The SCL30a SR protein regulates ABA-dependent seed traits and germination under stress.

SR proteins are conserved RNA-binding proteins best known as splicing regulators that have also been implicated in other steps of gene expression. Despite mounting evidence for a role in plant development and stress responses, the molecular pathways underlying SR protein regulation of these processes remain poorly understood. Here we show that the plant-specific SCL30a SR protein negatively regulates ABA signaling to control seed traits and stress responses during germination in Arabidopsis. Transcriptome-wide analyses revealed that loss of SCL30a function barely affects splicing, but largely induces ABA-responsive gene expression and genes repressed during germination. Accordingly, scl30a mutant seeds display delayed germination and hypersensitivity to ABA and high salinity, while transgenic plants overexpressing SCL30a exhibit reduced ABA and salt stress sensitivity. An ABA biosynthesis inhibitor rescues the enhanced mutant seed stress sensitivity, and epistatic analyses confirm that this hypersensitivity requires a functional ABA pathway. Finally, seed ABA levels are unchanged by altered SCL30a expression, indicating that the gene promotes seed germination under stress by reducing sensitivity to the phytohormone. Our results reveal a new player in ABA-mediated control of early development and stress response.

Regulation of alternative splicing by retrograde and light signals converges to control chloroplast proteins.

Retrograde signals sent by chloroplasts control transcription in the nucleus. These signals antagonistically converge with light signals to coordinate the expression of genes involved in chloroplast functioning and seedling development. Although significant advances have been made in understanding the molecular interplay between light and retrograde signals at the transcriptional level, little is known about their interconnection at the post-transcriptional level. By using different publicly available datasets, this study addresses the influence of retrograde signaling on alternative splicing and defines the molecular and biological functions of this regulation. These analyses revealed that alternative splicing mimics transcriptional responses triggered by retrograde signals at different levels. First, both molecular processes similarly depend on the chloroplast-localized pentatricopeptide-repeat protein GUN1 to modulate the nuclear transcriptome. Secondly, as described for transcriptional regulation, alternative splicing coupled with the nonsense-mediated decay pathway effectively downregulates expression of chloroplast proteins in response to retrograde signals. Finally, light signals were found to antagonistically control retrograde signaling-regulated splicing isoforms, which consequently generates opposite splicing outcomes that likely contribute to the opposite roles these signals play in controlling chloroplast functioning and seedling development.

Etiolated Hypocotyls: A New System to Study the Impact of Abiotic Stress on Cell Expansion.

Abiotic stress impacts a wide range of plant developmental processes. Among them, cell expansion is particularly important given its contribution to plant growth and morphogenesis. Here, we describe a new phenotypic system to quantify accurately the impact of different sources of abiotic stress on the cell's capacity to expand. This approach monitors hypocotyl growth in Arabidopsis thaliana etiolated seedlings, as in the dark this embryonic organ is known to grow solely by expanding its cells, without cell division.

BBX16 mediates the repression of seedling photomorphogenesis downstream of the GUN1/GLK1 module during retrograde signalling.

Plastid-to-nucleus retrograde signalling (RS) initiated by dysfunctional chloroplasts impact photomorphogenic development. We have previously shown that the transcription factor GLK1 acts downstream of the RS regulator GUN1 in photodamaging conditions to regulate not only the well established expression of photosynthesis-associated nuclear genes (PhANGs) but also to regulate seedling morphogenesis. Specifically, the GUN1/GLK1 module inhibits the light-induced phytochrome-interacting factor (PIF)-repressed transcriptional network to suppress cotyledon development when chloroplast integrity is compromised, modulating the area exposed to potentially damaging high light. However, how the GUN1/GLK1 module inhibits photomorphogenesis upon chloroplast damage remained undefined. Here, we report the identification of BBX16 as a novel direct target of GLK1. BBX16 is induced and promotes photomorphogenesis in moderate light and is repressed via GUN1/GLK1 after chloroplast damage. Additionally, we showed that BBX16 represents a regulatory branching point downstream of GUN1/GLK1 in the regulation of PhANG expression and seedling development upon RS activation. The gun1 phenotype in lincomycin and the gun1-like phenotype of GLK1OX are markedly suppressed in gun1bbx16 and GLK1OXbbx16. This study identified BBX16 as the first member of the BBX family involved in RS, and defines a molecular bifurcation mechanism operated by GLK1/BBX16 to optimise seedling de-etiolation, and to ensure photoprotection in unfavourable light conditions.

