The fourth internode from the apex (not counting the peduncle, see Supplementary Material 2A) was collected at the FBV stage (day 0), days 4 and 8 after the FBV stage on plants grown under high or low PPFD. Samples were collected 6 h after the beginning of the light period, immediately frozen in liquid nitrogen and stored at −80◦C. Then they were lyophilized and crushed. Sucrose, glucose, fructose, and starch (enzymatic digestion step) were determined by colorimetry (Konelab), as presented in Supplementary Material 3. CKs, free ABA, and free IAA concentrations were determined as in Li-Marchetti et al. (2015). Sugar and hormone contents were determined from four replicates with five plants per light treatment each.

#### Exogenous Supply of Chemicals Methods for Enriching Plant Tissues

For all chemicals and concentrations, except sugars at high concentrations, the cotton-wick method was used to supply the compounds directly into the stem, as described in Mahieu et al. (2009). The compounds were stored in the form of a liquid solution in a reservoir and brought to the plant by a cotton wick that went through the stem through an 0.5-mm hole positioned 5 mm below the fourth node (all the organs along the primary shoot are counted downward from the apex) (Supplementary Materials 2B,C). The wick was protected from desiccation and the reservoir was re-filled with the solution if needed.

At concentrations equal or above 300 mM, the sugars crystallized along the cotton wick, so they were supplied through the leaf rachis (Lin et al., 2011). The leaflets of the fourth leaf from the top were removed, except for the two proximal leaflets, and the rachis was rapidly immersed in a liquid solution in a 1.5 ml reservoir. After 1 week the rachis was cut 0.5 cm lower. The reservoirs were re-filled if necessary. Both methods supplied the exogenous compounds continuously.

#### Sugar, CK, and ABA Supply

All chemicals were supplied from the FBV stage onward. Sugars and CKs were provided to the plants grown under the low PPFD light treatment to test if they could relieve the bud outgrowth inhibition induced by low PPFD. Sugars were provided mainly as sucrose, which is the circulating form, but also as glucose because these two forms can trigger bud outgrowth in isolated rose nodes grown in vitro (Rabot et al., 2012). Under sucrose 50, 100, 200 mM and glucose 100 mM, mannitol 100 mM was used as an osmotic control; under sucrose 300, 600, 800 mM and glucose 600 mM, mannitol 300 mM served as a control. Mannitol is not metabolized by R. hybrida and does not stimulate bud outgrowth (Henry et al., 2011; Rabot et al., 2012). These concentrations are typical of phloem sap (Nadwodnik and Lohaus, 2008; Merchant et al., 2009) and are either equal to or higher than those previously shown to be able to trigger bud outgrowth in isolated rose internodes (Henry et al., 2011; Rabot et al., 2012; Barbier et al., 2015).

Synthetic CKs were supplied as 6-benzylaminopurine (BAP) at 50 or 500 µM, solubilized in water with NaOH 40 mM. The control consisted of NaOH 40 mM alone. The concentrations of 50 and 500 µM BAP were within the range of synthetic CKs used in pea and different woody species (Dun et al., 2012; Ni et al., 2015).

To test whether ABA could inhibit bud outgrowth in favorable conditions, ABA was supplied at 1.25 and 2.5 mM with NaOH 40 mM to plants grown continuously under a high PPFD of 350 µmol m−2 s −1 provided by white LED tubes. The control was an ABA-free solution of NaOH 40 mM. To test whether ABA could also inhibit bud outgrowth under low PPFD, ABA was supplied under low PPFD together with CKs, because buds grew out in plants supplied with CKs under the low PPFD (see "Results" section). ABA and CKs were supplied in the form of solutions containing BAP 500 µM + ABA 1.25, 2.5 or 5.0 mM + NaOH 40 mM. The control contained BAP 500 µM + NaOH 40 mM. High concentrations of ABA were applied as endogenous ABA contents were much higher than endogenous CK contents (see "Results" section).

# 13C-Labeled Sugar Uptake Studies

To check that sugars supplied by both the cotton-wick method and the rachis-feeding method were indeed delivered to the stem tissues, the plants grown under low PPFD were supplied with labeled sucrose. Labeled 13C-sucrose (ID 605417\_Aldrich, Sigma–Aldrich, Darmstadt, Germany) was incorporated at 3% in a solution of 200 mM sucrose supplied by a cotton wick just below node 4, or incorporated at 3.4% in a solution of 600 mM sucrose supplied through the rachis of leaf 4. Water supplied either by a

cotton wick or through the rachis served as a control. Exogenous supply started at the FBV stage onward. Days 4 and 8 after the FBV stage, the third, fourth, and fifth internodes from the apex (not counting the peduncle, Supplementary Material 2A) were sampled as described previously. Total carbon and the 13C:12C ratio were determined for each sample using an elemental analyzer (NA 1500 NCS; Carlo Erba, Milan, Italy) coupled with a Delta-S isotope ratio mass spectrometer (Finnigan-Mat; Thermoquest Corp., San Jose, CA, United States). δ 13C was calculated as:

$$\text{R}^{13}\text{C} \left(\% \right) \;= \left(\text{R sample}/\text{R reference} - 1\right) \times 1000 \qquad \text{(1)}$$

where R is the ratio of heavy-to-light isotope, and the R reference for 13C in the PDB standard is 0.0112372.

#### Growth Measurements of the Different Buds along the Primary Axis

Three to six times a week from FBV onward, the state (dormant or growing out) of each bud along the primary axis was noted, and bud length was measured with a ruler. Buds were considered to have grown out when at least one visible leaf protruded between the two bud scales (Girault et al., 2008; Roman et al., 2016). The mean bud lengths were calculated from the sizes of outgrowing and dormant buds from a same batch. Fourteen days after the FBV stage, buds were dissected using a stereomicroscope. Bud meristem organogenic activity, which results in the formation of new organs in the bud, was assessed by counting the number of leaves in the bud, leaves being here defined as young leaves plus foliar primordia, excluding the two bud scales. Measurements were performed on 8–16 plants per experimental modality (light treatment and/or chemical exogenous supply), with three biological replicates, except otherwise stated.

#### Photosynthesis Measurements

The CO2 net assimilation rate was measured as the quantity of CO2 assimilated per leaf surface unit per second, and represents the difference between photosynthetic assimilation and respiration. To assess the effect of the light treatments, CO2 net assimilation rates were measured both in a leaf still rapidly expanding and partially unfolded at the FBV stage (leaf 4 from the apex), and in a mature fully unfolded leaf (leaf 6 from the apex), on five plants, 3 h before and 3 h after the switch in light intensity, and then once a day for 14 days, 6 h after the beginning of the light period.

To test if the CK supply or the CK – ABA supply affected photosynthesis in plants grown under low PPFD, CO2 net assimilation rates were measured in leaves of plants supplied according to the following conditions: CK 500 µM + NaOH 40 mM, CK 500 µM + ABA 1.25, 2.5, 5.0 mM + NaOH 40 mM, control NaOH 40 mM. CO2 net assimilation rates were measured in the leaves positioned immediately above and below the supply point (leaves 4 and 5, respectively, Supplementary Materials 2A,B) on four plants per condition, every 2 days from FBV onward, 6 h after the beginning of the light period. Leaf positions were chosen so as to maximize the likelihood to observe an effect of the exogenous supply if it existed.

All measurements of CO2 net assimilation rates were performed using an infrared gas analyser, Li-Cor-6400 (Li-Cor Inc., Lincoln, NE, United States), at a temperature of 18◦C, a relative humidity of 64%, a cuvette air flow rate of 300 ml min−1 , and an ambient CO2 concentration of 400 µmol mol−1 . PPFDs of 540 and 90 µmol m−2 s −1 were provided to the measured leaves for the plants grown under high and low PPFD, respectively, by a mixture of red and blue LEDs.

#### Statistical Analysis

Statistical analyses were performed using R software for Windows (R Core Team, 2014). Groups were compared using one-way ANOVA (α = 0.05) followed by Tukey's test when the ANOVA indicated significant differences at P < 0.05 and there were more than two groups.

### RESULTS

#### Decreasing PPFD Inhibits Axillary Bud Outgrowth All along the Stem

The number of outgrown buds per plant was much lower under the low PPFD treatment than under the high PPFD treatment (**Figure 1A-inset** and Supplementary Figure S1). Under high PPFD buds grew out all along the stem, displaying an acrotonic pattern with a decreasing outgrowth rate from the apical to the basal part of the stem (**Figures 1A,B**). The time course of bud outgrowth was sequential, starting from the two uppermost apical buds 2 days after the FBV stage and proceeding downward (**Figure 1C**). The uppermost two apical buds did not respond to a reduction of PPFD because they are sylleptic buds whose outgrowth is independent of environmental conditions (Huché-Thélier et al., 2011; Demotes-Mainard et al., 2013b). But, the outgrowth rate of the third topmost bud was reduced as compared to high PPFD conditions (72% vs. 100%), and outgrowth of all the buds from the fourth topmost bud and below was totally inhibited (**Figures 1A,B**). Bud 4 displayed the most contrasted outgrowth behavior between PPFD treatments. While under high PPFD all buds 4 had grown out 8 days after the FBV stage on average, none grew out in low PPFD (**Figures 1A,C**). Elongation of bud 4 was rapid and continuous under high PPFD, whereas it was slow and stopped rapidly under low PPFD (**Figure 1D**).

#### Decreasing PPFD Reduces Sugar Contents in the Internode, but Local Sugar Supply Is Not Sufficient to Restore Bud Outgrowth

To determine if sugars are involved in mediating the effect of PPFD on bud outgrowth, we analyzed the sugar content of internode 4, which is adjacent to the bud displaying the most contrasted response to PPFD treatments (**Figures 1A,B**). Internode 4 displayed a lower content in sucrose, glucose, fructose, and starch under low PPFD than under high PPFD days

4 and 8 after the FBV stage (**Figure 2**). Day 8 was the time when bud 4 grew out under high PPFD (**Figure 1C**), and day 4 was the time when we presume that conditions may have affected the inducement of bud 4 outgrowth since 4 days are required for bud outgrowth after apical dominance has been relieved under favorable light conditions in this cultivar (Girault et al., 2010). The correlation between sugar content in internode 4 and bud 4 outgrowth thus suggests that a low sugar level in the node may be involved in bud outgrowth inhibition under low PPFD.

To investigate this hypothesis, we tested if local sugar supply could relieve bud inhibition under reduced PPFD. Sugars were supplied to plants either through a cotton wick (50, 100, and 200 mM) or through the rachis for the higher concentrations (300, 600, and 800 mM). Using labeled 13Csucrose, we demonstrated that sugars were indeed delivered into the stem tissues by these two methods (Supplementary Figures S2A,B). Sucrose supplied under low PPFD did not promote bud outgrowth, nor did it change the gradient of bud outgrowth along the stem as compared to the control, whatever the concentration (**Figures 3A,A-inset,D,D-inset**). Neither did it stimulate bud elongation (**Figures 3B,E**) or leaf formation into the bud (**Figures 3C,F**). Similar results were observed when glucose (100 or 800 mM) instead of sucrose was supplied (Supplementary Figures S3A–F). Altogether, these results show that although low PPFD leads to reduced sugar levels in the internode, the bud outgrowth inhibition induced by low PPFD cannot be solely relieved by local sugar supply. We therefore investigated whether CKs and ABA may limit bud outgrowth under low PPFD.

#### Decreasing PPFD Reduces CK Contents in the Internode, and CK Supply Can Relieve Low PPFD-Induced Bud Inhibition

Cytokinin (zeatin riboside-50 -monophosphate, ZRMP; isopentenyl adenosine-50 -monophosphate, iPRMP; isopentenyl adenine riboside, iPR) contents were measured in internode 4 under high and low PPFD treatments 4 and 8 days after the FBV stage. ZRMP and iPRMP are intermediate forms in the CK biosynthesis pathway, and iPR is an active form. The three CK forms followed different temporal dynamics under high PPFD:

average of four repetitions. Error bars represent standard error of the mean (SE). Different letters indicate significant differences between treatments and times

the content in ZRMP increased over the 8 days following the FBV stage, whereas the content in iPRMP was stable, and the content in iPR decreased over time (**Figures 4A–C**). Low PPFD reduced the content in all three CK forms in internode 4, 4 and 8 days after the FBV stage (**Figures 4A–C**), whereas the free IAA content in internode 4 was not affected by the PPFD level (**Figure 4D**).

(ANOVA followed by a Tukey test, P < 0.05).

In order to assess if CKs may be players in the regulation of the bud outgrowth pattern by PPFD, we tested if exogenous CKs could relieve the bud outgrowth inhibition induced by low PPFD. Synthetic CK BAP was supplied to internode 4 of plants grown under low PPFD using cotton wicks. When 50 and 500 µM CK were supplied, the number of buds that grew out per plant increased significantly as compared to the control (**Figure 5A-inset**). Increased outgrowth was observed locally close to the supply point for bud 4, but also at a distance for bud 3 (**Figure 5A**). Bud 4, whose outgrowth was totally inhibited under low PPFD in the absence of CK, displayed rates of 91 and 100% outgrowth when the plants were supplied with 50 and 500 µM of CKs, respectively, under low PPFD. Under low PPFD again, outgrowth of bud 4 was observed 7.78 ± 0.44 and 7.49 ± 0.39 days after the FBV stage in plants supplied with CKs at 50 and 500 µM, respectively, and bud 4 elongation was increased by exogenous CKs (**Figure 5B**), giving a similar date of outgrowth and dynamics of elongation as observed under high PPFD (**Figures 1C,D**). CK supply under low PPFD also increased the number of leaves formed into the bud (**Figure 5C**). Taken together, these results suggest that low PPFD inhibition of bud outgrowth is mediated at least partly through a lower stem CK content.

#### Low PPFD Increases the ABA Content, and ABA Supply Can Inhibit Bud Outgrowth under High PPFD and Antagonizes CK Stimulation of Bud Outgrowth under Low PPFD

Measurements of free ABA contents in the adjacent internode to bud 4 revealed that inhibition of bud outgrowth by low PPFD was also correlated to the maintenance of higher levels of ABA 4 and 8 days after the FBV stage (**Figure 6**).

To assess whether ABA could be involved in mediating PPFD control of bud outgrowth, we tested whether exogenous supply of ABA (0, 1.25, and 2.5 mM) to internode 4 could inhibit bud outgrowth under high PPFD (350 µmol m−2 s −1 ). ABA supply at both 1.25 and 2.5 mM had a strong inhibitory effect on the outgrowth and elongation of bud 4 (**Figures 7B,C**) but not on the number of leaves formed per bud (**Figure 7D**). Interestingly, outgrowth of buds located below the point of ABA supply increased (**Figure 7A**), so that the total number of outgrown buds per plant was not affected (**Figure 7A-inset**).

