The differentiation of chloroplasts in land plants depends foremost on light and developmental-dependent signals (Lopez-Juez and Pyke, 2005). A multitude of mutants perturbed in chloroplast development have been isolated by forward and reverse genetic approaches (Leister, 2003). Such mutants usually display uniform bleaching phenotypes, and are often embryonic lethal or seedling lethal. Some mutants have variegated leaves. Leaf variegation is based on the occurrence of tissue sectors that either contain fully developed chloroplasts or pale, aberrant plastids (Kirk and Tilney-Bassett, 1978; Sakamoto, 2003; Aluru et al., 2006). Recessive mutants that cause variegation have been isolated in monocot as well as in dicot plants (e.g., Hagemann and Scholz, 1962; Walbot and Coe, 1979; Carol et al., 1999; Wang et al., 2000, 2004). These are typically caused by nuclear-loci. By contrast, AmatK plants represent a chloroplast genetic alteration that is maternally inherited. Plastome-based variegation can be caused by segregation of two genetically distinct plastids, one of

which does not develop into fully green chloroplasts (Baur, 1909; Correns, 1909; Börner and Sears, 1986; Greiner and Bock, 2013). It has also been described for the segregation of two genetically distinct plastid chromosomes, for example segregation of chromosomes lacking a trnN-GUU gene versus wt chromosomes (Legen et al., 2007). Depletion of trnN–GUU pools during segregation of homoplastomic tissue was considered to cause leaf variegation. This cannot be the case with AmatK plants, since

these lines are homoplastomic according to Southern analyses. Thus, since AmatK plants are genetically uniform, variegation must be linked to a different, variable parameter. In AmatK cells, this parameter drops below a hypothetical threshold, which prevents chloroplast development. Three external factors, light, sugar and the antibiotic spectinomycin were shown to modulate the extent of variegation and are thus informative in terms of identifying the putative threshold. Light has been noted before as a booster of variegation (Rosso et al., 2009). Light intensity during growth correlates with pale leaf area in the variegation mutants immutans, spotty, var1, var3, and thf1 (Rosso et al., 2009). This effect has been attributed to increased excitation pressure and the resulting photo-oxidative damage under higher light fluences (Rosso et al., 2009; Huang et al., 2013). In analogy, we hypothesize that the AmatK insertion makes developing chloroplasts more vulnerable to increased excitation pressure. Given the role of MatK as a splicing factor, altered gene expression in AmatK plants might interfere with the correct expression of the photosynthetic apparatus and thus lead to detrimental changes in electron transport that are exacerbated under high light conditions. The nature of the primary defect remains to be determined.

Sugar as the second modifier of the AmatK phenotype has been shown to lead to the repression of nuclear genes for photosynthetic functions, e.g., the light harvesting proteins (Krapp et al., 1991; Price et al., 2004; Li et al., 2006). In addition, sugar represses plastid gene expression in liquid culture (Price et al., 2004; Gonzali et al., 2006; Osuna et al., 2007) and also in seedlings grown on sugar-containing MS media (Van Dingenen et al., 2016). Interestingly, the effect on chloroplast gene expression was observed as early as 3 h following sucrose treatment – roughly half of all repressed genes are from the chloroplast genome (Van Dingenen et al., 2016). Notably, matK and rps18 are the most strongly reacting non-photosynthetic chloroplast genes after sugar treatment (Table 1 in Van Dingenen et al., 2016). We hypothesize that the detrimental effect of AmatK on chloroplast gene expression adds up with the repressive effects of sugars on chloroplast gene expression (including on the endogenous matK), which ultimately results in increased variegation. This implies a threshold of plastid gene expression

during chloroplast development, below which no differentiation of chloroplasts is possible. This is further supported by our results from spectinomycin treatments, which showed an even more pronounced effect on variegation than light and sugar application. Spectinomycin blocks chloroplast translation, but should be detoxified by the expression of the aadA cassette in AmatK plants. This function is compromised given the almost complete bleaching of cotyledons on spectinomycincontaining medium. In conclusion, the expression of the aadA cassette is hampered in the AmatK background. Expression of the aadA cassette is driven by a PEP promoter and requires an active and intact plastid translational machinery. In summary, our data suggest that the AmatK insertion negatively affects plastid gene expression, which in some cotelydon cells is lowered beyond a point necessary for chloroplast development.

# AmatK Is Involved in Cotyledon-Specific Chloroplast Development

The main difference between cotyledons and primary leaves is their developmental origin. While all primary leaves are derived from the shoot apical meristem (SAM) post-germination, cotyledons emerge already at the late globular stage of embryo development. This is a time in development, when seedlings are submerged in the soil and live heterotrophically. The plastids in cotyledons develop into etioplasts and only when they emerge from the soil and are irradiated, etioplasts transform into chloroplasts (Mansfield and Briarty, 1996). This is a very different trajectory than plastids take in the SAM, where they differentiate from proplastids directly into chloroplasts during leaf primordia development. The genetic basis of chloroplast development in cotyledons is mostly unknown, but a smaller number of mutants have been described that display pale or even albino cotyledons, while the primary leaves are able to undergo normal greening. This includes white cotyledons (wco; Yamamoto et al., 2000), and snowy cotyledon 1, 2, and 3 (sco1,2,3; Albrecht et al., 2006, 2008, 2010; Ruppel and Hangarter, 2007; Shimada et al., 2007; Zagari et al., 2017). Other mutants, including sig6 and dg1 mutants, exert a dominant effect on cotyledons, but also lead to measureable alterations in chlorophyll content in primary leaves (Ishizaki et al., 2005; Chi et al., 2008). Only one mutant has been described to date that leads to cotyledon-specific variegation, caused by a disruption of the spd1 gene (Ruppel et al., 2011). All of the listed mutants are recessive and affect nuclear genes. AmatK is distinct in eliciting cotyledon variegation via a plastome modification. It is interesting that – like MatK – several of the mutated nuclear genes underlying variegation phenotypes are involved in plastid gene expression: WCO and SCO1 have been implicated in rRNA maturation and translation elongation, respectively. SIG6 and DG1 interact with each other and play a role for plastid transcription. This demonstrates that defects in chloroplast gene expression can selectively affect chloroplast development in cotyledons – a possible scenario also for the AmatK lines. SCO2 encodes a DNAJ-like protein and is required for the accumulation of photosystem II-LHCII complexes (Zagari et al., 2017). This mechanism for variegation is likely distinct from AmatK-induced variegation in that it does not involve plastid gene expression and since recent data indicate functions for SCO2 beyond cotyledons (Zagari et al., 2017). The functions of SPD1 and SCO3 are unknown. In sum, while the molecular mechanisms behind cotyledon-specific variegation remain unknown, it is intriguing that of the few factors known, most play a role for plastid gene expression. Thus, chloroplasts in cotyledons depend on a yet to be determined threshold of chloroplast gene expression, that is no longer crossed in all plastids during development in variegation mutants.

# A Gain of Function in AmatK Lines Impairs Chloroplast Development

The initial aim of our study was to generate a strong overexpressor of MatK. However, while AmatK mRNA is readily detected, we did not find any full-length MatK protein derived from the transgene, but instead only a weakly accumulating degradation product. Since the tag used for the detection of the degradation product is located at MatK's C-terminus, we conclude that translation proceeds to the stop codon, and that the protein is degraded co- or post-translationally. This is surprising, since the amino acid sequence of AmatK is identical to the stable, endogenously HA-tagged version of MatK (Zoschke et al., 2010). We speculate that translation dynamics of the AmatK mRNA are different from those of the endogenous matK mRNA since the two genes have massively different codon usage (**Supplementary Figure S1**). Changes in translation speed can lead to different folding kinetics of the nascent protein and thus to alternative degradation routes (Kimchi-Sarfaty et al., 2007; Gloge et al., 2014). We speculate that such a truncated protein could act as a dominant negative factor in the chloroplast. However, the truncated AmatK protein levels are not rising after theophylline treatment, while at the same time variegation increases. Thus, the N-terminally truncated AmatK cannot be the cause of increased defective chloroplast development after theophylline addition. We, however, cannot rule out that an AmatK protein fragment representing the N-terminal part of AmatKs exerts the effect. Such a fragment would, however, not be detectable, because it would lack the C-terminal HA tag and thus remains a speculation at present. Alternative explanations, i.e., that the AmatK gene or the AmatK mRNA cause the defect, cannot be ruled out, but are also not in line with the theophylline-dependence of the phenotype. While this needs to be tested in the future, our data suggest that the downstream effects represent a gain of function rather than a loss of function of MatK. First, splicing of MatK target introns appears unaffected in AmatK plants: If we agree to take MatK as a splicing factor (Zoschke et al., 2010), these data suggest that AmatK does not compete or interfere with the endogenous MatK. Second, we discovered a number of effects on the accumulation of RNAs that are dependent on inhibiting translation in AmatK plants. These RNAs are not associated with MatK in RIP-chip assays. For instance, the rpl33 operon accumulates more RNA in AmatK plants than in phenocopy controls, including the accumulation of antisense RNA. In the green alga Chlamydomonas reinhardtii, chloroplast

antisense RNAs are stabilized in the absence of translation by forming double-stranded RNA (Cavaiuolo et al., 2017). In general, over-accumulation of RNAs has been described for mutants with global defects in chloroplast translation and is caused at least in part by an increased activity of selected chloroplast RNA polymerases in albino tissue (Emanuel et al., 2004). We tried to control for such secondary effects by analyzing phenocopy controls, but in fact cannot be sure, whether the extent of translational inhibition in the control is truly mimicking the effect seen in AmatK plants. Therefore, whether the RNA defects seen in AmatK tissue are primary or secondary effects cannot be decided at present. Still, a defect in the expression of rpl33 and its neighboring genes, rps18 and rpl20, could very well explain the bleaching phenotype. rps18 and rpl20 encode essential ribosomal proteins (Rogalski et al., 2006; Rogalski et al., 2008). The antisense RNA detected here is long enough to also make hybrids with rps18 and rpl20 mRNAs. This could interfere with sense translation of both mRNAs and thus eventually to reduced ribosome biogenesis and translation capacity. That reduced translational capacity can lead to bleached tissue is exemplified by spectinomycin treatment and several mutants in genes for translational components (e.g., Bellaoui and Gruissem, 2004; Legen et al., 2007; Meurer et al., 2017). Future transplastomic expression of antisense RNA to selected mRNAs could test this hypothesis. Importantly, the option to modulate the level of variegation in cotelydons of AmatK lines makes these plants a valuable tool to determine the time-window and unravel the threshold at which chloroplast gene expression exerts its effect on plastid differentiation.

# MATERIALS AND METHODS

### Plant Material and Growth Conditions

Tobacco plants (N. tabacum cv. Petit Havana) were grown on agar medium containing 30 g/L sucrose for plastid transformation assays. Transplastomic AmatK or pRB70 plants were grown on the same medium or on medium supplemented with 500 mg/L spectinomycin or supplemented with various concentrations of theophylline. wt plants grown on medium with 17 mg/L spectinomycin served as phenocopy controls. For light treatments, plants were grown under standard conditions in a walk-in chamber (25◦C, humidity 55%, 16 h light/8 dark). Homoplastomy of the regenerates and seedlings of T1 generation were tested by Southern Blot Analysis and/or germination assay on a medium containing 500 mg/L spectinomycin.

## Construction of the Synthetic matK (AmatK) Overexpression Vector

The tobacco plastid rRNA operon promoter Prrn and riboswitch RS were amplified from plasmid pAV6 (Verhounig et al., 2010) with primer pair SacIf 50 -GAACAAAAGCTGGAGCTCG-3 0 (SacI site underlined) and AmatKRSr 50 - GTAACGCTGGATTTCTTCCATATGATCCTCTCCACGAGA G-30 (sequence overlap with AmatK underlined). The PCR product was ligated to the AmatK fragment via overlap PCR with primer pair SacIf and HA5AmatKr 50 - CATAATCAGGAACATCATAAGGATACTGGTAGTTTGCCA GGTC-30 (sequence overlap with HA underlined). The resulting PCR product was ligated to the 3XHA tag (Zoschke et al., 2010) via overlap PCR with primer pair SacIf and pAVHAr 50 - CCTTAATTGAATTTCTCTAGAGCCTCATTAAGCATAATCA GGTACATC-30 (XbaI site underlined). The resulting PCR product and pAV6 vector were digested with SacI and XbaI and ligated, resulting in the pRSAmatK vector.

# Plastid Transformation

Stable transformation of plastids was performed by particle bombardment followed by spectinomycin selection. The transformation protocol was carried out according to the protocols of Svab (Svab and Maliga, 1993) and Okuzaki (Okuzaki and Tabei, 2012). Two rounds of transformation were carried out; in the first round, two transplastomic lines with variegated cotyledons were retrieved, in the second round more than a dozen independent transplastomic lines with cotyledon variegation were isolated. Briefly, sterile tobacco leaves from wild type plants cultured on MS were cut into 0.5 cm × 0.5 cm squares. The leaf pieces were placed onto MS plates with the lower leaf side up and cultured overnight. The particle bombardment was carried out using the Biolistic PDS-1000/He Particle Delivery System (1,100 psi, L2 = 6 cm, 10 shots per construct, Bio-Rad) with gold particles (0.6 µM, Bio-Rad) loaded with the transformation plasmids. Spectinomycin resistant calli were regenerated on RMOP medium. Several putative transplastomic lines were obtained and further rounds of selection were carried out to generate homoplastomic lines. The correct integration of AmatK was verified by genotyping PCR and Southern blot. The PCR was performed with the primer pair AmatKseqf 50 -GTTCTCGCATTTGGATCTC-30 and trnMf 5 0 -TTCAAATCCTGTCTCCGCAA-30 .

# Isolation of DNA and Southern Blot

Genomic DNA was isolated from leaves according to standard protocols (Murray and Thompson, 1980). For Southern blot analysis, HindIII-digested total plant DNA was separated on a 0.8% agarose gel, blotted to a Nylon membrane and probed with a PCR product that was body-labeled using α-32P-CTP. The Decaprime DNA labeling Kit (ThermoFisher, Berlin, Germany) was used to label PCR-generated probe DNA fragments (see **Supplementary Table S1** for primers). Hybridization was carried out at 55◦C overnight using standard protocols (Sambrook et al., 1989).

