Phenotypic diversity and distinctiveness of the Belltall garlic landrace.

Among the Mediterranean horticultural landraces, garlic is one of the crops most threatened by genetic erosion. Due to its sexual sterility and to the incidence of seed-borne diseases, historical varieties have been widely replaced by commercial cultivars. In Catalonia, despite the historical relevance of the crop, solely the Belltall garlic landrace is cultivated for commercial purposes. To assess the genotypic and phenotypic diversity within the Belltall garlic, we evaluated sixteen local accessions and five recognized traditional and modern varieties as controls. Genetic analysis with SSR and InDel markers showed low genetic diversity within the Belltall population, grouping modern and traditional varieties separately. Farmers and consumers were involved in the definition of the landrace ideotype and classified the materials by means of projective mapping. Scant phenotypic diversity was found within the Belltall landrace, which is characterized by its color profile and the small size of bulb and cloves. The Belltall landrace grown outside its area of origin lost the distinctive quality signals that differentiate the landrace from the commercial cultivars (clove appearance), indicating that the high quality of the landrace is under genotype-by-environment effects (i.e. local adaptation). Moreover, the size of the Belltall sowing clove had a strong effect on the harvested bulb size. Our research represents a case study for the description of the variability within garlic landraces and an approach to quantify the phenomenon of local adaptation that currently drives their conservation.

Light signals generated by vegetation shade facilitate acclimation to low light in shade-avoider plants.

When growing in search for light, plants can experience continuous or occasional shading by other plants. Plant proximity causes a decrease in the ratio of R to far-red light (low R:FR) due to the preferential absorbance of R light and reflection of FR light by photosynthetic tissues of neighboring plants. This signal is often perceived before actual shading causes a reduction in photosynthetically active radiation (low PAR). Here, we investigated how several Brassicaceae species from different habitats respond to low R:FR and low PAR in terms of elongation, photosynthesis, and photoacclimation. Shade-tolerant plants such as hairy bittercress (Cardamine hirsuta) displayed a good adaptation to low PAR but a poor or null response to low R:FR exposure. In contrast, shade-avoider species, such as Arabidopsis (Arabidopsis thaliana), showed a weak photosynthetic performance under low PAR but they strongly elongated when exposed to low R:FR. These responses could be genetically uncoupled. Most interestingly, exposure to low R:FR of shade-avoider (but not shade-tolerant) plants improved their photoacclimation to low PAR by triggering changes in photosynthesis-related gene expression, pigment accumulation, and chloroplast ultrastructure. These results indicate that low R:FR signaling unleashes molecular, metabolic, and developmental responses that allow shade-avoider plants (including most crops) to adjust their photosynthetic capacity in anticipation of eventual shading by nearby plants.

MADS-Box and bHLH Transcription Factors Coordinate Transmitting Tract Development in Arabidopsis thaliana.

The MADS-domain transcription factor SEEDSTICK (STK) controls several aspects of plant reproduction. STK is co-expressed with CESTA (CES), a basic Helix-Loop-Helix (bHLH) transcription factor-encoding gene. CES was reported to control redundantly with the brassinosteroid positive signaling factors BRASSINOSTEROID ENHANCED EXPRESSION1 (BEE1) and BEE3 the development of the transmitting tract. Combining the stk ces-4 mutants led to a reduction in ovule fertilization due to a defect in carpel fusion which, caused the formation of holes at the center of the septum where the transmitting tract differentiates. Combining the stk mutant with the bee1 bee3 ces-4 triple mutant showed an increased number of unfertilized ovules and septum defects. The transcriptome profile of this quadruple mutant revealed a small subset of differentially expressed genes which are mainly involved in cell death, extracellular matrix and cell wall development. Our data evidence a regulatory gene network controlling transmitting tract development regulated directly or indirectly by a STK-CES containing complex and reveal new insights in the regulation of transmitting tract development by bHLH and MADS-domain transcription factors.

