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.

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.

Plant proximity perception dynamically modulates hormone levels and sensitivity in Arabidopsis.

The shade avoidance syndrome (SAS) refers to a set of plant responses initiated after perception by the phytochromes of light enriched in far-red colour reflected from or filtered by neighbouring plants. These varied responses are aimed at anticipating eventual shading from potential competitor vegetation. In Arabidopsis thaliana, the most obvious SAS response at the seedling stage is the increase in hypocotyl elongation. Here, we describe how plant proximity perception rapidly and temporally alters the levels of not only auxins but also active brassinosteroids and gibberellins. At the same time, shade alters the seedling sensitivity to hormones. Plant proximity perception also involves dramatic changes in gene expression that rapidly result in a new balance between positive and negative factors in a network of interacting basic helix-loop-helix proteins, such as HFR1, PAR1, and BIM and BEE factors. Here, it was shown that several of these factors act as auxin- and BR-responsiveness modulators, which ultimately control the intensity or degree of hypocotyl elongation. It was deduced that, as a consequence of the plant proximity-dependent new, dynamic, and local balance between hormone synthesis and sensitivity (mechanistically resulting from a restructured network of SAS regulators), SAS responses are unleashed and hypocotyls elongate.

The shade avoidance syndrome in Arabidopsis: a fundamental role for atypical basic helix-loop-helix proteins as transcriptional cofactors.

The shade avoidance syndrome (SAS) refers to a set of plant responses aimed at anticipating eventual shading by potential competitors. The SAS is initiated after perception of nearby vegetation as a reduction in the red to far-red ratio (R:FR) of the incoming light. Low R:FR light is perceived by the phytochromes, triggering dramatic changes in gene expression that, in seedlings, eventually result in an increased hypocotyl elongation to overgrow competitors. This response is inhibited by genes such as PHYTOCHROME RAPIDLY REGULATED 1 (PAR1), PAR2 and LONG HYPOCOTYL IN FR 1 (HFR1), which are transcriptionally induced by low R:FR. Although PAR1/PAR2 and HFR1 proteins belong to different groups of basic helix-loop-helix (bHLH) transcriptional regulators, they all lack a typical basic domain required for binding to E-box and G-box motifs in the promoter of target genes. By overexpressing derivatives of PAR1 and HFR1 we show that these proteins are actually transcriptional cofactors that do not need to bind DNA to directly regulate transcription. We conclude that protein-protein interactions involving the HLH domain of PAR1 and HFR1 are a fundamental aspect of the mechanism by which these proteins regulate gene expression, most likely through interaction with true transcription factors that do bind to the target genes and eventually unleash the observed SAS responses.

Interaction of shade avoidance and auxin responses: a role for two novel atypical bHLH proteins.

Plants sense the presence of potentially competing nearby individuals as a reduction in the red to far-red ratio of the incoming light. In anticipation of eventual shading, a set of plant responses known as the shade avoidance syndrome (SAS) is initiated soon after detection of this signal by the phytochrome photoreceptors. Here we analyze the function of PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and PAR2, two Arabidopsis thaliana genes rapidly upregulated after simulated shade perception. These genes encode two closely related atypical basic helix-loop-helix proteins with no previously assigned function in plant development. Using reverse genetic approaches, we show that PAR1 and PAR2 act in the nucleus to broadly control plant development, acting as negative regulators of a variety of SAS responses, including seedling elongation and photosynthetic pigment accumulation. Molecularly, PAR1 and PAR2 act as direct transcriptional repressors of two auxin-responsive genes, SMALL AUXIN UPREGULATED15 (SAUR15) and SAUR68. Additional results support that PAR1 and PAR2 function in integrating shade and hormone transcriptional networks, rapidly connecting phytochrome-sensed light changes with auxin responsiveness.

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.

Auxin promotion of seedling growth via ARF5 is dependent on the brassinosteroid-regulated transcription factors BES1 and BEH4.

Seedlings must continually calibrate their growth in response to the environment. Auxin and brassinosteroids (BRs) are plant hormones that work together to control growth responses during photomorphogenesis. We used our previous analysis of promoter architecture in an auxin and BR target gene to guide our investigation into the broader molecular bases and biological relevance of transcriptional co-regulation by these hormones. We found that the auxin-regulated transcription factor Auxin Responsive Factor 5 (ARF5) and the brassinosteroid-regulated transcription factor BRI1-EMS-Suppressor 1/Brassinazole Resistant 2 (BES1) co-regulated a subset of growth-promoting genes via conserved bipartite cis-regulatory elements. Moreover, ARF5 binding to DNA could be enriched by increasing BES1 levels. The evolutionary loss of bipartite elements in promoters results in loss of hormone responsiveness. We also identified another member of the BES1/BZR1 family called BEH4 that acts partially redundantly with BES1 to regulate seedling growth. Double mutant analysis showed that BEH4 and not BZR1 were required alongside BES1 for normal auxin response during early seedling development. We propose that an ARF5-BES1/BEH4 transcriptional module acts to promote growth via modulation of a diverse set of growth-associated genes.

Snap, crack and pop of explosive fruit.

There is an increasing appreciation for the role of physical forces in plant development. Mechanics are fundamental to how explosive fruit eject their seeds, and recent studies have successfully combined mechanics with developmental genetics to help explain how these dispersal traits are produced and how they evolved. Computational modeling is used more and more to address developmental questions, and explosive fruit are particularly good systems for combining biology and modeling approaches. Finite element models have been recently used to explore questions such as: Why do touch-me-not species with similar fruits, differ so much in how efficiently they transfer stored energy to eject seeds? And how do popping cress fruits use the expansive force of turgor pressure for tissue contraction?

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.

Photomorphogenesis.

As photoautotrophs, plants are exquisitely sensitive to their light environment. Light affects many developmental and physiological responses throughout plants' life histories. The focus of this chapter is on light effects during the crucial period of time between seed germination and the development of the first true leaves. During this time, the seedling must determine the appropriate mode of action to best achieve photosynthetic and eventual reproductive success. Light exposure triggers several major developmental and physiological events. These include: growth inhibition and differentiation of the embryonic stem (hypocotyl); maturation of the embryonic leaves (cotyledons); and establishment and activation of the stem cell population in the shoot and root apical meristems. Recent studies have linked a number of photoreceptors, transcription factors, and phytohormones to each of these events.

PAR1 and PAR2 integrate shade and hormone transcriptional networks.

PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and PAR2 are two negative regulators of shade avoidance syndrome (SAS) responses in Arabidopsis. PAR1 and PAR2 belong to the bHLH family of transcription factors and act as direct transcriptional repressors of auxin- and brassinosteroid-responsive genes. These observations led us to propose that PAR1 and PAR2 might integrate shade and hormone signals. After plant proximity perception by the phytochrome photoreceptors, the expression of PAR1, PAR2 and dozens of additional PAR genes is affected, initiating a complex web of transcriptional events instrumental for the establishment of the SAS responses. Studying the organization of this complex transcriptional network, that is, the interactions amongst the different PAR factors involved and how they are connected with the endogenous hormone-regulated transcriptional networks, seems therefore fundamental to understand how SAS is modulated.