## Isolation of RNA and RNA Gel Blot Hybridization

Total cellular RNA was extracted using TriZol (ThermoFisher, Berlin, Germany). 5 µg of total cellular RNA was separated on formaldehyde-containing 1.2% agarose gels and blotted to Hybond N membrane (GE healthcare, Munich, Germany). Strand-specific RNA probes were synthesized using T7 Polymerase (ThermoFisher, Berlin, Germany) with radioactive γ-32P-UTP (Hartmann Analytic) using PCR products as templates (see **Supplementary Table S1** for primer sequences). Strand-specific DNA probes were generated with the DecaLabel kit and PCR products according to the manufacturer's instructions (ThermoFisher, Berlin, Germany; see **Supplementary Table S1** for primer sequences). Alternatively, oligonucleotides were end-labeled using T4 polynucleotide kinase (ThermoFisher, Berlin, Germany) and γ-32P-UTP (Hartmann Analytic) and used as probes (see **Supplementary Table S1** for oligonucleotide sequences). Hybridizations were done according to standard procedures (Sambrook et al., 1989). Signals were detected using a PMI Imaging system (Bio-Rad, Munich, Germany).

### Immunoblot Analyses

fpls-09-01453 October 1, 2018 Time: 14:37 # 11

Protein extraction was performed according to standard protocols (Schagger and von Jagow, 1987). Protein fractions were separated on polyacrylamide gels and subsequently transferred in Towbin buffer (Towbin et al., 1979) to a PVDF membrane (0.2 µm pore size; GE Healthcare, Munich, Germany). Integrity and equal loading of proteins was detected by Ponceau S staining of the membrane. MatK:HA detection was performed with a rat HA-specific antibody (Roche, Mannheim, Germany). Chemoluminescent signals were detected using a ChemiDoc system (BioRad, Munich, Germany).

### Microarray Analysis

2 µg of total cellular RNA from 10 day old AmatK and phenocopy control plants with red- (Cy5) and green-fluorescing (Cy3) dyes, respectively, using the ULS RNA Fluorescent Labeling Kit according to the manufacturer's instructions (Kreatech, Amsterdam, Netherlands). The samples were hybridized to a whole-genome tobacco chloroplast tiling microarrays (Finster et al., 2013). Hybridization, array washings and signal detection were carried out as described previously (Kupsch et al., 2012).

Microarray analysis was done in R using the packages marray (version 1.52) and multtest (version 2.28). Namely, expression values were loaded in R using marray. After this, loess normalization at the probe level was applied using maNorm function from the marray package for the two biological replicates performed. The array contains 12 copies of each probe located at different spots to reduce any bias regarding its location on the array; the median log2 fold-change expression for the 12 copies was used to calculate the log2 fold-change

### REFERENCES

of the region represented by the probe for each biological replicate independently. A t-test was applied to calculate which regions show an average log2 fold-change different of zero. Multiple hypotheses correction was applied using the Benjamini & Hochberg method (BH) implemented in the multitest package.

### AUTHOR CONTRIBUTIONS

YQ designed the transformation vectors, performed chloroplast transformation and selection for homoplastomic lines, and performed genetic crosses. JA and AW analyzed transgene expression and splicing efficiency by RNA gel blot hybridization. JL performed genotyping PCRs and Southern analyses. SH and GH performed RNA gel blot analyses and microarray analyses. CT performed immunological analyses. GR participated in the screening for transplastomic plants. JM and UO evaluated the microarrays and contributed to manuscript preparation. GW supported the design of the transformation vector and immunological analyses. OO contributed to project design and manuscript preparation. CS-L conceived the study, prepared the figures, and wrote the manuscript.

### FUNDING

Generous funding by the DFG to CS-L (TR175; project A02) and by GIF to CS-L and OO (I-1213-319.13/2012) is gratefully acknowledged. YQ was supported by a fellowship from the China Scholarship Council.

### ACKNOWLEDGMENTS

Technical support by T. Börner (HU Berlin) is gratefully acknowledged. We are grateful to Ralph Bock and Stephanie Ruf for making available the pAV6 vector and pRB70 plants.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01453/ full#supplementary-material

photosynthetic parasitic plant genus Cuscuta (Convolvulaceae). BMC Biol. 5:55. doi: 10.1186/1741-7007-5-55

fpls-09-01453 October 1, 2018 Time: 14:37 # 13

**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 © 2018 Qu, Legen, Arndt, Henkel, Hoppe, Thieme, Ranzini, Muino, Weihe, Ohler, Weber, Ostersetzer and Schmitz-Linneweber. 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) and the copyright owner(s) 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.

# Alternative Splicing as a Regulator of Early Plant Development

Dóra Szakonyi\* and Paula Duque\*

Instituto Gulbenkian de Ciência, Oeiras, Portugal

Most plant genes are interrupted by introns and the corresponding transcripts need to undergo pre-mRNA splicing to remove these intervening sequences. Alternative splicing (AS) is an important posttranscriptional process that creates multiple mRNA variants from a single pre-mRNA molecule, thereby enhancing the coding and regulatory potential of genomes. In plants, this mechanism has been implicated in the response to environmental cues, including abiotic and biotic stresses, in the regulation of key developmental processes such as flowering, and in circadian timekeeping. The early plant development steps – from embryo formation and seed germination to skoto- and photomorphogenesis – are critical to both execute the correct body plan and initiate a new reproductive cycle. We review here the available evidence for the involvement of AS and various splicing factors in the initial stages of plant development, while highlighting recent findings as well as potential future challenges.

### Edited by:

Simon Gilroy, University of Wisconsin–Madison, United States

### Reviewed by:

Ligeng Ma, Capital Normal University, China Lydia Gramzow, Friedrich-Schiller-Universität Jena, Germany

### \*Correspondence:

Dóra Szakonyi<EMAIL_ADDRESS><EMAIL_ADDRESS>Paula Duque<EMAIL_ADDRESS>
### Specialty section:

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

Received: 03 April 2018 Accepted: 23 July 2018 Published: 15 August 2018

### Citation:

Szakonyi D and Duque P (2018) Alternative Splicing as a Regulator of Early Plant Development. Front. Plant Sci. 9:1174. doi: 10.3389/fpls.2018.01174 Keywords: alternative splicing, early seedling development, embryogenesis, photomorphogenesis, seed dormancy, seed maturation, seed germination, splicing factors

# mRNA PROCESSING AND ALTERNATIVE SPLICING

Accurate processing of precursor mRNAs (pre-mRNAs) is a major step in gene expression crucial for performing everyday housekeeping functions, executing developmental programs, and responding to intrinsic and environmental cues. It involves modification steps to remove noncoding sequences as well as add the cap and the poly(A) tail to the 50 and 30 ends of the mRNA, respectively (reviewed in Proudfoot, 2011; Shi and Manley, 2015; Ramanathan et al., 2016). Pre-mRNA splicing, the excision of introns followed by joining of exons, is catalyzed by the spliceosome, a large ribonucleoprotein complex. The spliceosomal subunits assemble at conserved nucleotides at the exon-intron boundaries also known as the 50 (or donor) and 30 (or acceptor) splice sites (SS), the branch point and the polypyrimidine tract. In addition to the core spliceosomal components, many RNA-binding proteins play key roles in mRNA processing, SS selection and splicing (reviewed in Meyer et al., 2015). In higher eukaryotes, intron-containing genes frequently give rise to multiple mRNAs through alternative splicing (AS) (**Figure 1A**), during which differential recognition of SS can lead to intron retention, exon skipping and/or alternative 5 0 /30 SS selection. AS can significantly enhance a genome's coding capacity by producing protein variants with altered function. It also often affects mRNA stability by introducing premature stop codons in the coding sequence, thus targeting these transcripts to degradation by nonsensemediated decay (NMD). Furthermore, AS can modify gene expression by modulating transcription elongation and/or translation efficiency (reviewed in Reddy et al., 2013; Laloum et al., 2017). It is hence not surprising that AS fulfills important biological functions. In plants, it has been found to control key processes like the circadian clock or flowering time as well as the response to environmental cues, including abiotic stress or pathogen attack (reviewed in Staiger and Brown, 2013; Yang et al., 2014; Laloum et al., 2017; Shang et al., 2017).

# EARLY PLANT DEVELOPMENT

The first stages of a plant's life are essential to establish the basic body pattern, develop different tissue types and initiate a new reproductive cycle (**Figure 1B**). Sexual reproduction of land plants involves the alternation of haploid and diploid stages. Angiosperms have a dominant diploid sporophyte and a relatively short haploid phase consisting of a few microscopic cells. Seeds are produced by double fertilization. One sperm cell fuses with the egg cell to form the diploid embryo, while a second sperm cell fertilizes the diploid central cell to give rise to the endosperm (reviewed in Raghavan, 2003; Berger et al., 2008). During embryogenesis, the one-cell zygote undergoes a tightly regulated developmental program to form a mature embryo. In dicots such as Arabidopsis thaliana (arabidopsis), this process includes distinct morphological stages, called globular, heart, torpedo, and bent cotyledon, leading to the establishment of the basic body plan and main tissue/organ initials including the shoot and root apical meristems (reviewed in Palovaara et al., 2016). Embryo morphogenesis is followed by seed maturation, which involves the accumulation of reserves, acquisition of desiccation tolerance, reduction of metabolic activities and induction of dormancy to enable survival of the embryo until favorable environmental conditions allow germination (reviewed in Graeber et al., 2012). Fresh seeds usually show high dormancy that gradually decreases over time in a process called after-ripening. The release from dormancy depends on environmental factors (e.g., light quality, day length, temperature, water availability, exposure to cold) and internal regulators (e.g., hormones, regulatory proteins, chromatin status) (reviewed in Kucera et al., 2005; Nee et al., 2017). Germination starts with water uptake (imbibition) and rapid expansion of the embryo, leading to rupture of the seed coat and emergence of the radicle. Seedlings growing in the dark display skotomorphogenic development (etiolated growth), characterized by elongated hypocotyls, apical hook, pale cotyledons and short roots. When exposed to light, the seedling undergoes photomorphogenesis to activate vegetative growth, displaying shorter and thicker hypocotyls as well as green and expanded cotyledons (reviewed in Wu, 2014). Hormones are important regulators of early plant development. Embryo formation is governed by auxins and cytokinins, while abscisic acid (ABA) is important for the completion of seed maturation and building up dormancy. ABA is also the major inhibitor of seed germination, with its effect being counteracted by gibberellic acid, ethylene, and brassinosteroids (reviewed in Palovaara et al., 2016).

# GLOBAL ALTERNATIVE SPLICING CHANGES DURING EARLY PLANT DEVELOPMENT

Next-generation sequencing has revolutionized transcriptomic studies. The latest RNA-seq data gathered in higher plants showed that traditional approaches largely underestimated the proportion of genes undergoing AS. Current assessments indicate that up to 70% of plant multiexon genes generate more than one transcript via this mechanism, with intron retention representing the predominant mode of AS (Lu et al., 2010; Zhang et al., 2010; Marquez et al., 2012; Shen et al., 2014; Thatcher et al., 2014; Chamala et al., 2015; Sun and Xiao, 2015; Iniguez et al., 2017; Zhang et al., 2017). In fact, increased sequencing coverage revealed a large number of non-annotated AS events and splice variants (Marquez et al., 2012; Zhang et al., 2017). Most of the plant AS events map to coding regions, thereby altering protein sequence and potentially function or compromising mRNA stability. Indeed, a significant proportion of intron-containing genes are potentially regulated by NMD (Zhang et al., 2010; Kalyna et al., 2012; Drechsel et al., 2013). Although thousands of alternatively spliced mRNAs are detected in genome-wide analyses, detailed genetic and molecular studies will be required to identify functionally relevant AS events.

Numerous plant large-scale studies have focused on gene expression and AS patterns in different tissues and during development, identifying many novel organ- or stage-specific mRNAs with dynamic expression changes and a stage-dependent switch in isoform dominance for many genes (Zhang et al., 2010; Thatcher et al., 2014; Klepikova et al., 2016; Vaneechoutte et al., 2017). Notably, genes encoding alternatively spliced transcripts are not necessarily differentially expressed during developmental transitions, suggesting that AS shapes the transcriptome independently from transcriptional regulation (Srinivasan et al., 2016). These findings are confirmed by deepsequencing studies tracking expression and AS changes during the first stages of plant development (Aghamirzaie et al., 2013; Lu et al., 2013; Sun and Xiao, 2015; Qu et al., 2016; Thatcher et al., 2016; Narsai et al., 2017). The detection of prominent AS switches and of development-specific splice variants corroborates an important regulatory layer of early plant development at the splicing level. Interestingly, RNA-processing factors themselves undergo AS resulting in a potential autoregulatory feedback loop.