BPC transcription factors and a Polycomb Group protein confine the expression of the ovule identity gene SEEDSTICK in Arabidopsis.

The BASIC PENTACYSTEINE (BPC) GAGA (C-box) binding proteins belong to a small plant transcription factor family. We previously reported that class I BPCs bind directly to C-boxes in the SEEDSTICK (STK) promoter and the mutagenesis of these cis-elements affects STK expression in the flower. The MADS-domain factor SHORT VEGETATIVE PHASE (SVP) is another key regulator of STK. Direct binding of SVP to CArG-boxes in the STK promoter are required to repress its expression during the first stages of flower development. Here we show that class II BPCs directly interact with SVP and that MADS-domain binding sites in the STK promoter region are important for the correct spatial and temporal expression of this homeotic gene. Furthermore, we show that class I and class II BPCs act redundantly to repress STK expression in the flower, most likely by recruiting TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 (TFL2/LHP1) and mediating the establishment and the maintenance of H3K27me3 repressive marks on DNA. We investigate the role of LHP1 in the regulation of STK expression. In addition to providing a better understanding of the role of BPC transcription factors in the regulation of STK expression, our results suggest the existence of a more general regulatory complex composed of BPCs, MADS-domain factors and Polycomb Repressive Complexes that co-operate to regulate gene expression in reproductive tissues. We believe that our data along with the molecular model described here could provide significant insights for a more comprehensive understanding of gene regulation in plants.

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.

Photoreceptor Activity Contributes to Contrasting Responses to Shade in Cardamine and Arabidopsis Seedlings.

Plants have evolved two major ways to deal with nearby vegetation or shade: avoidance and tolerance. Moreover, some plants respond to shade in different ways; for example, Arabidopsis (Arabidopsis thaliana) undergoes an avoidance response to shade produced by vegetation, but its close relative Cardamine hirsuta tolerates shade. How plants adopt opposite strategies to respond to the same environmental challenge is unknown. Here, using a genetic strategy, we identified the C. hirsuta slender in shade1 mutants, which produce strongly elongated hypocotyls in response to shade. These mutants lack the phytochrome A (phyA) photoreceptor. Our findings suggest that C. hirsuta has evolved a highly efficient phyA-dependent pathway that suppresses hypocotyl elongation when challenged by shade from nearby vegetation. This suppression relies, at least in part, on stronger phyA activity in C. hirsuta; this is achieved by increased ChPHYA expression and protein accumulation combined with a stronger specific intrinsic repressor activity. We suggest that modulation of photoreceptor activity is a powerful mechanism in nature to achieve physiological variation (shade tolerance versus avoidance) for species to colonize different habitats.

Shade Avoidance and Neighbor Detection.

Plants detect neighboring vegetation as potential competitors for resources. Vegetation proximity is perceived by changes in the red (R) to far-red (FR) ratio (R:FR) through the phytochrome photoreceptors. To face this challenge, many plants have evolved the strategy to avoid shade, displaying a series of responses known as the shade avoidance syndrome (SAS). The SAS responses have been mostly studied at the seedling stage, and cover hypocotyl elongation as well as cotyledon and primary leaf expansion. In adult stages, SAS responses include an increase in petiole elongation and a decrease in leaf expansion, and an increase in plant height. Thus, the analysis of these responses provides a valuable and simple way to study how vegetation proximity affects plant development in both seedlings and adult plants. Here we describe a simple protocol to simulate shade in the laboratory and to evaluate these responses. Overall, our protocol can be easily used to expand the set of SAS responses of plants at different stages of development.

Approaches to Study Light Effects on Brassinosteroid Sensitivity.

