A Solanum lycopersicum polyamine oxidase contributes to the control of plant growth, xylem differentiation, and drought stress tolerance.

Polyamines are involved in several plant physiological processes. In Arabidopsis thaliana, five FAD-dependent polyamine oxidases (AtPAO1 to AtPAO5) contribute to polyamine homeostasis. AtPAO5 catalyzes the back-conversion of thermospermine (T-Spm) to spermidine and plays a role in plant development, xylem differentiation, and abiotic stress tolerance. In the present study, to verify whether T-Spm metabolism can be exploited as a new route to improve stress tolerance in crops and to investigate the underlying mechanisms, tomato (Solanum lycopersicum) AtPAO5 homologs were identified (SlPAO2, SlPAO3, and SlPAO4) and CRISPR/Cas9-mediated loss-of-function slpao3 mutants were obtained. Morphological, molecular, and physiological analyses showed that slpao3 mutants display increased T-Spm levels and exhibit changes in growth parameters, number and size of xylem elements, and expression levels of auxin- and gibberellin-related genes compared to wild-type plants. The slpao3 mutants are also characterized by improved tolerance to drought stress, which can be attributed to a diminished xylem hydraulic conductivity that limits water loss, as well as to a reduced vulnerability to embolism. Altogether, this study evidences conservation, though with some significant variations, of the T-Spm-mediated regulatory mechanisms controlling plant growth and differentiation across different plant species and highlights the T-Spm role in improving stress tolerance while not constraining growth.

Histone Deacetylase Complex 1 and histone 1 epigenetically moderate stress responsiveness of Arabidopsis thaliana seedlings.

Early responses of plants to environmental stress factors prevent damage but can delay growth and development in fluctuating conditions. Optimising these trade-offs requires tunability of plant responsiveness to environmental signals. We have previously reported that Histone Deacetylase Complex 1 (HDC1), which interacts with multiple proteins in histone deacetylation complexes, regulates the stress responsiveness of Arabidopsis seedlings, but the underlying mechanism remained elusive. Here, we show that HDC1 attenuates transcriptome re-programming in salt-treated seedlings, and we identify two genes (LEA and MAF5) that inhibit seedling establishment under salt stress downstream of HDC1. HDC1 attenuates their transcriptional induction by salt via a dual mechanism involving H3K9/14 deacetylation and H3K27 trimethylation. The latter, but not the former, was also abolished in a triple knockout mutant of the linker histone H1, which partially mimics the hypersensitivity of the hdc1-1 mutant to salt stress. Although stress-induced H3K27me3 accumulation required both H1 and HDC1, it was not fully recovered by complementing hdc1-1 with a truncated, H1-binding competent HDC1 suggesting other players or independent inputs. The combined findings reveal a dual brake function of HDC1 via regulating both active and repressive epigenetic marks on stress-inducible genes. This natural 'anti-panic' device offers a molecular leaver to tune stress responsiveness in plants.

Correction: The bZIP transcription factor AREB3 mediates FT signalling and floral transition at the Arabidopsis shoot apical meristem.

[This corrects the article DOI: 10.1371/journal.pgen.1010766.].

Rice florigens control a common set of genes at the shoot apical meristem including the F-BOX BROADER TILLER ANGLE 1 that regulates tiller angle and spikelet development.

Rice flowering is triggered by transcriptional reprogramming at the shoot apical meristem (SAM) mediated by florigenic proteins produced in leaves in response to changes in photoperiod. Florigens are more rapidly expressed under short days (SDs) compared to long days (LDs) and include the HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) phosphatidylethanolamine binding proteins. Hd3a and RFT1 are largely redundant at converting the SAM into an inflorescence, but whether they activate the same target genes and convey all photoperiodic information that modifies gene expression at the SAM is currently unclear. We uncoupled the contribution of Hd3a and RFT1 to transcriptome reprogramming at the SAM by RNA sequencing of dexamethasone-inducible over-expressors of single florigens and wild-type plants exposed to photoperiodic induction. Fifteen highly differentially expressed genes common to Hd3a, RFT1, and SDs were retrieved, 10 of which still uncharacterized. Detailed functional studies on some candidates revealed a role for LOC_Os04g13150 in determining tiller angle and spikelet development and the gene was renamed BROADER TILLER ANGLE 1 (BRT1). We identified a core set of genes controlled by florigen-mediated photoperiodic induction and defined the function of a novel florigen target controlling tiller angle and spikelet development.

