Phosphoproteome analyses pinpoint the F-box protein SLOW MOTION as a regulator of warm temperature-mediated hypocotyl growth in Arabidopsis.

Hypocotyl elongation is controlled by several signals and is a major characteristic of plants growing in darkness or under warm temperature. While already several molecular mechanisms associated with this process are known, protein degradation and associated E3 ligases have hardly been studied in the context of warm temperature. In a time-course phosphoproteome analysis on Arabidopsis seedlings exposed to control or warm ambient temperature, we observed reduced levels of diverse proteins over time, which could be due to transcription, translation, and/or degradation. In addition, we observed differential phosphorylation of the LRR F-box protein SLOMO MOTION (SLOMO) at two serine residues. We demonstrate that SLOMO is a negative regulator of hypocotyl growth, also under warm temperature conditions, and protein-protein interaction studies revealed possible interactors of SLOMO, such as MKK5, DWF1, and NCED4. We identified DWF1 as a likely SLOMO substrate and a regulator of warm temperature-mediated hypocotyl growth. We propose that warm temperature-mediated regulation of SLOMO activity controls the abundance of hypocotyl growth regulators, such as DWF1, through ubiquitin-mediated degradation.

Functional annotation of proteins for signaling network inference in non-model species.

Molecular biology aims to understand cellular responses and regulatory dynamics in complex biological systems. However, these studies remain challenging in non-model species due to poor functional annotation of regulatory proteins. To overcome this limitation, we develop a multi-layer neural network that determines protein functionality directly from the protein sequence. We annotate kinases and phosphatases in Glycine max. We use the functional annotations from our neural network, Bayesian inference principles, and high resolution phosphoproteomics to infer phosphorylation signaling cascades in soybean exposed to cold, and identify Glyma.10G173000 (TOI5) and Glyma.19G007300 (TOT3) as key temperature regulators. Importantly, the signaling cascade inference does not rely upon known kinase motifs or interaction data, enabling de novo identification of kinase-substrate interactions. Conclusively, our neural network shows generalization and scalability, as such we extend our predictions to Oryza sativa, Zea mays, Sorghum bicolor, and Triticum aestivum. Taken together, we develop a signaling inference approach for non-model species leveraging our predicted kinases and phosphatases.

Functional analysis of Arabidopsis and maize transgenic lines overexpressing the ADP-ribose/NADH pyrophosphohydrolase, AtNUDX7.

The conserved poly(ADP-ribosyl)ation (PAR) pathway consists of three genetic components that are potential targets to modulate the plant's energy homeostasis upon stress with the aim to improve yield stability in crops and help secure food supply. We studied the role of the PAR pathway component ADP-ribose/NADH pyrophosphohydrolase (AtNUDX7) in yield and mild drought stress by using a transgenic approach in Arabidopsis thaliana and maize (Zea mays). Arabidopsis AtNUDX7 cDNA was overexpressed in Arabidopsis and maize by means of the constitutive Cauliflower Mosaic Virus 35S promoter and the strong constitutive Brachypodium distachyon pBdEF1α promoter, respectively. Overexpression of AtNUDX7 in Arabidopsis improved seed parameters that were measured by a novel, automated method, accelerated flowering and reduced inflorescence height. This combination of beneficial traits suggested that AtNUDX7 overexpression in Arabidopsis might enhance the ADP-ribose recycling step and maintain energy levels by supplying an ATP source in the poly(ADP-ribosyl)ation energy homeostasis pathway. Arabidopsis and maize lines with high, medium and low overexpression levels of the AtNUDX7 gene were analysed in automated platforms and the inhibition of several growth parameters was determined under mild drought stress conditions. The data showed that the constitutive overexpression of the Arabidopsis AtNUDX7 gene in Arabidopsis and maize at varying levels did not improve tolerance to mild drought stress, but knocking down AtNUDX7 expression did, however at the expense of general growth under normal conditions.

Histone 2B monoubiquitination complex integrates transcript elongation with RNA processing at circadian clock and flowering regulators.

