Christian S. Hardtke

Targeted destabilization of HY5 during light-regulated development of Arabidopsis.

Arabidopsis seedlings display contrasting developmental patterns depending on the ambient light. Seedlings grown in the light develop photomorphogenically, characterized by short hypocotyls and expanded green cotyledons. In contrast, seedlings grown in darkness become etiolated, with elongated hypocotyls and closed cotyledons on an apical hook. Light signals, perceived by multiple photoreceptors and transduced to downstream regulators, dictate the extent of photomorphogenic development in a quantitative manner. Two key downstream components, COP1 and HY5, act antagonistically in regulating seedling development. HY5 is a bZIP transcription factor that binds directly to the promoters of light-inducible genes, promoting their expression and photomorphogenic development. COP1 is a RING-finger protein with WD-40 repeats whose nuclear abundance is negatively regulated by light. COP1 interacts directly with HY5 in the nucleus to regulate its activity negatively. Here we show that the abundance of HY5 is directly correlated with the extent of photomorphogenic development, and that the COP1–HY5 interaction may specifically target HY5 for proteasome-mediated degradation in the nucleus.

HY5 stability and activity in arabidopsis is regulated by phosphorylation in its COP1 binding domain.

Arabidopsis HY5 is a bZIP transcription factor that promotes photomorphogenesis. Previous studies suggested that COP1, a negative regulator of photomorphogenesis, directly interacts with nuclear HY5 and targets it for proteasome‐mediated degradation. Light negatively regulates the nuclear level of COP1 and thus permits HY5 accumulation. Here we report that HY5 abundance peaks in early seedling development, consistent with its role in promoting photomorphogenesis. HY5 acts exclusively within a complex and exists in two isoforms, resulting from phosphorylation within its COP1 binding domain by a light‐ regulated kinase activity. Unphosphorylated HY5 shows stronger interaction with COP1, is the preferred substrate for degradation, has higher affinity to target promoters and is physiologically more active than the phosphorylated version. Therefore, HY5 phosphorylation provides an added level of light‐mediated regulation of HY5 stability and activity besides nuclear COP1 levels. Regulated HY5 phosphorylation not only provides abundant and physiologically more active unphosphorylated HY5 in the light, but also helps to maintain a small pool of less active phosphorylated HY5 in the dark, which could be essential for a rapid initial response during dark‐to‐light transition.

Overlapping and non-redundant functions of the Arabidopsis auxin response factors MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4.

Transcription factors of the auxin response factor (ARF) family have been implicated in auxin-dependent gene regulation, but little is known about the functions of individual ARFs in plants. Here, interaction assays, expression studies and combinations of multiple loss- and gain-of-function mutants were used to assess the roles of two ARFs, NONPHOTOTROPIC HYPOCOTYL 4 (NPH4/ARF7) and MONOPTEROS (MP/ARF5), in Arabidopsis development. Both MP and NPH4 interact strongly and selectively with themselves and with each other, and are expressed in vastly overlapping domains. We show that the regulatory properties of both genes are far more related than suggested by their single mutant phenotypes. NPH4 and MP are capable of controlling both axis formation in the embryo and auxin-dependent cell expansion. Interaction of MP and NPH4 in Arabidopsis plants is indicated by their joint requirement in a number of auxin responses and by synergistic effects associated with the co-overexpression of both genes. Finally, we demonstrate antagonistic interaction between ARF and Aux/IAA gene functions in Arabidopsis development. Overexpression of MP suppresses numerous defects associated with a gain-of-function mutation in BODENLOS (BDL)/IAA12. Together these results provide evidence for the biological relevance of ARF-ARF and ARF-Aux/IAA interaction in Arabidopsis plants and demonstrate that an individual ARF can act in both invariantly programmed pattern formation as well as in conditional responses to external signals.

BRX mediates feedback between brassinosteroid levels and auxin signalling in root growth.