During embryogenesis in soybean (Aghamirzaie et al., 2013), AS of 47,331 genes produced 217,371 different transcripts, most of which had not been previously identified. Nearly one third of the genes showed variations in transcript levels during embryo development, including those encoding enzymes involved in carbon or nitrogen metabolism and hormonemediated signaling pathways. Most AS events were detected during the later stages of embryogenesis, i.e., embryo maturation, dehydration, establishment of dormancy, and at the quiescent state. This induction of AS may be explained by the striking clustering of both splicing-related and ABA-associated factors observed at the late phases of seed development. Seed maturation and desiccation, which involve very specific developmental, hormonal, and biochemical processes, were also examined in arabidopsis (Srinivasan et al., 2016), where RNA-seq profiling was performed on developing and mature seeds. Interestingly, transcription and AS showed opposite trends, with transcription declining during seed maturation, while AS increased. Over a quarter of the loci undergoing AS expressed stage-specific splice variants or showed a marked isoform switch, with a striking 88% of the detected AS events being absent from the TAIR10 genome annotation. Again, there were no significant changes in total transcript levels of many alternatively spliced genes,

FIGURE 1 | Alternative splicing and early plant development. (A) Constitutive and alternative splicing. Nascent multi exonic mRNAs need to undergo pre-mRNA splicing. Constitutive splicing removes the non-coding introns, producing a mature mRNA that encodes the full-length protein or transcript with biological functions. The same pre-mRNA molecule can undergo alternative splicing (AS) and produce different transcript variants. AS events occurring in non-coding sequences often impact gene expression, but will result in a protein identical to the full-length isoform. In numerous cases, coding regions are affected by AS, thus originating markedly different mRNAs and potentially distinct proteins that can vary in virtually all functional aspects. (B) Embryogenesis, seed maturation and germination, and early seedling development. After fertilization, the zygote undergoes a rapid succession of highly coordinated cell divisions to form globular stage embryos, which show establishment of the apical–basal axis and a first distinction between outer and inner cells. The embryonic cells further differentiate during the heart stage, when many of the basic cell types (provasculature, endodermis, cortex, and protoderm) and organ primordia (cotyledons, hypocotyl, and primary root) are formed and a bilateral body pattern appears. Expanding cotyledons give the embryo a torpedo shape, and the formation of the shoot and root apical meristems is completed. The next steps involve further cell growth and divisions until the embryo reaches its final shape and size. Seed maturation is completed with the accumulation of reserves and the establishment of desiccation tolerance and seed dormancy. Dry seeds are released from dormancy in response to a combination of environmental cues and internal signals. After water uptake, key biochemical and molecular processes are restored, followed by the rupture of the seed coat and emergence of the radicle, marking the completion of germination. Under darkness, the buried seedling undergoes skotomorphogenesis characterized by a short root, elongated hypocotyl, apical hook and absence of photosynthetic pigments. Upon light exposure, photomorphogenesis is activated leading to inhibition of hypocotyl elongation, opening and expansion of the cotyledons, and initiation of photosynthesis after chloroplast maturation.

pointing to AS as an important regulatory mechanism operating independently from transcription. Most of the genes exhibiting differential splicing were involved in RNA processing, potentially amplifying the AS regulatory effect in preparation for seed germination.

Two recent studies addressed the AS contribution during seed germination. In barley embryos, 14–20% of multiexon genes expressed multiple mRNA isoforms, some of which displayed clear changes during early germination (Zhang et al., 2016). Surprisingly, the most prominent AS event was alternative 30 SS

selection, and there were no substantial alterations in total transcript levels for most genes. Assessment of the biological functions of the genes undergoing AS during germination indicated involvement in protein synthesis, energy and carbon metabolism as well as RNA transport and splicing. Overall, seed germination appears to require expression of a specific set of genes, with AS playing a widespread role. The regulatory potential of AS during germination is underscored by a subsequent report in arabidopsis (Narsai et al., 2017) confirming the expression of time- and tissue-specific mRNA variants, the occurrence of dynamic changes in isoform abundance, and that splicing regulators are major AS targets during this developmental process.

AS regulation during early plant growth is also relevant in the context of environmental responses. Light, which is perceived by various photoreceptors, strongly impacts the life cycle of plants, regulating among others early developmental steps such as seed germination and the transition to autotrophic growth. Genome-wide effects of light on plant AS were recently analyzed by RNA-seq (Wu et al., 2014; Mancini et al., 2016), including in very young seedlings (Shikata et al., 2014; Hartmann et al., 2016). Shikata et al. (2014) reported that, during the initial response of etiolated seedlings to red light, the number of genes showing phytochrome-mediated differential gene expression or changed AS pattern is comparable, while later transcription becomes the dominant regulatory mechanism. In the phytochrome-dependent AS dataset, splicing-related genes were overrepresented, including SR proteins and the U1 and U2 spliceosomal subunits, while transcription factors comprised the major group of differentially expressed genes. AS seemed to play a significant role in light-induced chloroplast differentiation, as photosynthesis- and plastid-related genes were also enriched in the differential AS sets. When Hartmann et al. (2016) analyzed the response of etiolated arabidopsis seedlings exposed blue, red, or white light treatments, ∼20% of genes were found to be differentially expressed, with ∼700 AS events being detected, most of which mapped to coding sequences. Again, gene ontology analysis revealed overrepresentation of the RNAbinding category, including many splicing factors. A link between light-induced AS and mRNA stability was also uncovered, with 77.2% of the detected mRNA isoforms more abundant in the dark samples being potential NMD targets. Remarkably, in most of AS events, an isoform switch from a putative instable mRNA variant to a protein-coding alternative occurred upon light exposure. Moreover, mutants lacking the major red or blue light receptors showed impaired AS mainly when subjected to monochromatic red or blue light, indicating that additional signaling pathways influence AS under white light. The authors suggested that metabolic signals, sugars in particular, are implicated in lightmediated AS regulation.

### SPLICING FACTORS REGULATING EARLY PLANT DEVELOPMENT

Compelling evidence from large-scale analyses pointing to an important role for AS during early plant development is being substantiated by accumulating in vivo genetic studies (**Table 1**).  Some studies have established a hormonal basis for the embryo and early seedling development defects caused by altered expression of splicing factors (Kalyna et al., 2003; Casson et al., 2009; Tsugeki et al., 2015), with abnormal spatial distribution of auxin arising from erroneous splicing and expression of auxin biosynthesis, transport, and signaling genes. A link between mRNA splicing and auxin signaling was also uncovered in flowers, where subcellular compartmentation of an auxin biosynthetic gene is regulated by AS (Kriechbaumer et al., 2012).

Seed dormancy and germination are also strongly affected in mRNA processing mutants. These effects were mostly reported to relate to splicing (Dolata et al., 2015) and polyadenylation (Cyrek et al., 2016) of the DOG1 gene, a key seed dormancy regulator and known AS target, and to changes in ABA signaling (Xiong et al., 2001; Sugliani et al., 2010; Jiang et al., 2012). Early seedling development can be affected as a manifestation of wider pleiotropic defects (Liu et al., 2010; Swaraz et al., 2011; Perea-Resa et al., 2012; Shikata et al., 2012; Hsieh et al., 2015; Yap et al., 2015) or in weak alleles of embryo lethal mutants (Kalyna et al., 2003; Gutierrez-Marcos et al., 2007; Fouquet et al., 2011; Tsugeki et al., 2015). Observed phenotypes include disturbed cotyledons, hypocotyls, vasculature patterning, roots and/or seedling viability and growth. Notably, mRNA splicing in plastids and mitochondria appears to be crucial for seed development and plant growth in both arabidopsis and maize (Schmitz-Linneweber et al., 2006; Gutierrez-Marcos et al., 2007; Liu et al., 2010; Hernando et al., 2015; Hsieh et al., 2015; Yap et al., 2015; Chen et al., 2017).

Genetic and molecular analyses have confirmed a role for splicing factors in photomorphogenesis, particularly in red-light responses. Phytochrome-dependent light signaling influences AS through specific splicing components, with additional splicing factors such as SR proteins being differentially processed in loss-of-function mutants of these effectors under various light conditions (Shikata et al., 2012; Hernando et al., 2015; Xin et al., 2017). Interestingly, Xin et al. (2017) demonstrated red lightdependent direct interaction and colocalization of a splicing factor and phytochrome B.

### ALTERNATIVE SPLICING TARGETS AFFECTING EARLY PLANT DEVELOPMENT

Despite massive transcriptome changes imposed by AS during early plant development, only a handful of alternatively spliced transcripts have had their functional significance analyzed in detail (**Table 1**). While one group, including the arabidopsis

### TABLE 1 | Splicing factors and targets functioning in early plant development.

 Moreover, PIF6 regulates both seed dormancy and light responses (Penfield et al., 2010). Despite the few individual events studied, AS is known to act via diverse mechanisms, as illustrated below.

In agreement with results from large-scale studies, the expression of numerous individual mRNA variants was found to be development- or tissue-specific (Zhou et al., 1998; Fan et al., 2007; Gagete et al., 2009; Sugliani et al., 2010; Gao et al., 2013; Wang et al., 2018), with some turning out to be non-functional, either because they did not produce an active protein or no phenotypic consequence was observed as a result of ectopic expression (Wang et al., 2018). In another study, genetic complementation tests indicated that the different splice variants perform functions equivalent to the constitutive form, even when lacking crucial amino acid sequences or domains (Li et al., 2017). Similarly, AS did not fundamentally influence DNA-binding or protein-protein interaction ability of the ABI3 and ABI5 transcription factors from different plant species, though the binding strength appeared to differ among the various isoforms (Zou et al., 2007; Gagete et al., 2009; Gao et al., 2013).

Alternative splice variants can also fulfill similar or distinct functions depending on developmental stage. The constitutive and alternative PIF6 mRNA variants similarly influenced light responses in seedlings, while only the short isoform displayed evident functions during seed germination (Penfield et al., 2010). In the case of COP1 and SPA3, ectopic overexpression of alternative splice forms phenocopied knock-out mutant phenotypes, indicating that some alternative forms can interfere with the function of the full-length protein (Zhou et al., 1998; Shikata et al., 2014). Strikingly, co-expression and direct protein interactions were found to be necessary for full DOG1 function (Nakabayashi et al., 2015). In genetic complementation assays, independent expression of individual DOG1 isoforms driven by the native promoter did not restore seed dormancy, whereas transgenic lines carrying two or more DOG1 variants showed improved dormancy. Detailed analysis of these results supported the hypothesis that, although single isoforms are active, the presence of multiple isoforms is required for adequate DOG1 function. On the other hand, AS-induced changes in protein sequence may lead not only to diminished biological function but, as demonstrated for an HYH isoform lacking a protein interaction domain for proteasomal degradation, also to a more stable and hence more active protein isoform (Sibout et al., 2006).

Subcellular targeting provides specialized locations for intracellular processes and can interfere with the regulatory and biochemical potential of proteins. AS of a pumpkin hydroxypyruvate reductase (HPR) acting in photorespiration affected the C-terminal targeting sequence, with one splice form localizing in the peroxisome and another in the cytosol (Mano et al., 1999). The two mRNAs were expressed at similar levels in darkness, while light promoted the production of the shorter, cytosol-localized variant. Most recently, retention of an mRNA variant of the arabidopsis SR30 splicing regulator in the nucleus was shown to influence mRNA stability by preventing the degradation of a potential NMD target in the cytoplasm and its association to the translation machinery (Hartmann et al., 2018).

### CONCLUSION AND PERSPECTIVES

Recent transcriptome-wide, genetic and molecular studies have demonstrated that regulation of the complex developmental steps from embryogenesis to establishment of a functional plant includes posttranscriptional control via AS. Seed maturation, establishment and maintenance of seed dormancy, and young seedling responses to light stand out as significant AS-regulated processes. The detection of time- and tissue-specific mRNA variants and of notable switches in splicing patterns substantiate crucial roles for AS in other early development processes. Further large-scale analyses in different tissue types using the latest sequencing technologies and single-cell approaches will be key to understand the full extent of AS events occurring during the initial stages of plant development. Improved standardization of data processing and analysis along with more meticulous experimental set-ups should also allow for more reliable comparative studies. Comprehensive publicly available databases, providing a detailed and up-to-date view of AS in plants are still lacking. These will be pivotal in pinpointing promising novel splice forms and assist in functional studies to distinguish biologically relevant AS contributing to proteomic diversity or gene expression regulation from nonfunctional AS events and splicing noise. Importantly, state-ofthe-art methodology such as iCLIP is proving successful in

### REFERENCES

plant systems and should allow identification of the mRNAs targeted directly by splicing factors to control early plant development.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### FUNDING

DS was supported by a Postdoctoral Fellowship (SFRH/BPD/94796/2013) from Fundação para a Ciência e a Tecnologia (FCT), which also finances research in our lab through Grant PTDC/BIA-PLA/1084/2014. Funding from the GREEN-it research unit (UID/Multi/04551/2013) is also acknowledged.

### ACKNOWLEDGMENTS

Our apologies to the authors whose work was not cited due to space restrictions.

multiple isoforms generated by alternative splicing. PLoS Genet. 11:e1005737. doi: 10.1371/journal.pgen.1005737

**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 © 2018 Szakonyi and Duque. 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) and the copyright owner(s) 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.

# Functional Characterization of SMG7 Paralogs in Arabidopsis thaliana

Claudio Capitao1† , Neha Shukla2† , Aneta Wandrolova2 , Ortrun Mittelsten Scheid1 and Karel Riha2 \*

1 Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna Biocenter, Vienna, Austria, 2 Central European Institute of Technology, Masaryk University, Brno, Czechia

SMG7 proteins are evolutionary conserved across eukaryotes and primarily known for their function in nonsense mediated RNA decay (NMD). In contrast to other NMD factors, SMG7 proteins underwent independent expansions during evolution indicating their propensity to adopt novel functions. Here we characterized SMG7 and SMG7 like (SMG7L) paralogs in Arabidopsis thaliana. SMG7 retained its role in NMD and additionally appears to have acquired another function in meiosis. We inactivated SMG7 by CRISPR/Cas9 mutagenesis and showed that, in contrast to our previous report, SMG7 is not an essential gene in Arabidopsis. Furthermore, our data indicate that the N-terminal phosphoserine-binding domain is required for both NMD and meiosis. Phenotypic analysis of SMG7 and SMG7L double mutants did not indicate any functional redundancy between the two genes, suggesting neofunctionalization of SMG7L. Finally, protein sequence comparison together with a phenotyping of T-DNA insertion mutants identified several conserved regions specific for SMG7 that may underlie its role in NMD and meiosis. This information provides a framework for deciphering the non-canonical functions of SMG7-family proteins.