Tailoring photomorphogenic markers to organ growth dynamics.

When a dark-germinated seedling reaches the soil surface and perceives sunlight for the first time, light signaling is activated to adapt the plant's development and transition to autotrophism. During this process, functional chloroplasts assemble in the cotyledons and the seedling's cell expansion pattern is rearranged to enhance light perception. Hypocotyl cells expand rapidly in the dark, while cotyledon cell expansion is suppressed. However, light reverses this pattern by activating cell expansion in cotyledons and repressing it in hypocotyls. The fact that light-regulated developmental responses, as well as the transcriptional mechanisms controlling them, are organ-specific has been largely overlooked in previous studies of seedling de-etiolation. To analyze the expansion pattern of the hypocotyl and cotyledons separately in a given Arabidopsis (Arabidopsis thaliana) seedling, we define an organ ratio, the morphogenic index (MI), which integrates either phenotypic or transcriptomic data for each tissue and provides an important resource for functional analyses. Moreover, based on this index, we identified organ-specific molecular markers to independently quantify cotyledon and hypocotyl growth dynamics in whole-seedling samples. The combination of these marker genes with those of other developmental processes occurring during de-etiolation will allow improved molecular dissection of photomorphogenesis. Along with organ growth markers, this MI contributes a key toolset to unveil and accurately characterize the molecular mechanisms controlling seedling growth.

Alternative splicing landscapes in Arabidopsis thaliana across tissues and stress conditions highlight major functional differences with animals.

BACKGROUND: Alternative splicing (AS) is a widespread regulatory mechanism in multicellular organisms. Numerous transcriptomic and single-gene studies in plants have investigated AS in response to specific conditions, especially environmental stress, unveiling substantial amounts of intron retention that modulate gene expression. However, a comprehensive study contrasting stress-response and tissue-specific AS patterns and directly comparing them with those of animal models is still missing. RESULTS: We generate a massive resource for Arabidopsis thaliana, PastDB, comprising AS and gene expression quantifications across tissues, development and environmental conditions, including abiotic and biotic stresses. Harmonized analysis of these datasets reveals that A. thaliana shows high levels of AS, similar to fruitflies, and that, compared to animals, disproportionately uses AS for stress responses. We identify core sets of genes regulated specifically by either AS or transcription upon stresses or among tissues, a regulatory specialization that is tightly mirrored by the genomic features of these genes. Unexpectedly, non-intron retention events, including exon skipping, are overrepresented across regulated AS sets in A. thaliana, being also largely involved in modulating gene expression through NMD and uORF inclusion. CONCLUSIONS: Non-intron retention events have likely been functionally underrated in plants. AS constitutes a distinct regulatory layer controlling gene expression upon internal and external stimuli whose target genes and master regulators are hardwired at the genomic level to specifically undergo post-transcriptional regulation. Given the higher relevance of AS in the response to different stresses when compared to animals, this molecular hardwiring is likely required for a proper environmental response in A. thaliana.

The photoperiodic response of hypocotyl elongation involves regulation of CDF1 and CDF5 activity.

