The E3 ubiquitin ligase SP1‐like 1 plays a positive role in peroxisome biogenesis in Arabidopsis

Peroxisomes are dynamic organelles crucial for a variety of metabolic processes during the development of eukaryotic organisms, and are functionally linked to other subcellular organelles, such as mitochondria and chloroplasts. Peroxisomal matrix proteins are imported by peroxins (PEX proteins), yet the modulation of peroxin functions is poorly understood. We previously reported that, besides its known function in chloroplast protein import, the Arabidopsis E3 ubiquitin ligase SP1 (suppressor of ppi1 locus1) also targets to peroxisomes and mitochondria, and promotes the destabilization of the peroxisomal receptor–cargo docking complex components PEX13 and PEX14. Here we present evidence that in Arabidopsis, SP1's closest homolog SP1‐like 1 (SPL1) plays an opposite role to SP1 in peroxisomes. In contrast to sp1, loss‐of‐function of SPL1 led to reduced peroxisomal β‐oxidation activity, and enhanced the physiological and growth defects of pex14 and pex13 mutants. Transient co‐expression of SPL1 and SP1 promoted each other's destabilization. SPL1 reduced the ability of SP1 to induce PEX13 turnover, and it is the N‐terminus of SP1 and SPL1 that determines whether the protein is able to promote PEX13 turnover. Finally, SPL1 showed prevalent targeting to mitochondria, but rather weak and partial localization to peroxisomes. Our data suggest that these two members of the same E3 protein family utilize distinct mechanisms to modulate peroxisome biogenesis, where SPL1 reduces the function of SP1. Plants and possibly other higher eukaryotes may employ this small family of E3 enzymes to differentially modulate the dynamics of several organelles essential to energy metabolism via the ubiquitin‐proteasome system.     

AtRAE1 is involved in degradation of ABA receptor RCAR1 and negatively regulates ABA signalling in Arabidopsis

The phytohormone abscisic acid (ABA) plays an important role in regulating plant growth, development, and adaption to various environmental stresses. Regulatory components of ABA receptors (RCARs, also known as PYR/PYLs) sense ABA and initiate ABA signalling through inhibiting the activities of protein phosphatase 2C in Arabidopsis. However, the way in which ABA receptors are regulated is not well known. A DWD protein AtRAE1 (for RNA export factor 1 in Arabidopsis), which may act as a substrate receptor of CUL4–DDB1 E3 ligase, is an interacting partner of RCAR1/PYL9. The physical interaction between RCAR1 and AtRAE1 is confirmed in vitro and in vivo. Overexpression of AtRAE1 in Arabidopsis causes reduced sensitivity of plants to ABA, whereas suppression of AtRAE1 causes increased sensitivity to ABA. Analysis of protein stability demonstrates that RCAR1 is ubiquitinated and degraded in plant cells and AtRAE1 regulates the degradation speed of RCAR1. Our findings indicate that AtRAE1 likely participates in ABA signalling through regulating the degradation of ABA receptor RCAR1.     

Arabidopsis C3HC4‐RING finger E3 ubiquitin ligase AtAIRP4 positively regulates stress‐responsive abscisic acid signaling

Degradation of proteins via the ubiquitin system is an important step in many stress signaling pathways in plants. E3 ligases recognize ligand proteins and dictate the high specificity of protein degradation, and thus, play a pivotal role in ubiquitination. Here, we identified a gene, named Arabidopsis thaliana abscisic acid (ABA)‐insensitive RING protein 4 (AtAIRP4), which is induced by ABA and other stress treatments. AtAIRP4 encodes a cellular protein with a C3HC4‐RING finger domain in its C‐terminal side, which has in vitro E3 ligase activity. Loss of AtAIRP4 leads to a decrease in sensitivity of root elongation and stomatal closure to ABA, whereas overexpression of this gene in the T‐DNA insertion mutant atairp4 effectively recovered the ABA‐associated phenotypes. AtAIRP4 overexpression plants were hypersensitive to salt and osmotic stresses during seed germination, and showed drought avoidance compared with the wild‐type and atairp4 mutant plants. In addition, the expression levels of ABA‐ and drought‐induced marker genes in AtAIRP4 overexpression plants were markedly higher than those in the wild‐type and atairp4 mutant plants. Hence, these results indicate that AtAIRP4 may act as a positive regulator of ABA‐mediated drought avoidance and a negative regulator of salt tolerance in Arabidopsis.     