### Germany

\*Correspondence: Karel Riha<EMAIL_ADDRESS>†Shared first co-authorship

Instituto Gulbenkian de Ciência (IGC),

Agricultural Biotechnology Institute,

Eberhard Karls Universität Tübingen,

### Specialty section:

Edited by: Dora Szakonyi,

Portugal Reviewed by: Daniel Silhavy,

Hungary Uener Kolukisaoglu,

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

Received: 31 May 2018 Accepted: 17 October 2018 Published: 06 November 2018

### Citation:

Capitao C, Shukla N, Wandrolova A, Mittelsten Scheid O and Riha K (2018) Functional Characterization of SMG7 Paralogs in Arabidopsis thaliana. Front. Plant Sci. 9:1602. doi: 10.3389/fpls.2018.01602 Keywords: nonsense mediated RNA decay, meiosis, SMG7, gene duplication, Arabidopsis

### INTRODUCTION

In eukaryotic cells, gene expression is controlled by several surveillance mechanisms that assure accurate and robust production of functional proteins. One such control mechanism is nonsense mediated RNA decay (NMD), which degrades aberrant RNAs (He and Jacobson, 2015; Lykke-Andersen and Jensen, 2015; Hug et al., 2016; Raxwal and Riha, 2016). NMD typically targets transcripts containing premature termination codons (PTC) that arise as a consequence of a missense mutation or alternative splicing, leading to the translation of a truncated protein. NMD is an evolutionary highly conserved pathway that may have co-evolved with the acquisition of introns (Farlow et al., 2010). Although NMD is one of the most well-studied RNA surveillance pathways, its exact molecular mechanism is not yet fully resolved. The consensus model, based on studies across a range of organisms, suggests that NMD is induced by the premature translation termination at stop codons in the absence of a full complement of canonical translation termination signals (He and Jacobson, 2015; Lykke-Andersen and Jensen, 2015).

The most detailed insights into the NMD mechanism were obtained in mammals (He and Jacobson, 2015; Lykke-Andersen and Jensen, 2015; Hug et al., 2016). The key step

is the phosphorylation and activation of the RNA helicase UPF1 by the SMG1 kinase in response to a ribosome stalled at a PTC. Mammalian NMD primarily targets transcripts harboring introns in their 30UTR and UPF1 activation is facilitated by the presence of an exon–exon junction 50–55 nucleotides downstream of the PTC. This is sensed via interaction with the exon junction complex, and is mediated by UPF3B and UPF2. Once phosphorylated, UPF1 is recognized by the phosphoserine-binding proteins SMG5, SMG6, and SMG7 that induce degradation of the aberrant mRNA. SMG5, SMG6, and SMG7 belong to the same protein family, which is characterized by a phosphoserine-binding domain that structurally resembles 14-3-3 proteins (Fukuhara et al., 2005). Their association with UPF1 defines two major RNA degradation pathways. SMG6 itself acts as an endonuclease cleaving mRNA in the vicinity of the PTC. SMG5 and SMG7 bind phosphorylated UPF1 as a heterodimer (Jonas et al., 2013; Nicholson et al., 2018). SMG7 recruits the CCR4-NOT deadenylase complex and induces deadenylation-dependent decapping, which is followed by 50 - 3 0mRNA degradation (Unterholzner and Izaurralde, 2004; Loh et al., 2013). NMD in human cells mainly relies on SMG6 mediated degradation, while the SMG5-SMG7 dimer seems to act as a back-up pathway.

The NMD mechanism described in mammals appears to be conserved in plants, although some aspects differ (Kerenyi et al., 2008; Shaul, 2015). NMD in plants acts efficiently on transcripts containing 30UTR introns, but also on transcripts that possess long 30UTR without introns (Kertesz et al., 2006; Kurihara et al., 2009; Kalyna et al., 2012; Drechsel et al., 2013; Degtiar et al., 2015). This mechanism, known as the faux 30UTR model, was described in budding yeast and postulates that the long distance between the PTC and poly(A)-tail fails to provide the proper context for translation termination and sensitizes transcripts to NMD (He and Jacobson, 2015; Hug et al., 2016). Functional studies in tobacco showed that both NMD pathways require UPF1, UPF2, and SMG7, while components of the exon junction complex are only involved in the intron-based mechanism (Kerenyi et al., 2008). As in mammals, phosphorylation of UPF1 is critical for late steps of plant NMD (Merai et al., 2012; Kerenyi et al., 2013). The SMG1 kinase is present in the majority of plants, but has been repeatedly lost in the Brassicaceae family, suggesting that its function can be substituted by an as yet unknown kinase (Lloyd and Davies, 2013; Causier et al., 2017). Phosphorylated UPF1 recruits SMG7 through its terminal 14-3-3 domain, which in turn mediates RNA degradation and UPF1 relocalization to P-bodies (Merai et al., 2012). Plants do not possess SMG5 and SMG6 paralogs, but the genomes of dicots encode an additional SMG7-like (SMG7L) protein (Riehs et al., 2008). Nevertheless, functional studies in grapevine indicate that SMG7L has lost its NMD activity (Benkovics et al., 2011).

Downregulation of NMD in Arabidopsis leads to a strong immune response that is caused by derepression of TIR-NB-LRR immune receptors (Jeong et al., 2011; Rayson et al., 2012; Riehs-Kearnan et al., 2012; Gloggnitzer et al., 2014; Raxwal and Riha, 2016). UPF1 and SMG7 are understood to act in subsequent steps of a linear pathway; therefore, their inactivation should have similar biological effects. However, the phenotypes of UPF1 and SMG7 in Arabidopsis differ substantially. A null upf1-3 mutation causes seedling lethality due to massive activation of the immune response (Yoine et al., 2006; Riehs-Kearnan et al., 2012). This lethality can be rescued by the inactivation of PAD4, a key component of pathogen signaling, but the upf1-3 pad4 double mutants still exhibit retarded growth and pleiotropic developmental defects. Analysis of plants carrying different T-DNA insertions in the SMG7 gene showed range of different phenotypes. The most N-terminal disruption in the 14- 3-3 domain (smg7-5) was proposed to cause embryonic lethality, as no mutant plants could be recovered (Riehs et al., 2008). Smg7- 1 and smg7-3 mutants carrying insertions in the central region of SMG7 are viable, but exhibit retarded growth caused by pathogen response activation and are infertile due to abortive meiosis (Riehs et al., 2008; Bulankova et al., 2010). While the vegetative growth defects are fully rescued in smg7-1 pad4 double mutants, meiosis is still defective. Furthermore, the meiotic defects are not observed in upf1-3 pad4 mutants, suggesting that the role of SMG7 in meiosis is not linked to NMD (Riehs-Kearnan et al., 2012).

Here, we aimed to clarify whether Arabidopsis SMG7 is an essential gene by characterizing SMG7-loss of function mutants generated by CRISPR-Cas9 mutagenesis. Furthermore, we investigated the requirements for the 14-3-3 domain in meiosis and the redundancy between SMG7 and SMG7L paralogs. Finally, we used phylogenetic analysis to search for motifs that may define SMG7 function in NMD and meiosis.

### MATERIALS AND METHODS

### Plant Material

Seeds of Arabidopsis thaliana were grown in soil in phytotrons at 20–22◦C with a photoperiod of 16 h light/8 h dark. All wild type and mutant plants were derived from A. thaliana ecotype Col-0. The smg7-1 and smg7-6 mutants used in this study were characterized previously (Riehs et al., 2008; Riehs-Kearnan et al., 2012) and the smg7l-1 mutation was obtained from the Arabidopsis Stock Centre (SAIL\_634H06).

### Vectors and Transgenic Plants

We used the CRISPR/Cas9 system developed for targeted mutagenesis in plants (Fauser et al., 2014). The CRISPR sequence guiding the Cas9 nuclease was designed to target the second exon of the SMG7 gene downstream of and near to the ATG codon. Briefly, the guide RNA was created by cloning the duplex oligonucleotide 50 -CTTGGCTCGCTCCCATGAAG-3 0 into the BbsI site of pEN-Chimera (Fauser et al., 2014). The region coding for the gRNA was transferred into pDe-CAS9 using the GatewayTM cloning system, creating the binary vector pCCC40. Transgenic plants were generated by Agrobacteriummediated floral-dip transformation of A. thaliana ecotype Col-0. Transgenic plants were screened in the T2 generation by high resolution melting (HRM) point analysis for the presence of sequence polymorphisms at the target site. The CRISPR/Cas9 transgene was out segregated in the T3 generation and plants carrying the smg7-7 allele in a heterozygous

constitution were identified by HRM analysis and sequencing. Plants were characterized in the T4 and T5 generations. The complementation constructs for functional significance of the 14- 3-3 domain of SMG7 were as follows: 6.9 kb of SMG7 genomic sequence including 1.5 kb promoter and 30UTR was PCR amplified using primers SMG7::SMG7-SbfI and SMG7::SMG7- SacI (**Supplementary Table S1**) and cloned into pJET1.2/blunt Cloning Vector (Thermo Fisher Scientific). For site directed mutagenesis, a smaller fragment of SMG7 gene was amplified with the primers F-SMG7-SpeI and R-SMG7-HpaI and the PCR product was subcloned in the pJET1.2/blunt Cloning Vector. Site directed mutagenesis of K77E and R185E was done in two rounds of PCR, first with primers F-SMG7-K77E and R-SMG7-K77E, and second with primers F-SMG7-R185E and R-SMG7-R185E. The mutated fragment was subcloned back into SpeI/HpaI of the SMG7 gene construct. Wild type and mutated versions of the SMG7 gene were subcloned into the PstI/SacI sites of the binary vector pCBK06 (Riha et al., 2002) and resulting constructs were transformed into Agrobacterium tumefaciens GV3101. Complementation lines were generated by transforming Arabidopsis heterozygous for the smg7-1 allele by floral dip technique.

### Fertility Assays

Pollen viability was determined by Alexander staining (Alexander, 1969). Meiosis in pollen mother cells was analyzed by staining whole anthers with DAPI followed by confocal microscopy according to (Brownfield et al., 2015).

### Gene Expression Analysis

Total RNA was isolated from plant leaf tissue using RNA Blue (Top-Bio) following the manufacturer's protocol. Quality and quantity of isolated RNA were checked on a 1.2% denaturing agarose gel and with the NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific), respectively. For each sample, 10 µg of total RNA was treated with DNase I (Roche) to remove genomic DNA and 2 µg DNA-free RNA was reverse transcribed using Superscript IV (Invitrogen) following the manufacturer's protocol. For qRT-PCR reactions, cDNA was diluted two times and all reactions were carried out using EvaGreen dye (Biotium), GoTaq polymerase (Promega, United States), and gene-specific primers on a LightCycler 96 (Roche). Expression was normalized to At2G28390 and relative fold change was calculated using the delta-delta Ct method (Livak and Schmittgen, 2001). All qRT-PCR reactions were performed on at least three biological replicates and 2–3 technical replicates. The sequence of all primers used in this study are listed in **Supplementary Table S1**.

### Protein Alignment

We downloaded the full-length protein sequences of SMG7 and SMG7L from 13 plant species (Physcomitrella patens, Selaginella moellendorffii, Amborella trichopoda, Medicago truncatula, Arabidopsis thaliana, Brassica oleracea, Cucumis sativus, Nicotiana attenuata, Helianthus annuus, Oryza sativa, Zea mays, Sorghum bicolor, Hordeum vulgare) from Ensembl Plants (release 38) and of human (Homo sapiens) from Ensembl (release 91). The following sequences were used for alignment: HsSMG7 (ENSP00000425133), PpSMG7a (PP1S80\_14V6.1), PpSMG7b (PP1S311\_73V6.1), SmSMG7a (EFJ27061), SmSMG7b (EFJ21470), AmtSMG7 (ERN18017), MtSMG7a (KEH28378), MtSMG7b (KEH16467), AtSMG7 (AT5G19400.1), AtSMG7L (AT1G28260.1), BoSMG7a (Bo9g153800.1), BoSMG7b (Bo2g018020.1), BoSMG7La (Bo5g054690.1), BoSMG7Lb (Bo3g143280.1), CsSMG7 (KGN66550), CsSMG7L (KGN64688), NaSMG7 (OIS97991), NaSMG7L (OIT28005), HaSMG7a (OTG27135), HaSMG7b (OTG30173), HaSMG7L (OTF97490), OsSMG7 (Os08t0305300-01), ZmSMG7a (Zm00001d019920\_P002), ZmSMG7b (Zm00001d005502\_P002), SbSMG7 (KXG35214), HvSMG7a (HORVU5Hr1G050800.5), HvSMG7b (HORVU0Hr1G029520.1). Protein alignment was performed with the MegAlign Pro function of DNASTAR Navigator (v<IP_ADDRESS>) using the MUSCLE (Multiple Sequence Comparison by Log-Expectation) method. The multiple sequence alignment (MSA) for selected sequence features of the SMG7 protein across the plant kingdom was used to generate a sequence logo using the online version of WebLogo3 (Crooks et al., 2004). SMG7 specific regions were identified by visually inspecting multiple sequence alignment of plant SMG7/SMG7L proteins for regions that are conserved in SMG7 paralogs of monocots and dicots, but diverged in SMG7L.

# RESULTS

# Full Inactivation of Arabidopsis SMG7 Does Not Result in Embryonic Lethality

In our attempts to genetically complement the smg7-5 mutation with a functional SMG7 gene, we failed to recover plants homozygous for the smg7-5 allele. This led us to ask whether the embryonic lethality observed in the smg7-5 line (Riehs et al., 2008) is indeed linked to the T-DNA insertion in the SMG7 gene. To clarify this issue, we used CRISPR/Cas9 targeted mutagenesis to generate another mutation that disrupts the conserved 14-3- 3 domain. We targeted the Cas9 nuclease to the second exon of the SMG7 gene and identified an allele that contains a frame shift mutation 13 amino acids downstream of the ATG codon and hence likely represents a full loss of function allele that we call smg7-7 (**Figure 1A**). We were able to recover viable plants homozygous for the smg7-7 allele that phenotypically resembled the growth-retarded smg7-1 mutants (**Figure 1B**). These plants were sterile and did not produce any pollen (**Figure 1C**). As the infertility of smg7-1 was caused by arrest of meiotic progression in anaphase of the second meiotic division (Riehs et al., 2008), we performed cytogenetic analysis of pollen mother cells in anthers of smg7-7 mutants. This revealed meiocytes with irregularly distributed chromosomes, which is typical for this anaphase II arrest (**Figure 1D**). To test the effect of smg7-7 on NMD, we used an assay that quantifies two alternatively spliced variants of the same transcript, one of which contains a PTC (Gloggnitzer et al., 2014; **Figure 1E**). We observed comparable upregulation of two different PTC containing transcripts in smg7-1 and smg7- 7, suggesting that NMD is impaired to the similar extent in both alleles. Thus, smg7-1 and smg7-7 lead to the same effect on NMD

indicated. (B) Approximately 6-week-old wild type and mutant plants. (C) Anthers with viable pollen visualized by Alexander staining. (D) Developing pollen mother cells within an anther stained by DAPI. Tetrads are apparent in wild type, while late smg7 meiocytes contain randomly distributed chromatids. (E) Effect of smg7 alleles on the relative abundance of two alternatively spliced variants of the same transcript as determined by real time RT-PCR. Error bars represent SEM of three biological replicas. Asterisks indicate statistical significance of difference from wild type (∗P < 0.5, ∗∗P < 0.01, ∗∗∗P < 0.001, two-tailed t-test).

and meiosis, demonstrating that the smg7-1 allele used in our previous studies likely represents a full loss of function mutation and that inactivation of SMG7 is not lethal, in contrast to the conclusion previously reported (Riehs et al., 2008).