Hypocotyl elongation relies on directional cell expansion, a process under light and circadian clock control. Under short photoperiods (SD), hypocotyl elongation in Arabidopsis thaliana follows a rhythmic pattern, a process in which circadian morning-to-midnight waves of the transcriptional repressors PSEUDO-RESPONSE REGULATORS (PRRs) jointly gate PHYTOCHROME-INTERACTING FACTOR (PIF) activity to dawn. Previously, we described CYCLING DOF FACTOR 5 (CDF5) as a target of this antagonistic PRR/PIF dynamic interplay. Under SD, PIFs induce CDF5 accumulation specifically at dawn, when it promotes the expression of positive cell elongation regulators such as YUCCA8 to induce growth. In contrast to SD, hypocotyl elongation under long days (LD) is largely reduced. Here, we examine whether CDF5 is an actor in this photoperiod specific regulation. We report that transcription of CDF5 is robustly induced in SD compared to LD, in accordance with PIFs accumulating to higher levels in SD, and in contrast to other members of the CDF family, whose expression is mainly clock regulated and have similar waveforms in SD and LD. Notably, when CDF5 was constitutively expressed under LD, CDF5 protein accumulated to levels comparable to SD but was inactive in promoting cell elongation. Similar results were observed for CDF1. Our findings indicate that both CDFs can promote cell elongation specifically in shorter photoperiods, however, their activity in LD is inhibited at the post-translational level. These data not only expand our understanding of the biological role of CDF transcription factors, but also identify a previously unrecognized regulatory layer in the photoperiodic response of hypocotyl elongation.

Phytochrome-imposed inhibition of PIF7 activity shapes photoperiodic growth in Arabidopsis together with PIF1, 3, 4 and 5.

Under photoperiodic conditions, Arabidopsis thaliana seedling growth is inhibited in long days (LDs), but promoted under the extended nights of short days (SDs). This behavior is partly implemented by phytochrome (phy)-imposed oscillations in the abundance of the growth-promoting, phy-interacting bHLH transcription factors PHY-INTERACTING FACTOR 1 (PIF1), PIF3, PIF4 and PIF5 (PIF quartet or PIFq). However, the observation that a pifq mutant is still stimulated to elongate when given a phy-inactivating end-of-day far-red pulse (EODFR), suggests that additional factors are involved in the phy-mediated suppression of growth during the subsequent dark period. Here, by combining growth-analysis of pif7 single- and higher-order mutants with gene expression analysis under SD, LD, SD-EODFR, and LD-EODFR, we show that PIF7 promotes growth during the dark hours of SD, by regulating growth-related gene expression. Interestingly, the relative contribution of PIF7 in promoting growth is stronger under EODFR, whereas PIF3 role is more important under SD, suggesting that PIF7 is a prominent target of phy-suppression. Indeed, we show that phy imposes phosphorylation and inactivation of PIF7 during the light hours in SD, and prevents full dephosphorylation during the night. This repression can be lifted with an EODFR, which correlates with increased PIF7-mediated gene expression and elongation. In addition, our results suggest that PIF7 function might involve heterodimerization with PIF3. Furthermore, our data indicate that a pifqpif7 quintuple mutant is largely insensitive to photoperiod for hypocotyl elongation. Collectively, the data suggest that PIF7, together with the PIFq, is required for the photoperiodic regulation of seasonal growth.

Circadian Waves of Transcriptional Repression Shape PIF-Regulated Photoperiod-Responsive Growth in Arabidopsis.

Plants coordinate their growth and development with the environment through integration of circadian clock and photosensory pathways. In Arabidopsis thaliana, rhythmic hypocotyl elongation in short days (SD) is enhanced at dawn by the basic-helix-loop-helix (bHLH) transcription factors PHYTOCHROME-INTERACTING FACTORS (PIFs) directly inducing expression of growth-related genes [1-6]. PIFs accumulate progressively during the night and are targeted for degradation by active phytochromes in the light, when growth is reduced. Although PIF proteins are also detected during the day hours [7-10], their growth-promoting activity is inhibited through unknown mechanisms. Recently, the core clock components and transcriptional repressors PSEUDO-RESPONSE REGULATORS PRR9/7/5 [11, 12], negative regulators of hypocotyl elongation [13, 14], were described to associate to G boxes [15], the DNA motifs recognized by the PIFs [16, 17], suggesting that PRR and PIF function might converge antagonistically to regulate growth. Here we report that PRR9/7/5 and PIFs physically interact and bind to the same promoter region of pre-dawn-phased, growth-related genes, and we identify the transcription factor CDF5 [18, 19] as target of this interplay. In SD, CDF5 expression is sequentially repressed from morning to dusk by PRRs and induced pre-dawn by PIFs. Consequently, CDF5 accumulates specifically at dawn, when it induces cell elongation. Our findings provide a framework for recent TIMING OF CAB EXPRESSION 1 (TOC1/PRR1) data [5, 20] and reveal that the long described circadian morning-to-midnight waves of the PRR transcriptional repressors (PRR9, PRR7, PRR5, and TOC1) [21] jointly gate PIF activity to dawn to prevent overgrowth through sequential regulation of common PIF-PRR target genes such as CDF5.

