A novel receptor‐like kinase involved in fungal pathogen defence in Arabidopsis thaliana

Plants are under constant attack from a variety of disease‐causing organisms. Lacking an adaptive immune system, plants repel pathogen attack via an array of pathogen recognition machinery. Receptor‐like kinases (RLKs) are involved in the recognition of pathogen‐associated molecular patterns (PAMPs) and activate resistance pathways against broad classes of pathogens. We have identified powdery mildew‐resistant kinase 1, an Arabidopsis gene encoding an RLK that is highly induced by chitin at early time points and localizes to the plasma membrane. Knockout mutants in pmrk1 are more susceptible to both Golovinomyces cichoracearum and Plectosphaerella cucumerina. Our data show that PMRK1 is essential in early stages of defence against fungi and provide evidence that PMRK1 may be unique to chitin‐induced signalling pathways. The results of this study indicate that PMRK1 is a critical component of plant innate immunity against fungal pathogens.     

Temperature‐dependent shade avoidance involves the receptor‐like kinase ERECTA

Plants detect the presence of neighbouring vegetation by monitoring changes in the ratio of red (R) to far‐red (FR) wavelengths (R:FR) in ambient light. Reductions in R:FR are perceived by the phytochrome family of plant photoreceptors and initiate a suite of developmental responses termed the shade avoidance syndrome. These include increased elongation growth of stems and petioles, enabling plants to overtop competing vegetation. The majority of shade avoidance experiments are performed at standard laboratory growing temperatures (>20°C). In these conditions, elongation responses to low R:FR are often accompanied by reductions in leaf development and accumulation of plant biomass. Here we investigated shade avoidance responses at a cooler temperature (16°C). In these conditions, Arabidopsis thaliana displays considerable low R:FR‐mediated increases in leaf area, with reduced low R:FR‐mediated petiole elongation and leaf hyponasty responses. In Landsberg erecta, these strikingly different shade avoidance phenotypes are accompanied by increased leaf thickness, increased biomass and an altered metabolite profile. At 16°C, low R:FR treatment results in the accumulation of soluble sugars and metabolites associated with cold acclimation. Analyses of natural genetic variation in shade avoidance responses at 16°C have revealed a regulatory role for the receptor‐like kinase ERECTA.     

An autophosphorylation site database for leucine‐rich repeat receptor‐like kinases in Arabidopsis thaliana

Leucine‐rich repeat receptor‐like kinases (LRR RLKs) form a large family of plant signaling proteins consisting of an extracellular domain connected by a single‐pass transmembrane sequence to a cytoplasmic kinase domain. Autophosphorylation on specific Ser and/or Thr residues in the cytoplasmic domain is often critical for the activation of several LRR RLK family members with proven functional roles in plant growth regulation, morphogenesis, disease resistance, and stress responses. While identification and functional characterization of in vivo phosphorylation sites is ultimately required for a full understanding of LRR RLK biology and function, bacterial expression of recombinant LRR RLK cytoplasmic catalytic domains for identification of in vitro autophosphorylation sites provides a useful resource for further targeted identification and functional analysis of in vivo sites. In this study we employed high‐throughput cloning and a variety of mass spectrometry approaches to generate an autophosphorylation site database representative of more than 30% of the approximately 223 LRR RLKs in Arabidopsis thaliana. We used His‐tagged constructs of complete cytoplasmic domains to identify a total of 592 phosphorylation events across 73 LRR RLKs, with 497 sites uniquely assigned to specific Ser (268 sites) or Thr (229 sites) residues in 68 LRR RLKs. Multiple autophosphorylation sites per LRR RLK were the norm, with an average of seven sites per cytoplasmic domain, while some proteins showed more than 20 unique autophosphorylation sites. The database was used to analyze trends in the localization of phosphorylation sites across cytoplasmic kinase subdomains and to derive a statistically significant sequence motif for phospho‐Ser autophosphorylation.     