The bZIP transcription factor AREB3 mediates FT signalling and floral transition at the Arabidopsis shoot apical meristem.

The floral transition occurs at the shoot apical meristem (SAM) in response to favourable external and internal signals. Among these signals, variations in daylength (photoperiod) act as robust seasonal cues to activate flowering. In Arabidopsis, long-day photoperiods stimulate production in the leaf vasculature of a systemic florigenic signal that is translocated to the SAM. According to the current model, FLOWERING LOCUS T (FT), the main Arabidopsis florigen, causes transcriptional reprogramming at the SAM, so that lateral primordia eventually acquire floral identity. FT functions as a transcriptional coregulator with the bZIP transcription factor FD, which binds DNA at specific promoters. FD can also interact with TERMINAL FLOWER 1 (TFL1), a protein related to FT that acts as a floral repressor. Thus, the balance between FT-TFL1 at the SAM influences the expression levels of floral genes targeted by FD. Here, we show that the FD-related bZIP transcription factor AREB3, which was previously studied in the context of phytohormone abscisic acid signalling, is expressed at the SAM in a spatio-temporal pattern that strongly overlaps with FD and contributes to FT signalling. Mutant analyses demonstrate that AREB3 relays FT signals redundantly with FD, and the presence of a conserved carboxy-terminal SAP motif is required for downstream signalling. AREB3 shows unique and common patterns of expression with FD, and AREB3 expression levels are negatively regulated by FD thus forming a compensatory feedback loop. Mutations in another bZIP, FDP, further aggravate the late flowering phenotypes of fd areb3 mutants. Therefore, multiple florigen-interacting bZIP transcription factors have redundant functions in flowering at the SAM.

Two florigens and a florigen-like protein form a triple regulatory module at the shoot apical meristem to promote reproductive transitions in rice.

Many plant species monitor and respond to changes in day length (photoperiod) for aligning reproduction with a favourable season. Day length is measured in leaves and, when appropriate, leads to the production of floral stimuli called florigens that are transmitted to the shoot apical meristem to initiate inflorescence development1. Rice possesses two florigens encoded by HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1)2. Here we show that the arrival of Hd3a and RFT1 at the shoot apical meristem activates FLOWERING LOCUS T-LIKE 1 (FT-L1), encoding a florigen-like protein that shows features partially differentiating it from typical florigens. FT-L1 potentiates the effects of Hd3a and RFT1 during the conversion of the vegetative meristem into an inflorescence meristem and organizes panicle branching by imposing increasing determinacy to distal meristems. A module comprising Hd3a, RFT1 and FT-L1 thus enables the initiation and balanced progression of panicle development towards determinacy.

A Method for Rapid and Reliable Molecular Detection of Drought-Response Genes in Sorghum bicolor (L.) Moench Roots.

Drought is a major environmental stress that limits growth and productivity in agricultural ecosystems limiting crop yield worldwide. Breeding crops for enhanced drought tolerance is a priority to preserve food security on the increasing world population. Recent work in Arabidopsis has shown that vascular brassinosteroid receptor BRL3 (Brassinosteroid insensitive like-3) transcriptionally controls the production of osmoprotectant metabolites that confer drought resistance without penalizing growth, offering new and exciting possibilities for biotechnological improvement of drought-resistant crops. In cereals, understanding transcriptional responses to drought is an essential step for the production of gene-edited drought-resistant cereals. In this chapter, we present a method to analyze the transcriptional responses to drought in Sorghum bicolor (L.) Moench, our cereal of choice. Among the genes we tested, we found that drought marker gene SbDHN1 has a 1000-fold increase only after 1 day of drought, bringing possibilities for the development of molecular sensors for testing drought. Overall, this analysis is useful to set up conditions of high-throughput transcriptomic analysis of drought stressed plants before drought phenotype is observed.

GIGANTEA Is a Negative Regulator of Abscisic Acid Transcriptional Responses and Sensitivity in Arabidopsis.