Brassinosteroid and auxin decisively influence plant development, and overlapping transcriptional responses to these phytohormones suggest an interaction between the two pathways. However, whether this reflects direct feedback or merely parallel inputs on common targets is unclear. Here we show that in Arabidopsis roots, this interaction is mediated by BREVIS RADIX (BRX), which is required for optimal root growth. We demonstrate that the brx phenotype results from a root-specific deficiency of brassinosteroid and is due to reduced, BRX-dependent expression of a rate-limiting enzyme in brassinosteroid biosynthesis. Unexpectedly, this deficiency affects the root expression level of ∼15% of all Arabidopsis genes, but the transcriptome profile can be restored to wild type by brassinosteroid treatment. Thus, proper brassinosteroid levels are required for the correct expression of many more genes than previously suspected. Moreover, embryonic or post-embryonic brassinosteroid application fully or partially, respectively, rescues the brx phenotype. Further, auxin-responsive gene expression is globally impaired in brx, demonstrating that brassinosteroid levels are rate-limiting for auxin-responsive transcription. BRX expression is strongly induced by auxin and mildly repressed by brassinolide, which means that BRX acts at the nexus of a feedback loop that maintains threshold brassinosteroid levels to permit optimal auxin action.

Vascular continuity and auxin signals

Plant vascular tissues form systems of interconnected cell files throughout the plant body. Vascular tissues usually differentiate at predictable positions but the wide range of functional patterns generated in response to abnormal growth conditions or wounding reveals partially self-organizing patterning mechanisms. Signals ensuring aligned cell differentiation within vascular strands are crucial in self-organized vascular patterning, and the apical-basal flow of indole acetic acid has been suspected to act as an orienting signal in this process. Several recent advances appear to converge on a more precise definition of the role of auxin flow in vascular tissue patterning.

Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1

Arabidopsis COP1 is a photomorphogenesis repressor capable of directly interacting with the photomorphogenesis‐promoting factor HY5. This interaction between HY5 and COP1 results in targeted deg radation of HY5 by the 26S proteasome. Here we characterized the WD40 repeat domain‐mediated interactions of COP1 with HY5 and two new proteins. Mutational analysis of those interactive partners revealed a conserved motif responsible for the interaction with the WD40 domain. This novel motif, with the core sequence V‐P‐E/D‐φ‐G (φ = hydrophobic residue) in conjunction with an upstream stretch of 4–5 negatively charged residues, interacts with a defined surface area of the β‐propeller assembly of the COP1 WD40 repeat domain through both hydrophobic and ionic interactions. Several residues in the COP1 WD40 domain that are critical for the interaction with this motif have been revealed. The fact that point mutations either in the COP1 WD40 domain or in the HY5 motif that abolish the interaction between COP1 and HY5 in yeast result in a dramatic reduction of HY5 degradation in transgenic plants validates the biological significance of this defined interaction.

Opposite root growth phenotypes of hy5 versus hy5 hyh mutants correlate with increased constitutive auxin signaling.

The Arabidopsis transcription factor HY5 controls light-induced gene expression downstream of photoreceptors and plays an important role in the switch of seedling shoots from dark-adapted to light-adapted development. In addition, HY5 has been implicated in plant hormone signaling, accounting for the accelerated root system growth phenotype of hy5 mutants. Mutants in the close HY5 homolog HYH resemble wild-type, despite the largely similar expression patterns and levels of HY5 and HYH, and the functional equivalence of the respective proteins. Moreover, the relative contribution of HYH to the overall activity of the gene pair is increased by an alternative HYH transcript, which encodes a stabilized protein. Consistent with the enhanced root system growth observed in hy5 loss-of-function mutants, constitutively overexpressed alternative HYH inhibits root system growth. Paradoxically, however, in double mutants carrying hy5 and hyh null alleles, the hy5 root growth phenotype is suppressed rather than enhanced. Even more surprisingly, compared to wild-type, root system growth is diminished in hy5 hyh double mutants. In addition, the double mutants display novel shoot phenotypes that are absent from either single mutant. These include cotyledon fusions and defective vasculature, which are typical for mutants in genes involved in the transcriptional response to the plant hormone auxin. Indeed, many auxin-responsive and auxin signaling genes are misexpressed in hy5 mutants, and at a higher number and magnitude in hy5 hyh mutants. Therefore, auxin-induced transcription is constitutively activated at different levels in the two mutant backgrounds. Our data support the hypothesis that the opposite root system phenotypes of hy5 single and hy5 hyh double mutants represent the morphological response to a quantitative gradient in the same molecular process, that is gradually increased constitutive auxin signaling. The data also suggest that HY5 and HYH are important negative regulators of auxin signaling amplitude in embryogenesis and seedling development.