HISTONE MONOUBIQUITINATION1 (HUB1) and its paralog HUB2 act in a conserved heterotetrameric complex in the chromatin-mediated transcriptional modulation of developmental programs, such as flowering time, dormancy, and the circadian clock. The KHD1 and SPEN3 proteins were identified as interactors of the HUB1 and HUB2 proteins with in vitro RNA-binding activity. Mutants in SPEN3 and KHD1 had reduced rosette and leaf areas. Strikingly, in spen3 mutants, the flowering time was slightly, but significantly, delayed, as opposed to the early flowering time in the hub1-4 mutant. The mutant phenotypes in biomass and flowering time suggested a deregulation of their respective regulatory genes CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and FLOWERING LOCUS C (FLC) that are known targets of the HUB1-mediated histone H2B monoubiquitination (H2Bub). Indeed, in the spen3-1 and hub1-4 mutants, the circadian clock period was shortened as observed by luciferase reporter assays, the levels of the CCA1α and CCA1ß splice forms were altered, and the CCA1 expression and H2Bub levels were reduced. In the spen3-1 mutant, the delay in flowering time was correlated with an enhanced FLC expression, possibly due to an increased distal versus proximal ratio of its antisense COOLAIR transcript. Together with transcriptomic and double-mutant analyses, our data revealed that the HUB1 interaction with SPEN3 links H2Bub during transcript elongation with pre-mRNA processing at CCA1 Furthermore, the presence of an intact HUB1 at the FLC is required for SPEN3 function in the formation of the FLC-derived antisense COOLAIR transcripts.

Interactive and noninteractive roles of histone H2B monoubiquitination and H3K36 methylation in the regulation of active gene transcription and control of plant growth and development.

Covalent modifications of histones are essential to control a wide range of processes during development and adaptation to environmental changes. With the establishment of reference epigenomes, patterns of histone modifications were correlated with transcriptionally active or silenced genes. These patterns imply the need for the precise and dynamic coordination of different histone-modifying enzymes to control transcription at a given gene. Classically, the influence of these enzymes on gene expression is examined separately and their interplays rarely established. In Arabidopsis, HISTONE MONOUBIQUITINATION2 (HUB2) mediates H2B monoubiquitination (H2Bub1), whereas SET DOMAIN GROUP8 (SDG8) catalyzes H3 lysine 36 trimethylation (H3K36me3). In this work, we crossed hub2 with sdg8 mutants to elucidate their functional relationships. Despite similar phenotypic defects, sdg8 and hub2 mutations broadly affect genome transcription and plant growth and development synergistically. Also, whereas H3K4 methylation appears largely dependent on H2Bub1, H3K36me3 and H2Bub1 modifications mutually reinforce each other at some flowering time genes. In addition, SDG8 and HUB2 jointly antagonize the increase of the H3K27me3 repressive mark. Collectively, our data provide an important insight into the interplay between histone marks and highlight their interactive complexity in regulating chromatin landscape which might be necessary to fine-tune transcription and ensure plant developmental plasticity.

The Elongator complex regulates hypocotyl growth in darkness and during photomorphogenesis.

The Elongator complex (hereafter Elongator) promotes RNA polymerase II-mediated transcript elongation through epigenetic activities such as histone acetylation. Elongator regulates growth, development, immune response and sensitivity to drought and abscisic acid. We demonstrate that elo mutants exhibit defective hypocotyl elongation but have a normal apical hook in darkness and are hyposensitive to light during photomorphogenesis. These elo phenotypes are supported by transcriptome changes, including downregulation of circadian clock components, positive regulators of skoto- or photomorphogenesis, hormonal pathways and cell wall biogenesis-related factors. The downregulated genes LHY, HFR1 and HYH are selectively targeted by Elongator for histone H3K14 acetylation in darkness. The role of Elongator in early seedling development in darkness and light is supported by hypocotyl phenotypes of mutants defective in components of the gene network regulated by Elongator, and by double mutants between elo and mutants in light or darkness signaling components. A model is proposed in which Elongator represses the plant immune response and promotes hypocotyl elongation and photomorphogenesis via transcriptional control of positive photomorphogenesis regulators and a growth-regulatory network that converges on genes involved in cell wall biogenesis and hormone signaling.This article has an associated First Person interview with the first author of the paper.

ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling.

The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical-basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity.

