling of light-signaling components in fleshy fruits might therefore be a legacy from the time when dinosaurs walked the earth.

### AUTHOR CONTRIBUTIONS

BL, LA, and MR-C searched and discussed the literature and wrote the article.

### ACKNOWLEDGMENTS

We acknowledge the support of grants from EC (CarotenActors, 300862), CYTED (Ibercarot, 112RT0445), MINECO (FPDI-2013-018882, BIO2011-23680, BIO2014-59092-P), MEC (AP2012-0189), and AGAUR (2014SGR-1434).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Llorente, D'Andrea and Rodríguez-Concepción. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# In silico Transcriptional Regulatory Networks Involved in Tomato Fruit Ripening

Stilianos Arhondakis, Craita E. Bita, Andreas Perrakis, Maria E. Manioudaki, Afroditi Krokida, Dimitrios Kaloudas and Panagiotis Kalaitzis \*

Department of Horticultural Genetics and Biotechnology, Mediterranean Agronomic Institute of Chania, Chania, Greece

Tomato fruit ripening is a complex developmental programme partly mediated by transcriptional regulatory networks. Several transcription factors (TFs) which are members of gene families such as MADS-box and ERF were shown to play a significant role in ripening through interconnections into an intricate network. The accumulation of large datasets of expression profiles corresponding to different stages of tomato fruit ripening and the availability of bioinformatics tools for their analysis provide an opportunity to identify TFs which might regulate gene clusters with similar co-expression patterns. We identified two TFs, a SlWRKY22-like and a SlER24 transcriptional activator which were shown to regulate modules by using the LeMoNe algorithm for the analysis of our microarray datasets representing four stages of fruit ripening, breaker, turning, pink and red ripe. The WRKY22-like module comprised a subgroup of six various calcium sensing transcripts with similar to the TF expression patterns according to real time PCR validation. A promoter motif search identified a cis acting element, the W-box, recognized by WRKY TFs that was present in the promoter region of all six calcium sensing genes. Moreover, publicly available microarray datasets of similar ripening stages were also analyzed with LeMoNe resulting in TFs such as SlERF.E1, SlERF.C1, SlERF.B2, SLERF.A2, SlWRKY24, SLWRKY37, and MADS-box/TM29 which might also play an important role in regulation of ripening. These results suggest that the SlWRKY22-like might be involved in the coordinated regulation of expression of the six calcium sensing genes. Conclusively the LeMoNe tool might lead to the identification of putative TF targets for further physiological analysis as regulators of tomato fruit ripening.

#### Keywords: tomato, fruit ripening, WRKY, ERF, regulatory network, transcriptome

### INTRODUCTION

Fleshy fruit development and ripening is a complex developmental process which is regulated by hormones and plethora of transcription factors (TFs) (Seymour et al., 2013). The evolution of this process requires the action of intricate regulatory networks of TFs (Seymour et al., 2013). In tomato fruit ripening, several TFs were demonstrated to play a central regulatory role such as the MADS box proteins RIPENING INHIBITOR (RIN) (Vrebalov et al., 2002), TOMATO AGAMOUS-LIKE1 (TAGL1) (Vrebalov et al., 2009) and FUL1/TDR4 and FUL2/MBP7 (Bemer et al., 2012). Additional classes of TFs were also shown to regulate tomato ripening such as the COLORLESS NON-RIPENING (CNR) which is a SBP TF (Manning et al., 2006), the NON-RIPENING (NOR) which was identified as a NAC domain TF (Martel et al., 2011) as well as the large class of ETHYLENE RESPONSE FACTORS (ERFs) which

Edited by:

Mario Pezzotti, University of Verona, Italy

#### Reviewed by:

Xinguang Zhu, University of Chinese Academy of Sciences, China Uener Kolukisaoglu, University of Tübingen, Germany

> \*Correspondence: Panagiotis Kalaitzis<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science

Received: 27 February 2016 Accepted: 03 August 2016 Published: 30 August 2016

#### Citation:

Arhondakis S, Bita CE, Perrakis A, Manioudaki ME, Krokida A, Kaloudas D and Kalaitzis P (2016) In silico Transcriptional Regulatory Networks Involved in Tomato Fruit Ripening. Front. Plant Sci. 7:1234. doi: 10.3389/fpls.2016.01234 belong to the AP2/ERF family mediating mostly ethylenedependent gene expression (Pirrello et al., 2012). Alterations in the expression of these TFs results in phenotypes with alterations in all aspects of fruit ripening including carotenoids and flavonoids biosynthesis, fruit softening, fruit size and shape, chloroplast degradation and chromoplast development (Klee and Giovannoni, 2014).

The physiological significance of other families of TFs such as the members of the WRKY gene family has not been investigated in tomato fruit development and ripening despite the fact that several of them are expressed in the fruit during various developmental stages (Huang et al., 2012). It was recently reported that five WRKY genes were upregulated in postclimacteric Chinese pear fruits suggesting association with fruit ripening development (Huang et al., 2014).

Despite the significant progress in the elucidation of the roles and interactions of the transcriptional regulators during tomato fruit ripening there are still unknown regulatory TFs and interactions which need to be investigated (Karlova et al., 2014). In this context, in silico analysis of large gene expression datasets has been used in the recent years in order to construct gene regulatory networks (Pan et al., 2013; Clevenger et al., 2015).

A tomato fruit gene regulatory network comprising TF gene expression profiles was generated using artificial network inference analysis to analyze Affymetrix GeneChip transcriptomic data from two different developmental and ripening stages, Mature Green (MG) and Breaker + 7 (Pan et al., 2013). A novel and fruit-related regulator of pigment accumulation in tomato was identified and its function was validated in transgenic plants indicating the significance of network analysis on the identification of regulatory TFs (Pan et al., 2013). In another report, transcriptome analysis of tomato fruit tissues expressing the tomato fruit shape gene SUN resulted in shifts of transcript profiles and metabolites according to gene regulatory network analysis and networks of metabolite correlations (Clevenger et al., 2015). The gene regulatory network analysis was based on the clustering of differentially expressed genes based on the log2 fold change using fuzzy C means (Clevenger et al., 2015). It was found that the main node represented genes related to calcium-regulated processes indicating involvement in calcium signaling.