Alternative Splicing Control of Abiotic Stress Responses.

Alternative splicing, which generates multiple transcripts from the same gene, is an important modulator of gene expression that can increase proteome diversity and regulate mRNA levels. In plants, this post-transcriptional mechanism is markedly induced in response to environmental stress, and recent studies have identified alternative splicing events that allow rapid adjustment of the abundance and function of key stress-response components. In agreement, plant mutants defective in splicing factors are severely impaired in their response to abiotic stress. Notably, mounting evidence indicates that alternative splicing regulates stress responses largely by targeting the abscisic acid (ABA) pathway. We review here current understanding of post-transcriptional control of plant stress tolerance via alternative splicing and discuss research challenges for the near future.

Genomic Analysis Reveals Contrasting PIFq Contribution to Diurnal Rhythmic Gene Expression in PIF-Induced and -Repressed Genes.

Members of the PIF quartet (PIFq; PIF1, PIF3, PIF4, and PIF5) collectively contribute to induce growth in Arabidopsis seedlings under short day (SD) conditions, specifically promoting elongation at dawn. Their action involves the direct regulation of growth-related and hormone-associated genes. However, a comprehensive definition of the PIFq-regulated transcriptome under SD is still lacking. We have recently shown that SD and free-running (LL) conditions correspond to "growth" and "no growth" conditions, respectively, correlating with greater abundance of PIF protein in SD. Here, we present a genomic analysis whereby we first define SD-regulated genes at dawn compared to LL in the wild type, followed by identification of those SD-regulated genes whose expression depends on the presence of PIFq. By using this sequential strategy, we have identified 349 PIF/SD-regulated genes, approximately 55% induced and 42% repressed by both SD and PIFq. Comparison with available databases indicates that PIF/SD-induced and PIF/SD-repressed sets are differently phased at dawn and mid-morning, respectively. In addition, we found that whereas rhythmicity of the PIF/SD-induced gene set is lost in LL, most PIF/SD-repressed genes keep their rhythmicity in LL, suggesting differential regulation of both gene sets by the circadian clock. Moreover, we also uncovered distinct overrepresented functions in the induced and repressed gene sets, in accord with previous studies in other examined PIF-regulated processes. Interestingly, promoter analyses showed that, whereas PIF/SD-induced genes are enriched in direct PIF targets, PIF/SD-repressed genes are mostly indirectly regulated by the PIFs and might be more enriched in ABA-regulated genes.

Phytochrome and retrograde signalling pathways converge to antagonistically regulate a light-induced transcriptional network.

Plastid-to-nucleus retrograde signals emitted by dysfunctional chloroplasts impact photomorphogenic development, but the molecular link between retrograde- and photosensory-receptor signalling has remained unclear. Here, we show that the phytochrome and retrograde signalling (RS) pathways converge antagonistically to regulate the expression of the nuclear-encoded transcription factor GLK1, a key regulator of a light-induced transcriptional network central to photomorphogenesis. GLK1 gene transcription is directly repressed by PHYTOCHROME-INTERACTING FACTOR (PIF)-class bHLH transcription factors in darkness, but light-activated phytochrome reverses this activity, thereby inducing expression. Conversely, we show that retrograde signals repress this induction by a mechanism independent of PIF mediation. Collectively, our data indicate that light at moderate levels acts through the plant's nuclear-localized sensory-photoreceptor system to induce appropriate photomorphogenic development, but at excessive levels, sensed through the separate plastid-localized RS system, acts to suppress such development, thus providing a mechanism for protection against photo-oxidative damage by minimizing the tissue exposure to deleterious radiation.