The Arabidopsis leucine‐rich repeat receptor‐like kinase MUSTACHES enforces stomatal bilateral symmetry in Arabidopsis

Stomata display a mirror‐like symmetry that is adaptive for shoot/atmosphere gas exchange. This symmetry includes the facing guard cells around a lens‐shaped and bilaterally symmetric pore, as well as radially arranged microtubule arrays that primarily originate at the pore and then grow outwards. Mutations in MUSTACHES (MUS), which encodes a leucine‐rich repeat receptor‐like kinase, disrupt this symmetry, resulting in defects ranging from skewed pores and abnormally focused and depolarized radial microtubule arrays, to paired guard cells that face away from each other, or a severe loss of stomatal shape. Translational MUSproMUS:tripleGFP fusions are expressed in cell plates in most cells types in roots and shoots, and cytokinesis and cell plates are mostly normal in mus mutants. However, in guard mother cells, which divide and then form stomata, MUS expression is notably absent from new cell plates, and instead is peripherally located. These results are consistent with a role for MUS in enforcing wall building and cytoskeletal polarity at the centre of the developing stoma via signalling from the vicinity of the guard cell membrane.     

The SERK1 receptor‐like kinase regulates organ separation in Arabidopsis flowers

Through a sensitized screen for novel components of pathways regulating organ separation in Arabidopsis flowers, we have found that the leucine‐rich repeat receptor‐like kinase SOMATIC EMBRYOGENESIS RECEPTOR‐LIKE KINASE1 (SERK1) acts as a negative regulator of abscission. Mutations in SERK1 dominantly rescue abscission in flowers without functional NEVERSHED (NEV), an ADP‐ribosylation factor GTPase‐activating protein required for floral organ shedding. We previously reported that the organization of the Golgi apparatus and location of the trans‐Golgi network (TGN) are altered in nev mutant flowers. Disruption of SERK1 restores Golgi structure and the close association of the TGN in nev flowers, suggesting that defects in these organelles may be responsible for the block in abscission. We have also found that the abscission zones of nev serk1 flowers are enlarged compared to wild‐type. A similar phenotype was previously observed in plants constitutively expressing a putative ligand required for organ separation, INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), suggesting that signalling through IDA and its proposed receptors, HAESA and HAESA‐LIKE2, may be deregulated in nev serk1 abscission zone cells. Our studies indicate that in addition to its previously characterized roles in stamen development and brassinosteroid perception, SERK1 plays a unique role in modulating the loss of cell adhesion that occurs during organ abscission.     

Identification of in vitro phosphorylation sites in the Arabidopsis thaliana somatic embryogenesis receptor‐like kinases

The Arabidopsis thaliana somatic embryogenesis receptor‐like kinase (SERK) family consists of five leucine‐rich repeat receptor‐like kinases (LRR‐RLKs) with diverse functions such as brassinosteroid insensitive 1 (BRI1)‐mediated brassinosteroid perception, development and innate immunity. The autophosphorylation activity of the kinase domains of the five SERK proteins was compared and the phosphorylated residues were identified by LC‐MS/MS. Differences in autophosphorylation that ranged from high activity of SERK1, intermediate activities for SERK2 and SERK3 to low activity for SERK5 were noted. In the SERK1 kinase the C‐terminally located residue Ser‐562 controls full autophosphorylation activity. Activation loop phosphorylation, including that of residue Thr‐462 previously shown to be required for SERK1 kinase activity, was not affected. In vivo SERK1 phosphorylation was induced by brassinosteroids. Immunoprecipitation of CFP‐tagged SERK1 from plant extracts followed by MS/MS identified Ser‐303, Thr‐337, Thr‐459, Thr‐462, Thr‐463, Thr‐468, and Ser‐612 or Thr‐613 or Tyr‐614 as in vivo phosphorylation sites of SERK1. Transphosphorylation of SERK1 by the kinase domain of the main brassinosteroid receptor BRI1 occurred only on Ser‐299 and Thr‐462. This suggests both intra‐ and intermolecular control of SERK1 kinase activity. Conversely, BRI1 was transphosphorylated by the kinase domain of SERK1 on Ser‐887. BRI1 kinase activity was not required for interaction with the SERK1 receptor in a pull down assay.     