Transcriptional reprogramming plays a key role in drought stress responses, preceding the onset of morphological and physiological acclimation. The best-characterized signal regulating gene expression in response to drought is the phytohormone abscisic acid (ABA). ABA-regulated gene expression, biosynthesis and signaling are highly organized in a diurnal cycle, so that ABA-regulated physiological traits occur at the appropriate time of day. The mechanisms that underpin such diel oscillations in ABA signals are poorly characterized. Here we uncover GIGANTEA (GI) as a key gatekeeper of ABA-regulated transcriptional and physiological responses. Time-resolved gene expression profiling by RNA sequencing under different irrigation scenarios indicates that gi mutants produce an exaggerated ABA response, despite accumulating wild-type levels of ABA. Comparisons with ABA-deficient mutants confirm the role of GI in controlling ABA-regulated genes, and the analysis of leaf temperature, a read-out for transpiration, supports a role for GI in the control of ABA-regulated physiological processes. Promoter regions of GI/ABA-regulated transcripts are directly targeted by different classes of transcription factors (TFs), especially PHYTOCHROME-INTERACTING FACTOR and -BINDING FACTOR, together with GI itself. We propose a model whereby diel changes in GI control oscillations in ABA responses. Peak GI accumulation at midday contributes to establishing a phase of reduced ABA sensitivity and related physiological responses, by gating DNA binding or function of different classes of TFs that cooperate or compete with GI at target regions.

Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2.

Free asparagine is the precursor for acrylamide, which forms during the baking, toasting and high-temperature processing of foods made from wheat. In this study, CRISPR/Cas9 was used to knock out the asparagine synthetase gene, TaASN2, of wheat (Triticum aestivum) cv. Cadenza. A 4-gRNA polycistronic gene was introduced into wheat embryos by particle bombardment and plants were regenerated. T1 plants derived from 11 of 14 T0 plants were shown to carry edits. Most edits were deletions (up to 173 base pairs), but there were also some single base pair insertions and substitutions. Editing continued beyond the T1 generation. Free asparagine concentrations in the grain of plants carrying edits in all six TaASN2 alleles (both alleles in each genome) were substantially reduced compared with wildtype, with one plant showing a more than 90 % reduction in the T2 seeds. A plant containing edits only in the A genome alleles showed a smaller reduction in free asparagine concentration in the grain, but the concentration was still lower than in wildtype. Free asparagine concentration in the edited plants was also reduced as a proportion of the free amino acid pool. Free asparagine concentration in the T3 seeds remained substantially lower in the edited lines than wildtype, although it was higher than in the T2 seeds, possibly due to stress. In contrast, the concentrations of free glutamine, glutamate and aspartate were all higher in the edited lines than wildtype. Low asparagine seeds showed poor germination but this could be overcome by exogenous application of asparagine.

Structural determinants for NF-Y subunit organization and NF-Y/DNA association in plants.

NF-Y transcription factor comprises three subunits: NF-YA, NF-YB and NF-YC. NF-YB and NF-YC dimerize through their histone fold domain (HFD), which can bind DNA in a non-sequence-specific fashion while serving as a scaffold for NF-YA trimerization. Upon trimerization, NF-YA specifically recognizes the CCAAT box sequence on promoters and enhancers. In plants, each NF-Y subunit is encoded by several genes giving rise to hundreds of potential heterotrimeric combinations. In addition, plant NF-YBs and NF-YCs interact with other protein partners to recognize a plethora of genomic motifs, as the CCT protein family that binds CORE sites. The NF-Y subunit organization and its DNA-binding properties, together with the NF-Y HFD capacity to adapt different protein modules, represent plant-specific features that play a key role in development, growth and reproduction. Despite their relevance, these features are still poorly understood at the molecular level. Here, we present the structures of Arabidopsis and rice NF-YB/NF-YC dimers, and of an Arabidopsis NF-Y trimer in complex with the FT CCAAT box, together with biochemical data on NF-Y mutants. The dimeric structures identify the key residues for NF-Y HFD stabilization. The NF-Y/DNA structure and the mutation experiments shed light on HFD trimerization interface properties and the NF-YA sequence appetite for the bases flanking the CCAAT motif. These data explain the logic of plant NF-Y gene expansion: the trimerization adaptability and the flexible DNA-binding rules serve the scopes of accommodating the large number of NF-YAs, CCTs and possibly other NF-Y HFD binding partners and a diverse audience of genomic motifs.