To test whether a high ABA stem content could inhibit bud outgrowth under low PPFD, ABA was supplied under low PPFD together with CK, a promoter of outgrowth. ABA antagonized the stimulating effect of CK in a dose-dependent manner and decreased bud 4 outgrowth (**Figures 8A,A-inset**). With the lowest ABA concentration, only 46% of these buds grew out and their outgrowth was delayed by 2 days, occurring only 10.2 ± 0.4 days after FBV, whereas with the highest ABA concentration only 4.7% of buds 4 grew out. In the presence of CKs, ABA also drastically limited the elongation of bud

FIGURE 3 | Impact of sucrose supply on bud outgrowth pattern of plants grown under the low PPFD treatment. Sucrose was supplied through the stem below node 4 at 50, 100, and 200 mM (A–C) or through the rachis of leaf 4 at 300, 600, and 800 mM (D–F) from FBV stage onward, and mannitol (100 mM A–C or 300 mM D–F) was used as non-metabolizable control. (A,D) Effect of sucrose supply on the percentage of individuals with outgrown buds 14 days after FBV according to node position along the shoot. (A-inset,D-inset) Total number of buds per plant that grew out 14 days after FBV. (B,E) Elongation of bud 4 along time. (C,F) Number of leaves in bud 4 at FBV stage (day 0) and 14 days after FBV (day 14). Buds are numbered basipetally along the stem. Each point represents the average of three repetitions. Error bars represent the standard error of the mean (SE). Different letters indicate significant differences between sugar supplies and times, ns indicates an absence of significant difference (ANOVA, followed by a Tukey test for C, F, P < 0.05).

4 (**Figure 8B**). With the highest ABA concentration supplied together with CK, the frequencies of bud 4 outgrowth and the kinetics of bud 4 elongation were very similar to those observed under low PPFD without any exogenous supply (**Figures 1**, **8**). ABA had no effect on the number of leaves formed into the bud (**Figure 8C**). The buds below the supply point did not grow out, whatever the ABA concentrations (**Figure 8A**).

Altogether, these results indicate that low PPFD maintains high ABA levels in the stem and that stem ABA can inhibit bud outgrowth under low and high PPFD in rose. They suggest that the high ABA stem contents induced by low PPFD may contribute to the inhibition of bud outgrowth, notably by antagonizing the promoting effect of CKs on bud outgrowth.

# Neither CK Stimulation of Bud Outgrowth under Low PPFD Nor ABA Antagonism of CK Stimulation under Low PPFD Require a Change in CO2 Net Assimilation

The decrease in PPFD from the FBV stage onward markedly and durably reduced photosynthetic CO2 net assimilation rates as compared to high PPFD, both in mature and in still rapidly expanding leaves (Supplementary Figure S4). The literature indicates that ABA may dampen photosynthesis (Raschke and Hedrich, 1985; Zhou et al., 2006), and that CKs alone or CKs and ABA together may affect photosynthesis in a way that varies with the species, the conditions of application and the plant growth conditions (Pospíšilová, 2003; Pospíšilová and Bat'ková, 2004; Shu-Qing et al., 2004). We studied whether the effect of CKs and ABA on bud outgrowth passed through an indirect effect on photosynthetic assimilation in our conditions. Over the 2 weeks following the FBV stage, CO2 net assimilation rates of leaves 4 and 5 of plants grown under low PPFD were affected neither by CK supply alone nor by combined CK and ABA supply, whatever the ABA concentration (**Figures 9A,B**). These results indicate that the effects of CKs and ABA on the bud outgrowth pattern under low PPFD are not directly mediated by changes in photosynthetic assimilation.

# DISCUSSION

## The Responses of Bud Outgrowth to PPFD Are Preceded by Changes in CK, ABA, and Sugar Stem Contents

The reduced PPFD applied from the FBV stage onward reduced the number of outgrowing buds, consistently with previous results in rose (Bredmose, 1995; Furet et al., 2014; Wubs et al., 2014). To address the question of how the CK, ABA, and sugar regulation pathways take part in the control of the branching pattern by light intensity, we focused on bud 4 that displayed the most contrasted fate with 100% or 0% outgrowth under high or low PPFD, respectively. Changes in PPFD led to variations in endogenous CK, ABA, and **s**ugar contents in internode 4, consistent with bud outgrowth response to PPFD: CKs and sugars, which promote bud outgrowth, had their levels decreased under low PPFD, while ABA, an outgrowth

inhibitor, had its content increased. The effect of PPFD on the CK stem content was consistent with the effect observed following exposure to light or darkness by Roman et al. (2016), but information on the effects of PPFD on the ABA stem content was lacking. The reduction in sugar stem content was consistent with the severe decrease in photosynthesis observed under low PPFD but may also have involved changes in partitioning. In addition, the variations in endogenous CK, ABA, and sugar contents in internode 4 preceded the outgrowth of the adjacent bud under high PPFD by at least 4 days; this supports the hypothesis that these chemicals could indeed contribute to the regulation of bud outgrowth. In rose, modifications in sugars and hormone (CK) levels and in expression of genes involved in bud outgrowth regulation were reported to be triggered before the occurrence of bud outgrowth (Girault et al., 2010; Henry et al., 2011; Rabot et al., 2012; Barbier et al., 2015; Roman et al., 2016).

### The CK Stem Content Appears to Be a Key Factor Mediating PPFD Regulation of Bud Outgrowth Pattern along the Stem in an Intact Plant

Despite the major role of CKs in promoting bud outgrowth (Dun et al., 2012; Müller et al., 2015), studies investigating their involvement in the regulation of bud outgrowth by light are scarce. Stem endogenous CKs dropped under low PPFD concomitantly to bud inhibition and conversely CK supply to

outgrown buds 14 days after FBV according to node position along the shoot. (A-inset) Total number of buds per plant that grew out 14 days after FBV. (B) Percentage of individuals whose buds 4 grew out 14 days after FBV stage. (C) Elongation of bud 4 along time. (D) Number of leaves in bud 4 at FBV stage (day 0) and 21 days after FBV (day 21). Buds are numbered basipetally along the stem. Each point represents the average of two repetitions. Error bars represent the standard error of the mean (SE). Different letters indicate significant differences between ABA supplies, ns indicates an absence of significant difference (ANOVA followed by a Tukey test, P < 0.05).

plants grown under low PPFD relieved the inhibition of bud outgrowth. CK supply stimulated bud meristem organogenic activity and bud elongation, that are the two fundamental processes of bud outgrowth. This stimulation happened in such a way that CK supply under low PPFD resulted in the same timings of bud 4 outgrowth and the same kinetics of bud 4 elongation as those observed under high PPFD. Altogether, these results are consistent with our previous findings showing that CKs are

initial targets of light in the regulation of bud outgrowth in decapitated defoliated plants (Roman et al., 2016). They further bring evidence that (i) CKs allow buds to be relieved from ecodormancy due to unfavorable environmental conditions (low PPFD) and from paradormancy (apical dominance and other correlative inhibitions, Zieslin and Halevy, 1976; Domagalska and Leyser, 2011); (ii) a reduction of CK levels not only mediates repression of bud outgrowth induced by total darkness but also by low PPFD, as can be encountered under normal daylight conditions.

The stimulating effect of CKs on bud meristem organogenic activity under low PPFD is consistent with the ability of CKs to rescue organogenic activity under darkness in shoot apical meristems of tomato and rose (Yoshida et al., 2011; Roman et al., 2016).

# The Changes in Stem CK Contents in Response to PPFD Are Not Mediated by Changes in IAA Contents

Knowing that stem IAA can downregulate stem CK contents (Tanaka et al., 2006; Shimizu-Sato et al., 2009), we wondered if the changes in stem CKs in response to PPFD may have resulted, at least partly, from a change in stem IAA. Knowledge on the effects of PPFD on stem auxin contents is poor, and the results obtained in various species and growth conditions are conflicting (Kurepin et al., 2006, 2007, 2008; Hersch et al., 2014). Here, the absence of difference in IAA contents in internode 4 between the two PPFD treatments indicates that the changes in stem CK content was not a consequence of changes in stem IAA levels. Roman et al. (2016) also observed changes in CK stem contents in response to light in the absence of variations in IAA content, but the stem IAA levels in their plant system were extremely low due to decapitation.

Both stem-synthesized CKs and root-derived CKs can contribute to bud outgrowth regulation, with conflicting results between species (Faiss et al., 1997; Tanaka et al., 2006; Müller et al., 2015). In our study we can assume that the regulation of the CK stem content by PPFD may result at least partly from local regulation of the CK metabolism in the stem, since CK synthesis genes are downregulated and CK degradation genes are upregulated in the stem of plants grown in darkness as compared to light (Roman et al., 2016). Regulation of the CK stem content may also partly result from changes in xylem-carried CKs coming from the roots, since in tobacco plants shade reduces xylem carried CKs by reducing the transpiration rate (Boonman et al., 2007).

### The High ABA Content in the Stem Induced by Reduced PPFD Can Inhibit Bud Outgrowth along the Stem and Antagonize CKs

Our results suggest that the high stem ABA content induced by low PPFD contributed to the regulation of bud outgrowth. Exogenous ABA supply to the stem inhibited bud outgrowth in rose under high PPFD, i.e., in environmental conditions favorable to outgrowth, in accordance with previous data in Arabidopsis, pea, Ipomoea nil, and tomato (Arney and Mitchell, 1969; Chatfield et al., 2000; Cline and Oh, 2006; Yao and Finlayson, 2015). To investigate further the role of ABA in mediating bud outgrowth regulation by PPFD, we showed that ABA could also inhibit bud outgrowth under low PPFD. ABA was able to antagonize the promotive effect of exogenous CKs on bud outgrowth in a dose-dependent manner under low PPFD. The antagonistic role of ABA and CKs had been evidenced in other processes such as stomatal regulation, seed germination or seedling development (Dodd, 2005; Wang et al., 2011; Guan et al., 2014), but such a dose-dependent antagonism was evidenced in this study for the first time in the regulation of bud outgrowth. Altogether, our results suggest that the effect of PPFD on bud outgrowth may be complex and that reduced PPFD inhibits bud outgrowth both by reducing the stem content in CKs (a branching inducer) and by maintaining a high content in stem ABA (a branching repressor). The high ABA content can dampen the effects of the remaining CKs. ABA was already known to mediate the response to the R:FR ratio or the photoperiod in Arabidopsis (Reddy et al., 2013; Yao and Finlayson, 2015; González-Grandío et al., 2017; Holalu and Finlayson, 2017). Our results extend its role to the mediation of environmental regulation of bud outgrowth by another light signal and species. In addition, they also point out the contribution of stem ABA in controlling bud outgrowth, and complement the previous works that focused on bud ABA.

Interestingly, ABA inhibited bud outgrowth and impaired bud elongation, but did not affect meristem organogenic activity in the bud, irrespective of the PPFD level. In contrast, CKs stimulated bud meristem organogenic activity, which indicates that the antagonistic effect of ABA and CKs was limited to certain bud outgrowth-related processes.

### Supplying Sugars Close to the Bud Cannot Alone Relieve Low PPFD-Induced Bud Inhibition

Under low PPFD, exogenous supply of both sucrose and glucose close to the bud failed to promote bud outgrowth in intact plants. These results suggest that sugars are not the main limiting factor

FIGURE 10 | Schematic representation of the relationships between CKs, ABA, IAA, and sugars in the regulation of bud outgrowth by PPFD in an intact rose plant. Low PPFD reduces stem CK content, which appears to be a key limiting factor of bud outgrowth. The level of CKs transported from the stem to the bud, where they stimulate outgrowth, is likely to be dampened under low PPFD. The CK stimulation of bud sugar sink strength, shown in decapitated rose plants (Roman et al., 2016), may also be lowered. Low PPFD maintains a high stem ABA content, ABA presumably enters the bud where it inhibits outgrowth, antagonizing the effects of CKs. Low PPFD also reduces photosynthesis and sugar stem content. However, sugar stem content is not the main limiting factor for bud outgrowth since sugar supply is not able to override bud outgrowth inhibition under low PPFD. The reduced bud sugar sink strength under low PPFD may prevent the use of sugars by the bud for outgrowth. Although IAA is known to dampen stem CK content, low PPFD effect on CK is not mediated through changes in stem IAA content.

for bud outgrowth in intact plants under low PPFD, but would act downward other factors, at least CKs and ABA. These results are consistent with those obtained in the upper bud of decapitated defoliated plants grown under darkness (Roman et al., 2016).

Cytokinins have positive effects on source-sink organ activities (Cowan et al., 2005; Peleg et al., 2011). In our case the CK supply that promoted outgrowth under low PPFD is unlikely to have acted on the global sugar content per plant as it did not change photosynthetic assimilation. CK supply to the stem is known to increase the sugar sink strength of the bud (Roman et al., 2016), in agreement with CK-mediated upregulation of sugar sink strength in other tissues (Roitsch and González, 2004). Thus, we can hypothesize that one action of CK supply in the vicinity of the bud under low PPFD was to increase bud sugar sink strength and allow buds to better compete for sugars at the expense of other plant organs. Unlike CKs, ABA affects negatively sink strength by limiting the availability of nutrients and energy (Garciarrubio et al., 1997; Parish et al., 2012). It would be of great interest to investigate in future works if ABA negatively affects the sink strength of buds and how it interacts with CKs in response to PPFD. Mason et al. (2014) demonstrated that sugar supply alone can promote bud outgrowth when plants are grown in a non-limiting light environment in which the shoot tip's high demand for sugars is the limiting factor for bud access to sugars. In our experiments the apical organs, that were in rapid expansion (Demotes-Mainard et al., 2013a), most probably intensively competed with buds for sugars too. However, sugar supply to the stem could not trigger outgrowth under low PPFD because PPFD controlled bud outgrowth through an upstream mechanism, involving CK and presumably ABA regulation. We hypothesize that at least part of this control is due to low stem CK content that restricts sugar import into the bud under low PPFD.

#### Action of Exogenous CKs and ABA on Bud Outgrowth at Different Locations along the Stem

Exogenous CK supply had an effect on the directly treated bud (bud 4) and on the first bud located above it (bud 3). The absence of effect on the buds located below the supply point is consistent with results on isolated nodes and decapitated plants (Ohkawa, 1984; Chatfield et al., 2000).

Abscisic acid supply in the stem inhibited only the local bud. We did not expect exogenous ABA supply to inhibit lower buds since ABA inhibits outgrowth in isolated nodes only when supplied from the basal side of the stem (Chatfield et al., 2000). The absence of inhibition of bud 3, located apically, may result from the dose of ABA that reached bud 3 and/or from a lower sensitivity of this bud to ABA due to its physiological state. Bud 3 was indeed much less sensitive to PPFD than the buds below, as shown by the outgrowth patterns under low and high PPFD.