The Arabidopsis A4 subfamily of lectin receptor kinases negatively regulates abscisic acid response in seed germination

Abscisic acid (ABA) is an important plant hormone for a wide array of growth and developmental processes and stress responses, but the mechanism of ABA signal perception on the plasma membrane remains to be dissected. A previous GeneChip analysis revealed that a member of the A4 subfamily of lectin receptor kinases (LecRKs) of Arabidopsis (Arabidopsis thaliana), At5g01540 (designated LecRKA4.1), is up-regulated in response to a low dose of ABA in the rop10-1 background. Here, we present functional evidence to support its role in ABA response. LecRKA4.1 is expressed in seeds and leaves but not in roots, and the protein is localized to the plasma membrane. A T-DNA knockout mutant, lecrka4.1-1, slightly enhanced ABA inhibition of seed germination. Interestingly, LecRKA4.1 is adjacent to two other members of the A4 subfamily of LecRK genes, At5g01550 (LecRKA4.2) and At5g01560 (LecRKA4.3). We found that loss-of-function mutants of LecRKA4.2 and LecRKA4.3 exhibited similarly weak enhancement of ABA response in seed germination inhibition. Furthermore, LecRKA4.2 suppression by RNA interference in lecrka4.1-1 showed stronger ABA inhibition of seed germination than lecrka4.1-1, while the response to gibberellic acid was not affected in lecrka4.1-1 and lecrka4.1-1; LecRKA4.2 (RNAi) lines. Expression studies, together with network-based analysis, suggest that LecRKA4.1 and LecRKA4.2 regulate some of the ABA-responsive genes. Taken together, our results demonstrate that the A4 subfamily of LecRKs has a redundant function in the negative regulation of ABA response in seed germination.     

A pair of receptor‐like kinases is responsible for natural variation in shoot growth response to mannitol treatment in Arabidopsis thaliana

Growth is a complex trait that adapts to the prevailing conditions by integrating many internal and external signals. Understanding the molecular origin of this variation remains a challenging issue. In this study, natural variation of shoot growth under mannitol‐induced stress was analyzed by standard quantitative trait locus mapping methods in a recombinant inbred line population derived from a cross between the Col‐0 and Cvi‐0 Arabidopsis thaliana accessions. Cloning of a major QTL specific to mannitol‐induced stress condition led to identification of EGM1 and EGM2, a pair of tandem‐duplicated genes encoding receptor‐like kinases that are potentially involved in signaling of mannitol‐associated stress responses. Using various genetic approaches, we identified two non‐synonymous mutations in the EGM2[Cvi] allele that are shared by at least ten accessions from various origins and are probably responsible for a specific tolerance to mannitol. We have shown that the enhanced shoot growth phenotype contributed by the Cvi allele is not linked to generic osmotic properties but instead to a specific chemical property of mannitol itself. This result raises the question of the function of such a gene in A. thaliana, a species that does not synthesize mannitol. Our findings suggest that the receptor‐like kinases encoded by EGM genes may be activated by mannitol produced by pathogens such as fungi, and may contribute to plant defense responses whenever mannitol is present.     

A rice lectin receptor‐like kinase that is involved in innate immune responses also contributes to seed germination

Seed germination and innate immunity both have significant effects on plant life spans because they control the plant's entry into the ecosystem and provide defenses against various external stresses, respectively. Much ecological evidence has shown that seeds with high vigor are generally more tolerant of various environmental stimuli in the field than those with low vigor. However, there is little genetic evidence linking germination and immunity in plants. Here, we show that the rice lectin receptor‐like kinase OslecRK contributes to both seed germination and plant innate immunity. We demonstrate that knocking down the OslecRK gene depresses the expression of α–amylase genes, reducing seed viability and thereby decreasing the rate of seed germination. Moreover, it also inhibits the expression of defense genes, and so reduces the resistance of rice plants to fungal and bacterial pathogens as well as herbivorous insects. Yeast two‐hybrid and co‐immunoprecipitation experiments revealed that OslecRK interacts with an actin‐depolymerizing factor (ADF) in vivo via its kinase domain. Moreover, the rice adf mutant exhibited a reduced seed germination rate due to the suppression of α–amylase gene expression. This mutant also exhibited depressed immune responses and reduced resistance to biotic stresses. Our results thus provide direct genetic evidence for a common physiological pathway connecting germination and immunity in plants. They also partially explain the common observation that high‐vigor seeds often perform well in the field. The dual effects of OslecRK may be indicative of progressive adaptive evolution in rice.     