Functional Analysis of the Arabidopsis TETRASPANIN Gene Family in Plant Growth and Development.

TETRASPANIN (TET) genes encode conserved integral membrane proteins that are known in animals to function in cellular communication during gamete fusion, immunity reaction, and pathogen recognition. In plants, functional information is limited to one of the 17 members of the Arabidopsis (Arabidopsis thaliana) TET gene family and to expression data in reproductive stages. Here, the promoter activity of all 17 Arabidopsis TET genes was investigated by pAtTET::NUCLEAR LOCALIZATION SIGNAL-GREEN FLUORESCENT PROTEIN/ß-GLUCURONIDASE reporter lines throughout the life cycle, which predicted functional divergence in the paralogous genes per clade. However, partial overlap was observed for many TET genes across the clades, correlating with few phenotypes in single mutants and, therefore, requiring double mutant combinations for functional investigation. Mutational analysis showed a role for TET13 in primary root growth and lateral root development and redundant roles for TET5 and TET6 in leaf and root growth through negative regulation of cell proliferation. Strikingly, a number of TET genes were expressed in embryonic and seedling progenitor cells and remained expressed until the differentiation state in the mature plant, suggesting a dynamic function over developmental stages. The cis-regulatory elements together with transcription factor-binding data provided molecular insight into the sites, conditions, and perturbations that affect TET gene expression and positioned the TET genes in different molecular pathways; the data represent a hypothesis-generating resource for further functional analyses.

Plant Elongator regulates auxin-related genes during RNA polymerase II transcription elongation.

In eukaryotes, transcription of protein-encoding genes is strongly regulated by posttranslational modifications of histones that affect the accessibility of the DNA by RNA polymerase II (RNAPII). The Elongator complex was originally identified in yeast as a histone acetyltransferase (HAT) complex that activates RNAPII-mediated transcription. In Arabidopsis thaliana, the Elongator mutants elo1, elo2, and elo3 with decreased leaf and primary root growth due to reduced cell proliferation identified homologs of components of the yeast Elongator complex, Elp4, Elp1, and Elp3, respectively. Here we show that the Elongator complex was purified from plant cell cultures as a six-component complex. The role of plant Elongator in transcription elongation was supported by colocalization of the HAT enzyme, ELO3, with euchromatin and the phosphorylated form of RNAPII, and reduced histone H3 lysine 14 acetylation at the coding region of the SHORT HYPOCOTYL 2 auxin repressor and the LAX2 auxin influx carrier gene with reduced expression levels in the elo3 mutant. Additional auxin-related genes were down-regulated in the transcriptome of elo mutants but not targeted by the Elongator HAT activity showing specificity in target gene selection. Biological relevance was apparent by auxin-related phenotypes and marker gene analysis. Ethylene and jasmonic acid signaling and abiotic stress responses were up-regulated in the elo transcriptome and might contribute to the pleiotropic elo phenotype. Thus, although the structure of Elongator and its substrate are conserved, target gene selection has diverged, showing that auxin signaling and influx are under chromatin control.

The ang3 mutation identified the ribosomal protein gene RPL5B with a role in cell expansion during organ growth.

The role of translation in the regulation of higher plant growth and development is not well understood. Mutational analysis is a powerful tool to identify and study the function of genes related to a biological process, such as growth. Here we analyzed functionally the angusta3 (ang3) narrow leaf mutant. The AG3 gene was cloned by fine mapping combined with candidate gene sequencing and it corresponded to the ribosomal protein gene RPL5B. Based on amino acid sequence homology, promoter DNA sequence homology and in silico gene expression analysis, RPL5B was found to be putatively functionally redundant with RPL5A. The morphological analysis of ang3 mutants showed that the leaf lamina area was significantly reduced from the third rosette leaf on, mainly because of decreased width. Cellular analysis of the abaxial epidermal cell layer of the third leaf indicated that the cell number in the mutant was similar to that of the wild type, but the cell size was significantly reduced. We postulate that the reduced cell expansion in the epidermis contributes to the narrow shape of ang3 leaves. Growth was also significantly impaired in hypocotyls and primary roots, hinting at a general role for RPL5B in organ growth, unrelated to dorsiventral axis formation. Comparison of the transcriptome of the shoot apices of the mutant and the wild type revealed a limited number of differentially expressed genes, such as MYB23 and MYB5, of which the lower expression in the ang3 mutant correlated with reduced trichome density. Our data suggest that translation is an important level of control of growth and development in plants.