When exogenous ABA totally inhibited bud 4 outgrowth under high PPFD, the outgrowth rate of the buds just below it (buds 5 and 6) increased. This increase in outgrowth may be an indirect effect of bud 4 inhibition under high PPFD: bud 4 inhibition by ABA may have both reduced the correlative inhibition exerted on lower buds (Domagalska and Leyser, 2011) and made sugars more available for these lower buds, thereby facilitating their outgrowth under high PPFD (Mason et al., 2014). In isolated internodes of Arabidopsis, ABA supplied from the apical side of the stem reduced inhibition by IAA (Chatfield et al., 2000). Such an effect cannot be ruled out but was not observed in Ipomoea nil or in sunflower (Cline and Oh, 2006).

# CONCLUSION

This study provides evidence that in intact rose plants reduced PPFD controls bud outgrowth pattern both by reducing the stem CK content and by maintaining a high stem ABA content. These two hormones regulate bud outgrowth antagonistically,

but their antagonistic effect is limited to certain bud outgrowthrelated processes since bud meristem organogenic activity is solely regulated by CKs. Although the stem sugar content was severely reduced under low PPFD, this drop did not appear to be the triggering factor of bud outgrowth inhibition under low PPFD. A model based on our results and literature is presented in **Figure 10**. Further work will contribute to investigate the relationships between CKs and ABA in the regulation cascade of the bud outgrowth pattern in response to PPFD by investigating at the molecular level regulation pathways between buds showing different sensitivity levels to PPFD variations.

### AUTHOR CONTRIBUTIONS

AC, NL, SS, SD-M conceived and designed the research. AC, OD, HA, M-DP-G, and SC carried the experimental work, collected and prepared the data. AC conducted statistical analyses. NL, SS, and SD-M directed the work. AC, HR, JB, SS, NL, and SD-M interpreted the data and wrote the paper.

### FUNDING

This research was conducted in the framework of the regional program "Objectif Végétal, Research, Education and Innovation in Pays de la Loire," supported by the French Region Pays de la Loire, Angers Loire Métropole, and the European Regional Development Fund.

# REFERENCES

#### ACKNOWLEDGMENTS

We thank the certified facility in Functional Ecology (PTEF OC 081) from UMR 1137 EEF and UR 1138 BEF in research center INRA Nancy-Lorraine for the isotopic analyzes. The PTEF facility is supported by the French National research Agency through the laboratory of Excellence ARBRE (ANR-11-LABX-0002-01). We thank Bénédicte Dubuc (UMR IRHS) and the ImHorPhen team (Phenotic platform, UMR IRHS), particularly Christian Cattanéo, Jacky Granger, Dominique Honoré, and Rémi Gardet, for rose cutting production, plant care and the maintenance of experimental facilities; Anne-Claire Grosbois (trainee UMR IRHS and Université d'Angers) for technical assistance with the ABA experiments; Rachid Boumaza (UMR IRHS) for the advice provided in statistical analyses; Jérémy Lothier, Thomas Péron (UMR IRHS), Wim van den Ende (KU Leuven), Christophe Godin (INRIA Virtual Plant), Didier Combes (INRA, UR P3F), and Elzbieta Frak (INRA, UR P3F) for fruitful discussions; PAIGE team (UMR IRHS) for the administrative work, Annie Buchwalter (a scientific translator) for reviewing the paper for English usage.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2017.01724/ full#supplementary-material

growth potential in apple trees (Malus domestica [L.] Borkh.). J. Exp. Bot. 53, 2143–2149. doi: 10.1093/jxb/erf063

dominance. Plant J. 45, 1028–1036. doi: 10.1111/j.1365-313X.2006. 02656.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 © 2017 Corot, Roman, Douillet, Autret, Perez-Garcia, Citerne, Bertheloot, Sakr, Leduc and Demotes-Mainard. 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.

# Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control

Mareike Kessenbrock1 , Simone M. Klein2 , Lena Müller1 , Mauricio Hunsche2,3 , Georg Noga2 and Georg Groth1,4 \*

1 Institute of Biochemical Plant Physiology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany, 2 Institute of Crop Science and Resource Conservation – Horticultural Science, University of Bonn, Bonn, Germany, 3 COMPO EXPERT GmbH, Münster, Germany, 4 Bioeconomy Science Center, Forschungszentrum Jülich, Jülich, Germany

#### Edited by:

Chi-Kuang Wen, Shanghai Institutes for Biological Sciences (CAS), China

#### Reviewed by:

Bram Van De Poel, KU Leuven, Belgium Jie Le, Key Laboratory of Plant Molecular Physiology, Institute of Botany (CAS), China

\*Correspondence:

Georg Groth<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

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

Received: 19 June 2017 Accepted: 21 August 2017 Published: 05 September 2017

#### Citation:

Kessenbrock M, Klein SM, Müller L, Hunsche M, Noga G and Groth G (2017) Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control. Front. Plant Sci. 8:1528. doi: 10.3389/fpls.2017.01528 Ethylene signaling is decisive for many plant developmental processes. Among these, control of senescence, abscission and fruit ripening are of fundamental relevance for global agriculture. Consequently, detailed knowledge of the signaling network along with the molecular processes of signal perception and transfer are expected to have high impact on future food production and agriculture. Recent advances in ethylene research have demonstrated that signaling of the plant hormone critically depends on the interaction of the ethylene receptor family with the NRAMP-like membrane protein ETHYLENE INSENSITIVE 2 (EIN2) at the ER membrane, phosphorylation-dependent proteolytic processing of ER-localized EIN2 and subsequent translocation of the cleaved EIN2 C-terminal polypeptide (EIN2-CEND) to the nucleus. EIN2 nuclear transport, but also interaction with the receptors sensing the ethylene signal, both, depend on a nuclear localization signal (NLS) located at the EIN2 C-terminus. Loss of the tight interaction between receptors and EIN2 affects ethylene signaling and impairs plant ethylene responses. Synthetic peptides derived from the NLS sequence interfere with the EIN2–receptor interaction and have utility in controlling plant ethylene responses such as ripening. Here, we report that a synthetic peptide (NOP-1) corresponding to the NLS motif of Arabidopsis EIN2 (aa 1262–1269) efficiently binds to tomato ethylene receptors LeETR4 and NR and delays ripening in the post-harvest phase when applied to the surface of sampled green fruits pre-harvest. In particular, degradation of chlorophylls was delayed by several days, as monitored by optical sensors and confirmed by analytical methods. Similarly, accumulation of β-carotene and lycopene in the fruit pulp after NOP-1 application was delayed, without having impact on the total pigment concentration in the completely ripe fruits. Likewise, the peptide had no negative effects on fruit quality. Our molecular and phenotypic studies reveal that peptide biologicals could contribute to the development of a novel family of ripening inhibitors and innovative ripening control in climacteric fruit.

Keywords: ethylene signaling, ethylene receptors, peptide, ripening control, Solanum lycopersicum (tomato), post-harvest application

# INTRODUCTION

fpls-08-01528 September 2, 2017 Time: 15:8 # 2

Worldwide, a tremendous amount of food produced for human consumption is lost or wasted until the product reaches the consumer, with about 50% of those food losses being valuable vegetables and fruits (Blanke, 2014). The tomato fruit is one of the most important climacteric fruits (Jackman et al., 1990; Vidoz et al., 2010) and has worldwide high economic and nutritional importance, mainly because of its high concentrations of carotenoids such as lycopene, β-carotene and pro-vitamin A (Canene-Adams et al., 2005; Vidoz et al., 2010) which accumulate during fruit ripening. The ripening process of climacteric fruits is characterized by a strong increase in cell respiration which is mainly regulated by the plant hormone ethylene (Alexander and Grierson, 2002; Guo and Ecker, 2004). Ripening is initiated by a burst of an auto-stimulated ethylene synthesis, with following activation of ripening related genes (Abano and Buah, 2015). This ethylene related gene expression leads to physiological, morphological and biochemical changes. In the process of fruit ripening, fruits change color, texture, firmness, flavor and aroma (Brady, 1987; Alexander and Grierson, 2002) due to degradation of pectins, cellulose and chlorophyll as well as due to a decreasing content of organic acids and increasing concentration of soluble sugars, carotenes and aroma volatiles (Brady, 1987).

Besides the traditional quality analysis of firmness and content of sugars, acids, vitamins and pigments, changes in fruit ripening and fruit quality might be evaluated by non-destructive optical methods (Abbott, 1999; Hoffmann et al., 2015). Analogous to that, consumers usually estimate fruit quality based on fruit skin color. Color development of tomatoes from green to red can be measured by monitoring chlorophyll degradation as well as lycopene and β-carotene accumulation (Alexander and Grierson, 2002). Typically, the total content of these pigments is analyzed using wet chemical procedures (Barros et al., 2007; Azeez et al., 2012; Kalogeropoulos et al., 2012), whereas nondestructive optical sensors that evaluate overall changes in fruit color based on reflection and fluorescence properties provide monitoring parameters that strongly correlate with the analytical values (McGuire, 1992; Hoffmann et al., 2015).

Control of ripening is important to ensure quality and to reduce post-harvest losses of climacteric fruits. At commercial scales, fruits are usually stored at low temperatures under controlled atmosphere to limit ethylene production and ethylene response (Watkins et al., 2000; Saltveit, 2005; Passam et al., 2007). Alternative approaches, such as genetic engineering of ethylene biosynthesis to decrease endogenous ethylene production, are under development in science and research despite of ongoing discussion in Europe about genetic engineering in general. At the production scale, fruit maturation can be delayed with aminoethoxyvinylglycine (AVG), an inhibitor of ACC-synthase, the key enzyme of ethylene biosynthesis (Saltveit, 2005). For postharvest treatment, 1-methylcyclopropene (1-MCP), a gaseous chemical with the ability to inhibit ethylene receptors and receptor-triggered ethylene response can be applied (Watkins et al., 2000; Yuan and Carbaugh, 2007).

Pathways and mechanisms for biosynthesis, perception and signal transduction of the plant hormone ethylene have been extensively studied in the model plant Arabidopsis thaliana. These studies disclosed that the ethylene signal is perceived by a family of five receptor proteins, which form homo- and heterodimers at the ER membrane and function as negative regulators of the ethylene response (Bleecker et al., 1988; Chang et al., 1993; Hua et al., 1995, 1998; Hua and Meyerowitz, 1998; Grefen et al., 2008). Although the exact output of the receptors is still obscure, genetic studies demonstrate that in the absence of ethylene, receptors activate the Raf-like protein kinase CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1), another negative regulator of the pathway (Kieber et al., 1993). Downstream of the receptors and the ER associated CTR1 kinase the membrane protein ETHYLENE INSENSITIVE 2 (EIN2), which contains a highly conserved NLS (Bisson and Groth, 2011; Qiao et al., 2012) shown to mediate interaction with the up-stream receptors (Bisson and Groth, 2015; Bisson et al., 2016), implements a positive regulatory role on ethylene signaling. In the presence of ethylene, the receptors bind the hormone and become inactivated. CTR1 cannot be activated by the receptors, and the lack of CTR1 activation cannot phosphorylate EIN2. Subsequently, the C-terminal end of EIN2 (C-END) containing the NLS-motif is cleaved off by an unknown mechanism and translocated to the nucleus (Ju et al., 2012; Qiao et al., 2012; Wen et al., 2012). In the nucleus, the EIN2 C-terminus directly or indirectly stabilizes the transcription factor EIN3 (Wen et al., 2012; Li et al., 2015) and its paralogous, the EIN3-like proteins (EILs), and transcription of ethylene response genes is activated (Chao et al., 1997; Solano et al., 1998).

In analogy to the model plant Arabidopsis, tomato contains a multigene family of the ethylene receptors. In total, seven isoforms named LeETR1, LeETR2, NR, LeETR4, LeETR5, LeETR6, and LeETR7 have been identified (Wilkinson et al., 1995; Zhou et al., 1996a,b; Lashbrook et al., 1998; Tieman and Klee, 1999) which are structurally diverse sharing at the most extreme less than 50% sequence identity. Similar to their Arabidopsis relatives the tomato receptors cluster in two subfamilies. LeETR1, LeETR2 and NR forming the subfamily I are characterized by a functional histidine kinase domain and a sensor domain consisting of three transmembrane helices. An additional putative membrane-spanning domain is present in LeETR5, LeETR6, LeETR7, and possibly in LeETR4 of subfamily II which are further characterized by a degenerated histidine kinase domain. All receptors except for NR contain a C-terminal response regulator domain (Lashbrook et al., 1998; Tieman and Klee, 1999). Expression patterns vary among the different receptor isoforms. While LeETR1 is expressed constitutively in all tissues, expression of LeETR2 is bound to seed germination and leaf senescence. NR, LeETR4 and to a lower extent LeETR5 are found at high expression levels in ripening fruit (Payton et al., 1996; Lashbrook et al., 1998; Tieman and Klee, 1999), but are rapidly degraded in the presence of ethylene by a 26S proteasome dependent pathway (Kevany et al., 2007). Due to this strong post-translational regulation of their protein level by the

**Abbreviations:** ACC, 1-aminocyclopropane-1-carboxylic acid; LeETR, ethylene receptor from tomato; NLS, Nuclear localization signal; NR, NeverRipe.

plant hormone and the observed correlation of receptor content and fruit ripening (Kevany et al., 2007), these receptors are of particular interest for studying the molecular effect of ripening inhibitors targeting ethylene signaling.

Recent insights in the ethylene signaling pathway propose a novel way to interfere with fruit ripening based on a yet unknown function of the NLS in the ethylene signaling protein EIN2. Peptides such as the synthetic octapeptide LKRYKRRL (NOP-1) mimicking this NLS motif were shown to block the interaction of EIN2 and ETR1 receptors and reduce plant ethylene responses (Bisson and Groth, 2015; Bisson et al., 2016).

In this study, we demonstrate that the NOP-1 octapeptide also efficiently binds to the ripening related tomato receptors NR and LeETR4 structurally divergent from ETR1. Moreover, we provide quantitative measures of the ripening delay related to NOP-1 treatment such as pigment content, overall color analysis and fruit firmness. Our data show that surface application of NOP-1 on tomato fruits can delay ripening without impairment of fruit quality.