Abscisic Acid and Flowering Regulation: Many Targets, Different Places.

Plants can react to drought stress by anticipating flowering, an adaptive strategy for plant survival in dry climates known as drought escape (DE). In Arabidopsis, the study of DE brought to surface the involvement of abscisic acid (ABA) in controlling the floral transition. A central question concerns how and in what spatial context can ABA signals affect the floral network. In the leaf, ABA signaling affects flowering genes responsible for the production of the main florigen FLOWERING LOCUS T (FT). At the shoot apex, FD and FD-like transcription factors interact with FT and FT-like proteins to regulate ABA responses. This knowledge will help separate general and specific roles of ABA signaling with potential benefits to both biology and agriculture.

The Four FAD-Dependent Histone Demethylases of Arabidopsis Are Differently Involved in the Control of Flowering Time.

In Arabidopsis thaliana, four FAD-dependent lysine-specific histone demethylases (LDL1, LDL2, LDL3, and FLD) are present, bearing both a SWIRM and an amine oxidase domain. In this study, a comparative analysis of gene structure, evolutionary relationships, tissue- and organ-specific expression patterns, physiological roles and target genes for the four Arabidopsis LDL/FLDs is reported. Phylogenetic analysis evidences a different evolutionary history for the four LDL/FLDs, while promoter activity data show that LDL/FLDs are strongly expressed during plant development and embryogenesis, with some gene-specific expression patterns. Furthermore, phenotypical analysis of loss-of-function mutants indicates a role of all four Arabidopsis LDL/FLD genes in the control of flowering time, though for some of them with opposing effects. This study contributes toward a better understanding of the LDL/FLD physiological roles and may provide biotechnological strategies for crop improvement.

Drought Resistance by Engineering Plant Tissue-Specific Responses.

Drought is the primary cause of agricultural loss globally, and represents a major threat to food security. Currently, plant biotechnology stands as one of the most promising fields when it comes to developing crops that are able to produce high yields in water-limited conditions. From studies of Arabidopsis thaliana whole plants, the main response mechanisms to drought stress have been uncovered, and multiple drought resistance genes have already been engineered into crops. So far, most plants with enhanced drought resistance have displayed reduced crop yield, meaning that there is still a need to search for novel approaches that can uncouple drought resistance from plant growth. Our laboratory has recently shown that the receptors of brassinosteroid (BR) hormones use tissue-specific pathways to mediate different developmental responses during root growth. In Arabidopsis, we found that increasing BR receptors in the vascular plant tissues confers resistance to drought without penalizing growth, opening up an exceptional opportunity to investigate the mechanisms that confer drought resistance with cellular specificity in plants. In this review, we provide an overview of the most promising phenotypical drought traits that could be improved biotechnologically to obtain drought-tolerant cereals. In addition, we discuss how current genome editing technologies could help to identify and manipulate novel genes that might grant resistance to drought stress. In the upcoming years, we expect that sustainable solutions for enhancing crop production in water-limited environments will be identified through joint efforts.

Antagonistic Transcription Factor Complexes Modulate the Floral Transition in Rice.

Plants measure day or night lengths to coordinate specific developmental changes with a favorable season. In rice (Oryza sativa), the reproductive phase is initiated by exposure to short days when expression of HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1) is induced in leaves. The cognate proteins are components of the florigenic signal and move systemically through the phloem to reach the shoot apical meristem (SAM). In the SAM, they form a transcriptional activation complex with the bZIP transcription factor OsFD1 to start panicle development. Here, we show that Hd3a and RFT1 can form transcriptional activation or repression complexes also in leaves and feed back to regulate their own transcription. Activation complexes depend on OsFD1 to promote flowering. However, additional bZIPs, including Hd3a BINDING REPRESSOR FACTOR1 (HBF1) and HBF2, form repressor complexes that reduce Hd3a and RFT1 expression to delay flowering. We propose that Hd3a and RFT1 are also active locally in leaves to fine-tune photoperiodic flowering responses.

