The ARP2/3 complex, a highly conserved nucleator of F-actin, and its activator, the SCAR complex, are essential for growth in plants and animals. In this article, we present a pathway through which roots of Arabidopsis thaliana directly perceive light to promote their elongation. The ARP2/3-SCAR complex and the maintenance of longitudinally aligned F-actin arrays are crucial components of this pathway. The involvement of the ARP2/3-SCAR complex in light-regulated root growth is supported by our finding that mutants of the SCAR complex subunit BRK1/HSPC300, or other individual subunits of the ARP2/3-SCAR complex, showed a dramatic inhibition of root elongation in the light, which mirrored reduced growth of wild-type roots in the dark. SCAR1 degradation in dark-grown wild-type roots by constitutive photomorphogenic 1 (COP1) E3 ligase and 26S proteasome accompanied the loss of longitudinal F-actin and reduced root growth. Light perceived by the root photoreceptors, cryptochrome and phytochrome, suppressed COP1-mediated SCAR1 degradation. Taken together, our data provide a biochemical explanation for light-induced promotion of root elongation by the ARP2/3-SCAR complex.

Genetically encoded filamentous actin (F-actin) reporters designed based on fluorescent protein fusions to F-actin binding domains of actin regulatory proteins have emerged as powerful tools to decipher the role of the actin cytoskeleton in plant growth and development. However, these probes could interfere with the function of endogenous actin binding proteins and in turn impact actin organization and plant growth. We therefore surveyed F-actin labeling and compared organ growth in Arabidopsis thaliana lines expressing a variety of F-actin markers. Here we show that the variant of fluorescent protein, type of actin binding domain, and the promoter that drives reporter expression can influence the quality of F-actin labeling particularly in stable plant lines. For example, older red fluorescent protein (RFP)-based probes such as DsRed2 and mOrange induced more aberrant labeling compared to the newer RFP-based, mCherry, GFP, and GFP-derived fluorophores such as YFP and CFP. Moreover, qualitative and quantitative analyses revealed differences in F-actin organization in seedlings expressing Talin- and Lifeact-based reporters in some cell types compared to the fimbrin actin binding domain 2 (ABD2)-based reporters. Finally, the use of the ubiquitin10 (UBQ10) promoter to drive expression of the GFP-ABD2-GFP probe minimized loss of fluorescence and growth defects observed in the 35S-driven version. Taken together, this study shows that care must be taken in the interpretation of data derived from stable expression of certain F-actin reporters and that using alternative promoters such as UBQ10 can overcome some of the pitfalls that accompany the use of in vivo F-actin probes in plants. © 2014 Wiley Periodicals, Inc.

Before plants can be effectively utilised as a component of enclosed life-support systems for space exploration, it is important to understand the molecular mechanisms by which they develop in microgravity. Using the Biological Research in Canisters (BRIC) hardware on board the second to the last flight of the Space Shuttle Discovery (STS-131 mission), we studied how microgravity impacts root growth in Arabidopsis thaliana. Ground-based studies showed that the actin cytoskeleton negatively regulates root gravity responses on Earth, leading us to hypothesise that actin might also be an important modulator of root growth behaviour in space. We investigated how microgravity impacted root growth of wild type (ecotype Columbia) and a mutant (act2-3) disrupted in a root-expressed vegetative actin isoform (ACTIN2). Roots of etiolated wild-type and act2-3 seedlings grown in space skewed vigorously toward the left, which was unexpected given the reduced directional cue provided by gravity. The left-handed directional root growth in space was more pronounced in act2-3 mutants than wild type. To quantify differences in root orientation of these two genotypes in space, we developed an algorithm where single root images were converted into binary images using computational edge detection methods. Binary images were processed with Fast Fourier Transformation (FFT), and histogram and entropy were used to determine spectral distribution, such that high entropy values corresponded to roots that deviated more strongly from linear orientation whereas low entropy values represented straight roots. We found that act2-3 roots had a statistically stronger skewing/coiling response than wild-type roots, but such differences were not apparent on Earth. Ultrastructural studies revealed that newly developed cell walls of space-grown act2-3 roots were more severely disrupted compared to space-grown wild type, and ground control wild-type and act2-3 roots. Collectively, our results provide evidence that, like root gravity responses on Earth, endogenous directional growth patterns of roots in microgravity are suppressed by the actin cytoskeleton. Modulation of root growth in space by actin could be facilitated in part through its impact on cell wall architecture.