#### MATERIALS AND METHODS

### Cloning of Tomato Receptors LeETR4 and NR into Expression Vector pET16b

Full-length codon optimized cDNA sequences encoding tomato ethylene receptors LeETR4 and NR (UniProt ID: LeETR4 Q9XET8; NR Q41341) were ordered at GenScript United States according to published sequences (NCBI ID: LeETR4 NM\_001247276.2; NR NM\_001246965.2). Construction of expression vector pET16b (Novagen, Madison, WI, United States) carrying the target DNA sequence, an ampicillin resistance and a deca-histidine tag were performed by Gibson Assembly (Gibson et al., 2009). For amplification of linearized vector forward primer 50 -GGATCCGGCTGCTAA CAAAGC-30 and reverse primer 50 -ATGACGACCTTCGATA TGGC-30 were used. LeETR4 was amplified using forward primer 5 0 -ATCGAAGGTCGTCATATGCTGCGTACCCTGGCGAG-30 and reverse primer sequence 50 -TTAGCAGCCGCCTTACAT CAGAGCTGGATTACGGCTACCACGCA-30 . For amplification of NR forward primer 50 -ATCGAAGGTCGTCATATGGACGA TTGCATT-30 and reverse primer 50 -TTAGCAGCCGGATCC TTACAGGCTACGCTGATAACGCT-30 were used. Amplified fragments were added to Gibson Assembly Master Mix containing an exonuclease, a DNA polymerase and a ligase to assemble a circular plasmid with LeETR4 and NR coding sequence, respectively. Reaction assays were incubated at 50◦C for 10 min and at 40◦C for 60 min. Assembled plasmids were transformed into E. coli strain XL 1-blue and sequenced by Seqlab (Göttingen, Germany) to verify correctness.

# Expression of Recombinant Tomato Receptors LeETR4 and NR in E. coli

For expression of recombinant LeETR4 and NR the related pET16b expression vectors were transformed into E. coli strains C43 and BL21 (DE3), respectively. Cells were grown in 2YT medium [1.6% (w/v) peptone, 1% (w/v) yeast extract and 0.5% (w/v) NaCl] with 2% ethanol and 100 µg/mL ampicillin at 30◦C. At OD600 = 0.4 temperature was reduced to 16◦C. Expression of tomato receptors was induced at OD600 = 0.6 by the addition of 0.5 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG). Cells were grown and harvested after 20 h (LeETR4) or 6 h (NR) by centrifugation for 15 min at 7,000 × g and 4◦C. Expression of tomato receptors was analyzed by SDS–PAGE (Laemmli, 1970) and detected by Western blotting (Towbin et al., 1979).

## Solubilization and Purification of Recombinant Tomato Receptors LeETR4 and NR

The resulting cell pellet after expression was resuspended in PBS pH 8, 10% (w/v) glycerol, 1 mM dithiothreitol and 0.002% (w/v) phenylmethylsulfonyl fluoride (PMSF). DNase I (10 µg/mL) was added before cells were broken with Constants Cell Disruption System (Constant Systems, Daventry, United Kingdom) at 2.4 kbar and 5◦C. Cell lysate was centrifuged for 30 min at 14,000 × g and 4◦C. The resulting supernatant was centrifuged again for 30 min at 40,000 × g and 4◦C. The pellet was resuspended in PBS buffer and centrifuged for 30 min at 34,000 × g and 4 ◦C. For solubilization the pellet was resuspended in 50 mM Tris/HCl pH 8, 200 mM NaCl, 1.2% (w/v) FosCholine-16, 0.002% (w/v) PMSF (buffer S) and stirred at RT and 700 rpm for 1 h. Membrane fragments were isolated by ultracentrifugation (229,600 × g, 4◦C, 30 min). The supernatant was loaded to a 5 mL HisTrap FF column operated by an ÄKTAprime plus (both GE Healthcare Life Sciences) at 4◦C equilibrated with buffer A [buffer S containing 0.015% (w/v) FosCholine-16], followed by an ATP washing step of 20 column volumes [50 mM Tris/HCl pH 8, 200 mM NaCl, 50 mM KCl, 20 mM MgCl2, 10 mM ATP and 0.002% (w/v) PMSF]. The column was washed with 50 mM imidazole and receptors eluted with 250 mM imidazole. Purified proteins were concentrated to 2.5 mL and buffer was changed to 100 mM potassium phosphate buffer pH 7.3, 300 mM NaCl, 0.015% (w/v) FosCholine-16, 0.002% (w/v) PMSF for labeling with Alexa Fluor 488-Maleimide (Thermo Fisher Scientific) on a PD-10 column (GE Healthcare Life Sciences). Alexa Fluor 488-Maleimide was applied to the protein in 2.5-fold excess and incubated for 30 min at RT. Then, buffer was changed to 50 mM Tris/HCl pH 8, 300 mM NaCl, 5% (w/v) glycerol, 0.015% (w/v) FosCholine-16, 0.002% (w/v) PMSF. Purity of LeETR4 and NR was analyzed by SDS–PAGE (Laemmli, 1970) with colloidal Coomassie staining (Dyballa and Metzger, 2009) and Western blotting (Towbin et al., 1979) using a directly conjugated Anti-His-HRP monoclonal antibody (Miltenyi Biotech, Bergisch Gladbach, Germany). Proper folding of receptors was verified by CD-spectroscopy (Classen and Groth, 2012; Kessenbrock and Groth, 2017).

### CD Spectroscopy of Recombinant Tomato Receptors

CD measurements were performed in a Jasco J715 spectropolarimeter (Jasco GmbH, Gross-Umstadt, Germany). For the far UV spectra a cylindrical quartz cuvette from Hellma

Analytics (Muellheim, Germany) with 1-mm-path-length was used. Purified tomato receptors LeETR4 and NR were dissolved to a final concentration of 0.2 mg ml−1 in 10 mM potassium phosphate pH 8.0 and 0.0075% (w/v) FosCholine-16. Protein and FosCholine-16 concentrations were determined by a Direct Detect Infrared Spectrometer (Merck Chemicals GmbH, Darmstadt, Germany) (Strug et al., 2014). For detailed information on protein preparation see Kessenbrock and Groth (2017). Measurements were run at ambient temperature. Each protein sample was recorded in the range of 260–185 nm. The CD spectra were obtained by averaging ten individual spectra using a bandwidth of 1 nm at 50 nm min−1 . Secondary structure content of purified proteins were calculated from the spectra by CDSSTR and CONTINLL (Provencher and Gloeckner, 1981; Johnson, 1999).

#### Binding Studies of NOP-1 at Tomato Receptors LeETR4 and NR by Microscale Thermophoresis

Binding of the NOP-1 octapeptide to purified recombinant tomato receptors LeETR4 and NR was analyzed by microscale thermophoresis (MST) (Duhr and Braun, 2006; Wienken et al., 2010; Jerabek-Willemsen et al., 2011; Seidel et al., 2013). Receptors (100 nM) labeled with Alexa Fluor 488-Maleimide (Thermo Fisher Scientific) were titrated with peptide ligand NOP-1 dissolved in 50 mM Tris–HCl pH 8.0, 300 mM NaCl at concentrations from 500 µM to 61.04 nM. Then samples were transferred into standard glass capillaries and thermophoresis was measured using a Monolith NT.115 (NanoTemper Technologies GmbH, München, Germany). MST measurements were recorded at 20% MST power for LeETR4- NOP-1 and 60% MST power for NR-NOP-1, respectively. Receptors were chemically denatured by incubation with the strong ionic detergent SDS [4% (w/v)] and the small-molecule redox reagent DTT (40 mM) for 5 min in the dark at RT and served as control to confirm specific and selective binding of the ligand. All measurements were run in triplicate.

### Fruit Material, Treatments and Storage Conditions

Tomato fruits (Solanum lycopersicum L.) of the cultivar 'Lyterno' (Rijk Zwaan, De Lier, Netherlands) were harvested at the maturity stage "green" (USDA; 2005) from tomato plants which where cultivated in a commercial-like greenhouse at the research station Campus Klein-Altendorf (University of Bonn, Germany). At the early development stages, the trusses were manually thinned to six fruits per truss, according to the common practice aiming standardized fruit size and quality. For the experiment, the last two fruits of the fourth truss, counted from the bottom, were chosen for evaluations. At the beginning of the experiment, 150 fruits showing similar color and size were divided into four treatments (n = 25 fruits per treatment). On each fruit, a transparent polyethylene film was placed at the equatorial zone to demark the four evaluation points of 2.8 cm diameter each.

The four treatments were as follows: (1) control; (2) NOP-1, 400 µM; (3) NOP-1, 1000 µM; (4) NOP-1, 2000 µM. The NOP-1 peptide (GenScript, Piscataway, NJ, United States) was dissolved in 5 ml deionized water. On each fruit, a total of 200 microdroplets (0.5 µL each) of the peptide solution were gently deposited (50 micro droplets on each marked area of the tomato fruit) with a Hamilton microdispenser (Hamilton Bonaduz AG, Bonaduz, Switzerland). Fruits of the control treatment received an equal number of microdroplets of deionized water. After application of the droplets fruits of each treatment were allocated in storage boxes thereby avoiding fruit-to-fruit contact, and stored at room temperature (19 ± 2 ◦C).

### Non-destructive Measurements of Fruit Color at Ripening

Starting at the beginning of the experiment until 28 days after treatments (DAT), fruit ripening was evaluated and monitored twice a week with two non-destructive sensors using the principles of light reflectance and fluorescence emission. Evaluations were done on the marked fruit zones (n = 4 areas/fruit, n = 25 fruits per treatment).

Changes of the surface color over time were determined with a portable spectrophotometer (CM-503d, Konica Minolta Inc., Tokyo, Japan), which has a sensing area of 7 mm2 . Based on the CIELAB model (McGuire, 1992), the recorded parameters are converted into the hue◦ index. Hue◦ was calculated according to the following formula:

$$\text{hue}^{\circ} = \tan^{-1} \left( \frac{b^\*}{a^\*} \right),$$

where 'a ∗ ' and 'b ∗ ' are defined as color coordinates, provided that 'a ∗ ' is the point of the green-red axis and 'b ∗ ' is defined as the point of the yellow-blue axis. On the basis of the above, the resulting angle is converted into the corresponding color value.

#### Fluorescence Based Analysis of Fruit Maturity

Pigment fluorescence was applied as second non-destructive technique to address fruit maturation. To this end, a handheld device (Multiplex R 3, Force-A, Orsay, France) equipped with light-emitting diodes (LEDs) with UV (375 nm), blue (475 nm), green (510 nm) and red (635 nm) excitation was used. Fluorescence was detected in the blue-green (BGF, 425–475 nm), red (RF, 680–690 nm) and near-infrared (FRF, 720–755 nm) spectral regions (Ben Ghozlen et al., 2010). Based on the absolute fluorescence signals recorded in a detection diameter of approximately 2 cm, simple and complex fluorescence ratios were calculated. The parameter Simple Fluorescence Ratio excited with red light (SFR\_R) as a measure of the chlorophyll content is as follows:

SFR\_R=(FRF\_R/RF\_R) (according to Ben Ghozlen et al., 2010).

#### Determination of Chlorophyll, β-Carotene and Lycopene

Determination of the pigment content in the fruits was done weekly on five fruits each treatment. The concentrations

of β-carotene, lycopene and chlorophyll were analyzed from freeze-dried and ground fruit samples, as described below. Concentrations of chlorophyll, β-carotene and lycopene were analyzed according to the method of Nagata and Yamashita (1992) as described by Barros et al. (2007), Azeez et al. (2012) and Kalogeropoulos et al. (2012) with the following modifications. Briefly, 1.5 ml of the solvent Aceton:Hexane (4:6) was added to 0.1 g of the freeze-dried and ground material, homogenized and centrifuged for 10 min at 16,100 × g (CENTRIFUGE 5415 R, Eppendorf AG, Hamburg, Germany). Next, 2 ml of the solvent was added to the supernatant and the absorption of the solutions was determined at 435, 505, 645, and 663 nm in a LAMBDA 35 spectrophotometer (PerkinElmer R , Waltham, MA, United States). Concentrations of chlorophyll, β-carotene and lycopene were calculated according to Nagata and Yamashita (1992) by the following equations:

chlorophyll a - mg/100 ml = 0.999A663 − 0.0989A645

chlorophyll b - mg/100 ml = −0.328A663 + 1.77A645

β − carotene - mg/100 ml = 0.216A663 − 1.22A645 − 0.304A505 − 0.452A453

lycopene - mg/100 ml = −0.0458A663 + 0.204A645 + 0.372A505 − 0.0806A453

Total chlorophyll was calculated by adding chlorophyll a and chlorophyll b content.

#### Statistical Analysis

All results are expressed as mean ± SE. Analyses of variance were determined with one-way ANOVA (α ≤ 0.05). In case of statistical significance, the Tukey's HSD (α ≤ 0.05) was applied to establish the differences among means. Statistical analyses were carried out using SPSS 22.0.

#### RESULTS

#### Expression and Purification of Tomato Receptors LeETR4 and NR

Codon optimized synthetic genes encoding full-length ethylene receptors LeETR4 and NR were each cloned into expression vector pET16b by Gibson Assembly Cloning (Gibson et al., 2009). Expression vectors encoding the tomato receptors were transformed into cells of E. coli strains BL21 (DE3) and C43 (DE3) which have been successfully applied for the expression of different members of the ethylene receptor family from A. thaliana, Lycopersicon esculentum and Physcomitrella patens in previous studies (Voet-van-Vormizeele and Groth, 2008; Classen and Groth, 2012). Protein expression was induced by the addition of 0.5 mM IPTG. Optimum expression was obtained for LeETR4 after 20 h in C43 (DE3) at 16◦C, while systematic analysis of expression parameter for NR showed best expression after 6 h in BL21 (DE3) at 16◦C (**Figure 1**).

Receptors were localized in the membrane fractions of the host and solubilized from these membranes by the mild detergent Fos-Choline-16. After solubilization, receptors were purified in a single chromatography step on Ni–NTA agarose (GE Healthcare Life Sciences, Munich, Germany). Purification of the recombinant tomato receptors was analyzed by SDS–PAGE. The related protein gels (**Figure 2**) show prominent bands at 90 and 70 kDa corresponding to the molecular weight of LeETR4 (88 kDa) and NR (74 kDa). Besides, two minor contaminations were detected in the lower MW range at 55 and 32 kDa, respectively. Hence, both receptors have been successfully purified from their heterologous host. Identity of the receptors was confirmed by antibodies directed against the deca-histidine tag in both proteins.

#### Secondary Protein Structure and Functional Folding of Purified LeETR4 and NR

Folding and protein secondary structure of purified recombinant tomato receptors were probed by CD spectroscopy. The corresponding spectra of LeETR4 and NR shown in **Figure 3** are typical of partially helical proteins, displaying two minima at approximately 209 and 222 nm with an isosbestic point at 202 nm. Overall, the spectra of both receptor proteins are highly similar and correspond to previous CD data on receptor orthologs from Arabidopsis and Physcomitrella (Classen and Groth, 2012). Secondary structure calculations by CDSSTR and CONTINLL suggest an α-helix content of 34% and a β-sheet percentage of 18–20% for LeETR4. Similar numbers of 41– 42% α-helix and 14% β-sheet structure were obtained for NR. Consequently, CD spectroscopic measurements verify that the purified receptors adopt a well-folded structure and are indicative for a native conformation of the recombinant tomato proteins.