# Arabidopsis SMG7L Does Not Compensate for the Loss of SMG7

Our observation that complete loss of SMG7 does not cause embryonic lethality implies that the consequence of SMG7 inactivation is milder than that of UPF1. This led us to ask whether SMG7L can partially compensate for the loss of SMG7 in NMD. To address this question, we generated plants with mutations in both genes. We combined smg7l-1, which carries a T-DNA insertion in the N-terminal 14-3-3 like domain, with either the severe smg7-1, or the hypomorphic smg7-6 (**Figure 2A**). smg7-6 contains a T-DNA insertion in the C-terminus and was reported to be proficient for NMD but partially impaired in meiosis (Riehs-Kearnan et al., 2012). Quantitative RT-PCR analysis showed that mRNA encoding the

N-terminal portion of the SMG7 protein is expressed at the same level in smg7-6 as in wild type plants, suggesting the production of a truncated, partially functional protein (**Figure 2B**). Plants deficient for SMG7L did not show any obvious phenotype and were indistinguishable from wild type plants (**Figure 2C**).

We anticipated that if SMG7L acts redundantly with SMG7, it should exacerbate the phenotypes of both smg7 alleles. However, neither smg7l-1 smg7-1 nor smg7l-1 smg7-6 double mutants exhibited any difference from the respective smg7 single mutants with regards to growth defects and fertility (**Figures 2C**, **3**). While smg7-1 is infertile, smg7-6 plants produce a reduced amount of viable pollen and are partially fertile (**Figures 3A,B**). Usually, the first 15–20 flowers on the main inflorescence bolt are infertile, while later flowers produce viable seeds (**Figure 3C**). Quantification of pollen and seed production did not reveal any difference between smg7-6 and smg7l-1 smg7-6, arguing that SMG7L does not compensate for the meiotic function of SMG7. We assessed the efficiency of NMD by quantifying three different endogenous transcripts known to be degraded by NMD (**Figure 4**; Gloggnitzer et al., 2014). The smg7-1 null mutation displayed increased amounts of all transcripts targeted by NMD (**Figure 4**). Although we have previously reported that the hypomorphic smg7-6 allele is NMD proficient (Riehs-Kearnan et al., 2012), we observed a slight but reproducible increase of the three NMD-targeted transcripts in this study (**Figure 4**) suggesting a very mild NMD defect. Importantly, the smg7l-1 smg7-1 and smg7l-1 smg7-6 double mutants did not show any additional increase in NMD-regulated transcripts. Together, these data allow the conclusion that SMG7L does not act redundantly with SMG7 either in meiosis or in NMD.

### The N-Terminal 14-3-3 Domain of SMG7 Is Important for Meiosis

The genetic separation of NMD and meiotic functions in smg7-6 mutants together with the fact that UPF1 and UPF3 deficient plants lack a meiotic phenotype (Riehs-Kearnan et al., 2012) indicate that SMG7 acts in meiosis through a different

mechanism than in NMD. Thus, we wanted to know whether the conserved 14-3-3 domain that mediates interaction with phosphorylated UPF1 in NMD is also required for meiosis. We complemented the smg7-1 with a copy of the Arabidopsis SMG7 gene carrying mutations in the conserved K77 and R185 residues that form the phosphoserine-binding pocket and are required for NMD in tobacco (**Figure 5A**; Fukuhara et al., 2005; Merai et al., 2012). We transformed plants heterozygous for smg7-1 with constructs carrying either wild type SMG7 or the mutant SMG7K77E R185E and identified transformants homozygous for smg7-1 in segregating T2 populations. Three independent lines were analyzed for each construct. While the wild type construct readily complemented the growth phenotype, fertility, and pollen production, SMG7K77E R185E plants were

indistinguishable from smg7-1 mutants despite higher level of SMG7 mRNA expression (**Figures 5B,C**). As expected, the SMG7K77E R185E plants were deficient in NMD, but they also failed to produce any pollen (**Figures 5C,D**). This indicates that the phosphoserine-binding activity mediated by the conserved N-terminal 14-3-3 motif is important not only for NMD, but also for the meiotic function of SMG7.

## The Meiotic Function of SMG7 May Depend on Conserved Motifs in Its C-Terminus

Studies in grapevine using virus induced gene silencing (Benkovics et al., 2011) together with our phenotypic characterization of Arabidopsis mutants demonstrated that SMG7 and SMG7L paralogs functionally diverged in dicots. While SMG7 retained its function in NMD, SMG7L apparently acquired a novel function(s) unrelated to NMD and meiosis. We hypothesized that this neofunctionalization would be accompanied by amino acid sequence divergence in regions exclusively important for NMD. According to this hypothesis, SMG7 should have retained motifs involved in NMD whereas some of these motifs may have been lost in SMG7L.

We performed a sequence comparison of multiple plant SMG7 and SMG7L proteins to identify regions that are conserved across the plant kingdom but which diverged during the evolution of SMG7L in dicots. Five such regions were identified in the evolutionary conserved N-terminal half of SMG7, and three additional SMG7-specific motifs were found at the C-terminus (**Figure 6**). While all 6 amino acids residues that form the phosphoserine-binding pocket of the 14-3-3 domain are retained in both SMG7 and SMG7L, two regions in the 14-3-3 domain diverged between these paralogs. When mapped over the structure of human SMG7 (Fukuhara et al., 2005), the plant SMG7-specific regions 1 and 2 correspond to alpha helices α2 and α4 with the connecting loop to α5, respectively. The α2 and α4 helices are aligned on the convex surface of the 14-3-3 domain, and α4 together with the extended loop was reported to form an interaction interface with SMG5 (Jonas et al., 2013). Thus, it is likely that these regions form a protein interaction surface and their divergence in SMG7L reflects altered protein-binding specificity. The SMG7-specific region 3 is located in the loop that connects the 14-3-3 domain with the helical domain and is larger in SMG7 than in SMG7L paralogs (**Figure 6**). The SMG7 specific regions 4 and 5 span helices α16 and α18 at the end of the helical domain. The C-terminal portion of SMG7 is generally less conserved than the N-terminus. Nevertheless, we found three motifs at the C-terminus of SMG7 proteins (motifs 6, 7, and 8) that are shared among monocots, dicots, and moss, but were lost in dicot SMG7L (**Figure 6**).

# DISCUSSION

SMG7 is an ancient phosphoserine-binding protein that is present in most eukaryotes. Its primary function is linked to

NMD as its physical and functional interactions with UPF1 are conserved from plants to animals (Unterholzner and Izaurralde, 2004; Kerenyi et al., 2013). Nevertheless, proteins of the SMG7 family have acquired additional functions during evolution. The best known example is Est1p in budding yeast that recruits telomerase to chromosome ends via binding to phosphorylated Cdc13 (Li et al., 2009; Chen et al., 2018). Hence, SMG7 acts as an adaptor protein that can recruit different molecular machines to the sites of their action in a phosphorylation dependent manner. Arabidopsis SMG7 is involved in at least two independent molecular processes, NMD and meiotic progression. Here we show that its N-terminal 14-3-3 domain is required for both NMD and meiosis. While UPF1 is the substrate that defines the role of SMG7 in NMD (Kerenyi et al., 2013), the meiotic function is likely mediated by another binding protein as Arabidopsis deficient for UPF1 does not exhibit the meiotic defects described for smg7 mutants (Riehs-Kearnan et al., 2012).

Proteins of the SMG7 gene family underwent independent multiplications during evolution that were accompanied by functional diversification. In vertebrates SMG5, SMG6, and SMG7 paralogs play distinct roles in NMD that define two separate RNA degradation pathways. Multiple copies of SMG7 have also independently arisen in plants including maize, poplar, grapevine, or the moss P. patens (Benkovics et al., 2011; Li et al., 2015). In the majority of cases, these are relatively recent species-specific duplications that only occasionally span larger phylogenetic units. SMG7L represents the most ancient plant duplication, originating at the root of dicots (Riehs et al., 2008). Here we demonstrate that SMG7L does not act redundantly with SMG7 in either NMD or meiosis implying that it evolved a novel, yet unknown function. Domain swapping experiments indicated that the N-terminal portion of SMG7L retained its capability to bind UPF1 and complement NMD, whereas the C-terminus lost its capability to trigger RNA degradation in a tethering assay (Benkovics et al., 2011). Nevertheless, it is unlikely that UPF1 is a physiological substrate of SMG7L; sequence divergence in the loops and helices that form the surface interfaces of the conserved N-terminal domains rather suggest that SMG7L binds other proteins.

Phylogenetic comparison of SMG7 and SMG7L protein sequences revealed several regions that were conserved in SMG7, but diverged in SMG7L. Positions of the SMG7-specific motifs

in respect to mutations that have been functionally characterized (**Figure 6A**) may provide clues on their function. Arabidopsis smg7-1 and smg7-3 represent T-DNA insertions that truncate SMG7 at amino acid positions 355 and 675, and both alleles are deficient in NMD and meiosis (Riehs et al., 2008; Riehs-Kearnan et al., 2012). The smg7-6 disruption is only 43 amino acids downstream from smg7-3 at position 718 yet is only very mildly impaired in NMD, implying a functional significance of this region. Nevertheless, the region between amino acids 675 and 718 is highly divergent among plant SMG7 homologs (**Supplementary Figure S1**), which speaks against a specific role for this region in NMD. Since smg7-3 is disrupted shortly after a motif conserved in both SMG7 and SMG7L paralogs, this mutation may affect the proper folding or stability of the truncated protein. Experiments using a transient expression in tobacco showed that the Arabidopsis SMG7 C-terminal domain is required for NMD and that tethering a C-terminal fragment (amino acids 517–1059) to RNA is sufficient to trigger its degradation (Benkovics et al., 2011). Thus, the region responsible for RNA degradation must be located between amino acids 517 and 718, which overlaps with the SMG7 specific region 5 (**Figure 6**). The impaired fertility of smg7-6 indicates a requirement for the SMG7 C-terminus in meiosis. It is likely that the meiotic function of SMG7 is mediated additionally through conserved regions 6, 7, and 8. In support of this notion, Arabidopsis smg7-4 which carry a T-DNA disruption at amino acid position 1013 immediately after the SMG7-specific region 8 are fully fertile (Riehs-Kearnan et al., 2012).

Whether the conserved function of these motifs is specifically linked to reproduction, or whether they underlie a general molecular function, for which deficiency in Arabidopsis is manifested as a meiotic defect, remains to be seen.

# AUTHOR CONTRIBUTIONS

CC, NS, KR, and OMS designed the experiments. CC performed the experiments shown in **Figures 1–5**. NS performed the

### REFERENCES

experiments shown in **Figures 1**, **6**. AW generated the smg7-7 mutant plants. KR and NS wrote the manuscript.

## FUNDING

The authors are grateful for financial support by the Austrian Science Fund (FWF W 1238, DK "Chromosome Dynamics" to KR and OMS), by the Ministry of Education, Youth and Sports of the Czech Republic, European Regional Development Fund-Project "REMAP" (No. CZ.02.1.01/0.0/0.0/15\_003/0000479 to KR), and by the Czech Science Foundation (16-18578S to KR).

# ACKNOWLEDGMENTS

We acknowledge the Plant Science core facility and the core facility CELLIM of CEITEC supported by the Czech-BioImaging large RI project (LM2015062 funded by MEYS CR) for their support with obtaining scientific data presented in this paper.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01602/ full#supplementary-material

FIGURE S1 | Sequence alignment of the following SMG7 proteins: HsSMG7 (ENSP00000425133), PpSMG7a (PP1S80\_14V6.1), PpSMG7b (PP1S311\_73V6.1), SmSMG7a (EFJ27061), SmSMG7b (EFJ21470), AmtSMG7 (ERN18017), MtSMG7a (KEH28378), MtSMG7b (KEH16467), AtSMG7 (AT5G19400.1), AtSMG7L (AT1G28260.1), BoSMG7a (Bo9g153800.1), BoSMG7b (Bo2g018020.1), BoSMG7La (Bo5g054690.1), BoSMG7Lb (Bo3g143280.1), CsSMG7 (KGN66550), CsSMG7L (KGN64688), NaSMG7 (OIS97991), NaSMG7L (OIT28005), HaSMG7a (OTG27135), HaSMG7b (OTG30173), HaSMG7L (OTF97490), OsSMG7 (Os08t0305300-01), ZmSMG7a (Zm00001d019920\_P002), ZmSMG7b (Zm00001d005502\_P002), SbSMG7 (KXG35214), HvSMG7a (HORVU5Hr1G050800.5), HvSMG7b (HORVU0Hr1G029520.1).

TABLE S1 | Primers used in this study.

throughout eukaryotic evolution. Sci. Rep. 7:16692. doi: 10.1038/s41598-0 17-16942-w

**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 © 2018 Capitao, Shukla, Wandrolova, Mittelsten Scheid and Riha. 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) and the copyright owner(s) 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.