Light perception and hormone signaling in plants are likely connected at multiple points. Light conditions, perceived by photoreceptors, control plant responses by altering hormone concentration, tissue sensitivity, or a combination of both. Whereas it is relatively straightforward to assess the light effects on hormone levels, hormone sensitivity is subjected to interpretation. In Arabidopsis thaliana seedlings, hypocotyl length is strongly affected by light conditions. As hypocotyl elongation also depends on brassinosteroids (BRs), assaying this response provides a valuable and easy way to measure the responsiveness of seedlings to BRs and the impact of light. We describe a simple protocol to evaluate the responsiveness of hypocotyls to commercial BRs and/or BR inhibitors under a range of light conditions. These assays can be used to establish whether light affects BR sensitivity or whether BRs affect light sensitivity. Overall, our protocol can be easily applied for deetiolation (under polychromatic or monochromatic light) and simulated shade treatments combined with BR treatments.

Seed abscission and fruit dehiscence required for seed dispersal rely on similar genetic networks.

Seed dispersal is an essential trait that enables colonization of new favorable habitats, ensuring species survival. In plants with dehiscent fruits, such as Arabidopsis, seed dispersal depends on two processes: the separation of the fruit valves that protect the seeds (fruit dehiscence) and the detachment of the seeds from the funiculus connecting them to the mother plant (seed abscission). The key factors required to establish a proper lignin pattern for fruit dehiscence are SHATTERPROOF 1 and 2 (SHP1 and SHP2). Here, we demonstrate that the SHP-related gene SEEDSTICK (STK) is a key factor required to establish the proper lignin pattern in the seed abscission zone but in an opposite way. We show that STK acts as a repressor of lignin deposition in the seed abscission zone through the direct repression of HECATE3, whereas the SHP proteins promote lignin deposition in the valve margins by activating INDEHISCENT. The interaction of STK with the SEUSS co-repressor determines the difference in the way STK and SHP proteins control the lignification patterns. Despite this difference in the molecular control of lignification during seed abscission and fruit dehiscence, we show that the genetic networks regulating these two developmental pathways are highly conserved.

Plant Responses to Vegetation Proximity: A Whole Life Avoiding Shade.

In high density of vegetation, plants detect neighbors by perceiving changes in light quality through phytochrome photoreceptors. Close vegetation proximity might result in competition for resources, such as light. To face this challenge, plants have evolved two alternative strategies: to either tolerate or avoid shade. Shade-avoiding species generally adapt their development by inducing hypocotyl, stem, and petiole elongation, apical dominance and flowering, and decreasing leaf expansion and yield, a set of responses collectively known as the shade avoidance syndrome (SAS). The SAS responses have been mostly studied at the seedling stage, centered on the increase of hypocotyl elongation. After compiling the main findings about SAS responses in seedlings, this review is focused on the response to shade at adult stages of development, such as petioles of adult leaves, and the little information available on the SAS responses in reproductive tissues. We discuss these responses based on the knowledge about the molecular mechanisms and components with a role in regulating the SAS response of the hypocotyls of Arabidopsis thaliana. The transcriptional networks involved in this process, as well as the communication among the tissues that perceive the shade and the ones that respond to this stimulus will also be briefly commented.

DRACULA2 is a dynamic nucleoporin with a role in regulating the shade avoidance syndrome in Arabidopsis.

When plants grow in close proximity basic resources such as light can become limiting. Under such conditions plants respond to anticipate and/or adapt to the light shortage, a process known as the shade avoidance syndrome (SAS). Following genetic screening using a shade-responsive luciferase reporter line (PHYB:LUC), we identified DRACULA2 (DRA2), which encodes an Arabidopsis homolog of mammalian nucleoporin 98, a component of the nuclear pore complex (NPC). DRA2, together with other nucleoporins, participates positively in the control of the hypocotyl elongation response to plant proximity, a role that can be considered dependent on the nucleocytoplasmic transport of macromolecules (i.e. is transport dependent). In addition, our results reveal a specific role for DRA2 in controlling shade-induced gene expression. We suggest that this novel regulatory role of DRA2 is transport independent and that it might rely on its dynamic localization within and outside of the NPC. These results provide mechanistic insights in to how SAS responses are rapidly established by light conditions. They also indicate that nucleoporins have an active role in plant signaling.