#### Binding of NOP-1 to Purified Recombinant Tomato Receptors LeETR4 and NR

Analysis of protein–ligand interactions by MST was used to monitor and to quantify the interaction of synthetic NOP-1 octapeptide with purified recombinant LeETR4 and NR, respectively. Ligand binding and the related dissociation constant with the isolated receptors were deduced from changes in thermophoresis upon addition of the NOP-1 octapeptide (**Figure 4**). In analogy to previous studies on receptors from Arabidopsis and constitutively expressed tomato receptor LeETR1 (Bisson et al., 2016) clear changes of the thermophoretic signal were observed upon addition of the synthetic peptide with purified recombinant LeETR4 and NR receptors that are expressed at high levels at fruit ripening. Selectivity of the peptide–receptor interaction was probed in MST studies with chemically denatured receptor proteins. In these experiments, no change in thermophoresis was detected upon addition of NOP-1 (**Figure 4**). The apparent dissociation constant (Kd) calculated from the changes in thermophoresis induced by different

amounts of NOP-1 added to fluorescently labeled tomato receptors was 4.15 ± 0.85 µM for LeETR4 and 23.52 ± 1.99 µM for NR, respectively (**Figure 4**). Both numbers are in the lower micromolar range and together with the negative controls on denatured receptor proteins are indicative of efficient and specific binding of NOP-1 to tomato receptors LeETR4 and NR.

### Impact of NOP-1 on Fruit Ripening and Fruit Quality

For many fruits and vegetables color development is the most important external characteristic to assess ripeness and postharvest life. Color change from green to red was slowed down in tomato fruits treated with NOP-1 (1000 µM), as indicated by the hue◦ index which was significantly higher

on DAT 4, 7, and 9 in this treatment group as compared to control (untreated) fruits (**Figure 5A**). For treatment with 400 or 2000 µM NOP-1 slower color change as compared to control fruits were observed only at DAT 4, and to a smaller extent at DAT 7. Thereafter, color change of treated fruits was similar to controls. From DAT 14 onwards, there were no significant differences in hue◦ among all evaluated treatments. Similar to the observed effects on the hue index, application of 1000 µM NOP-1 also resulted in a significantly higher Simple Fluorescence Index (SFR\_R, estimating chlorophyll concentration) on DAT 4, 7, and 9 (**Figure 5B**). A slight increase in SFR\_R compared to non-treated fruits was further observed

for fruits treated with 400 and 2000 µM NOP-1 at DAT 7. Finally, all treatments reached similar values on DAT 14 and no further changes were observed until the end of the experiment (DAT 23).

Color development is another sensitive but invasive measure to monitor ripening. The chlorophyll content of tomato fruits over time is shown in **Figure 6A**. Fruits treated with 1000 µM NOP-1 showed higher concentrations of total chlorophyll compared to non-treated controls throughout the experiment. While total chlorophyll decreased rapidly to about 30% after DAT 9 in controls, only 50% of the pigment originally present was degraded in fruits treated with 1000 µM at this time. However, over time chlorophyll breakdown in fruits treated with 1000 µM NOP-1 converged to chlorophyll degradation in controls and had essentially ceased after DAT

16. Surprisingly, fruits treated with 400 and 2000 µM NOP-1 showed a more pronounced chlorophyll breakdown on DAT 9 than non-treated controls. However, with further progression of the experiment chlorophyll degradation in these fruits ceased and chlorophyll levels adapted to controls on DAT 16–24.

In contrast to the observed degradation in chlorophyll concentration of β-carotene increased in all treatments throughout the experiment (**Figure 6B**). However, fruits treated with 1000 µM NOP-1 showed a slower increase in β-carotene concentration and revealed significantly lower levels of this pigment at DAT 9 when compared to all other treatments which essentially showed the same pattern for the increase of this carotenoid during ripening. Over time β-carotene in the fruits treated with 1000 µM NOP-1 increased to control levels. All treatments showed comparable levels of this carotene on DAT 16 and concentration of this pigment remained constant throughout

FIGURE 6 | (A) Total chlorophyll content of control tomato fruits ( ) and tomato fruits treated with NOP-1 400 µM (#), 1000 µM (H) or 2000 µM (1). Concentration of β-carotene (B) and lycopene (C) of control tomato fruits ( ) and tomato fruits treated with NOP-1 400 µM (#), 1000 µM (H) or 2000 µM (1). Data are means ± SE, n = 5, different letters indicate significant differences between treatments on each measurement day (Tukey's HSD, α ≤ 0.05).

the further experiment. Slightly higher concentrations of β-carotene were observed for the 400 µM treatment at DAT 23–24.

The concentration of lycopene principally responsible for the characteristic deep-red color of ripe tomato fruits, increased significantly in all treatment groups (**Figure 6C**). Similar to the pattern observed for β-carotene, treatment with 1000 µM NOP-1 showed the largest delay in pigment accumulation at DAT 9 where only 33% of the lycopene level of non-treated controls was measured. Treatment with 400 µM of the synthetic peptide still resulted in a delay of lycopene accumulation of about 69% compared to non-treated controls. According to the pattern observed with β-carotene, lycopene levels in all treatments adjusted to similar concentrations after DAT 16 and stayed constant for the rest of the experiment. Highest concentrations of lycopene were measured for all treatments in completely ripe tomatoes at DAT 24. Total color development of fruits treated at different concentrations of NOP-1 is illustrated by visual images of whole fruits (**Figure 7**).

Fruit soften during ripening due to biochemical processes resulting in the breakdown of cell-wall polymers. Hence, firmness is an indirect measurement of ripeness and represents one of the most important variables for fruit quality. Consequently, we determined fruit firmness in tomato fruits treated with NOP-1 and non-treated controls using a non-destructive sensor and a shore scale ranging from 0 to 100 units. Firmness decreased from 80–89 shore to 55–50 shore in the course of the experiment (**Figure 8**). Firmest fruits were observed at the beginning of the experiment, softest fruits were measured at the end. Control, treatment with NOP-1 at concentrations of 400 and 2000 µM showed a continuous decrease in fruit firmness over time, whereas firmness was unaffected at early stages (DAT 4–9) in fruits treated with 1000 µM NOP-1. However, after the initial lag phase firmness decreased to similar levels in these fruits as observed for the other treatments on DAT 16 and later stages. All treatments showed similar numbers for fruit firmness and thereby comparable fruit quality at the end of the experiment on DAT 25.

#### DISCUSSION

Previous studies on Arabidopsis demonstrated that the small basic peptide NOP-1 derived from the natural NLS-sequence of the ethylene regulator protein EIN2 is able to disrupt ethylene signaling and inhibit plant ethylene responses. Protein–protein interaction studies on recombinant purified proteins EIN2 and ETR1 and related FRET studies in planta suggest that the inhibitory peptide competes for binding of EIN2 at the receptors offering a novel way to interfere with ethylene signal transduction and ethylene responses in planta (Bisson and Groth, 2015; Bisson et al., 2016). The high conservation of the NLS-motif among the plant kingdom (see Supplementary Figure S1 in Bisson et al., 2016) and the level of homology in ethylene receptors open up new avenues for ripening control of fruits and vegetables by biological peptides in modern agriculture and horticulture. Initial studies on tomato, a climacteric fruit of high economic and nutritional impact serving as model to study fruit ripening, support these ideas and confirm the results obtained with the Arabidopsis genetic model.

FIGURE 7 | Visual images of fruits treated at different concentrations of NOP-1. Representative photos of whole fruits treated with 400, 1000 and 2000 µM NOP-1 on DAT 1, 7, 10, 14, and 16. Control fruits are depicted in the upper row.

In order to further evaluate the potential of the inhibitory peptide identified in previous studies, we have further analyzed molecular and physiological effects of the basic NLS-derived peptide NOP-1 on tomato. Our studies with purified recombinant receptors LeETR4 and NR, which are both highly expressed in ripening fruit, reveal efficient binding of the peptide to both receptors and thereby confirm that the NOP-1 octapeptide may interact with receptors from both receptor subfamilies. Both receptors interact with the peptide at affinities in the lower µM-range, but interaction with LeETR4 seems to be stronger.

Noteworthy, previous studies demonstrate an even stronger binding affinity of NOP-1 at tomato receptor LeETR1 – a receptor of subfamily I continuously expressed throughout the plant. However, LeETR4 and NR are quite different from LeETR1 with respect to kinase activity as well as in the number and their type of phosphorylation sites (Kamiyoshihara et al., 2012). Keeping further in mind the actual sequence identities of Arabidopsis ETR1 and tomato LeETR1/NR/LeETR4 of 81, 69, and 41%, the observed range in affinities of different receptors and receptor subfamilies for NOP-1 is not surprising at all. Bearing in mind that NOP-1 was derived from the NLS motif in EIN2 the different binding affinities observed with the peptide may also suggest that receptors have different affinities for the EIN2 central hub.

In associated post-harvest studies, we have evaluated changes in color and texture development of tomato fruits treated with different concentrations of NOP-1 and the impact of the

NLS-derived peptide on fruit ripening and fruit quality. Our studies show clear effects on fruit ripening at concentrations of 1000 µM, whereas effects on color development at 400 µM are substantially less pronounced and manifest only at early ripening stages (DAT 9 – see **Figure 6C**). In total, a concentration of 400 µM NOP-1 applied to the fruit surface as microdroplets seems to be too low for significant inhibition of the ethylene transduction cascade, whereas 1000 µM allows a ripening delay, as expressed by the chlorophyll degradation and lycopene and β-carotene accumulation (**Figure 6**). When applied at 2000 µM concentration no effect of NOP-1 was observed which might be related to concentration-dependent aggregation or changes in secondary structure (Garg et al., 2013), which may impair uptake of the peptide by the fruit surface. Alternatively, the fact that no significant positive effect on ripening delay was observed at 2000 µM concentration in contrast to 1000 µM NOP-1 may be explained by differences in droplet–surface–uptake interactions at the different concentrations. In this case, the solution concentration of 1000 µM showed higher uptake of NOP-1 at the same contact surface area (i.e., tomato fruit surfaces), possibly due to the optimum dose/concentration/interfacial area which apparently causes the maximum penetration. For 2000 µM droplet the NOP-1 loading was twice as high at the same water volume. Consequently, this may have reduced the droplet viscosity on the cuticle, also apparent by its modified macroscopic appearance and altered biomechanical properties (Domínguez et al., 2011; Burkhardt and Hunsche, 2013). In consequence, penetration and uptake of NOP1 may have been reduced, resulting in a less effective 2000 µM treatment.

Improving shelf life and nutritional quality of tomato fruits is difficult to achieve with the methods currently in use. Maintaining adequate storage conditions is expensive and might cause chilling injury if used improperly (Passam et al., 2007). Even though genetic modifications reducing gene expression of proteins involved in ethylene synthesis are possible in principle (Abano and Buah, 2015) these procedures are banned by law throughout Europe and have low acceptance among European

#### REFERENCES

consumers. Chemical methods such as application of AVG to inhibit ethylene biosynthesis or treatment with 1-MCP to inhibit ethylene response significantly delay ripening and slow down lycopene synthesis and chlorophyll breakdown (Saltveit, 2005; Passam et al., 2007). The drawbacks of these methods correlating with the restricted use of both chemicals on tomatoes in Europe also relate to quality losses in taste development or to complete arrest in maturation, as observed in some cases (Passam et al., 2007).

In summary, our study shows that the NOP-1 octapeptide derived from the NLS-motif at the EIN2 C-terminus is a potent inhibitor of the maturation process in tomato. The peptide efficiently binds to different receptor isoforms and, when applied to the surface of immature fruit, successfully delays the ripening process without impairment of final overall fruit quality at the fully mature stage. This novel approach to delay fruit ripening is making use of a synthetic peptide that corresponds to the highly conserved NLS-motif in all known EIN2 sequences and holds great promise to control processes such as ripening or senescence in horticultural and agricultural applications.

#### AUTHOR CONTRIBUTIONS

GG conceived the project. GG, GN, and MH planned, designed and supervised the research. MK, SK, and LM performed the experiments and contributed equally to this work. All authors contributed to data analysis and the writing of the manuscript.

#### ACKNOWLEDGMENTS

This work was funded in part by a grant from the Ministry of Innovation, Science and Research within the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-002 13) and by the Deutsche Forschungsgemeinschaft, CRC 1208, project B06 to GG.

Canene-Adams, K., Campbell, J. K., Zaripheh, S., Jeffery, E. H., and Erdmam, J. W. Jr. (2005). The tomato as a functional food. J. Nutr. 135, 1226–1230.

ethylene signaling. Methods Mol. Biol. 1573, 141–159. doi: 10.1007/978-1-4939- 6854-1-12

Zhou, D., Mattoo, A., and Tucker, M. (1996b). Molecular cloning of a tomato cDNA encoding an ethylene receptor. Plant Physiol. 110, 1435–1436.

**Conflict of Interest Statement:** MH is associate professor at the University of Bonn and Global Head of R&D of the company COMPO EXPERT GmbH.

The other 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 © 2017 Kessenbrock, Klein, Müller, Hunsche, Noga and Groth. 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.

# Diverse Roles of Ethylene in Regulating Agronomic Traits in Rice

Cui-Cui Yin1 , He Zhao1,2, Biao Ma1 , Shou-Yi Chen1 and Jin-Song Zhang1,2 \*

1 State Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China, 2 University of Chinese Academy of Sciences, Beijing, China

Gaseous hormone ethylene has diverse effects in various plant processes. These processes include seed germination, plant growth, senescence, fruit ripening, biotic and abiotic stresses responses, and many other aspects. The biosynthesis and signaling of ethylene have been extensively studied in model Arabidopsis in the past two decades. However, knowledge about the ethylene signaling mechanism in crops and roles of ethylene in regulation of crop agronomic traits are still limited. Our recent findings demonstrate that rice possesses both conserved and diverged mechanism for ethylene signaling compared with Arabidopsis. Here, we mainly focused on the recent advances in ethylene regulation of important agronomic traits. Of special emphasis is its impact on rice growth, flowering, grain filling, and grain size control. Similarly, the influence of ethylene on other relevant crops will be compared. Additionally, interactions of ethylene with other hormones will also be discussed in terms of crop growth and development. Increasing insights into the roles and mechanisms of ethylene in regulating agronomic traits will contribute to improvement of crop production through precise manipulation of

#### Edited by:

Chi-Kuang Wen, Shanghai Institutes for Biological Sciences (CAS), China

#### Reviewed by:

Caren Chang, University of Maryland, College Park, United States Rongfeng Huang, Institute of Biotechnology (CAAS), China

#### \*Correspondence:

Jin-Song Zhang<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science

Received: 30 June 2017 Accepted: 12 September 2017 Published: 26 September 2017

#### Citation:

Yin C-C, Zhao H, Ma B, Chen S-Y and Zhang J-S (2017) Diverse Roles of Ethylene in Regulating Agronomic Traits in Rice. Front. Plant Sci. 8:1676. doi: 10.3389/fpls.2017.01676 ethylene actions in crops.