# Under a New Light: Regulation of Light-Dependent Pathways by Non-coding RNAs

### Camila Sánchez-Retuerta1 , Paula Suaréz-López1 and Rossana Henriques1,2,3 \*

1 Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB-UB, Barcelona, Spain, 2 School of Biological, Earth and Environmental Sciences, University College Cork, Cork, Ireland, 3 Environmental Research Institute, University College Cork, Cork, Ireland

The biological relevance of non-protein coding RNAs in the regulation of critical plant processes has been firmly established in recent years. This has been mostly achieved with the discovery and functional characterization of small non-coding RNAs, such as small interfering RNAs and microRNAs (miRNAs). However, recent next-generation sequencing techniques have widened our view of the non-coding RNA world, which now includes long non-coding RNAs (lncRNAs). Small and lncRNAs seem to diverge in their biogenesis and mode of action, but growing evidence highlights their relevance in developmental processes and in responses to particular environmental conditions. Light can affect MIRNA gene transcription, miRNA biogenesis, and RNA-induced silencing complex (RISC) activity, thus controlling not only miRNA accumulation but also their biological function. In addition, miRNAs can mediate several light-regulated processes. In the lncRNA world, few reports are available, but they already indicate a role in the regulation of photomorphogenesis, cotyledon greening, and photoperiod-regulated flowering. In this review, we will discuss how light controls MIRNA gene expression and the accumulation of their mature forms, with a particular emphasis on those miRNAs that respond to different light qualities and are conserved among species. We will also address the role of small non-coding RNAs, particularly miRNAs, and lncRNAs in the regulation of light-dependent pathways. We will mainly focus on the recent progress done in understanding the interconnection between these non-coding RNAs and photomorphogenesis, circadian clock function, and photoperiod-dependent flowering.

Keywords: microRNAs, long non-coding RNAs, light, circadian clock, photoperiod

# INTRODUCTION

Light is fundamental for plant life. It not only provides the energy necessary for photosynthesis but also functions as an indicator of time and place. Light quality and duration inform a plant about its neighbors in a process described as shade avoidance, which ultimately shapes its architecture and development. Light cues also function as indicators of time of the day and season [e.g., short days (SDs) in autumn and winter versus long days (LDs) in spring and summer]. Last but not least, light, together with temperature, is an input to the circadian clock, providing circadian entrainment and

### Edited by:

Dora Szakonyi, Instituto Gulbenkian de Ciência (IGC), Portugal

### Reviewed by:

Santiago Mora-Garcia, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina Umesh K. Reddy, West Virginia State University, United States

> \*Correspondence: Rossana Henriques<EMAIL_ADDRESS>
### Specialty section:

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

Received: 23 March 2018 Accepted: 14 June 2018 Published: 26 July 2018

### Citation:

Sánchez-Retuerta C, Suaréz-López P and Henriques R (2018) Under a New Light: Regulation of Light-Dependent Pathways by Non-coding RNAs. Front. Plant Sci. 9:962. doi: 10.3389/fpls.2018.00962

**63**

contributing to maintain rhythmicity and robustness of the clock. Proper clock resetting is critical for the adequate timing of biological processes, which will then reflect in plant fitness.

In parallel with seasonal and positional information, light also provides important developmental cues. For instance, during photomorphogenesis, when the young seedling emerges through the soil reaching for light, hypocotyl elongation is inhibited, while roots grow, cotyledons open and expand, and the first true leaves develop (Leivar and Monte, 2014). These morphological changes are accompanied by the development of chloroplasts and chlorophyll accumulation. Later in development, light will also regulate the time of flowering. Depending on each species requirements, specific photoperiods (the relative duration of the daily light and dark periods) will provide molecular cues, such as the accumulation of specific regulators, required for the transition of shoot apical meristems into inflorescence meristems (Andrés and Coupland, 2012).

Therefore, considering all light-dependent and/or lightregulated processes, it is not surprising that light signals promote massive transcriptional changes affecting both protein coding genes as well as non-protein coding transcripts (López-Juez et al., 2008; Wang H. et al., 2014). Although light regulation of (proteincoding) gene expression has been studied in extreme detail, the role of light in the regulation of non-protein coding RNAs is less known.

Non-coding RNAs have been divided into different functional groups based on the size of their active forms. Non-coding RNAs shorter than 200 base pairs (bp) fall within the category of small non-coding RNAs, whereas longer ones constitute long non-coding RNAs (lncRNAs). Historically, small noncoding RNAs were the first identified and characterized in great detail. Within this group, microRNAs (miRNAs) are transcribed from MIRNA genes as longer precursors (primary miRNAs, pri-miRNAs) by RNA polymerase II. Plant pri-miRNAs fold into stem-loop structures and are processed by a complex containing DICER-LIKE1 (DCL1), HYPONASTIC LEAVES1 (HYL1), and SERRATE (SE), which will first release the stemloop (pre-miRNA) structure, and then cleave it giving rise to a mature miRNA:miRNA∗ duplex, normally 20–22 nucleotides (nt) long, with the rarer cases of 23–25 nt, with 2-nt 30 overhangs (Voinnet, 2009; Borges and Martienssen, 2015; Yu et al., 2017). This duplex is methylated at its 30 ends by the methyltransferase HUA ENHANCER1 (HEN1) and this methylation is necessary to stabilize miRNAs (Yu et al., 2005). The mature miRNA strand is then selectively bound by ARGONAUTE1 (AGO1) and loaded into the RNA-induced silencing complex (RISC), which recognizes transcripts with full or partial complementarity to the miRNA and cleaves and/or represses translation of these miRNA target transcripts, therefore silencing them (Voinnet, 2009; Yu et al., 2017) (**Figure 1**). miRNAs act in particular developmental processes and/or in response to certain environmental cues, sometimes in specific tissues where they accumulate at lower levels than their targets, protein-coding transcripts.

lncRNAs, on the other hand, can exert their biological function without further processing and accumulate to similar levels as miRNAs (Liu et al., 2012). lncRNAs are classified according to their genomic location into intergenic (expressed in between coding regions), intronic (expressed from introns), or natural antisense transcripts (NATs, expressed from the complementary strand to a protein-coding gene or another lncRNA; Liu et al., 2012; Wang H. et al., 2014). Comprehensive screening approaches have revealed thousands of lncRNAs that display spatial-, temporal-, and developmental-specific patterns of expression. Interestingly, among the few lncRNAs whose biological function is known, two are involved in lightregulated processes. HIDDEN TREASURE 1 (HID1) is involved in photomorphogenesis and seedling greening (Wang Y. et al., 2014; Wang et al., 2018), whereas CDF5 LONG NON-CODING RNA (FLORE) mediates the circadian regulation of flowering time (Henriques et al., 2017).

Here, we review the role of miRNAs and lncRNAs associated with, or regulated by light-dependent pathways. We will discuss light regulation of miRNA expression, biogenesis, processing, and function, as well as their role in light-related processes, such as photomorphogenesis and photoperiod-dependent flowering. We will also discuss the available reports characterizing lncRNA function in light-dependent pathways.

# MicroRNAs

Light regulation of plant miRNAs can occur through at least three different mechanisms: regulation of MIRNA gene transcription and its levels, regulation of miRNA processing via effects on components of the miRNA biogenesis machinery, and regulation of miRNA function through control of RISC factors. In turn, miRNAs can target genes encoding components of the light signaling pathways, thereby affecting light responses such as photomorphogenesis and photoperiod-dependent flowering. Below we discuss the recent progresses made in these fields of research. Due to the interconnection between light and the circadian clock, we will also discuss the available reports on circadian-regulated ncRNAs.

### Light Regulation of miRNA Levels

Light, photoperiod, and the circadian clock modulate the expression levels of MIRNA genes. This regulation is achieved by the presence of light-responsive elements in their promoters, and can be triggered by specific light qualities (red, blue, far-red, UV-A, and UV-B). To identify the miRNA families under this control, several screening approaches have been developed, resulting in extensive listings of light-responsive miRNAs.

For instance, the comparison of miRNAs between wild-type rice (Oryza sativa) plants and a phytochrome B (phyB) mutant identified 135 differentially expressed miRNAs, of which 97 were upregulated and 38 were downregulated. These miRNAs include conserved and novel miRNAs, with sizes varying between 21-nt and 24-nt, such as miR156, miR166, miR171, and miR408 (Sun et al., 2015). Differently from a previous report showing that miR172 is regulated by PHYB in potato (Martin et al., 2009), Sun et al. (2015) did not find any miR172 family member that responded to PHYB in rice, suggesting that PHYB-mediated regulation of miRNAs may differ between plant species. Using degradome sequencing, it was found that 70 rice genes were

targeted by 32 differentially expressed miRNAs between the wildtype and the phyB mutant (Sun et al., 2015). A large proportion of them (42%) encode transcription factors, suggesting that regulation of gene expression by miRNA target genes may play an important role in PHYB-mediated light signaling. Members of the transcription factor families identified by Sun et al. (2015) are involved in light signaling in Arabidopsis. It is worth noting that one of the identified miRNA target genes is a homolog of the Arabidopsis circadian-regulated TANDEM ZINC KNUCKLE PROTEIN (TZP) gene, which is involved in blue lightassociated growth (Loudet et al., 2008). However, further research is required to fully understand the role of these rice miRNAs and target genes in light-regulated processes (Sun et al., 2015).

complementarity to the miRNA and represses the translation (8) or cleaves (9) these miRNA targets.

Moreover, ELONGATED HYPOCOTYL 5 (HY5), a major regulator of photomorphogenesis that acts downstream of several photoreceptors, was shown to bind to the promoters and regulate the expression of several MIRNA genes, such as MIR156D, MIR402, MIR408, and MIR858A in Arabidopsis (Zhang et al., 2011). This, together with the effect of PHYB on miRNA levels (Sun et al., 2015), strongly suggests that light affects miRNA abundance. In support of this, small RNA sequencing indicates that the miRNA population of the seedling apical hook changes after treating dark-grown soybean (Glycine max) seedlings with far-red light. The levels of several miRNAs (miR166, miR167, miR168, miR394, miR396, miR530, miR1507, miR1508, miR1509, and miR2218) respond to far-red light in different parts of the soybean seedling, in most cases showing upregulation by far-red light (Li et al., 2014). Therefore, light seems to upregulate miRNAs in the apical hook, leading to downregulation of target genes that presumably repress apical hook and cotyledon opening.

Light has strong effects on plant metabolism, especially in the regulation of fundamental plant processes such as photosynthesis (Kooke and Keurentjes, 2012). To understand the involvement of miRNAs in light regulation of potato metabolism, Qiao et al. (2017) used high-throughput sequencing to study the expression levels of miRNAs and mRNAs in dark-treated and red-lighttreated leaves and detached tuber skin in potato. Among the 69 known and novel miRNAs that were differentially regulated, most were upregulated in leaves, whereas in tuber skin, about half of them were upregulated and half downregulated (Qiao et al., 2017). Notably, the miR399 family and the novel miRNA miRn55 showed opposite light responses in leaves (upregulation) and tuber skin (downregulation). General gene expression analyses revealed that most differentially expressed mRNAs (67%) were upregulated in tuber skin, whereas in leaves, about half were upregulated and half downregulated. Only 14% of differentially expressed miRNAs and 12% of differentially expressed genes were common to leaves and tuber skin, indicating that the molecular response to red light differs between these tissues. Nevertheless, there was an effect of light on genes involved in primary and secondary metabolism in both tissues, especially in carbohydrate metabolism, which is downregulated, and alkaloid metabolism, which is upregulated. Repression of carbohydrate

metabolism was supported by anti-correlation between miRNAs and mRNAs, which suggests downregulation of putative miRNA targets involved in this process (Qiao et al., 2017). Although confirmation of these findings using additional methods is required, this work points to a role of miRNAs in the regulation of metabolic pathways affected by red light in potato.

Additional support to the effect of red light on miRNA levels has been obtained in Arabidopsis. In this species, the most differentially expressed miRNAs in red light versus darkness are miR160, miR163, miR319, miR394, miR779, miR851, miR854, and miR2111 (Sun et al., 2018). At least part of this effect is mediated by PHYTOCHROME-INTERACTING FACTOR4 (PIF4), a transcription factor that is a negative regulator of PHYB-mediated red light signaling. In a pif4 mutant, 22 mature miRNAs, representing 16 miRNA families, show altered levels relative to wild-type plants under red light. PIF4 promotes the expression of genes encoding miR156/157, miR160, miR165/166, miR167, miR170/171, and miR394 and represses the expression of genes encoding miR172 and miR319 by binding to the promoters of several of these genes. In addition to acting as a transcription factor for MIRNA genes, PIF4 regulates miRNA processing, as discussed below (Sun et al., 2018).

Besides light exposure, photoperiod can also control miRNA accumulation. Li et al. (2015) used high-throughput sequencing to identify miRNAs regulated by photoperiod in soybean. Comparing miRNA levels in seedlings grown under LDs and SDs revealed 37 miRNA families to be day-length-responsive. Five members of the miR156 family were induced under LD conditions and this correlated with downregulation of four miR156-target SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) genes. Given that miR156 overexpression delays flowering in soybean (Cao et al., 2015), lower miR156 levels under SD than LD may explain the fact that soybean flowering is induced by SD. Conversely, the expression of six members of the miR172 family was induced under SD conditions and this was coordinated with the repression of ten predicted targets belonging to the APETALA2 (AP2)-like family (Li et al., 2015). In addition to miR156 and miR172, other conserved miRNAs, such as miR159/319, miR166, miR167, miR395, and miR408 were also shown to be photoperiod regulated in soybean.

The simplified model of the circadian clock places light as an input that confers clock entrainment. Similarly to light, the clock also controls the expression of a vast set of genes, especially at the dark/light transition (Michael et al., 2008), thus allowing plants to anticipate certain daily processes. Interestingly, the circadian clock can also regulate the expression of proteincoding as well as non-coding transcripts (Hazen et al., 2009). To uncover circadian-regulated miRNAs, Siré et al. (2009) compared mature and pri-miRNA transcript levels under light/dark cycles or under free-running (continuous light) conditions for two consecutive days. It was found that miR167, miR168, miR171, and miR398 levels oscillated under light/dark cycles, peaking around the light/dark transition. However, for certain miRNAs, the waving pattern was not accompanied by a clear antiphasic expression of their targets but rather by a phase delay. This was the case of miR171/SCARECROW-LIKE 6 (SCL6, also named SCL6-IV, HAM3, and LOM3) and miR398/Cu/Zn SUPEROXIDE DISMUTASE 2 (CSD2) pairs, where target gene expression is reduced as the miRNA levels increase. However, in the case of the miR168/AGO1 and miR167/AUXIN RESPONSE FACTOR 6 (ARF6) pairs, an in-phase pattern of accumulation was found, suggesting that miRNAs could regulate their targets by incorporating feedback loops. However, when these modules were tested under free-running conditions, a clear oscillation was not found, an indication that these miRNAs are most likely light regulated.