Evaluation and classification of RING-finger domains encoded by the Arabidopsis genome

BackgroundIn computational analysis, the RING-finger domain is one of the most frequently detected domains in the Arabidopsis proteome. In fact, it is more abundant in Arabidopsis than in other eukaryotic genomes. However, computational analysis might classify ambiguous domains of the closely related PHD and LIM motifs as RING domains by mistake. Thus, we set out to define an ordered set of Arabidopsis RING domains by evaluating predicted domains on the basis of recent structural data.ResultsInspection of the proteome with a current InterPro release predicts 446 RING domains. We evaluated each detected domain and as a result eliminated 59 false positives. The remaining 387 domains were grouped by cluster analysis and according to their metal-ligand arrangement. We further defined novel patterns for additional computational analyses of the proteome. They were based on recent structural data that enable discrimination between the related RING, PHD and LIM domains. These patterns allow us to predict with different degrees of certainty whether a particular domain is indeed likely to form a RING finger.ConclusionsIn summary, 387 domains have a significant potential to form a RING-type cross-brace structure. Many of these RING domains overlap with predicted PHD domains; however, the RING domain signature mostly prevails. Thus, the abundance of PHD domains in Arabidopsis has been significantly overestimated. Cluster analysis of the RING domains defines groups of proteins, which frequently show significant similarity outside the RING domain. These groups document a common evolutionary origin of their members and potentially represent genes of overlapping functionality.

Mobile Gibberellin Directly Stimulates Arabidopsis Hypocotyl Xylem Expansion

In this study, the authors find that the expansion of the xylem in Arabidopsis hypocotyls observed upon flowering is directly triggered by signaling through the plant hormone gibberellin. The authors also demonstrate that this involves mobile gibberellin, which therefore can act as a long-distance signal. Secondary growth of the vasculature results in the thickening of plant structures and continuously produces xylem tissue, the major biological carbon sink. Little is known about the developmental control of this quantitative trait, which displays two distinct phases in Arabidopsis thaliana hypocotyls. The later phase of accelerated xylem expansion resembles the secondary growth of trees and is triggered upon flowering by an unknown, shoot-derived signal. We found that flowering-dependent hypocotyl xylem expansion is a general feature of herbaceous plants with a rosette growth habit. Flowering induction is sufficient to trigger xylem expansion in Arabidopsis. By contrast, neither flower formation nor elongation of the main inflorescence is required. Xylem expansion also does not depend on any particular flowering time pathway or absolute age. Through analyses of natural genetic variation, we found that ERECTA acts locally to restrict xylem expansion downstream of the gibberellin (GA) pathway. Investigations of mutant and transgenic plants indicate that GA and its signaling pathway are both necessary and sufficient to directly trigger enhanced xylogenesis. Impaired GA signaling did not affect xylem expansion systemically, suggesting that it acts downstream of the mobile cue. By contrast, the GA effect was graft transmissible, suggesting that GA itself is the mobile shoot-derived signal.

Strigolactone Promotes Degradation of DWARF14, an α/β Hydrolase Essential for Strigolactone Signaling in Arabidopsis

A mechanism was identified by which degradation of D14, the putative receptor of the plant hormone strigolactone, is promoted by this hormone. This mechanism, which is mediated by protein degradation via the proteasome machinery and requires the activity of the F-box protein MAX2, should cause a substantial drop in strigolactone perception, thereby limiting hormone signaling duration and intensity. Strigolactones (SLs) are phytohormones that play a central role in regulating shoot branching. SL perception and signaling involves the F-box protein MAX2 and the hydrolase DWARF14 (D14), proposed to act as an SL receptor. We used strong loss-of-function alleles of the Arabidopsis thaliana D14 gene to characterize D14 function from early axillary bud development through to lateral shoot outgrowth and demonstrated a role of this gene in the control of flowering time. Our data show that D14 distribution in vivo overlaps with that reported for MAX2 at both the tissue and subcellular levels, allowing physical interactions between these proteins. Our grafting studies indicate that neither D14 mRNA nor the protein move over a long range upwards in the plant. Like MAX2, D14 is required locally in the aerial part of the plant to suppress shoot branching. We also identified a mechanism of SL-induced, MAX2-dependent proteasome-mediated degradation of D14. This negative feedback loop would cause a substantial drop in SL perception, which would effectively limit SL signaling duration and intensity.