Metabolomics of red‐light‐induced stomatal opening in Arabidopsis thaliana: Coupling with abscisic acid and jasmonic acid metabolism

Environmental stimuli‐triggered stomatal movement is a key physiological process that regulates CO₂ uptake and water loss in plants. Stomata are defined by pairs of guard cells that perceive and transduce external signals, leading to cellular volume changes and consequent stomatal aperture change. Within the visible light spectrum, red light induces stomatal opening in intact leaves. However, there has been debate regarding the extent to which red‐light‐induced stomatal opening arises from direct guard cell sensing of red light versus indirect responses as a result of red light influences on mesophyll photosynthesis. Here we identify conditions that result in red‐light‐stimulated stomatal opening in isolated epidermal peels and enlargement of protoplasts, firmly establishing a direct guard cell response to red light. We then employ metabolomics workflows utilizing gas chromatography mass spectrometry and liquid chromatography mass spectrometry for metabolite profiling and identification of Arabidopsis guard cell metabolic signatures in response to red light in the absence of the mesophyll. We quantified 223 metabolites in Arabidopsis guard cells, with 104 found to be red light responsive. These red‐light‐modulated metabolites participate in the tricarboxylic acid cycle, carbon balance, phytohormone biosynthesis and redox homeostasis. We next analyzed selected Arabidopsis mutants, and discovered that stomatal opening response to red light is correlated with a decrease in guard cell abscisic acid content and an increase in jasmonic acid content. The red‐light‐modulated guard cell metabolome reported here provides fundamental information concerning autonomous red light signaling pathways in guard cells.     

The role of Arabidopsis thaliana RASD1 gene in ABA‐dependent abiotic stress response

• Abiotic stress is one of the key parameters affecting plant productivity. Drought and soil salinity, in particular, challenge plants to activate various response mechanisms to withstand these adverse growth conditions. While the molecular events that take place are complex and to a large extent unclear, the plant hormone abscisic acid (ABA) is considered a major player in mediating the adaptation of plants to stress.
• Here we report the identification of an ABA‐insensitive mutant from Arabidopsis thaliana. A combination of molecular, genetic and physiology approaches were implemented, to characterise the AtRASD1 locus (A BA D ROUGHT 相似文献   

Quantitative phosphoproteomics of early elicitor signaling in Arabidopsis

Perception of general elicitors by plant cells initiates signal transduction cascades that are regulated by protein phosphorylation. The earliest signaling events occur within minutes and include ion fluxes across the plasma membrane, activation of MAPKs, and the formation of reactive oxygen species. The phosphorylation events that regulate these signaling cascades are largely unknown. Here we present a mass spectrometry-based quantitative phosphoproteomics approach that identified differentially phosphorylated sites in signaling and response proteins from Arabidopsis cells treated with either flg22 or xylanase. Our approach was sensitive enough to quantitate phosphorylation on low abundance signaling proteins such as calcium-dependent protein kinases and receptor-like kinase family members. With this approach we identified one or more differentially phosphorylated sites in 76 membrane-associated proteins including a number of defense-related proteins. Our data on phosphorylation indicate a high degree of complexity at the level of post-translational modification as exemplified by the complex modification patterns of respiratory burst oxidase protein D. Furthermore the data also suggest that protein translocation and vesicle traffic are important aspects of early signaling and defense in response to general elicitors. Our study presents the largest quantitative Arabidopsis phosphoproteomics data set to date and provides a new resource that can be used to gain novel insight into plant defense signal transduction and early defense response.     