Transcriptional and Post-transcriptional Mechanisms Limit Heading Date 1 (Hd1) Function to Adapt Rice to High Latitudes.

Rice flowering is controlled by changes in the photoperiod that promote the transition to the reproductive phase as days become shorter. Natural genetic variation for flowering time has been largely documented and has been instrumental to define the genetics of the photoperiodic pathway, as well as providing valuable material for artificial selection of varieties better adapted to local environments. We mined genetic variation in a collection of rice varieties highly adapted to European regions and isolated distinct variants of the long day repressor HEADING DATE 1 (Hd1) that perturb its expression or protein function. Specific variants allowed us to define novel features of the photoperiodic flowering pathway. We demonstrate that a histone fold domain scaffold formed by GRAIN YIELD, PLANT HEIGHT AND HEADING DATE 8 (Ghd8) and several NF-YC subunits can accommodate distinct proteins, including Hd1 and PSEUDO RESPONSE REGULATOR 37 (PRR37), and that the resulting OsNF-Y complex containing Hd1 can bind a specific sequence in the promoter of HEADING DATE 3A (Hd3a). Artificial selection has locally favored an Hd1 variant unable to assemble in such heterotrimeric complex. The causal polymorphism was defined as a single conserved lysine in the CCT domain of the Hd1 protein. Our results indicate how genetic variation can be stratified and explored at multiple levels, and how its description can contribute to the molecular understanding of basic developmental processes.

A plant spermine oxidase/dehydrogenase regulated by the proteasome and polyamines.

Polyamine oxidases (PAOs) are flavin-dependent enzymes involved in polyamine catabolism. In Arabidopsis five PAO genes (AtPAO1-AtPAO5) have been identified which present some common characteristics, but also important differences in primary structure, substrate specificity, subcellular localization, and tissue-specific expression pattern, differences which may suggest distinct physiological roles. In the present work, AtPAO5, the only so far uncharacterized AtPAO which is specifically expressed in the vascular system, was partially purified from 35S::AtPAO5-6His Arabidopsis transgenic plants and biochemically characterized. Data presented here allow AtPAO5 to be classified as a spermine dehydrogenase. It is also shown that AtPAO5 oxidizes the polyamines spermine, thermospermine, and N(1)-acetylspermine, the latter being the best in vitro substrate of the recombinant enzyme. AtPAO5 also oxidizes these polyamines in vivo, as was evidenced by analysis of polyamine levels in the 35S::AtPAO5-6His Arabidopsis transgenic plants, as well as in a loss-of-function atpao5 mutant. Furthermore, subcellular localization studies indicate that AtPAO5 is a cytosolic protein undergoing proteasomal control. Positive regulation of AtPAO5 expression by polyamines at the transcriptional and post-transcriptional level is also shown. These data provide new insights into the catalytic properties of the PAO gene family and the complex regulatory network controlling polyamine metabolism.

Compensatory expression and substrate inducibility of gamma-glutamyl transferase GGT2 isoform in Arabidopsis thaliana.

γ-Glutamyl transferases (GGT; EC 2.3.2.2) are glutathione-degrading enzymes that are represented in Arabidopsis thaliana by a small gene family of four members. Two isoforms, GGT1 and GGT2, are apoplastic, sharing broad similarities in their amino acid sequences, but they are differently expressed in the tissues: GGT1 is expressed in roots, leaves, and siliques, while GGT2 was thought to be expressed only in siliques. It is demonstrated here that GGT2 is also expressed in wild-type roots, albeit in very small amounts. GGT2 expression is enhanced in ggt1 knockout mutants, suggesting a compensatory effect to restore GGT activity in the root apoplast. Supplementation with 100 µM glutathione (GSH) resulted in the up-regulation of GGT2 gene expression in wild-type and ggt1 knockout roots, and of GGT1 gene expression in wild-type roots. Glutathione recovery was hampered by the GGT inhibitor serine/borate, suggesting a major role for apoplastic GGTs in this process. These findings can explain the ability of ggt1 knockout mutants to retrieve exogenously added glutathione from the growth medium.