In another report, Li et al. (2016) addressed the mechanism by which the circadian clock regulates carbon and nitrogen metabolism in rice plants grown in field conditions. Using a comprehensive approach, the authors investigated how light/dark cycles control certain biological processes. They investigated metabolic and enzymatic activities, as well as gene expression and alternative splicing events that could show rhythmic behavior. In their search for oscillatory miRNAs associated with carbon and/or nitrogen metabolism, Li et al. (2016) uncovered miR1440b, miR1877, miR2876-5p, and miR5799 as possible rice miRNA candidates. These miRNAs oscillate under light/dark cycles and negatively correlate with their targets at the majority of time points analyzed. However, it still remains to be seen whether these are bona fide circadian miRNAs oscillating under free-running conditions.

### UV-Responsive miRNAs

Since different light qualities can trigger diverse developmental responses, some of which can be detrimental, several screening approaches have been designed to understand how light, especially high light, UV-A, and UV-B radiation can modulate accumulation of miRNAs and their targets. Zhou et al. (2016), using seedlings from Brassica rapa exposed to blue light and UV-A, identified 15 conserved and 226 novel differentially expressed miRNAs that could target genes encoding regulators of plant growth, development, and photomorphogenesis (Zhou et al., 2016). Blue light and UV-A exposure slightly downregulated miR156/157 expression, which correlated with upregulation of their targets, SPL9 (Bra004674) and SPL15 (Bra003305), which encode transcription factors involved in the regulation of many processes, including the juvenile-to-adult transition, flowering, and secondary metabolism, especially anthocyanin biosynthesis (Wu et al., 2009; Gou et al., 2011; Wang and Wang, 2015). In Arabidopsis, miR156 and SPLs are involved in complex feedback loops (Wu et al., 2009; Yant et al., 2010). As plants age, miR156 levels decrease and SPLs (e.g., SPL9) accumulate, inhibiting anthocyanin biosynthesis (Gou et al., 2011). Zhou et al. (2016) hypothesize that blue and UV-A light activate genes involved in light signaling and some of them promote anthocyanin biosynthesis. When anthocyanin levels increase, then the regulatory feedback loops involving miR156/157 would act to balance its metabolism.

Similar to UV-A, UV-B radiation affects plant growth, physiology, and metabolism (Dotto and Casati, 2017). In one of the first reports on the effect of light on miRNAs, Zhou et al. (2007) used a computational approach to predict MIRNA genes induced by UV-B light in Arabidopsis. miRNAs belonging to 11

miRNA families were identified as putatively upregulated by UV-B light (Zhou et al., 2007). Although these results have not been experimentally confirmed in Arabidopsis, several reports have shown the effect of UV-B on miRNA levels in aspen (Populus tremula), maize (Zea mays), and wheat (Triticum aestivum). Jia et al. (2009) found that the level of eight miRNA families increased and that of seven families decreased in response to UV-B light stress in aspen. In maize, from the 16 UV-Bregulated miRNA families detected, 6 were upregulated and 10 downregulated (Casati, 2013). UV-B-treated wheat plants showed upregulation of three and downregulation of another three known miRNAs, in addition to a novel wheat miRNA, which was slightly upregulated shortly after UV-B exposure and then was downregulated (Wang et al., 2013). Thirteen UV-B-responsive miRNA families, i.e., miR156/157, miR159, miR160, miR164, miR165/166, miR167, miR169, miR170/171, miR172, miR393, miR395 miR398, and miR399, are common to at least two plant species, indicating substantial conservation in the miRNAs that respond to UV-B light. Although the direction of the effect (upregulation or downregulation) on several miRNAs differs between species, others are consistently promoted (miR165/166, miR167, and miR398) or repressed (miR395) by UV-B light in three species. This conservation probably reflects important roles of these miRNAs in plant responses to UV-B radiation.

The upstream regulatory regions of the UV-B-regulated MIRNA genes contain numerous light-related motifs, such as GT-1 sites, G boxes, I-boxes, CCAAT-boxes, and GATA-boxes (Zhou et al., 2007; Jia et al., 2009; Wang et al., 2013), consistent with the effect of light on these genes. Stress-responsive ciselements were also found in several of those promoters (Jia et al., 2009; Wang et al., 2013). Given that some of these miRNAs are involved in responses to abiotic and biotic stresses (Casati, 2013; Li et al., 2017), we hypothesize that these miRNAs might coordinate adaptive responses to diverse threats.

Among the targets of the UV-B-regulated miRNAs are transcription factors, several factors involved in auxin signaling and factors involved in responses to other stresses (Zhou et al., 2007; Jia et al., 2009; Casati, 2013). In most cases, as would be expected, there is an inverse correlation between the effect of UV-B on miRNAs and on their corresponding target transcripts (Zhou et al., 2007; Jia et al., 2009; Casati, 2013; Wang et al., 2013). For instance, in maize, an increase in miR165/166 correlated with inhibition of their target gene, encoding a HD-ZIP III transcription factor, ROLLED LEAF1 (RLD1; Casati, 2013). In agreement with the role of these miRNAs in delimitating HD-ZIP III transcripts to the adaxial side of leaf primordia (Juarez et al., 2004), UV-B-dependent repression of RLD1 was associated with leaf arching, suggesting a role for miR165/166 in this UV-B response (Casati et al., 2006). In aspen and wheat, miR395 downregulation is associated with upregulation of its targets encoding ATP sulfurylases (APS), enzymes involved in inorganic sulfate assimilation (Jia et al., 2009; Wang et al., 2013). miR395 and APS are inversely regulated by sulfate starvation, and miR395 overexpression reduces APS transcript levels and affects the response of Arabidopsis to abiotic stress conditions (Jones-Rhoades and Bartel, 2004; Kim et al., 2010), linking again a UV-B-regulated miRNA with other stress responses.

This is also the case of miR164, whose downregulation in UV-B-treated maize leaves is associated with increased levels of two stress-responsive NAC-DOMAIN PROTEIN target transcripts, as well as EXOSTOSIN PROTEIN-LIKE and an ASPARTYL PROTEASE (Hegedus et al., 2003; Casati, 2013). Interestingly, other miRNAs (miR171, miR172, miR156, and miR529) that target genes involved in critical developmental transitions (juvenile-to-adult transition and/or flowering) were also downregulated by UV-B light (Lauter et al., 2005; Xie et al., 2006; Chuck et al., 2008; Cuperus et al., 2011). This suggests that UV-B exposure promotes miRNA-mediated responses that delay certain developmental transitions, allowing plants either to repair UV-B-induced damage or to adapt to these stressful conditions.

Overall, the analysis of UV-B-regulated miRNAs in several plant species indicates that there is a core set of conserved UV-Bresponsive miRNAs. Nevertheless, the findings also suggest that different species may recruit distinct miRNAs in order to cope with UV-B stress. Understanding the role of these miRNAs in UV-B-regulated processes awaits further investigation.

### miRNAs Accumulated in High Light Conditions

Besides UV light, high light can also be detrimental for the development of different plant species. To address how light, especially high light, controls miRNA expression in ma bamboo (Dendrocalamus latiflorus) grown under LD conditions, Zhao et al. (2013) generated small RNA libraries and determined how miRNA families were regulated. Several conserved miRNAs were identified, miR168 being the most abundant family, followed by miR156/157, miR535, miR165/166, and miR167. Interestingly, miR172 was not found, probably due to the unique flowering cycle of ma bamboo. Using stem-loop RT-qPCR, Zhao et al. (2013) determined the level of novel miRNAs in plants grown under regular white light, high light, or dark. However, the role of these miRNAs in high-light-mediated processes still remains to be determined.

A global analysis of the light-regulated miRNAs reported so far reveals that numerous miRNA families are affected by diverse light conditions (**Table 1**). Some of them, such as miR156/157, miR159/319, miR164, miR165/miR166, miR167, miR170/171, miR172, miR395, miR398, miR399, and miR408, have been identified as responsive to several light conditions in at least five plant species, strongly suggesting that they may play roles in light responses (**Table 1**). However, light could also modulate miRNA processing and/or activity toward its targets. Below we present the most recent reports addressing this type of regulation.

## Regulation of miRNA Biogenesis, Processing, and Function by Light

Besides regulating MIRNA gene expression, light can also modulate the levels and the activity of mature miRNAs. This can be achieved by a direct connection between light and regulators of the miRNA biogenesis pathway. HYL1 is a RNA-binding protein involved in miRNA processing (Yu et al., 2017). In Arabidopsis, HYL1 protein levels are regulated by CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), an E3 ubiquitin ligase that mediates the proteasomal degradation of light signaling factors, such as photoreceptors

### TABLE 1 | miRNA families differentially regulated by light.

(Continued)

### TABLE 1 | Continued

In this table, we present a summary of the different miRNAs that respond to two or more light treatments. 1miRNA families reported to be regulated by light in at least two papers have been included. miRNA families are ordered according to the number of papers reporting their light regulation. 2W, white light; FR, far-red light; R, red light; B, blue light; L/D, light/dark; LD/SD, long days/short days. 3Ath, Arabidopsis thaliana; Gma, Glycine max (soybean); Mdo, Malus x domestica (apple); Osa, Oryza sativa (rice); Ppy, Pyrus pyrifolia (Chinese sand pear); Ptr, Populus tremula (aspen); Stu, Solanum tuberosum (potato); Tae, Triticum aestivum (wheat); Zma, Zea mays (maize).

(Cho et al., 2014). It was found that the reduction of miRNA levels in cop1 mutants relative to wild-type plants greatly overlapped with that observed in the hyl1 mutant. Further analyses showed that COP1 regulates HYL1 protein levels but not through direct ubiquitination. Combining protein synthesis blockers with either autophagy or 26S proteasome inhibitors revealed that HYL1 was degraded by an unknown protease, which removed its N-terminal fragment, where two RNAbinding domains, essential for miRNA processing, are located (Cho et al., 2014). Light regulates COP1 nucleocytoplasmic shuttling (Jang et al., 2010) and, therefore, during the day, light stabilizes HYL1 due to cytoplasmic accumulation of COP1, which would target the protease acting on HYL1 degradation (**Figure 2**). This regulation could control miRNA processing and thus help understand the light/dark accumulation pattern of several mature miRNAs, such as miR167, miR168, miR171, and miR398, previously described by Siré et al. (2009).

HYPONASTIC LEAVES1 protein levels are also controlled by PIF4, another protein involved in photomorphogenesis (Sun et al., 2018). Both HYL1 and DCL1 interact with PIF4, which destabilizes them under red light and stabilizes them in the dark. The mechanism by which PIF4 controls the levels of HYL1 and DCL1 is unknown, but it does not involve transcriptional regulation or the ubiquitin-proteasome pathway. Given that PIF4 interacts with the photoreceptor PHYB and the miRNA processing factors DCL1 and HYL1, the question arises as to whether DCL1 and HYL1 affect light responses (**Figure 2**). Indeed, dcl1 and hyl1 mutants show shorter hypocotyls than wildtype plants under red light, indicating that DCL1 and HYL1 play negative roles in photomorphogenesis (Sun et al., 2018).

Another miRNA processing factor regulated by light signaling pathways is HEN1. Light induction of HEN1 expression results from the coordinated action of HY5 and HY5 homolog (HYH; **Figure 2**), which positively regulate HEN1 downstream of several photoreceptors (Tsai et al., 2014). Consistent with this regulation, HEN1 and HYL1 are negative regulators of photomorphogenesis, in the case of HEN1, this is due to its role as a repressor of key transcription factors of this process (Tsai et al., 2014). In addition, alternative splicing of transcripts encoding several miRNA biogenesis and processing factors, such as DCL1, HYL1, and HEN1, is affected by different light qualities, adding a new layer to the regulation of miRNAs by light (reviewed in Hernando et al., 2017).

The de-etiolation process can also affect miRNA activity toward its targets. Lin et al. (2017) matched small RNA profiling with degradome sequencing in Arabidopsis and found that, although light exposure did not change dramatically the level of most miRNAs, it could modulate their target cleavage activity. In fact, with the exception of miR163, most miRNAs showed little fluctuations in their amount upon light transition. This did not correspond with the analysis of degradome signatures, implying that de-etiolation especially promoted the degradation of miR156/157 and miR396 targets, and thus suggesting higher cleavage activity for these families (Lin et al., 2017). However, although Lin et al. (2017) report that miR168 regulates AGO1

levels under light and could modulate RISC activity and the efficiency of miRNA target cleavage, the mechanisms underlying this regulation by light still need to be demonstrated.

Interestingly, another report has shown AGO1 involvement in light regulation of adventitious rooting and hypocotyl elongation. Arabidopsis ago1 mutants are hypersensitive to light, probably due to deregulation of the PHYA-dependent light signal transduction pathway (Sorin et al., 2005). Moreover, deetiolation of an Arabidopsis hypomorphic ago1-27 mutant in farred light is impaired, indicating that normal photomorphogenesis requires AGO1 (Li et al., 2014). Given that AGO1 is an essential component of the RISC, these findings suggest that miRNA function is involved in far-red light responses.

Therefore, several components of the miRNA machinery are involved in certain light responses. On the other hand, light modulates MIRNA gene expression, accumulation of mature miRNAs by regulating components of the biogenesis pathway, as well as miRNA activity. How miRNAs participate in light as well as photoperiod signaling events will be discussed next.

# miRNAs Involved in Light and Photoperiod Signaling

Our analysis revealed several miRNA families that accumulate upon certain light treatments in different species. Most of these miRNAs mediate critical biological processes necessary for plants to adjust to these changes. They can regulate metabolism, plant development, and certain responses to stress.