Current perspectives on the hormonal control of seed development in Arabidopsis and maize: a focus on auxin.

The seed represents the unit of reproduction of flowering plants, capable of developing into another plant, and to ensure the survival of the species under unfavorable environmental conditions. It is composed of three compartments: seed coat, endosperm and embryo. Proper seed development depends on the coordination of the processes that lead to seed compartments differentiation, development and maturation. The coordination of these processes is based on the constant transmission/perception of signals by the three compartments. Phytohormones constitute one of these signals; gradients of hormones are generated in the different seed compartments, and their ratios comprise the signals that induce/inhibit particular processes in seed development. Among the hormones, auxin seems to exert a central role, as it is the only one in maintaining high levels of accumulation from fertilization to seed maturation. The gradient of auxin generated by its PIN carriers affects several processes of seed development, including pattern formation, cell division and expansion. Despite the high degree of conservation in the regulatory mechanisms that lead to seed development within the Spermatophytes, remarkable differences exist during seed maturation between Monocots and Eudicots species. For instance, in Monocots the endosperm persists until maturation, and constitutes an important compartment for nutrients storage, while in Eudicots it is reduced to a single cell layer, as the expanding embryo gradually replaces it during the maturation. This review provides an overview of the current knowledge on hormonal control of seed development, by considering the data available in two model plants: Arabidopsis thaliana, for Eudicots and Zea mays L., for Monocots. We will emphasize the control exerted by auxin on the correct progress of seed development comparing, when possible, the two species.

Ovule development, a new model for lateral organ formation.

In spermatophytes the ovules upon fertilization give rise to the seeds. It is essential to understand the mechanisms that control ovule number and development as they ultimately determine the final number of seeds and, thereby, the yield in crop plants. In Arabidopsis thaliana, ovules arise laterally from a meristematic tissue within the carpel referred to as placenta. For a correct determination of the number of ovules, a precise establishment of the positions where ovule primordia emerge is needed, and a tight definition of the boundaries between ovules is therefore also required. In the last decades, few factors have been identified to be involved in the determination of ovule number. Recently, plant hormones have also been revealed as fundamental players in the control of the initiation of ovule formation. In this review we summarize the current knowledge about both the molecular and hormonal mechanisms that control ovule formation in Arabidopsis thaliana.

The bHLH proteins BEE and BIM positively modulate the shade avoidance syndrome in Arabidopsis seedlings.

The shade avoidance syndrome (SAS) refers to a set of plant responses initiated after perception by the phytochromes of light with a reduced red to far-red ratio, indicative of vegetation proximity or shade. These responses, including elongation growth, anticipate eventual shading from potential competitor vegetation by overgrowing neighboring plants or flowering to ensure production of viable seeds for the next generation. In Arabidopsis thaliana seedlings, the SAS includes dramatic changes in gene expression, such as induction of PHYTOCHROME RAPIDLY REGULATED 1 (PAR1), encoding an atypical basic helix-loop-helix (bHLH) protein that acts as a transcriptional co-factor to repress hypocotyl elongation. Indeed, PAR1 has been proposed to act fundamentally as a dominant negative antagonist of conventional bHLH transcription factors by forming heterodimers with them to prevent their binding to DNA or other transcription factors. Here we report the identification of PAR1-interacting factors, including the brassinosteroid signaling components BR-ENHANCED EXPRESSION (BEE) and BES1-INTERACTING MYC-LIKE (BIM), and characterize their role as networked positive regulators of SAS hypocotyl responses. We provide genetic evidence that these bHLH transcriptional regulators not only control plant growth and development under shade and non-shade conditions, but are also redundant in the control of plant viability. Our results suggest that SAS responses are initiated as a consequence of a new balance of transcriptional regulators within the pre-existing bHLH network triggered by plant proximity, eventually causing hypocotyls to elongate.

Maternal control of PIN1 is required for female gametophyte development in Arabidopsis.