Molecular convergence of clock and photosensory pathways through PIF3-TOC1 interaction and co-occupancy of target promoters.

A mechanism for integrating light perception and the endogenous circadian clock is central to a plant's capacity to coordinate its growth and development with the prevailing daily light/dark cycles. Under short-day (SD) photocycles, hypocotyl elongation is maximal at dawn, being promoted by the collective activity of a quartet of transcription factors, called PIF1, PIF3, PIF4, and PIF5 (phytochrome-interacting factors). PIF protein abundance in SDs oscillates as a balance between synthesis and photoactivated-phytochrome-imposed degradation, with maximum levels accumulating at the end of the long night. Previous evidence shows that elongation under diurnal conditions (as well as in shade) is also subjected to circadian gating. However, the mechanism underlying these phenomena is incompletely understood. Here we show that the PIFs and the core clock component Timing of CAB expression 1 (TOC1) display coincident cobinding to the promoters of predawn-phased, growth-related genes under SD conditions. TOC1 interacts with the PIFs and represses their transcriptional activation activity, antagonizing PIF-induced growth. Given the dynamics of TOC1 abundance (displaying high postdusk levels that progressively decline during the long night), our data suggest that TOC1 functions to provide a direct output from the core clock that transiently constrains the growth-promoting activity of the accumulating PIFs early postdusk, thereby gating growth to predawn, when conditions for cell elongation are optimal. These findings unveil a previously unrecognized mechanism whereby a core circadian clock output signal converges immediately with the phytochrome photosensory pathway to coregulate directly the activity of the PIF transcription factors positioned at the apex of a transcriptional network that regulates a diversity of downstream morphogenic responses.

Branching of the PIF3 regulatory network in Arabidopsis: roles of PIF3-regulated MIDAs in seedling development in the dark and in response to light.

Plants need to accurately adjust their development after germination in the underground darkness to ensure survival of the seedling, both in the dark and in the light upon reaching the soil surface. Recent studies have established that the photoreceptors phytochromes and the bHLH phytochrome interacting factors PIFs regulate seedling development to adjust it to the prevailing light environment during post-germinative growth. However, complete understanding of the downstream regulatory network implementing these developmental responses is still lacking. In a recent work, published in The Plant Cell, we report a subset of PIF3-regulated genes in dark-grown seedlings that we have named MIDAs (MISREGULATED IN DARK). Analysis of their functional relevance using mutants showed that four of them present phenotypic alterations in the dark, and that each affected a particular facet of seedling development, suggesting organ-specific branching in the signal that PIF3 relays downstream. Furthermore, our results also showed an altered response to light in seedlings with an impaired PIF3/MIDA regulatory network, indicating that these factors might also be essential to initiate and optimize the developmental adjustment of the seedling to the light environment.

Functional profiling identifies genes involved in organ-specific branches of the PIF3 regulatory network in Arabidopsis.

The phytochrome (phy)-interacting basic helix-loop-helix transcription factors (PIFs) constitutively sustain the etiolated state of dark-germinated seedlings by actively repressing deetiolation in darkness. This action is rapidly reversed upon light exposure by phy-induced proteolytic degradation of the PIFs. Here, we combined a microarray-based approach with a functional profiling strategy and identified four PIF3-regulated genes misexpressed in the dark (MIDAs) that are novel regulators of seedling deetiolation. We provide evidence that each one of these four MIDA genes regulates a specific facet of etiolation (hook maintenance, cotyledon appression, or hypocotyl elongation), indicating that there is branching in the signaling that PIF3 relays. Furthermore, combining inferred MIDA gene function from mutant analyses with their expression profiles in response to light-induced degradation of PIF3 provides evidence consistent with a model where the action of the PIF3/MIDA regulatory network enables an initial fast response to the light and subsequently prevents an overresponse to the initial light trigger, thus optimizing the seedling deetiolation process. Collectively, the data suggest that at least part of the phy/PIF system acts through these four MIDAs to initiate and optimize seedling deetiolation, and that this mechanism might allow the implementation of spatial (i.e., organ-specific) and temporal responses during the photomorphogenic program.