### miR396 and UV-B Regulation of Cell Proliferation

For instance, UV-B radiation affects cell proliferation in several species (Dotto and Casati, 2017). In Arabidopsis, UV-B light upregulates miR396 levels, leading to repression of the miR396 target genes GROWTH REGULATING FACTOR1 (GRF1), GRF2, and GRF3, with consequent inhibition of cell proliferation in leaves (Casadevall et al., 2013). In fact, either reducing miR396 activity or expressing miR396-resistant forms of GRF3 or GRF2 results in reduced sensitivity of leaf growth to UV-B irradiation, confirming that GRFs mediate this phenotype. Nevertheless, downregulation of GRF3 by UV-B light also seems to occur through miR396-independent mechanisms. In addition, miR396-mediated repression of leaf cell proliferation by UV-B radiation is dependent on the mitogen-activated protein kinase MPK3, known to be involved in the response to UV-B stress (Casadevall et al., 2013). Interestingly, in UV-B-treated maize leaves, miR396 inhibition had the opposite effect on GRFs, which were upregulated (Casati, 2013). This could be due to the different developmental stages analyzed in Arabidopsis and maize, although further studies are necessary to clarify the role of

miR396 in the regulation of UV-B-dependent responses in dicots and monocots.

### miRNAs Regulating Metabolism

As mentioned before, light controls many metabolic pathways in plants. Within this context, light-regulated miRNAs were shown to modulate metabolic events, such as methylation of specific signaling molecules (e.g., hormones), nutrient allocation, and pigment synthesis/accumulation.

miR163 as well as pri-miR163 were recently shown to be highly induced by light in Arabidopsis (Chung et al., 2016; Lin et al., 2017). Moreover, miR163 may target PXMT1 (At1g66700), a neighbor gene encoding a putative 1,7 paraxanthine methyltransferase that has been implicated in the methylation of natural chemicals such as hormones (Chung et al., 2016). Whereas pri-miR163 is induced in red, blue, and white light, and PXMT1 expression is inhibited upon light exposure, light does not affect pri-miR163 maturation and processing. Both pri-miR163 and its target PXMT1 accumulate in roots, where miR163 accumulation increases root length, especially in the elongation/differentiation zone, most likely by inhibiting PXMT1. Interestingly, the miR163/PXMT1 pair also seems to regulate germination, probably by controlling the early stages of plant development since radicle emergence to seedling de-etiolation. However, to fully understand the role of miR163 in this process, a more extensive functional characterization of PXMT1 would be necessary.

Plant metabolism requires proper nutrient allocation, especially in the case of nutrients that constitute co-factors and are present in different proteins and biological processes. This is the case of copper, since it is required for electron transport in photosynthesis, defense against reactive-oxygen species as well as ethylene perception (Zhang et al., 2014). Maintenance of copper homeostasis is critical to photosynthesis efficiency and thus plant growth. This is achieved by the SPL7 transcription factor, which functions as a copper sensor able to adjust target gene expression upon changing copper levels. Surprisingly, light and copper seem to regulate gene expression of many shared biological processes, suggesting a direct connection between the transcriptional regulators SPL7 and HY5 (Zhang et al., 2014). Using chromatin immunoprecipitation and RNA sequencing, Zhang et al. (2014) characterized the SPL7 regulon and found it to overlap with HY5. This analysis revealed MIR408 as a SPL7/HY5 common target, with SPL7 being the predominant transcription factor determining MIR408 levels, thus confirming previous work showing that HY5 regulates MIR408 expression (Zhang et al., 2011). Nevertheless, HY5/SPL7 coordinated regulation allowed miR408 accumulation upon copper deficiency and high light. Confirming this regulation, MIR408 silencing phenotypes were similar to those of the hy5 spl7 double mutant, whereas miR408 constitutive expression partially rescued them, suggesting that miR408 is a critical component of the SPL7/HY5 network (Zhang et al., 2014). Further analysis of the expression levels of the miR408 targets, LACCASE12 (LAC12) and LAC13, showed that the SPL7/HY5/miR408 module allows differential regulation of very closely related genes. Therefore, this signaling network constitutes a molecular mechanism that integrates light and copper signals to maintain copper homeostasis and consequently efficient photosynthetic rates that promote plant growth.

### **miRNAs and pigment synthesis**

Flavonoids are a group of phenolic compounds that include flavonols, flavones, isoflavones, and anthocyanins (Sharma et al., 2016). Due to their molecular diversity, flavonoids can confer protection against biotic and abiotic stress, as well as regulate plant growth and development. Anthocyanins, for instance, include a wide range of pigments (blue, red, and purple), which can not only protect against UV radiation but can also attract pollinators (Wang et al., 2016). Due to their relevance to plant life, the biosynthesis and regulation of all these compounds have been extensively studied. The transcription factors MYBlike 2 (MYBL2) and SPL9 are negative regulators of anthocyanin biosynthesis in Arabidopsis (Dubos et al., 2008; Matsui et al., 2008; Gou et al., 2011). On the other hand, overexpression of miR156 increases anthocyanin levels in Arabidopsis, since some miR156 target genes, including SPL9, repress anthocyanin biosynthesis (Gou et al., 2011; Cui et al., 2014). In Chinese sand pear (Pyrus pyrifolia) fruits, bagging and subsequent re-exposure to light causes anthocyanin accumulation in fruit peel, giving red color to the fruits (Qian et al., 2013). Qian et al. (2017) hypothesized that miR156 and SPLs would also be involved in anthocyanin accumulation in pears in response to bagging treatments. They detected miR156 in peels of bagging-treated pear fruits and found that the promoters of two pear PpMIR156 genes contained lightresponsive elements. After bag removal, miR156 levels increased, whereas PpSPL transcripts decreased. This was accompanied by upregulation of homologs of genes encoding transcription factors that control anthocyanin biosynthesis, and followed by upregulation of anthocyanin biosynthesis genes (Qian et al., 2017). Although functional analyses of miR156 and PpSPL genes in pear have not yet been reported, these results point to a possible role of miR156 and PpSPL proteins in light-induced anthocyanin biosynthesis in bagging-treated pear fruits.

In a related report, Qu et al. (2016) investigated the miRNA profiling of "Granny Smith" apple peels upon re-exposure to sunlight after fruit bagging, using high-throughput sequencing of small RNA libraries. They found that over 67% of differentially expressed miRNAs were downregulated in the bagged group as compared to the unbagged control. These findings suggest the existence of a light-regulated pool of miRNAs in apple peel. miR156, miR160, miR171, miR172, miR395, and miR398 were differentially expressed upon sunlight re-exposure. Interestingly, these miRNAs families had previously been identified as UV-B-regulated miRNAs in several plant species (see Section "UV-Responsive miRNAs"). In Arabidopsis, in addition to miR156, miR858 positively regulates anthocyanin biosynthesis by targeting specific repressors of this process (Gou et al., 2011; Qian et al., 2013; Cui et al., 2014; Wang et al., 2016). In tomato, miR858 also promotes anthocyanin biosynthesis (Jia et al., 2015). Interestingly, miR156, miR828, and miR858 expression levels varied upon apple fruit debagging, suggesting their involvement in anthocyanin biosynthesis in apple peels. In fact, miR156 correlated with anthocyanin accumulation only in "Granny Smith," whereas miR5072 did it only in the "Starkrimson"

cultivar. These results highlight the high specificity of certain miRNA families to control particular responses, such as pigment accumulation to protect against light stress.

As previously mentioned, miR858 is a positive regulator of anthocyanin biosynthesis (Wang et al., 2016). In Arabidopsis, MIR858A expression is induced by light, especially high light, in a HY5-dependent manner. Upon transfer from dark to high light, MIR858A level oscillates, suggesting also circadian regulation. Interestingly, miR858a overexpression partially complements the long hypocotyl phenotype of the hy5 mutant and rescues the anthocyanin levels of this mutant. miR858a and miR858b can promote anthocyanin biosynthesis in two ways: (1) by indirectly inducing the expression of genes encoding components of the anthocyanin biosynthetic pathway and (2) by inhibiting protein accumulation of their target gene encoding the repressor MYBL2 protein, most likely at the translational level. HY5 directly binds and represses the MYBL2 promoter probably by a mechanism that involves the loss of H3K9Ac and H3K4me3-positive histone marks (Wang et al., 2016). Therefore, HY5 has a dual effect in promoting anthocyanin biosynthesis: it binds and represses MYBL2, and it promotes MIR858A expression in response to light (e.g., high light) signals.

It has been shown that hen1 mutants, which have reduced levels of mature miRNAs, accumulate high anthocyanin levels (Tsai et al., 2014), a result somehow contradictory with miR156 and miR858 promoting anthocyanin biosynthesis. However, Sharma et al. (2016) propose a different function for miR858 in the regulation of flavonoid biosynthesis. They found that miR858 targets the positive regulators of flavonoid biosynthesis MYB11, MYB12, and MYB111. Moreover, transcriptional profiling of miR858-overexpressing lines (miR858-OX) revealed MYB20, MYB42, and MYB83 as new targets of miR858. Since these transcription factors regulate several growth responses, the authors characterized growth phenotypes of miR858-OX and miR858a target mimic lines (MIM858), in which miR858a activity is silenced. While miR858-OX plants showed enhanced growth, early flowering, and an increase in seed size, MIM858 plants displayed reduced growth, late flowering, and smaller seeds (Sharma et al., 2016). The metabolic profiling of these lines confirmed their opposite effect on flavonoid accumulation, since miR858 depletion resulted in the accumulation of major flavonoids, whereas they were significantly reduced in miR858- OX. This was accompanied by an opposite effect on lignin biosynthesis, since miR858-OX showed increased lignification and upregulation of lignin biosynthetic genes. These results indicate that miR858 could differently regulate anthocyanin, other flavonoids, and lignin biosynthesis. This could be achieved by targeting different MYB transcription factors in response to distinct light conditions (white light versus low or high light). This differential regulation could then help maintain a metabolic flux balance between the different biosynthetic pathways.

Chlorophylls are critical plant pigments involved in light absorption and energy transfer during photosynthesis. However, this fundamental process for plant life also generates reactive oxygen species, which are detrimental for plant growth and development. Considering that chlorophyll biosynthesis precursors are also sources for reactive oxygen species, it is of paramount importance to tightly regulate this pathway. One of such mechanisms includes miR171 and miR171 targeted SCL genes, also known as HAIRY MERISTEM (HAM) or LOST MERISTEMS (LOM). In Arabidopsis, miR171 positively regulates chlorophyll biosynthesis in the light through downregulation of miR171-targeted SCLs, leading to upregulation of protochlorophyllide oxidoreductase (POR), a key enzyme in chlorophyll biosynthesis (Ma et al., 2014). MIR171C-overexpressing plants and scl6 scl22 scl27 mutants show higher chlorophyll and POR levels than wild-type plants, whereas plants expressing a miR171-resistant form of SCL27 (rSCL27) show reduced chlorophyll and POR levels (Wang et al., 2010; Ma et al., 2014). On the other hand, downregulation of POR expression reduces the chlorophyll content of wild-type, MIR171C-OX, and scl triple mutants (Ma et al., 2014), indicating that the role of miR171c and SCLs on chlorophyll regulation is mediated by PORC. SCL27 represses PORC expression by directly binding and inhibiting its promoter.

Moreover, the miR171-SCL module also mediates gibberellindependent effects on chlorophyll biosynthesis due to its regulation of DELLA proteins and PORC expression in the light, but not in the dark. In fact, SCL27 interacts with the DELLA protein RGA and this interaction reduces the ability of SCL27 to bind to the PORC promoter. Finally, SCLs induce the expression of MIR171 genes, revealing the existence of a regulatory feedback loop (Ma et al., 2014). Ma et al. (2014) propose that this feedback loop may also contribute to maintain the diurnal oscillation of miR171.

Taken together, these findings emphasize the role of miRNAs in diverse light-regulated processes. In addition, miRNAs can also directly target major regulators of the light signaling pathways and/or the photoperiod pathway, thus modulating photomorphogenesis or the time to flower, respectively. The most recent findings on miRNAs involved in these processes are discussed below.

### Photomorphogenesis

Within the group of light-responsive miRNAs, the miR156/157 family occupies a prominent position (**Table 1**). In Arabidopsis, miR157d and miR319 promote the degradation of their target transcripts, encoding the positive and negative key photomorphogenesis regulators HY5 and TCP (TEOSINTE BRANCHED1, CYCLOIDEA, and PCF) transcription factors, respectively. Interestingly, both induction and stabilization of miR157d and miR319 were shown to depend on HEN1, with HEN1 accumulation increasing miR157d and miR319 levels in de-etiolating seedlings (Tsai et al., 2014). In turn, HY5 induces HEN1 expression in a light-dependent manner. Therefore, HEN1 and HY5 constitute a negative feedback regulatory loop that is mediated by miR157, since this miRNA will ultimately target HY5 transcripts for cleavage. In a hen1 mutant, both HY5 transcript and protein levels are increased, probably due to the decrease in miR157d, resulting in a light-hypersensitive phenotype. Oppositely, miR157 constitutive expression reduced HY5 transcript and protein levels, resulting in seedlings displaying a light hyposensitivity phenotype. Nevertheless, the light-hypersensitive phenotype displayed by hen1 mutants may

also be dependent on miR319, which promoted the cleavage of TCP mRNAs and repressed hypocotyl elongation (Tsai et al., 2014). The role of miR319 in photomorphogenesis has been confirmed using a loss of function mutant, which shows longer hypocotyls than the wild type under red light (Sun et al., 2018). Perhaps, the HEN1/HY5 feedback loop may help explain the inconsistencies found in the effect of HEN1, miR156, and miR858 on anthocyanin accumulation.

Three additional miRNAs, miR160, miR167, and miR848, affect Arabidopsis hypocotyl elongation under red light (Sun et al., 2018). It is worth mentioning that miR160 and miR167 respond to different light conditions in several species (**Table 1**). Further understanding of the role of these miRNAs in light responses will undoubtedly provide novel insights into how miRNAs contribute to adaptation to different light environments.