Keywords: ethylene, agronomic traits, rice, crops, stress

# INTRODUCTION

Ethylene is a simple gaseous phytohormone present in plants and regulates plant growth and developmental processes ranging from germination to senescence (Bleecker and Kende, 2000). Dark-grown Arabidopsis seedlings treated with saturated ethylene exhibit triple response that consists of inhibiton of hypocotyls and roots and exaggeration of the curvature apical hooks (Guzmán and Ecker, 1990). Significant progress has been made in the ethylene signal pathway after the discovery of ethylene-insensitive and constitutive ethylene response mutants from the dicotyledonous model Arabidopsis. In Arabidopsis, ethylene is perceived by its five receptors (Hua and Meyerowitz, 1998). Then receptors and another negative regulator, CONSTITUTIVE TRIPLE-RESPONSE1 (CTR1) are inactivated, and the central signal transducer ETHYLENE-INSENSITIVE 2 (EIN2) C-terminal end (CEND) (Lin et al., 2009a) is dephosphorylated and cleaved. The cleaved CEND is translocated into the nucleus (Qiao et al., 2012; Wen et al., 2012) and the P-body. EIN2 CEND mediates translation repression of ETHYLENE-INSENSITIVE (EIN3) Binding F-Box1/2 (EBF1/2) at P-body and consequently activates ethylene response (Li et al., 2015). In the nucleus, the master transcription factors EIN3 and ETHYLENE INSENSITIVE-LIKE 1 (EIL1) lead to the ethylene-induced transcription activation and ethylene response (Chao et al., 1997). Interestingly, the EIN2 CEND also contributes to the downstream signaling through elevating the acetylation at H3K4 and H3K23 (Zhang et al., 2016a). Although these studies shed better light on how ethylene regulates plant growth and development and adaption to environment, there are still limitations due to the difference between dicotyledonous and monocotyledonous plants.

Semi-aquatic rice has a "double response" (inhibition of root growth but promotion of coleoptile elongation) of darkgrown seedling upon ethylene treatment (Ma et al., 2013). The ethylene growth response kinetics in monocots, including millet, barley and rice, is distinct from that in the dicots such as Arabidopsis and tomato (Kim et al., 2012). There are five ethylene receptors in rice genome. OsERS1 and OsERS2 belong to subfamily I with a conserved histidine kinase domain, whereas OsETR2, OsETR3, and OsETR4 belong to subfamily II with a diverged kinase domain (Yang et al., 2015a). But rice and many other monocotyledonous plants do not have ETR1-type ethylene receptor and OsETR2 have Ser/Thr kinase activity (Wuriyanghan et al., 2009). In rice, OsRTH1, OsRTH2, and OsRTH3 are identified and they are homologous to AtRTH1 in Arabidopsis. OsRTH1 and AtRTE1 have the highest sequence identity, and ectopic transgenic analysis indicates that only OsRTH1 is able to mimic the function of AtRTE1 (Zhang et al., 2012). AtCTR1 is encoded by a single gene in Arabidopsis. Its loss-of-function mutation leads to a constitutive ethylene response (Huang et al., 2003). In rice, three homologs OsCTR1, OsCTR2 and OsCTR3 were identified, and OsCTR2 is most closely related to AtCTR1 (Wang et al., 2013; Yang et al., 2015a). Similar to that in Arabidopsis, OsEIN2 is a positive regulator in ethylene signal pathway of rice (Ma et al., 2013). EILs are the master transcription factors in ethylene signaling pathway, with six members found in rice (Yang et al., 2015a). However, only OsEIL1 and OsEIL2 regulate root and coleoptile ethylene response respectively, with the left members having no role in ethylene response (Yang et al., 2015b). These results indicate that OsEIL1 and OsEIL2 have divergent function in growth and development in rice. Moreover, both OsEIL1 and OsEIL2 negatively affect rice salt tolerance by promoting transcription of HIGH-AFFINITY K + TRANSPORTER 2;1 (HKT2;1) and Na+ absorption in roots (Yang et al., 2015b). Carotenoid isomerase (CRTISO) catalyzes the conversion of prolycopene to all-trans-lycopene (Fang et al., 2008). ABA4 drives the conversion of zeaxanthin to neoxanthin but no enzyme activity is detected in Arabidopsis (North et al., 2007). Its homologous gene MHZ4/ABA4 mutation reduced abscisic acid (ABA) production in rice (Ma et al., 2014). Both MHZ5/CRTISO and MHZ4/ABA4 are involved in ABA biosynthesis. The study of the two ethylene response mutants mhz4/aba4 and mhz5/crtiso indicates that ethylene inhibits root growth through ABA pathway in rice (Ma et al., 2014; Yin et al., 2015). In contrast, in Arabidopsis, ethylene inhibition of root elongation is ABA-independent (Beaudoin et al., 2000). Besides, MHZ5/CRTISO and MHZ4/ABA4 mediated ABA pathway inhibits rice coleoptile elongation likely through downregulating ethylene signaling pathway (Ma et al., 2014; Yin et al., 2015). These studies indicate that the ethylene signal transduction has conserved and divergent aspects between Arabidopsis and rice (**Figure 1**). Furthermore, rice ethylene response is also different from other monocots such as maize, wheat, sorghum, and Brachypodium distachyon (Yang et al., 2015a). Therefore, identification of rice ethylene signal transduction components and analysis of the interactions between ethylene and other phytohormones will shed better

FIGURE 1 | A diagram of ethylene signal transduction pathway in rice. In the dark, rice etiolated seedlings have "double response" with ethylene treatment. Ethylene promotes coleoptile elongation but inhibits root growth in rice. A linear ethylene signaling pathway has been found in rice etiolated seedlings which is similar to that in Arabidopsis. Rice has homologs of Arabidopsis ethylene signaling pathway, such as ethylene receptors, OsCTRs, MHZ7/OsEIN2, and MHZ6/OsEIL1 and OsEIL2. In contrast to that in Arabidopsis, the AtEIN3/EIL1 homologs OsEIL1 and OsEIL2 have divergent functions in rice coleoptile and root growth. (A) Ethylene promotes coleoptile/mesocotyl elongation through OsEIL2 which inhibits GY1/EG1-mediated jasmonate (JA) biosynthesis. JA pathway acts downstream of ethylene signaling pathway to inhibit cell elongation. On the other hand, MHZ5/CRTISO and MHZ4/ABA4-mediated ABA pathway acts upstream of ethylene signaling pathway to inhibit transcription of MHZ7/OsEIN2 and inhibit coleoptile elongation. (B) Ethylene regulates root growth through MHZ6/OsEIL1 function. In Arabidopsis, AtEIN3 and AtEIL1 have functional redundancy in root inhibition. Moreover, ethylene inhibits root growth partially through MHZ5/CRTISO and MHZ4/ABA4-mediated ABA pathway. Arrows and T-bars indicate direct or indirect activation and inhibition, respectively. Dotted lines indicate several steps involved that are not shown in the diagram.

light on how ethylene is specifically involved in rice growth and adaptation.

Rice, as a major staple crop, feeds more than half of the world's population (Yu et al., 2016). To satisfy the increasing global demand of the growing population, a 50% increase in rice production will be required (Alexandratos and Bruinsma, 2012). Rice shares close synteny and collinearity with other important cereal crops (Gale and Devos, 1998). Rice has the smallest genome of the major cereals and rich genetic diversity (Sasaki et al., 2005). In addition, the sequence of rice entire genome (Sasaki et al., 2005; Du et al., 2017) provides basis for identification of homologous genes for other crops. Rice is an annual grass, and can finish a life cycle (**Figure 2**) in 5 months or less. Ethylene regulates several stages of life cycle of rice.

Previous research indicates that three phytohormones including gibberellins (GA), cytokinins, and brassinosteroids regulate several agronomically important traits of rice, such as plant height (Sasaki et al., 2002), grain number (Ashikari et al., 2005) and leaf erectness. Ethylene plays a subtle role in plant growth, particularly in adaption to stressful environmental conditions (Peng et al., 2014; Tao et al., 2015). Abiotic and biotic stresses affect growth of crops at various stages and ultimately result in loss of yield. Reproductive processes including flowering, grain filling, and maturation are highly affected by abiotic stresses. Ethylene-insensitive mutants offer a great opportunity for understanding ethylene signal transduction in rice (Ma et al., 2013; Yang et al., 2015b; Yin et al., 2015). Then, how does the ethylene signaling affect agronomic traits? Ethylene impacts on fruit ripening have been studied in a series of plant species, including tomato and apple, etc. (Alexander and Grierson, 2002; Lee et al., 2010; Li et al., 2017). Jasmonate (JA) promotes ethylene biosynthesis to promote apple fruit ripening (Li et al., 2017). This review will highlight the impact of ethylene on crop (especially for rice) agronomic traits, emphasizing that the topics should be further investigated.

# ETHYLENE AND SEEDLING GROWTH

Ethylene has a biphasic role, stimulating and inhibiting growth depending on the species, organs/tissue, developmental stages, and environmental conditions (Hattori et al., 2009; Zhong et al., 2012; Yu et al., 2013). In Arabidopsis, ethylene inhibits hypocotyl elongation through activating the transcription factors WAVED-DAMPENED 5 (WDL5) (Sun et al., 2015) and ETHYLENE RESPONSE FACTOR 1 (ERF1) (Zhong et al., 2012; Shi et al., 2016a,b) in the dark or low light intensities. Transcription factor HYPOCOTYL 5 (HY5) also participates in this process which is degraded by the E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) (Osterlund et al., 2000). In the light, AtEIN3 acts upstream of COP1 to influence COP1 localization and HY5 stabilization, and balances the equilibrium between ethylene and light signaling and ultimately promotes

hypocotyl growth (Yu et al., 2013; Shi et al., 2016a,b). These analyses indicate that ethylene signaling pathway is indispensable for plant emergence from the soil.

In rice, coleoptile and mesocotyl are essential protection structures for seedling emergence from the soil. Ethylene promotes coleoptile elongation not only in the dark but also in the light (Ma et al., 2013; Wang et al., 2013; Yin et al., 2015). This fact is different from that in Arabidopsis where ethylene plays an opposite role in hypocotyl elongation in dark and in light. In addition, ethylene promotes the curvature of rice coleoptile in the light (Wang et al., 2013). Obviously, identification and characterization of the component of ethylene signaling pathway in rice will benefit us in unraveling its mechanisms in rice. Progress has been made in the function of ethylene on rice growth and development and agronomic traits using the forward and reverse genetic approaches.

The rice ethylene receptor loss-of-function mutants Osers2, Osetr2, and Osetr3 in Zhonghua background all exhibited mild enhanced ethylene response in coleoptile elongation (Wuriyanghan et al., 2009). However, loss-offunction Osers1, Osers2, and Osetr2 mutants in the strain Dongjin (DJ) background showed no apparent phenotype in coleoptile but an enhanced ethylene response in root growth inhibition (Ma et al., 2014; Yin et al., 2015). These results suggest that the rice ethylene receptors may have functional redundancy and background/subspecies-specific features. In Arabidopsis, the ethylene insensitivity phenotype of REVERSION-TO-ETHYLENE SENSITIVITY 1 (RTE1) overexpression lines depends on ETR1 (Zhou et al., 2007). The OsRTH1 overexpression lines exhibited ethylene insensitivity in coleoptile elongation in dark-grown seedlings and coleoptile curvature in light-grown seedlings (Zhang et al., 2012). As the key negative component, CTR's loss of function mutant Osctr2 had a stronger exaggerated coleoptile curvature on ethylene treatment and this phenotype is probably not due to different ethylene production but resulted from enhanced ethylene sensitivity (Wang et al., 2013). Loss-of-function mutant Osein2/mhz7 exhibited complete coleoptile and root insensitivity to ethylene. The OsEIN2-overexpression lines showed constitutive ethylene response without ethylene treatment and enhanced ethylene response in the presence of ethylene (Ma et al., 2013). OsEIL2- RNAi transgenic lines exhibited coleoptile insensitivity to ethylene, and loss of function in Oseil1/mhz6 led to ethylene insensitivity in roots (Yang et al., 2015b). These features indicate that OsEIL1/MHZ6 and OsEIL2 have divergent functions in seedling growth in rice, which is different from those in Arabidopsis where EIN3/EIL1 showed no apparent organ specificity in ethylene responses.

Upon ethylene perception, OsEIL2 directly bound to the promoter of GAOYAO/EXTRA GLUME (GY1/EG1) (Zhang et al., 2016b) and inhibited GY1/EG1-mediated jasmonic acid biosynthesis and ultimately promoted mesocotyl and coleoptile elongation in etiolated rice seedlings (Xiong et al., 2017). In addition, the promotion of ethylene responses in the coleoptile is correlated with a decrease in the levels of OsEIL2 and an increase in rice F-box protein OsEBF2 transcripts upon treatment with 10 ppm ethylene for 3 h, whereas no significant change was observed in the transcript levels of OsEIL1 (Wang X. et al., 2009; Kim et al., 2012). Whether OsEIL2 exerts its effects on mesocotyl/coleoptile growth through regulation at levels of transcriptional repression activity, its own gene transcription and/or protein degradation need further investigation in rice. Besides, the MHZ4/ABA4 and MHZ5/CRTISO-mediated ABA pathway likely inhibits coleoptile elongation through decreasing the transcription of OsEIN2 and acts upstream of ethylene signaling pathway (Ma et al., 2014; Yin et al., 2015).

Taken together, the crosstalk between ethylene and light signaling pathway is extensive and complex in plant emergence from soil (Shi et al., 2016a,b; Xiong et al., 2017; Yu and Huang, 2017). The growth of rice coleoptile plays a vital role in earlystage rice seedling growth and development, and ethylene has a major role in this process. What is the molecular mechanism of ethylene in promoting rice coleoptile elongation through OsEIL2? Are there any specific downstream factors, e.g., OsERFs, involved in this process? How does ethylene collaborate with light in regulating these processes? Are the mechanisms different from that in Arabidopsis? Revealing the molecular mechanism of ethylene control on the elongation of rice coleoptile/mesocotyl will provide new genetic resources and theoretical basis for breeding new cultivars suitable for growth in dry land.