Land plants are characterised by haplo-diploid life cycles, and developing ovules are the organs in which the haploid and diploid generations coexist. Recently it has been shown that hormones such as auxin and cytokinins play important roles in ovule development and patterning. The establishment and regulation of auxin levels in cells is predominantly determined by the activity of the auxin efflux carrier proteins PIN-FORMED (PIN). To study the roles of PIN1 and PIN3 during ovule development we have used mutant alleles of both genes and also perturbed PIN1 and PIN3 expression using micro-RNAs controlled by the ovule specific DEFH9 (DEFIFICENS Homologue 9) promoter. PIN1 down-regulation and pin1-5 mutation severely affect female gametophyte development since embryo sacs arrest at the mono- and/or bi-nuclear stages (FG1 and FG3 stage). PIN3 function is not required for ovule development in wild-type or PIN1-silenced plants. We show that sporophytically expressed PIN1 is required for megagametogenesis, suggesting that sporophytic auxin flux might control the early stages of female gametophyte development, although auxin response is not visible in developing embryo sacs.

Basic pentacysteine proteins mediate MADS domain complex binding to the DNA for tissue-specific expression of target genes in Arabidopsis.

Basic pentacysteine (BPC) transcription factors have been identified in a large variety of plant species. In Arabidopsis thaliana there are seven BPC genes, which, except for BPC5, are expressed ubiquitously. BPC genes are functionally redundant in a wide range of developmental processes. Recently, we reported that BPC1 binds to guanine and adenine (GA)-rich consensus sequences in the seedstick (STK) promoter in vitro and induces conformational changes. Here we show by chromatin immunoprecipitation experiments that in vivo BPCs also bind to the consensus boxes, and when these were mutated, expression from the STK promoter was derepressed, resulting in ectopic expression in the inflorescence. We also reveal that short vegetative phase (SVP) is a direct regulator of STK. SVP is a floral meristem identity gene belonging to the MADS box gene family. The SVP-APETALA1 (AP1) dimer recruits the SEUSS (SEU)-LEUNIG (LUG) transcriptional cosuppressor to repress floral homeotic gene expression in the floral meristem. Interestingly, we found that GA consensus sequences in the STK promoter to which BPCs bind are essential for recruitment of the corepressor complex to this promoter. Our data suggest that we have identified a new regulatory mechanism controlling plant gene expression that is probably generally used, when considering BPCs' wide expression profile and the frequent presence of consensus binding sites in plant promoters.

A dual mechanism controls nuclear localization in the atypical basic-helix-loop-helix protein PAR1 of Arabidopsis thaliana.

PAR1 is an atypical basic-helix-loop-helix (bHLH) protein that negatively regulates the shade avoidance syndrome in Arabidopsis thaliana acting as a transcriptional cofactor. Consistently with this function, PAR1 has to be in the nucleus to display biological activity. Previous structure-function analyses revealed that the N-terminal region of PAR1 drives the protein to the nucleus. However, truncated forms of PAR1 lacking this region still display biological activity, implying that PAR1 has additional mechanisms to localize into the nucleus. In this work, we compared the primary structure of PAR1 and various related and unrelated plant bHLH proteins, which led us to suggest that PAR1 contains a non-canonical nuclear localization signal (NLS) in the N-terminal region. By overexpressing truncated and mutated derivatives of PAR1, we have also investigated the importance of other regions of PAR1, such as the acidic and the extended HLH dimerization domains, for its nuclear localization. We found that, in the absence of the N-terminal region, a functional HLH domain is required for nuclear localization. Our results suggest the existence of a dual mechanism for PAR1 nuclear localization: (1) one mediated by the N-terminal non-consensus NLS and (2) a second one that involves interaction with other proteins via the dimerization domain.

A novel high-throughput in vivo molecular screen for shade avoidance mutants identifies a novel phyA mutation.