The single‐subunit RING‐type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling

Membrane‐delimited events play a crucial role for ABA signaling and PYR/PYL/RCAR ABA receptors, clade A PP2Cs and SnRK2/CPK kinases modulate the activity of different plasma membrane components involved in ABA action. Therefore, the turnover of PYR/PYL/RCARs in the proximity of plasma membrane might be a step that affects receptor function and downstream signaling. In this study we describe a single‐subunit RING‐type E3 ubiquitin ligase RSL1 that interacts with the PYL4 and PYR1 ABA receptors at the plasma membrane. Overexpression of RSL1 reduces ABA sensitivity and rsl1 RNAi lines that impair expression of several members of the RSL1/RFA gene family show enhanced sensitivity to ABA. RSL1 bears a C‐terminal transmembrane domain that targets the E3 ligase to plasma membrane. Accordingly, bimolecular fluorescent complementation (BiFC) studies showed the RSL1–PYL4 and RSL1–PYR1 interaction is localized to plasma membrane. RSL1 promoted PYL4 and PYR1 degradation in vivo and mediated in vitro ubiquitylation of the receptors. Taken together, these results suggest ubiquitylation of ABA receptors at plasma membrane is a process that might affect their function via effect on their half‐life, protein interactions or trafficking.     

A novel calmodulin-binding protein functions as a negative regulator of osmotic stress tolerance in Arabidopsis thaliana seedlings

A clone for a novel Arabidopsisthaliana calmodulin (CaM)-binding protein of 25 kDa (AtCaMBP25) has been isolated by using a radiolabelled CaM probe to screen a cDNA expression library derived from A. thaliana cell suspension cultures challenged with osmotic stress. The deduced amino acid sequence of AtCaMBP25 contains putative nuclear localization sequences and shares significant degree of similarity with hypothetical plant proteins only. Fusion of the AtCaMBP25 coding sequence to reporter genes targets the hybrid protein to the nucleus. Bacterially expressed AtCaMBP25 binds, in a calcium-dependent manner, to a canonical CaM but not to a less conserved isoform of the calcium sensor. AtCaMBP25 is encoded by a single-copy gene, whose expression is induced in Arabidopsis seedlings exposed to dehydration, low temperature or high salinity. Transgenic plants overexpressing AtCaMBP25 exhibits an increased sensitivity to both ionic (NaCl) and non-ionic (mannitol) osmotic stress during seed germination and seedling growth. By contrast, transgenic lines expressing antisense AtCaMBP25 are significantly more tolerant to mannitol and NaCl stresses than the wild type. Thus, the AtCaMBP25 gene functions as a negative effector of osmotic stress tolerance and likely participates in stress signal transduction pathways.     

LTP3 contributes to disease susceptibility in Arabidopsis by enhancing abscisic acid (ABA) biosynthesis

Several plant lipid transfer proteins (LTPs) act positively in plant disease resistance. Here, we show that LTP3 (At5g59320), a pathogen and abscisic acid (ABA)‐induced gene, negatively regulates plant immunity in Arabidopsis. The overexpression of LTP3 (LTP3‐OX) led to an enhanced susceptibility to virulent bacteria and compromised resistance to avirulent bacteria. On infection of LTP3‐OX plants with Pseudomonas syringae pv. tomato, genes involved in ABA biosynthesis, NCED3 and AAO3, were highly induced, whereas salicylic acid (SA)‐related genes, ICS1 and PR1, were down‐regulated. Accordingly, in LTP3‐OX plants, we observed increased ABA levels and decreased SA levels relative to the wild‐type. We also showed that the LTP3 overexpression‐mediated enhanced susceptibility was partially dependent on AAO3. Interestingly, loss of function of LTP3 (ltp3‐1) did not affect ABA pathways, but resulted in PR1 gene induction and elevated SA levels, suggesting that LTP3 can negatively regulate SA in an ABA‐independent manner. However, a double mutant consisting of ltp3‐1 and silent LTP4 (ltp3/ltp4) showed reduced susceptibility to Pseudomonas and down‐regulation of ABA biosynthesis genes, suggesting that LTP3 acts in a redundant manner with its closest homologue LTP4 by modulating the ABA pathway. Taken together, our data show that LTP3 is a novel negative regulator of plant immunity which acts through the manipulation of the ABA–SA balance.     

The phs1-3 mutation in a putative dual-specificity protein tyrosine phosphatase gene provokes hypersensitive responses to abscisic acid in Arabidopsis thaliana

The plant hormone abscisic acid (ABA) controls numerous physiological traits: dormancy and germination of seeds, senescence and resistance to abiotic stresses. In order to get more insight into the role of protein tyrosine phosphatase (PTP) in ABA signalling, we obtained eight homozygous T-DNA insertion lines in Arabidopsis thaliana PTP genes. One mutant, named phs1-3, exhibited a strong ABA-induced inhibition of germination as only 26% of its seeds germinated after 3 days instead of 92% for the Columbia (Col-0) line. Genetic and molecular analyses of phs1-3 showed that it bears a unique T-DNA insertion in the promoter of the gene and that the mutation is recessive. PHS1 expression in the mutant is about half that of the Col-0 line. The upregulation of two ABA-induced genes (At5g06760, RAB18) and the downregulation of two ABA-repressed genes (AtCLC-A, ACL) are enhanced in the phs1-3 mutant compared with the wild-type. The 'in planta' aperture of phs1-3 stomata is reduced and the inhibition of the light-induced opening of stomata by ABA is stronger in phs1-3 leaves than in Col-0 leaves. Finally, PHS1 expression is upregulated in the presence of ABA in both phs1-3 and Col-0 but more intensively in the mutant. Thus, phs1-3 is hypersensitive to ABA. Taken together, these results show that PHS1, which encodes a dual-specificity PTP, is a negative regulator of ABA signalling.     