Phototropin‐ and photosynthesis‐dependent mitochondrial positioning in Arabidopsis thaliana mesophyll cells

Mitochondria are frequently observed in the vicinity of chloroplasts in photosynthesizing cells, and this association is considered necessary for their metabolic interactions. We previously reported that, in leaf palisade cells of Arabidopsis thaliana, mitochondria exhibit blue‐light‐dependent redistribution together with chloroplasts, which conduct accumulation and avoidance responses under the control of blue‐light receptor phototropins. In this study, precise motility analyses by fluorescent microscopy revealed that the individual mitochondria in palisade cells, labeled with green fluorescent protein, exhibit typical stop‐and‐go movement. When exposed to blue light, the velocity of moving mitochondria increased in 30 min, whereas after 4 h, the frequency of stoppage of mitochondrial movement markedly increased. Using different mutant plants, we concluded that the presence of both phototropin1 and phototropin2 is necessary for the early acceleration of mitochondrial movement. On the contrary, the late enhancement of stoppage of mitochondrial movement occurs only in the presence of phototropin2 and only when intact photosynthesis takes place. A plasma‐membrane ghost assay suggested that the stopped mitochondria are firmly adhered to chloroplasts. These results indicate that the physical interaction between mitochondria and chloroplasts is cooperatively mediated by phototropin2‐ and photosynthesis‐dependent signals. The present study might add novel regulatory mechanism for light‐dependent plant organelle interactions.     

Wounding of Arabidopsis leaves induces indole‐3‐carbinol‐dependent autophagy in roots of Arabidopsis thaliana

In cruciferous plants insect attack or physical damage induce the synthesis of the glucosinolate breakdown product indole‐3‐carbinol, which plays a key role in the defense against attackers. Indole‐3‐carbinol also affects plant growth and development, acting as an auxin antagonist by binding to the TIR1 auxin receptor. Other potential functions of indole‐3‐carbinol and the underlying mechanisms in plant biology are unknown. Here we show that an indole‐3‐carbinol‐dependent signal induces specific autophagy in root cells. Leaf treatment with exogenous indole‐3‐carbinol or leaf‐wounding induced autophagy and inhibited auxin response in the root. This induction is lost in glucosinolate‐defective mutants, indicating that the effect of indole‐3‐carbinol is transported in the plants. Thus, indole‐3‐carbinol is not only a defensive metabolite that repels insects, but is also involved in long‐distance communication regulating growth and development in plants.     

Hydrotropism in abscisic acid,wavy, and gravitropic mutants of Arabidopsis thaliana

We have developed experimental systems to study hydrotropism in seedling roots of Arabidopsis thaliana (L.) Heynh. Arabidopsis roots showed a strong curvature in response to a moisture gradient, established by applying 1% agar and a saturated solution of KCl or K(2)CO(3) in a closed chamber. In this system, the hydrotropic response overcame the gravitropic response. Hydrotropic curvature commenced within 30 min and reached 80-100 degrees within 24 h of hydrostimulation. When 1% agar and agar containing 1 MPa sorbitol were placed side-by-side in humid air, a water potential gradient formed at the border between the two media. Although the gradient changed with time, it still elicited a hydrotropic response in Arabidopsis roots. The roots curved away from 0.5-1.5 MPa of sorbitol agar. Various Arabidopsis mutants were tested for their hydrotropic response. Roots of aba1-1 and abi2-1 mutants were less sensitive to hydrotropic stimulation. Addition of abscisic acid restored the normal hydrotropic response in aba1-1 roots. In comparison, mutants that exhibit a reduced response to gravity and auxin, axr1-3 and axr2-1, showed a hydrotropic response greater than that of the wild type. Wavy mutants, wav2-1 and wav3-1, showed increased sensitivity to the induction of hydrotropism by the moisture gradient. These results suggest that auxin plays divergent roles in hydrotropism and gravitropism, and that abscisic acid plays a positive role in hydrotropism. Furthermore, hydrotropism and the wavy response may share part of a common molecular pathway controlling the directional growth of roots.