The Arabidopsis thaliana homolog of yeast BRE1 has a function in cell cycle regulation during early leaf and root growth.

Chromatin modification and transcriptional activation are novel roles for E3 ubiquitin ligase proteins that have been mainly associated with ubiquitin-dependent proteolysis. We identified HISTONE MONOUBIQUITINATION1 (HUB1) (and its homolog HUB2) in Arabidopsis thaliana as RING E3 ligase proteins with a function in organ growth. We show that HUB1 is a functional homolog of the human and yeast BRE1 proteins because it monoubiquitinated histone H2B in an in vitro assay. Hub knockdown mutants had pale leaf coloration, modified leaf shape, reduced rosette biomass, and inhibited primary root growth. One of the alleles had been designated previously as ang4-1. Kinematic analysis of leaf and root growth together with flow cytometry revealed defects in cell cycle activities. The hub1-1 (ang4-1) mutation increased cell cycle duration in young leaves and caused an early entry into the endocycles. Transcript profiling of shoot apical tissues of hub1-1 (ang4-1) indicated that key regulators of the G2-to-M transition were misexpressed. Based on the mutant characterization, we postulate that HUB1 mediates gene activation and cell cycle regulation probably through chromatin modifications.

The TORNADO1 and TORNADO2 genes function in several patterning processes during early leaf development in Arabidopsis thaliana.

In multicellular organisms, patterning is a process that generates axes in the primary body plan, creates domains upon organ formation, and finally leads to differentiation into tissues and cell types. We identified the Arabidopsis thaliana TORNADO1 (TRN1) and TRN2 genes and their role in leaf patterning processes such as lamina venation, symmetry, and lateral growth. In trn mutants, the leaf venation network had a severely reduced complexity: incomplete loops, no tertiary or quaternary veins, and vascular islands. The leaf laminas were asymmetric and narrow because of a severely reduced cell number. We postulate that the imbalance between cell proliferation and cell differentiation and the altered auxin distribution in both trn mutants cause asymmetric leaf growth and aberrant venation patterning. TRN1 and TRN2 were epistatic to ASYMMETRIC LEAVES1 with respect to leaf asymmetry, consistent with their expression in the shoot apical meristem and leaf primordia. TRN1 codes for a large plant-specific protein with conserved domains also found in a variety of signaling proteins, whereas TRN2 encodes a transmembrane protein of the tetraspanin family whose phylogenetic tree is presented. Double mutant analysis showed that TRN1 and TRN2 act in the same pathway.

The rotunda2 mutants identify a role for the LEUNIG gene in vegetative leaf morphogenesis.

Leaf development in Arabidopsis thaliana is considered to be a two-step process. In the first step, a leaf primordium is formed that involves a switch from indeterminate to leaf developmental fate in the shoot apical meristem cells. The second step, known as leaf morphogenesis, consists of post-initiation developmental events such as patterned cell proliferation, cell expansion, and cell differentiation. The results are presented of the molecular and genetic analyses of the rotunda2 (ron2) mutants of Arabidopsis, which were isolated based on their wide and serrated vegetative leaf lamina. The RON2 gene was positionally cloned and was identical to LEUNIG (LUG); it encodes a transcriptional co-repressor that has been described to affect flower development. Morphological and histological analyses of expanded leaves indicated that RON2 (LUG) acts at later stages of leaf development by restricting cell expansion during leaf growth. Real-time reverse-transcription polymerase chain reaction was used to quantify the expression of KNOX, WUSCHEL, YABBY3, LEAFY, ASYMMETRIC LEAVES, and GIBBERELLIN OXIDASE genes in expanding and fully expanded rosette leaf laminas of the wild type and ron2 and lug mutants. SHOOTMERISTEMLESS was expressed in wild-type leaves and down-regulated in the mutants. The results indicate that RON2 (LUG) has a function in later stages of leaf development.