The heterotrimeric G‐protein β subunit,AGB1, plays multiple roles in the Arabidopsis salinity response

Salinity stress includes both osmotic and ionic toxicity. Sodium homeostasis is influenced by Na⁺ uptake and extrusion, vacuolar Na⁺ compartmentation and root to shoot Na⁺ translocation via transpiration. The knockout mutant of the Arabidopsis heterotrimeric G‐protein Gβ subunit, agb1, is hypersensitive to salt, exhibiting a leaf bleaching phenotype. We show that AGB1 is mainly involved in the ionic toxicity component of salinity stress and plays roles in multiple processes of Na⁺ homeostasis. agb1 mutants accumulate more Na⁺ and less K⁺ in both shoots and roots of hydroponically grown plants, as measured by inductively coupled plasma atomic emission spectrometry. agb1 plants have higher root to shoot translocation rates of radiolabelled ²⁴Na⁺ under transpiring conditions, as a result of larger stomatal apertures and increased stomatal conductance. ²⁴Na⁺ tracer experiments also show that ²⁴Na⁺ uptake rates by excised roots of agb1 and wild type are initially equal, but that agb1 has higher net Na⁺ uptake at 90 min, implicating possible involvement of AGB1 in the regulation of Na⁺ efflux. Calcium alleviates the salt hypersensitivity of agb1 by reducing Na⁺ accumulation to below the toxicity threshold. Our results provide new insights into the regulatory pathways underlying plant responses to salinity stress, an important agricultural problem.     

The Arabidopsis STRESS RESPONSE SUPPRESSOR DEAD‐box RNA helicases are nucleolar‐ and chromocenter‐localized proteins that undergo stress‐mediated relocalization and are involved in epigenetic gene silencing

DEAD‐box RNA helicases are involved in many aspects of RNA metabolism and in diverse biological processes in plants. Arabidopsis thaliana mutants of two DEAD‐box RNA helicases, STRESS RESPONSE SUPPRESSOR1 (STRS1) and STRS2 were previously shown to exhibit tolerance to abiotic stresses and up‐regulated stress‐responsive gene expression. Here, we show that Arabidopsis STRS‐overexpressing lines displayed a less tolerant phenotype and reduced expression of stress‐induced genes confirming the STRSs as attenuators of Arabidopsis stress responses. GFP–STRS fusion proteins exhibited localization to the nucleolus, nucleoplasm and chromocenters and exhibited relocalization in response to abscisic acid (ABA) treatment and various stresses. This relocalization was reversed when stress treatments were removed. The STRS proteins displayed mis‐localization in specific gene‐silencing mutants and exhibited RNA‐dependent ATPase and RNA‐unwinding activities. In particular, STRS2 showed mis‐localization in three out of four mutants of the RNA‐directed DNA methylation (RdDM) pathway while STRS1 was mis‐localized in the hd2c mutant that is defective in histone deacetylase activity. Furthermore, heterochromatic RdDM target loci displayed reduced DNA methylation and increased expression in the strs mutants. Taken together, our findings suggest that the STRS proteins are involved in epigenetic silencing of gene expression to bring about suppression of the Arabidopsis stress response.     

Arabidopsis ROP‐interactive CRIB motif‐containing protein 1 (RIC1) positively regulates auxin signalling and negatively regulates abscisic acid (ABA) signalling during root development

Auxin and abscisic acid (ABA) modulate numerous aspects of plant development together, mostly in opposite directions, suggesting that extensive crosstalk occurs between the signalling pathways of the two hormones. However, little is known about the nature of this crosstalk. We demonstrate that ROP‐interactive CRIB motif‐containing protein 1 (RIC1) is involved in the interaction between auxin‐ and ABA‐regulated root growth and lateral root formation. RIC1 expression is highly induced by both hormones, and expressed in the roots of young seedlings. Whereas auxin‐responsive gene induction and the effect of auxin on root growth and lateral root formation were suppressed in the ric1 knockout, ABA‐responsive gene induction and the effect of ABA on seed germination, root growth and lateral root formation were potentiated. Thus, RIC1 positively regulates auxin responses, but negatively regulates ABA responses. Together, our results suggest that RIC1 is a component of the intricate signalling network that underlies auxin and ABA crosstalk.