In addition to regulating etiolated seedlings, ethylene influences rice seedling growth in light. OsRTH1 overexpression had an inhibitory effect on ethylene-induced leaf elongation and adventitious root growth in rice (Zhang et al., 2012). Osctr2 produced more adventitious root regardless of ethylene than control cultivar DJ (Wang et al., 2013). When grown in water, compared to the curved roots in control seedlings, the root of Osein2/mhz7 mutant was longer and straight, while the shoots of Osein2/mhz7 were significantly shorter than control cultivar (Ma et al., 2013). This short shoot phenotype is consistent with that observed in OsEIN2-RNAi plants by Jun et al. (2004). The phenotype of Oseil1/mhz6 seedling was similar to that of Osein2/mhz7 mutants above. However, it should be noted that, at mature stage, the plant height of Oseil1/mhz6 mutant, OsEIL1-overexpressing plants and OsEIL2-RNAi lines showed no significant difference compared to control plants, while the OsEIL2-overexpressing transgenic plants were apparently shorter than control plants (Yang et al., 2015b). The mhz4/aba4 mutant plants were taller than WT and MHZ4-overexpression transgenic lines were shorter than WT. Double mutant analysis revealed that MHZ4/ABA4 negatively regulates plant height in an OsEIN2 dependent manner (Ma et al., 2014). In contrast, the plant height of mhz5/crtiso is shorter than WT due to the shorter length of all internodes (Yin et al., 2015). Double mutant analysis revealed that MHZ5/CRTISO regulate plant height in an OsEIN2-independent manner.

#### ETHYLENE AND FLOWERING

The control of plant flowering time is essential to produce sufficient seed for propagation and can be influenced by environmental factors (e.g., day length, temperature, etc.) and endogenous developmental cues of plants. The major genes

controlling flowering time are conserved and divergent between Arabidopsis and rice (Higgins et al., 2010). The flowering time regulatory networks of the two model plants were compared in the review provided by Shrestha et al. (2014). The two evolutionarily distant plant species had different photoperiod requirement. The B-type response regulator EARLY HEADING DATE 1 (EHD1) in rice has no ortholog genes in Arabidopsis and it acts independent of HEADING DATE1 (Hd1). This is a unique flowering pathway in rice (Doi et al., 2004).

Ethylene regulates plant flowering (Abeles et al., 1992; Iqbal et al., 2017). In Arabidopsis, the ethylene-overproducer mutant eto1 exhibited early flowering, whereas the dominant gain-offunction etr1 mutants, and loss-of-function ein2-1 and ein3-1 mutants showed delayed flowering (Ogawara et al., 2003). However, the loss-of-function mutation of the key negative regulator Ser/Thr kinase gene AtCTR1 led to delayed flowering (Achard et al., 2007). The discrepancy of the role of ethylene signaling components in flowering requires further investigation. In rice, the OsETR2-RNAi lines and loss-of- function Osetr2 mutant exhibited early flowering and heading time, and the latter showed enhanced ethylene sensitivity compared to that of control. Overexpression of OsETR2 transgenic lines exhibited reduced ethylene sensitivity and showed late flowering time compared with that of WT. The delayed flowering is associated with the higher transcript level of OsGI (OsGIGANTEA) and RCN1 (TERMINAL FLOWER1/CENTRORADIALIS-like). Recessive loss-of-function Osetr3 mutant also showed early flowering (Wuriyanghan et al., 2009). At mature stage, the OsETR2-overexpression lines matured later than controls and RNAi-lines (Wuriyanghan et al., 2009). Both loss-of-function Osctr2 mutants and the 35S:OsCTR21−513 lines exhibited late flowering compared with the control plants (Dongjin and ZH11) (Wang et al., 2013). The panicles of Osein2/mhz7 mutant seemed to have more green grains and matured later than control plants (Ma et al., 2013). These studies indicate that ethylene signaling likely leads to early flowering in Arabidopsis and in rice. Alterations of ABA-biosynthesis pathway in mhz4/aba4, MHZ4 overexpressing plants, and mhz5/crtiso resulted in delayed heading time in comparison with control plants (Ma et al., 2014; Yin et al., 2015).

Ethylene affects plant flowering time by interaction with other classical phytohormones. It has been reported that the inhibitory effect of auxin in Pharbitis nil flowering results from the stimulation of ethylene production (Kesy et al., 2008). It is likely that ethylene inhibits flowering probably depending on the ABA level which is influenced by ethylene in Pharbitis nil (Wilmowicz et al., 2008). In Arabidopsis, gibberellin plays an important role in initiating flowering through promoting specific transcription factors under short days and long days, respectively (Wilson et al., 1992; Porri et al., 2012). DELLA-domain proteins are transcription factors and function to repress gibberellin (GA) responses in plants (Fleet and Sun, 2005). In Arabidopsis, ethylene promotes EIN3 accumulation and results in delayed flowering in a DELLA-dependent manner under short day conditions (Achard et al., 2007). These findings suggest that mechanism of ethylene regulation of plant flowering time is complex and needs further investigation.

# ETHYLENE AND GRAIN FILLING

Seed maturation process usually involves grain filling and fruit ripening. Previous studies mainly focus on the mechanism of ethylene on climacteric plant fruit ripening (Barry and Giovannoni, 2007). Ethylene has an auto-inhibitory effect during vegetative growth and an auto-stimulatory effect during fruit ripening (Lelièvre et al., 1998). However, ethylene effect on grain filling of crops is largely obscure and needs elucidation. Rice grain mainly consists of starch, protein, and other metabolites. The two major reserves of starch and protein largely determine the quality and yield of rice (Duan and Sun, 2005). Increasing the number of spikelets per panicle would likely expand yield sink capacity but might result in poor grain filling and low yield. Effect of ethylene on filling of compact-panicle and laxpanicle in rice has been studied. The differential grain filling of the superior and inferior spikelets probably resulted from the higher content of ethylene in compact-panicle rice cultivar (Yang et al., 2006b; Panda et al., 2015, 2016). The basal spikelets produce more ethylene at anthesis, and the expression of ethylene receptors and ethylene signaling transducers retained longer at post-anthesis compared to apical spikelets (Sekhar et al., 2015). Higher ethylene concentration negatively correlated with cell division rate, grain filling and starch concentration, but positively correlated with soluble sugar concentration of the growing endosperm (Panda et al., 2009; Kuanar et al., 2010; Sekhar et al., 2015). Ethylene affects the activity of the enzymes for starch synthesis (Naik and Mohapatra, 2000). The downregulation of essential proteins for rice cell progression and division, such as importin-α, elongation factor 1-β and cell division control protein 48 (CDC48), might limit the sink capacity and lead to poor grain filling in the inferior spikelets compared to that in the superior spikelets (Das et al., 2016). Ethylene negatively regulated grain filling by decreasing the activities of sucrose synthase (SuSase) and starch synthase (StSase) (Zhao et al., 2007). These analyses suggest a negative impact of ethylene on grain filling. Inhibitors including 1-MCP, AVG (aminoethoxyvinylglycine), and STS of ethylene biosynthesis or signaling have been useful in investigating the function of ethylene in rice grain filling. 1-MCP and AVG treatment increased cell number and size, and starch synthesizing enzyme gene expression, and ultimately improved grain filling (Mohapatra and Mohapatra, 2006; Panda et al., 2016). Furthermore, 1-MCP treatment enhanced expression of cell cycle regulators such as cyclin dependent kinase (CDK), cyclin (CYC) and cyclin dependent kinase inhibitor (CKI) and ultimately increased grain filling (Panda et al., 2016).

Using rice lines altered in ethylene signaling, regulation of grain filling by ethylene can be characterized. Ethylene receptor OsETR2 delays the flowering time and influences starch accumulation in the grains through decreasing transcription level of a monosaccharide transporter gene, ultimately leading to a lower seed-setting rate. In addition, both loss-of-function mutants Osetr2 and Osetr3 reduced starch accumulation in stems. The seeds of OsETR2-RNAi lines ripe earlier than that of seeds from control and overexpression lines (Wuriyanghan et al., 2009). The higher expression level of OsETR4 at 6 days after anthesis during spikelet development probably correlated with

grain-filling (Sekhar et al., 2015). The weight of grains of Osctr2 is slightly lower than that of DJ plants (Wang et al., 2013). Compared to WT, the total grain weight per plant decreased in Osein2/mhz7 mutant and OsEIN2-overexpression lines (Ma et al., 2013). OsEILs, acting downstream of OsEIN2, induce ethylene responsive factors (ERF) and/or other transcription activation/inhibition events (Hattori et al., 2009; Ma et al., 2013; Yang et al., 2015b). Sekhar et al. (2015) found that OsEIL1 is negatively associated with grain filling in the inferior spikelets of lax-panicles. Our recent research found that ethylene induced the expression of MHZ5/CRTISO and MHZ4/ABA4 (Ma et al., 2014; Yin et al., 2015). Moreover, grain filling is hindered in mhz4/aba4 mutant (Ma et al., 2014). Carotenoid metabolism is tightly related to tomato fruit ripening. The ethylene-induced carotenoid biosynthesis may also be beneficial for rice grain filling. These results indicate that alteration of ethylene signaling pathway affects rice grain filling.

Ethylene may work in coordination with other hormones to influence rice /grain filling. Numerous data suggest that ethylene negatively regulates rice inferior grain filling through interaction with ABA and other hormones. Under moderate water-stressed condition, higher ratio of ABA to ethylene/ACC concentrations was beneficial for the grain-filling rate in wheat and rice (Yang et al., 2002b, 2004, 2006a). Our results indicate that mutants of mhz4/aba4 and mhz5/crtiso have decreased ABA contents but higher ethylene production (Ma et al., 2014; Yin et al., 2015). The high ratio of ethylene to ABA inhibits the expression of starch synthesis genes and their enzyme activities and leads to poor grain filling (Zhu et al., 2011). Similar results have also been obtained in wheat. When treated with spermidine and spermine, the endogenous ethylene content decreased and led to poor the grain filling and lower grain weight (Liu et al., 2013). Furthermore, under drought conditions, spermidine and spermine significantly increased zeatin, zeatin riboside and ABA content, decreased ethylene content in grains, and improved grain-filling rate in wheat (Liu et al., 2016). Furthermore, the balance of ethylene and ABA plays a pivotal role in grain-filling rate (Yang et al., 2004). Besides, an increase in ABA content but a decrease in GA content, and the altered balance of hormones in rice grains controlled by moderate water deficit, could enhance the weight of the grains (Yang et al., 2001). Cytokinins accelerate cell division during the early phase of rice grain development and consequently influence grain filling (Yang et al., 2000, 2002a). In addition, ethylene inhibited the filling of grains in the basal spikelets while ABA and auxin stimulated this process (Zhang et al., 2009; Kuanar et al., 2010). IAA content is also associated with cell division at the early grain filling stage (Yang et al., 2001).

Taken together, the grain filling is a complicated process and may be regulated by ethylene and other hormones through interactions (Zhao et al., 2007; Liu et al., 2013, 2016). Roles of the hormones from exogenous application studies and from the endogenous determination studies have their own limitations and advantages. Genetic approaches should be more reliable among these, and mutants and transgenic materials should be more valuable in elucidating roles of hormones in grain filling. However, the molecular mechanisms by which ethylene regulates grain filling are largely obscure and need further investigation.

# ETHYLENE AND GRAIN SIZE

As one of the factors determining grain weight, grain size is specified by grain length, grain width, length-to-width ratio, and grain thickness. On the other hand, 1000-grain weight (KGW) is the most reliable trait in assessing grain weight (Huang et al., 2013; Zuo and Li, 2014). Previous studies highlight the pivotal roles of cytokinin (Ashikari et al., 2005), brassinosteroid (BR) (Hong et al., 2003; Tanabe et al., 2005; Sakamoto et al., 2006; Che et al., 2015), and auxin (Ishimaru et al., 2013) on rice grain production. Next the genetic and molecular regulation of ethylene on grain size is discussed.

Rice ethylene response mutants are ideal tools to study the effects of ethylene on agronomic traits. The 1000-grain weights of OsETR2-RNAi lines were substantially higher than that of control; however, this parameter was reduced or similar in ETR2-overexpression plants (Wuriyanghan et al., 2009). As a central component of ethylene signal transduction, OsEIN2/MHZ7 regulates several agronomic traits and plays a pivotal role in rice production. Compared to WT, the total number of panicles was significantly reduced and the KGW of total grains decreased in OsEIN2/MHZ7-overexpression lines. More precisely, OsEIN2/MHZ7 also affects rice grain size. In four allelic mutants of mhz7, the length and width of wellfilled grains decreased compared to WT grains except the allelic mutant mhz7-1. In MHZ7-overexpressing plants, grain length was increased compared to that in WT. Ratio of grain length/grain width in mhz7 mutants was higher or similar with that of WT (Ma et al., 2013). Similarly, loss-of-function Oseil1/mhz6 mutant exhibits a significant reduction in grain length and width compared to WT grains. OsEIL1/MHZ6 overexpression increases grain size and 1000-grain weight (Yang et al., 2015b). The grain size and KGW of OsEIL2-OX lines were smaller than those of WT plants whereas the two parameters of the OsEIL2-RNAi plants exhibited some fluctuation compared with that of WT (Yang et al., 2015b). From these analyses, we propose that ethylene regulates rice agronomic traits under a subtle control. Substantially, ethylene insensitivity may lead to reduced grain size and grain weight, and enhanced ethylene response may be related to larger grains and/or more grain weight, depending on the individual signaling genes. The seedsetting rate and KGW of mhz4/aba4 and mhz5/crtiso were all significantly reduced. Besides, the two mutants showed an increase in grain length and the mhz4 mutant exhibited a decrease in grain thickness (Ma et al., 2014; Yin et al., 2015). The mhz5-3 Osein2 double mutant showed significant decreases in both seed-setting rate and 1000-grain weight, which is similar to that of mhz5/crtiso mutant plants (Yin et al., 2015). These observations suggest that MHZ5/CRTISO regulates seed-setting rate and 1000-grain weight in rice in an OsEIN2-independent manner.

Compared to other cereals, our knowledge about the molecular mechanism that regulates grain size in rice is more than that of wheat, sorghum and maize (Li et al., 2010; Zuo and Li, 2014). ZmIPT2, an isopentenyl transferase involved in cytokinin biosynthesis, is associated with kernel weight and final grain yield (Weng et al., 2013). Several key genes associated with seed size

have been cloned and studied in Arabidopsis. AtAP2 is involved in the increase of total protein and seed oil contents and ultimately regulating Arabidopsis seed size although it negatively affects plant fertility and growth (Jofuku et al., 2005). The mutation of NIMA-related kinase NEK6 leads to increased ethylene production and rounder seeds, while AtNEK6 overexpression suppressed expression of several ethylene biosynthesis- and signaling-related genes and resulted in smaller seeds and reduced 1000-grain weight. These results indicate that NEK6 negatively regulates Arabidopsis seed size (Zhang et al., 2011). Endosperm is the main component of a rice grain, whereas an Arabidopsis seed contains mostly the embryo. This difference suggests that grain size control may be different in various plant species.