Root hairs (RHs) function in nutrient and water acquisition, root metabolite exudation, soil anchorage and plant-microbe interactions. Longer or more abundant RHs are potential breeding traits for developing crops that are more resource-use efficient and can improve soil health.While many genes are known to promote RH elongation, relatively little is known about genes and mechanisms that constrain RH growth.Here we demonstrate that a DOMAIN OF UNKNOWN FUNCTION 506 (DUF506) protein, AT3G25240, negatively regulates Arabidopsis thaliana RH growth. The AT3G25240 gene is strongly and specifically induced during phosphorus (P)-limitation. Mutants of this gene, which we call REPRESSOR OF EXCESSIVE ROOT HAIR ELONGATION 1 (RXR1), have much longer RHs, higher phosphate content and seedling biomass, while overexpression of the gene exhibits opposite phenotypes. Co-immunoprecipitation, pull-down and bimolecular fluorescence complementation (BiFC) analyses reveal that RXR1 physically interacts with a RabD2c GTPase in nucleus, and a rabd2c mutant phenocopies the rxr1 mutant. Furthermore, N-terminal variable region of RXR1 is crucial for inhibiting RH growth. Overexpression of a Brachypodium distachyon RXR1 homolog results in repression of RH elongation in Brachypodium.Taken together, our results reveal a novel DUF506-GTPase module with a prominent role in repression of plant RH elongation especially under P stress.

No abstract

The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. Here, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of a hlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants.

Physical dormancy in seeds exists widely in seed plants and plays a vital role in maintaining natural seed banks. The outermost cuticle of the seed coat forms a water-impermeable layer, which is critical for establishing seed physical dormancy. We previously set up the legume plant Medicago truncatula as an excellent model for studying seed physical dormancy, and our studies revealed that a class II KNOTTED-like homeobox, KNOX4, is a transcription factor critical for controlling hardseededness. Here we report the function of a seed coat β-ketoacyl-CoA synthase, KCS12. The expression level of KCS12 is significantly downregulated in the knox4 mutant. The KCS12 gene is predominantly expressed in the seed coat, and seed development in the M. truncatula kcs12 mutant is altered. Further investigation demonstrated that kcs12 mutant seeds lost physical dormancy and were able to absorb water without scarification treatment. Chemical analysis revealed that concentrations of C24:0 lipid polyester monomers are significantly decreased in mutant seeds, indicating that KCS12 is an enzyme that controls the production of very long chain lipid species in the seed coat. A ChIP assay demonstrated that the expression of KCS12 in the seed coat is directly regulated by the KNOX4 transcription factor. These findings define a molecular mechanism by which KNOX4 and KCS12 control formation of the seed coat and seed physical dormancy.

Diverse leaf forms ranging from simple to compound leaves are found in plants. It is known that the final leaf size and shape vary greatly in response to developmental and environmental changes. However, changes in leaf size and shape have been quantitatively characterized only in a limited number of species. Here, we report development of LeafletAnalyzer, an automated image analysis and classification software to analyze and classify blade and serration characteristics of trifoliate leaves in Medicago truncatula. The software processes high quality leaf images in an automated or manual fashion to generate size and shape parameters for both blades and serrations. In addition, it generates spectral components for each leaflets using elliptic Fourier transformation. Reconstruction studies show that the spectral components can be reliably used to rebuild the original leaflet images, with low, and middle and high frequency spectral components corresponding to the outline and serration of leaflets, respectively. The software uses artificial neutral network or k-means classification method to classify leaflet groups that are developed either on successive nodes of stems within a genotype or among genotypes such as natural variants and developmental mutants. The automated feature of the software allows analysis of thousands of leaf samples within a short period of time, thus facilitating identification, comparison and classification of leaf groups based on leaflet size, shape and tooth features during leaf development, and among induced mutants and natural variants.