The shade avoidance syndrome (SAS) allows plants to anticipate and avoid shading by neighbouring plants by initiating an elongation growth response. The phytochrome photoreceptors are able to detect a reduction in the red:far red ratio in incident light, the result of selective absorption of red and blue wavelengths by proximal vegetation. A shade-responsive luciferase reporter line (PHYB::LUC) was used to carry out a high-throughput screen to identify novel SAS mutants. The dracula 1 (dra1) mutant, that showed no avoidance of shade for the PHYB::LUC response, was the result of a mutation in the PHYA gene. Like previously characterized phyA mutants, dra1 showed a long hypocotyl in far red light and an enhanced hypocotyl elongation response to shade. However, dra1 additionally showed a long hypocotyl in red light. Since phyB levels are relatively unaffected in dra1, this gain-of-function red light phenotype strongly suggests a disruption of phyB signalling. The dra1 mutation, G773E within the phyA PAS2 domain, occurs at a residue absolutely conserved among phyA sequences. The equivalent residue in phyB is absolutely conserved as a threonine. PAS domains are structurally conserved domains involved in molecular interaction. Structural modelling of the dra1 mutation within the phyA PAS2 domain shows some similarity with the structure of the phyB PAS2 domain, suggesting that the interference with phyB signalling may be the result of non-functional mimicry. Hence, it was hypothesized that this PAS2 residue forms a key distinction between the phyA and phyB phytochrome species.

Overlapping and antagonistic activities of BASIC PENTACYSTEINE genes affect a range of developmental processes in Arabidopsis.

The BASIC PENTACYSTEINE (BPC) proteins are a plant-specific transcription factor family that is present throughout land plants. The Arabidopsis BPC proteins have been categorized into three classes based on sequence similarity, and we demonstrate that there is functional overlap between classes. Single gene mutations produce no visible phenotypic effects, and severe morphological phenotypes occur only in higher order mutants between members of classes I and II, with the most severe phenotype observed in bpc1-1 bpc2 bpc4 bpc6 plants. These quadruple mutants are dwarfed and display small curled leaves, aberrant ovules, altered epidermal cells and reduced numbers of lateral roots. Affected processes include coordinated growth of cell layers, cell shape determination and timing of senescence. Disruption of BPC3 function rescues some aspects of the bpc1-1 bpc2 bpc4 bpc6 phenotype, indicating that BPC3 function may be antagonistic to other members of the family. Ethylene response is diminished in bpc1-1 bpc2 bpc4 bpc6 plants, although not all aspects of the phenotype can be explained by reduced ethylene sensitivity. Our data indicate that the BPC transcription factor family is integral for a wide range of processes that support normal growth and development.

Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae.

Basic helix-loop-helix proteins (bHLHs) are found throughout the three eukaryotic kingdoms and constitute one of the largest families of transcription factors. A growing number of bHLH proteins have been functionally characterized in plants. However, some of these have not been previously classified. We present here an updated and comprehensive classification of the bHLHs encoded by the whole sequenced genomes of Arabidopsis (Arabidopsis thaliana), Populus trichocarpa, Oryza sativa, Physcomitrella patens, and five algae species. We define a plant bHLH consensus motif, which allowed the identification of novel highly diverged atypical bHLHs. Using yeast two-hybrid assays, we confirm that (1) a highly diverged bHLH has retained protein interaction activity and (2) the two most conserved positions in the consensus play an essential role in dimerization. Phylogenetic analysis permitted classification of the 638 bHLH genes identified into 32 subfamilies. Evolutionary and functional relationships within subfamilies are supported by intron patterns, predicted DNA-binding motifs, and the architecture of conserved protein motifs. Our analyses reveal the origin and evolutionary diversification of plant bHLHs through differential expansions, domain shuffling, and extensive sequence divergence. At the functional level, this would translate into different subfamilies evolving specific DNA-binding and protein interaction activities as well as differential transcriptional regulatory roles. Our results suggest a role for bHLH proteins in generating plant phenotypic diversity and provide a solid framework for further investigations into the role carried out in the transcriptional regulation of key growth and developmental processes.