### ETHYLENE AND LEAF SENESCENCE

Senescence of vegetative organs is essential for reserve remobilization to developing grain. The main function of leaf senescence is nutrient recycling and this confers an adaptive advantage. To some extent, leaf senescence is beneficial for flowering and seed production. Ethylene is a positive regulator of leaf and flower senescence (Bleecker and Kende, 2000). Studies show that ethylene is one of the most important hormones that affects plant leaf senescence (reviewed by Iqbal et al., 2017). In Arabidopsis, the two mutant alleles of EIN2 oresara2 (ore2) and (ore3) showed delayed senescence, indicating that ethylene affects developmental leaf senescence (Oh et al., 1997). The AtEIN2 positively accelerates leaf senescence by repressing microRNA164 (miRNA164) and regulating the transcription of ORE1/NAC2. The transcription factor ORE1/NAC2 is one of the targets of miRNA164 (Kim et al., 2009). AtEIN3 acts downstream of AtEIN2 to directly repress the expression of miRNA164 and increase ORE1/NAC2 gene transcription to promote leaf senescence (Li et al., 2013). In addition, AtEIN2 affects leaf senescence by regulating the senescence-associated NAC transcription factors (TFs), including ANAC019, AtNAP, ANAC047, ANAC055, ORS1, and ORE1/NAC2. AtEIN3 acts downstream of AtEIN2 and activates ORE1/NAC2 and AtNAP. Furthermore, ORE1/NAC2 and AtNAP affect leaf senescence by activating transcript of common and/or distinct downstream NAC TF factors (Kim et al., 2014). Recent research indicates that EIN3, ORE1/NAC2 and chlorophyll catabolic gene (CCGs) formed a coherent feed-forward loop in the process of ethyleneregulated chlorophyll degradation (Qiu et al., 2015). These results suggest that ethylene regulation of leaf senescence is a highly intricate process and further studies are required to reveal the role of ethylene in leaf senescence.

It is reported that an F-box protein (containing a Kelch repeat motif) OsFBK12 interacted with Oryza sativa S-PHASE KINASE-ASSOCIATED PROTEIN-LIKE PROTEIN (OSK1) to form an SCF complex and degrade its substrate, such as S-ADENOSYL-L-METHIONINE SYNTHETASE1 (SAMS1). The degradation of OsSAMS1 results in a lower content of ethylene, ultimately suppressing leaf senescence and increasing seed size and grain number (Chen et al., 2013). Overexpression of OsRTH1 prevented ethylene-induced leaf senescence through repressing the expression of ethylene-inducible gene, such as submergence 1C (Sub1c), alcohol dehydrogenase 2 (ADH2), and glutathione S-transferase (SC129) (Zhang et al., 2012). Loss-of-function Osctr2 mutant leaves exhibited a much stronger senescence phenotype than Dongjin with or without ethylene treatment. 1-MCP treatment could delay the ethylene-induced senescence in Dongjin but not in Osctr2 (Wang et al., 2013). Osein2/mhz7 mutant leaf remained green whereas the overexpression lines turned yellow under the treatment of dark-induced and natural senescence. Some senescence-associated genes (SAGs) were upregulated, including OsL36, OsL43, OsL85, OsL55, OsNAC1/2 (Ma et al., 2013). Overexpression of the submergence tolerance gene SUB1A delayed dark-induced flag-leaf senescence by limiting ethylene production and responsiveness to the other two positive senescence regulatory hormones JA and salicylic acid (Fukao et al., 2012). Ethylene regulates plant leaf senescence through interactions with other hormones. Ethylene interacts with JA to regulate rice leaf senescence by a direct regulatory cascade of OsCOI1b-OsJAZ-OsEIN3-OsORE1 (Lee et al., 2015). In rice, the AtNAP homologous gene OsNAP promotes leaf senescence and OsNAP is induced by ABA but not 1-aminocyclopropane-1-carboxylic acid (Liang et al., 2014). These studies suggest that the mechanism of NAC TFs involved in leaf senescence may be different in rice and Arabidopsis and need to be further explored.

#### ETHYLENE AND ARCHITECTURE ESTABLISHMENT

Leaf angle is an important morphological trait of plant architecture and influences rice cultivation and grain yield (Sinclair and Sheehy, 1999). Several plant hormones, e.g., ethylene, GA, and auxin are involved in leaf angle formation, and most of these phytohormones regulate the leaf angle through interaction with BRs (Cao and Chen, 1995; Wang L. et al., 2009). In addition, Cao and Chen (1995) suggested that ethylene may be involved in BR-induced rice leaf epinasty. However, Takeno and Pharis (1982) suggest that ethylene is not effective in rice lamina inclination in the intact leaves. Further study is required to reveal the role of ethylene in the BR-induced rice leaf epinasty. Rice ethylene response mutant may be useful for studying the relationship between leaf angle and ethylene. At mature stage, the OsETR2-overexpression lines had erect panicles compared with controls and RNAi-lines (Wuriyanghan et al., 2009). Osein2/mhz7 mutants were more erect than WT plants (Ma et al., 2013).

Great progress has been achieved in identifying important genes associated with rice tiller and panicle branches (for review Liang et al., 2014). Effective tillers produce panicles and play an important role in determining rice yield. The number of panicles per plant is one of the major factors determining rice yield. Previous studies showed that cytokinin (Ashikari et al., 2005), auxin (Xia et al., 2012), and SL (Lin et al., 2009b), and BR (Tong et al., 2009) regulate rice tiller and affect yield. The OsETR2 overexpression lines had reduced effective panicles and seed setting rates whereas the RNAi lines had no significant difference

FIGURE 3 | Diverse roles of ethylene in regulating agronomic traits in rice. In rice, ethylene regulates a wide variety of major agronomic traits ranging from emerging from the soil to grain filling and senescence. Ethylene promotes growth of coleoptiles/mesocotyls by partially inhibiting JA biosynthesis. In contrast, JA promotes rice leaf senescence through a cascade of OsCOI1b-OsJAZ-OsEIN3-OsORE1. JA and ethylene synergistically accelerate rice leaf senescence by activating common and differential SAGs. Ethylene positively regulates rice grain size/weight, flowering, tillering and leaf angle but negatively affects rice grain filling and salt tolerance. The lower ratio of ethylene to ABA is beneficial for rice grain filling. Upon submergence, SUB1A negatively regulates GA response by inhibiting the degradation of SLR1/SLRL1 to restrict plant growth in submergence-tolerance species. On the contrary, ethylene promotes internode elongation through increasing transcription of SK1/2 and GA production to escape flooding in deepwater rice. OsGI1, OsGIGANTEA1; RCN1, TERMINAL FLOWER1/CENTRORADIALIS-like; GY1/EG1, GAOYAO/EXTRA GLUME; HKT2;1, HIGH-AFFINITY K+ TRANSPORTER2;1; SLR1, Slender Rice-1; SLRL1 SLR-like 1; ABA, abscisic acid; GA, gibberellin; JA, jasmonate; COI1, coronatine insensitive 1; JAZ, jasmonate ZIM-domain protein. Blue lines indicate the leaf senescence regulatory pathway. Purple lines indicate the submergence tolerance regulatory pathway. Arrows and T-bars indicate direct or indirect activation and suppression, respectively. Dotted lines indicate several or unknown steps involved in the pathway.

compared with the controls (Wuriyanghan et al., 2009). Both Osctr2 and 35S:OsCTR21−513 transgenic lines produced more effective tillers than the wild type (Dongjin and ZH11) (Wang et al., 2013). The ABA-deficient mutant mhz4/aba4 plants had the same effective tiller number compared to WT, whereas the MHZ4-overexpressing lines produced more tillers than WT (Ma et al., 2014). In contrast, the other ethylene abnormal mutant mhz5/crtiso had excessive tillers, smaller panicles, and fewer primary and secondary branches in panicles compared with WT plants. The tiller number of double mutant mhz5-3 Osein2 was the same as mhz5-3, indicating that MHZ5/CRTISO regulates plant tiller in an OsEIN2-independent manner (Yin et al., 2015). In addition, the strigolactone biosynthesis is also impaired in mhz5/crtiso, and the SL mainly regulates plant tiller/branch (Lin et al., 2009b; Yin et al., 2015). These results suggest that ethylene directly or indirectly regulates rice tiller number.

# ETHYLENE AND SUBMERGENCE

Ethylene is the primary signal for rice and other semi-aquatic plants to adapt to flooding. Concentration changes of abscisic acid, gibberellin, and auxin are also required to gain fast growth under water (Raskin and Kende, 1984; Cox et al., 2004; Saika et al., 2007; Jackson, 2008). Rice has an abundant genetic diversity and evolved two mechanisms to escape flooding. First, increased ethylene upregulates the expression of snorkel1/2 (SK1/2) via OsEIL1b/OsEIL2 binding to the SK1 and SK2 promoters and GA content to promote internode elongation in deepwater rice during flooding (Hattori et al., 2009). Second, SUB1A, an ERF transcription factor existed in limited rice accessions, negatively regulates GA response through restriction of Slender Rice-1 (SLR1) and SLR-like 1 (SLRL1) degradation to inhibit plant elongation during flash floods at the seedling stage in water-tolerance rice (Fukao and Bailey-Serres, 2008).

SUB1A promotes plant recovery from drought at the vegetative stage through decrease of leaf water loss and lipid peroxidation, and increase of gene transcript level associated with acclimation to dehydration. In addition, overexpression of SUB1A could augment ABA responsiveness to enhance plant tolerance to drought (Fukao et al., 2011). Fukao and Xiong (2013) have reviewed the intricate regulatory mechanisms of rice plants acclimating the submergence and drought stress, and ethylene plays a key role in this process. Furthermore, the SUB1A is beneficial for plant physiological recovery upon desubmergence due to the greater capability for non-photochemical quenchingmediated photoprotection (Alpuerto et al., 2016). These results suggest that plant had orchestrating regulatory mechanisms to enhance plant adaptation.

#### CONCLUSION AND PERSPECTIVES

In this review, we summarized roles of ethylene-related factors and genes in regulation of seedling growth, flowering, grain filling, grain size, leaf senescence, leaf angle, tiller and submergence in rice (summarized in **Figure 3**). At present, several environmental cues, including global warming, heat stress, drought, chilling and salinity, affect crop productivity drastically and threaten global food security. Ethylene signaling is indispensable for plant response and adaptation. It has been proven that EIN3/EIL1 is the integration node of several hormones in plant growth and development and adaption to biotic and abiotic stresses (Kazan, 2015; Wawrzynska and Sirko, ´ 2016; Quan et al., 2017). Ethylene promotes salt tolerance in Arabidopsis (Lei et al., 2011). However, the research of Oseil1/mhz6 mutants and OsEIL2-RNAi lines indicates that ethylene signaling negatively regulates salt tolerance in rice (Yang et al., 2015b; **Figure 3**). The roles of ethylene in plant response to salinity has been reviewed by Tao and the divergence between Arabidopsis and rice in the regulation of salinity response by ethylene probably due to the evolutionary divergence under different growing conditions or different plant species (Tao et al., 2015).

In conclusion, higher concentrations of ethylene impair rice grain filling. Similar results were found in wheat. In mature wheat plants, increased ethylene production is associated with decreased 1000-grain weight and hastened maturity (Beltrano et al., 1999). Down-regulating the ethylene biosynthesis pathway

#### REFERENCES

can significantly improve the maize grain yield under abiotic stress (Habben et al., 2014). It should be noted that while higher production of ethylene negatively affects several agronomic traits in wheat and rice, overexpression of OsEIL1/MHZ6 increased grain size and 1000-grain weight (Yang et al., 2015b). OsEIN2/MHZ7 overexpression increased grain size but not grain weight (Ma et al., 2013), and OsETR2-RNAi lines and loss-offunction mutant Osetr2 produced higher 1000-grain weight than control (Wuriyanghan et al., 2009). This discrepancy suggests that the ethylene biosynthesis and signaling pathways may have different functions in grain size control or the ethylene effect is governed not only by the ethylene production, but also by the tissue sensitivity to ethylene. These discrepancies may also be due to the fact that different plants/stages/methods were used. Although each study has its own contribution toward a special area, genetic analysis should be regarded as an effective approach. While progress has been made in recent years in understanding the molecular basis of ethylene signaling in rice, many fundamental questions remain unanswered. Further investigation is needed to confirm the effects of ethylene on crop agronomic traits, especially grain/yield-related traits, using ethylene biosynthesis and signaling mutant or transgenic plants with an alteration of hormone action. How does ethylene signaling connect to grain filling, storage accumulation, and grain size control? The integration and crosstalk points should be identified. Elucidation of how ethylene influences rice grain development may contribute to the advancement of comparative biological studies in other cereals, and assist in breeding of novel high-yield/quality cultivars.

#### AUTHOR CONTRIBUTIONS

C-CY, BM, S-YC, and J-SZ conceived the topic. C-CY wrote the manuscript. J-SZ revised the final version. HZ drew the picture of rice life cycle.

### ACKNOWLEDGMENTS

This work is supported by the National Natural Science Foundation of China (31530004, 31600980, and 31670274), the 973 project (2015CB755702) and the State Key Lab of Plant Genomics.

**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 © 2017 Yin, Zhao, Ma, Chen and Zhang. 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.

fpls-08-01676 September 23, 2017 Time: 15:18 # 12

# Comparative Physiological, Biochemical, and Genetic Responses to Prolonged Waterlogging Stress in Okra and Maize Given Exogenous Ethylene Priming

Emuejevoke Vwioko1 , Onyekachukwu Adinkwu1 and Mohamed A. El-Esawi 2, 3 \*

*1 Department of Plant Biotechnology, Faculty of Life Sciences, University of Benin, Benin, Nigeria, 2 Botany Department, Faculty of Science, Tanta University, Tanta, Egypt, 3 The Sainsbury Laboratory, University of Cambridge, Cambridge, United Kingdom*

#### Edited by:

*Chi-Kuang Wen, Shanghai Institutes for Biological Sciences (CAS), China*

#### Reviewed by:

*Rakesh Kumar Shukla, Central Institute of Medicinal and Aromatic Plants (CIMAP), India Islam Abd El-Daim, Agricultural Research Center, Egypt*

\*Correspondence:

*Mohamed A. El-Esawi mohamed.elesawi@ science.tanta.edu.eg*

#### Specialty section:

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

Received: *04 May 2017* Accepted: *14 August 2017* Published: *25 September 2017*

#### Citation: