Core clock genes adjust growth cessation time to day-night switches in poplar.

Poplar trees use photoperiod as a precise seasonal indicator, synchronizing plant phenology with the environment. Daylength cue determines FLOWERING LOCUS T 2 (FT2) daily expression, crucial for shoot apex development and establishment of the annual growing period. However, limited evidence exists for the molecular factors controlling FT2 transcription and the conservation with the photoperiodic control of Arabidopsis flowering. We demonstrate that FT2 expression mediates growth cessation response quantitatively, and we provide a minimal data-driven model linking core clock genes to FT2 daily levels. GIGANTEA (GI) emerges as a critical inducer of the FT2 activation window, time-bound by TIMING OF CAB EXPRESSION (TOC1) and LATE ELONGATED HYPOCOTYL (LHY2) repressions. CRISPR/Cas9 loss-of-function lines validate these roles, identifying TOC1 as a long-sought FT2 repressor. Additionally, model simulations predict that FT2 downregulation upon daylength shortening results from a progressive narrowing of this activation window, driven by the phase shift observed in the preceding clock genes. This circadian-mediated mechanism enables poplar to exploit FT2 levels as an accurate daylength-meter.

Functionally Divergent Splicing Variants of the Rice AGAMOUS Ortholog OsMADS3 Are Evolutionary Conserved in Grasses.

Within the MADS-box gene family, the AGAMOUS-subfamily genes are particularly important for plant reproduction, because they control stamen and carpel identity. A number of studies in the last three decades have demonstrated that the AGAMOUS (AG) function has been conserved during land plant evolution. However, gene duplication events have led to subfunctionalization and neofunctionalization of AG-like genes in many species. Here we show that alternative splicing in Oryza sativa produces two variants of the AG ortholog OsMADS3 which differ in just one serine residue, S109. Interestingly, this alternative splicing variant is conserved and specific to the grass family. Since in eudicots the S109 residue is absent in AG proteins, stamen and carpel identity determination activity of the two rice isoforms was tested in Arabidopsis thaliana. These experiments revealed that only the eudicot-like OsMADS3 isoform, lacking the serine residue, had ability to specify stamens and carpels in ag mutant flowers, suggesting an important functional role for the serine residue at position 109 in AG proteins of grasses.

Dual function of HYPONASTIC LEAVES 1 during early skotomorphogenic growth in Arabidopsis.

Seeds germinating underground display a specific developmental programme, termed skotomorphogenesis, to ensure survival of the emerging seedlings until they reach the light. They rapidly elongate the hypocotyl and maintain the cotyledons closed, forming a hook with the hypocotyl in order to protect apical meristematic cells from mechanical damage. Such crucial events for the fate of the seedling are tightly regulated and although some transcriptional regulators and phytohormones are known to be implicated in this regulation, we are still far from a complete understanding of these biological processes. Our work provides information on the diverse roles in skotomorphogenesis of the core components of microRNA biogenesis in Arabidopsis, HYL1, DCL1, and SE. We show that hypocotyl elongation is promoted by all these components, probably through the action of specific miRNAs. Hook development also depends on these proteins however, remarkably, HYL1 exerts its role in an opposite way to DCL1 and SE. Interestingly, we found that a specific HYL1 domain involved in protein-protein interaction is required for this function. Genetic evidences also point to the phosphorylation status of HYL1 as important for this function. We propose that HYL1 help maintain the hook closed during early skotomorphogenesis in a microprocessor-independent manner by repressing the activity of HY5, the transcriptional master regulator that triggers light responses. This work uncovers a previously unnoticed link between components of the miRNA biogenesis machinery, the skotomorphogenic growth, and hook development in Arabidopsis.

Early cold stress responses in post-meiotic anthers from tolerant and sensitive rice cultivars.

BACKGROUND: Rice grain production is susceptible to a changing environment that imposes both biotic and abiotic stress conditions. Cold episodes are becoming more frequent in the last years and directly affect rice yield in areas with a temperate climate. Rice is particularly susceptible to cold stress during the reproductive phase, especially in anthers during post-meiotic stages which, in turn, affect pollen production. However, a number of rice cultivars with a certain degree of tolerance to cold have been described, which may represent a good breeding resource for improvement of susceptible commercial varieties. Plants experiencing cold stress activate a molecular response in order to reprogram many metabolic pathways to face these hostile conditions. RESULTS: Here we performed RNA-seq analysis using cold-stressed post-meiotic anther samples from a cold-tolerant, Erythroceros Hokkaido (ERY), and a cold-susceptible commercial cultivar Sant'Andrea (S.AND). Both cultivars displayed an early common molecular response to cold, although the changes in expression levels are much more drastic in the tolerant one. Comparing our datasets, obtained after one-night cold stress, with other similar genome-wide studies showed very few common deregulated genes, suggesting that molecular responses in cold-stressed anthers strongly depend on conditions and the duration of the cold treatments. Cold-tolerant ERY exhibits specific molecular responses related to ethylene metabolism, which appears to be activated after cold stress. On the other hand, S.AND cold-treated plants showed a general downregulation of photosystem I and II genes, supporting a role of photosynthesis and chloroplasts in cold responses in anthers, which has remained elusive. CONCLUSIONS: Our study revealed that a number of ethylene-related transcription factors, as putative master regulators of cold responses, were upregulated in ERY providing promising candidates to confer tolerance to susceptible cultivars. Our results also suggest that the photosynthesis machinery might be a good target to improve cold tolerance in anthers. In summary, our study provides valuable candidates for further analysis and molecular breeding for cold-tolerant rice cultivars.

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.

Genome-Wide Transcriptome Analysis During Anthesis Reveals New Insights into the Molecular Basis of Heat Stress Responses in Tolerant and Sensitive Rice Varieties.

Rice is one of the main food crops in the world. In the near future, yield is expected to be under pressure due to unfavorable climatic conditions, such as increasing temperatures. Therefore, improving rice germplasm in order to guarantee rice production under harsh environmental conditions is of top priority. Although many physiological studies have contributed to understanding heat responses during anthesis, the most heat-sensitive stage, molecular data are still largely lacking. In this study, an RNA-sequencing approach of heat- and control-treated reproductive tissues during anthesis was carried out using N22, one of the most heat-tolerant rice cultivars known to date. This analysis revealed that expression of genes encoding a number of transcription factor families, together with signal transduction and metabolic pathway genes, is repressed. On the other hand, expression of genes encoding heat shock factors and heat shock proteins was highly activated. Many of these genes are predominantly expressed at late stages of anther development. Further physiological experiments using heat-tolerant N22 and two sensitive cultivars suggest that reduced yield in heat-sensitive plants may be associated with poor pollen development or production in anthers prior to anthesis. In parallel, induction levels of a set of heat-responsive genes in these tissues correlated well with heat tolerance. Altogether, these findings suggest that proper expression of protective chaperones in anthers is needed before anthesis to overcome stress damage and to ensure fertilization. Genes putatively controlling this process were identified and are valuable candidates to consider for molecular breeding of highly productive heat-tolerant cultivars.

Comparative functional analysis of full-length and N-terminal fragments of phytochrome C, D and E in red light-induced signaling.

Phytochromes (phy) C, D and E are involved in the regulation of red/far-red light-induced photomorphogenesis of Arabidopsis thaliana, but only limited data are available on the mode of action and biological function of these lesser studied phytochrome species. We fused N-terminal fragments or full-length PHYC, D and E to YELLOW FLUORESCENT PROTEIN (YFP), and analyzed the function, stability and intracellular distribution of these fusion proteins in planta. The activity of the constitutively nuclear-localized homodimers of N-terminal fragments was comparable with that of full-length PHYC, D, E-YFP, and resulted in the regulation of various red light-induced photomorphogenic responses in the studied genetic backgrounds. PHYE-YFP was active in the absence of phyB and phyD, and PHYE-YFP controlled responses, as well as accumulation, of the fusion protein in the nuclei, was saturated at low fluence rates of red light and did not require functional FAR-RED ELONGATED HYPOCOTYL1 (FHY-1) and FHY-1-like proteins. Our data suggest that PHYC-YFP, PHYD-YFP and PHYE-YFP fusion proteins, as well as their truncated N-terminal derivatives, are biologically active in the modulation of red light-regulated photomorphogenesis. We propose that PHYE-YFP can function as a homodimer and that low-fluence red light-induced translocation of phyE and phyA into the nuclei is mediated by different molecular mechanisms.

Phytochrome-imposed oscillations in PIF3 protein abundance regulate hypocotyl growth under diurnal light/dark conditions in Arabidopsis.

Arabidopsis seedlings display rhythmic growth when grown under diurnal conditions, with maximal elongation rates occurring at the end of the night under short-day photoperiods. Current evidence indicates that this behavior involves the action of the growth-promoting bHLH factors PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) at the end of the night, through a coincidence mechanism that combines their transcriptional regulation by the circadian clock with control of protein accumulation by light. To assess the possible role of PIF3 in this process, we have analyzed hypocotyl responses and marker gene expression in pif single- and higher-order mutants. The data show that PIF3 plays a prominent role as a promoter of seedling growth under diurnal light/dark conditions, in conjunction with PIF4 and PIF5. In addition, we provide evidence that PIF3 functions in this process through its intrinsic transcriptional regulatory activity, at least in part by directly targeting growth-related genes, and independently of its ability to regulate phytochrome B (phyB) levels. Furthermore, in sharp contrast to PIF4 and PIF5, our data show that the PIF3 gene is not subject to transcriptional regulation by the clock, but that PIF3 protein abundance oscillates under diurnal conditions as a result of a progressive decline in PIF3 protein degradation mediated by photoactivated phyB, and consequent accumulation of the bHLH factor during the dark period. Collectively, the data suggest that phyB-mediated, post-translational regulation allows PIF3 accumulation to peak just before dawn, at which time it accelerates hypocotyl growth, together with PIF4 and PIF5, by directly regulating the induction of growth-related genes.

Potato CONSTANS is involved in photoperiodic tuberization in a graft-transmissible manner.

CONSTANS (CO) is involved in the photoperiodic control of plant developmental processes, including flowering in several species and seasonal growth cessation and bud set in trees. It has been proposed that CO could also affect the day-length regulation of tuber induction in Solanum tuberosum (potato), a plant of great agricultural relevance. To address this question, we examined the role of CO in potato. A potato CO-like gene, StCO, was identified and found to be highly similar to a previously reported potato gene of unknown function. Potato plants overexpressing StCO tuberized later than wild-type plants under a weakly inductive photoperiod. StCO silencing promoted tuberization under both repressive and weakly inductive photoperiods, but did not have any effect under strongly inductive short days, demonstrating that StCO represses tuberization in a photoperiod-dependent manner. The effect of StCO on tuber induction was transmitted through grafts. In addition, StCO affected the mRNA levels of StBEL5 - a tuberization promoter, the mRNA of which moves long distances in potato plants - and StFT/StSP6A, a protein highly similar to FLOWERING LOCUS T (FT), which is a key component of systemic flowering signals in other species. We also found that StFT/StSP6A transcript levels correlate with the induction of tuber formation in wild-type plants. These results show that StCO plays an important role in photoperiodic tuberization and, together with the recent demonstration that StFT/StSP6A promotes tuberization, indicate that the CO/FT module participates in controlling this process. Moreover, they support the notion that StCO is involved in the expression of long-distance regulatory signals in potato, as CO does in other species.

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.

Graft-transmissible induction of potato tuberization by the microRNA miR172.

The photoreceptor phytochrome B (PHYB) and the homeodomain protein BEL5 are involved in the response of potato tuber induction to the photoperiod. However, whether they act in the same tuberization pathway is unknown. Here we show the effect of a microRNA, miR172, on this developmental event. miR172 levels are higher under tuber-inducing short days than under non-inductive long days and are upregulated in stolons at the onset of tuberization. Overexpression of this microRNA in potato promotes flowering, accelerates tuberization under moderately inductive photoperiods and triggers tuber formation under long days. In plants with a reduced abundance of PHYB, which tuberize under long days, both BEL5 mRNA and miR172 levels are reduced in leaves and increased in stolons. This, together with the presence of miR172 in vascular bundles and the graft transmissibility of its effect on tuberization, indicates that either miR172 might be mobile or it regulates long-distance signals to induce tuberization. Consistent with this, plants overexpressing miR172 show increased levels of BEL5 mRNA, which has been reported to be transmissible through grafts. Furthermore, we identify an APETALA2-like mRNA containing a miR172 binding site, which is downregulated in plants overexpressing miR172 and plants in which PHYB is silenced. Altogether, our results suggest that miR172 probably acts downstream of the tuberization repressor PHYB and upstream of the tuberization promoter BEL5 and allow us to propose a model for the control of tuberization by PHYB, miR172 and BEL5.

Ectopic expression of mitochondrial gamma carbonic anhydrase 2 causes male sterility by anther indehiscence.

Plant mitochondria include gamma-type carbonic anhydrases (gammaCAs) of unknown function. In Arabidopsis, the gammaCAs form a gene family of five members which all are attached to the NADH dehydrogenase complex (complex I) of the respiratory chain. Here we report a functional analysis of gamma carbonic anhydrase 2 (CA2). The gene encoding CA2 is constitutively expressed in all plant organs investigated but it is ten fold induced in flowers, particularly in tapetal tissue. Ectopic expression of CA2 in Arabidopsis causes male sterility in transgenic plants. In normal anther development, secondary thickenings of the endothecial cell wall cause anthers to open upon dehydration. Histological analyses revealed that abnormal secondary thickening prevents anther opening in 35S::CA2 transgenic plants. CA2 abundance in transgenic plants is increased 2-3 fold compared to wild-type plants as revealed by Western blotting analyses. Moreover, abundance of other members of the CA family, termed CA3 and CAL2, is increased in transgenic plants. Oxygen uptake measurements revealed that respiration in transgenic plants is mainly based on NADH reduction by the alternative NADH dehydrogenases present in plant mitochondria. Furthermore, the formation of reactive oxygen species (ROS) is very low in transgenic plants. We propose that reduction in ROS inhibits H(2)O(2) dependent lignin polymerization in CA2 over-expressing plants, thereby causing male sterility.

Gamma carbonic anhydrases in plant mitochondria.

Three genes from Arabidopsis thaliana with high sequence similarity to gamma carbonic anhydrase (gammaCA), a Zn containing enzyme from Methanosarcina thermophila (CAM), were identified and characterized. Evolutionary and structural analyses predict that these genes code for active forms of gammaCA. Phylogenetic analyses reveal that these Arabidopsis gene products cluster together with CAM and related sequences from alpha and gamma proteobacteria, organisms proposed as the mitochondrial endosymbiont ancestor. Indeed, in vitro and in vivo experiments indicate that these gene products are transported into the mitochondria as occurs with several mitochondrial protein genes transferred, during evolution, from the endosymbiotic bacteria to the host genome. Moreover, putative CAM orthologous genes are detected in other plants and green algae and were predicted to be imported to mitochondria. Structural modeling and sequence analysis performed in more than a hundred homologous sequences show a high conservation of functionally important active site residues. Thus, the three histidine residues involved in Zn coordination (His 81, 117 and 122), Arg 59, Asp 61, Gin 75, and Asp 76 of CAM are conserved and properly arranged in the active site cavity of the models. Two other functionally important residues (Glu 62 and Glu 84 of CAM) are lacking, but alternative amino acids that might serve to their roles are postulated. Accordingly, we propose that photosynthetic eukaryotic organisms (green algae and plants) contain gammaCAs and that these enzymes codified by nuclear genes are imported into mitochondria to accomplish their biological function.

Gamma carbonic anhydrase like complex interact with plant mitochondrial complex I.

We report the identification by two hybrid screens of two novel similar proteins, called Arabidopsis thaliana gamma carbonic anhydrase like1 and 2 (AtgammaCAL1 and AtgammaCAL2), that interact specifically with putative Arabidopsis thaliana gamma Carbonic Anhydrase (AtgammaCA) proteins in plant mitochondria. The interaction region that was located in the N-terminal 150 amino acids of mature AtgammaCA and AtgammaCA like proteins represents a new interaction domain. In vitro experiments indicate that these proteins are imported into mitochondria and are associated with mitochondrial complex I as AtgammaCAs. All plant species analyzed contain both AtgammaCA and AtgammaCAL sequences indicating that these genes were conserved throughout plant evolution. Structural modeling of AtgammaCAL sequences show a deviation of functionally important active site residues with respect to gammaCAs but could form active interfaces in the interaction with AtgammaCAs. We postulate a CA complex tightly associated to plant mitochondrial complex.

A mitochondrial dysfunction induces the expression of nuclear-encoded complex I genes in engineered male sterile Arabidopsis thaliana.

To study the effect of a mitochondrial dysfunction induced by the expression of the unedited form of the subunit 9 of ATP synthase gene (u-atp9) in Arabidopsis, we constructed transgenic plants expressing u-atp9 under the control of three different promoters: CaMV 35S, apetala 3 and A9. The size and shape of transgenic plants bearing the apetala3::u-atp9 and A9::u-atp9 genes looked normal while the 35S::u-atp9 transformed plants showed a dwarf morphology. All u-atp9 expressing plants, independent of the promoter used, exhibited a male sterile phenotype. Molecular analysis of male sterile plants revealed the induction of the mitochondrial nuclear complex I (nCI) genes, psst, tyky and nadh binding protein (nadhbp), associated with a mitochondrial dysfunction. These results support the hypothesis that the expression of u-atp9 can induce male sterility and reveal that the apetala3::u-atp9 and A9::u-atp9 plants induced the sterile phenotype without affecting the vegetative development of Arabidopsis plants. Moreover, male sterile plants produced by this procedure are an interesting model to study the global changes generated by an engineered mitochondrial dysfunction at the transcriptome and proteome levels in Arabidopsis plants.