Arabidopsis EXTRA-LARGE G PROTEIN 1 (XLG1) functions together with XLG2 and XLG3 in PAMP-triggered MAPK activation and immunity.

Pattern-triggered immunity (PTI) is an essential strategy used by plants to deploy broad-spectrum resistance against pathogen attacks. Heterotrimeric G proteins have been reported to contribute to PTI. Of the three non-canonical EXTRA-LARGE G PROTEINs (XLGs) in Arabidopsis thaliana, XLG2 and XLG3 were shown to positively regulate immunity, but XLG1 was not considered to function in defense, based on the analysis of a weak xlg1 allele. In this study, we characterized the xlg1 xlg2 xlg3 triple knockout mutants generated from an xlg1 knockout allele. The strong xlg1 xlg2 xlg3 triple mutants compromised pathogen-associated molecular pattern (PAMP)-triggered activation of mitogen-activated protein kinases (MAPKs) and resistance to pathogen infection. The three XLGs interacted with MAPK cascade proteins involved in defense signaling, including the MAPK kinase kinases MAPKKK3 and MAPKKK5, the MAPK kinases MKK4 and MKK5, and the MAPKs MPK3 and MPK6. Expressing a constitutively active form of MKK4 restored MAPK activation and partially recovered the compromised disease resistance seen in the strong xlg1 xlg2 xlg3 triple mutant. Furthermore, mutations of all three XLGs largely restored the phenotype of the autoimmunity mutant bak1-interacting receptor-like kinase 1. Our study reveals that all three XLGs function redundantly in PAMP-triggered MAPK activation and plant immunity.

Redox-sensitive bZIP68 plays a role in balancing stress tolerance with growth in Arabidopsis.

Perturbation of the cellular redox state by stress conditions is sensed by redox-sensitive proteins so that the cell can physiologically respond to stressors. However, the mechanisms linking sensing to response remain poorly understood in plants. Here we report that the transcription factor bZIP68 underwent in vivo oxidation in Arabidopsis cells under oxidative stress which is dependent on its redox-sensitive Cys320 residue. bZIP68 is primarily localized to the nucleus under normal growth conditions in Arabidopsis seedlings. Oxidative stress reduces its accumulation in the nucleus and increases its cytosolic localization. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed that bZIP68 primarily binds to promoter regions containing the core G-box (CACGTG) or G-box-like motif of the genes involved in abiotic and biotic stress responses, photosynthesis, biosynthetic processes, and transcriptional regulation. The bzip68 mutant displayed slower growth under normal conditions but enhanced tolerance to oxidative stress. The results from the ChIP-seq and phenotypic and transcriptome comparison between the bzip68 mutant and wildtype indicate that bZIP68 normally suppresses expression of stress tolerance genes and promotes expression of growth-related genes, whereas its inactivation enhances stress tolerance but suppresses growth. bZIP68 might balance stress tolerance with growth through the extent of its oxidative inactivation according to the environment.

Arabidopsis CPK6 positively regulates ABA signaling and drought tolerance through phosphorylating ABA-responsive element-binding factors.

Abscisic acid (ABA) regulates numerous developmental processes and drought tolerance in plants. Calcium-dependent protein kinases (CPKs) are important Ca2+ sensors playing crucial roles in plant growth and development as well as responses to stresses. However, the molecular mechanisms of many CPKs in ABA signaling and drought tolerance remain largely unknown. Here we combined protein interaction studies, and biochemical and genetic approaches to identify and characterize substrates that were phosphorylated by CPK6 and elucidated the mechanism that underlines the role of CPK6 in ABA signaling and drought tolerance. The expression of CPK6 is induced by ABA and dehydration. Two cpk6 T-DNA insertion mutants are insensitive to ABA during seed germination and root elongation of seedlings; in contrast, overexpression of CPK6 showed the opposite phenotype. Moreover, CPK6-overexpressing lines showed enhanced drought tolerance. CPK6 interacts with and phosphorylates a subset of core ABA signaling-related transcription factors, ABA-responsive element-binding factors (ABFs/AREBs), and enhances their transcriptional activities. The phosphorylation sites in ABF3 and ABI5 were also identified through MS and mutational analyses. Taken together, we present evidence that CPK6 mediates ABA signaling and drought tolerance through phosphorylating ABFs/AREBs. This work thus uncovers a rather conserved mechanism of calcium-dependent Ser/Thr kinases in ABA signaling.

Rapeseed calcineurin B-like protein CBL4, interacting with CBL-interacting protein kinase CIPK24, modulates salt tolerance in plants.

Calcium is a ubiquitous intracellular secondary messenger in eukaryotes. Upon stress challenge, cytosolic Ca(2+) fluctuation could be sensed and bound by calcineurin B-like proteins (CBLs), which further regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs) to relay the signal and induce cellular responses. Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in rapeseed. In the present study, we characterized CBL4 gene from rapeseed. We found that CBL4 is localized at the plasma membrane and it interacted with CIPK24 in both yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Unlike the orthologs in Arabidopsis, rapeseed CIPK24 did not interact with CBL10. Furthermore, expression of rapeseed CBL4 rescued the salt-sensitive phenotype of sos3-1 mutant and overexpression of rapeseed CBL4 in Arabidopsis showed enhanced tolerance of salt stress than wild-type. Overall, the results clarified the function of CBL4 in rapeseed.

Mitogen-activated protein kinase kinase kinase (MAPKKK) 4 from rapeseed (Brassica napus L.) is a novel member inducing ROS accumulation and cell death.

MAPKKK is the largest family of MAPK cascade, which is known to play important roles in plant growth, development and immune responses. So far, only a few have been functionally characterized even in the model plant, Arabidopsis due to the potential functional redundancy of MAPKKK. We previously identified and cloned a few MAPKKK family genes from rapeseed. In this study, BnaMAPKKK4 was characterized as a member in eliciting accumulation of reactive oxygen species (ROS) and hypersensitive response (HR)-like cell death. This is accompanied with accumulation of malondialdehyde (MDA), anthocyanin as well as nuclear DNA fragmentation. The transcript abundance of a series of ROS accumulation, cell death, and defense response related genes were up-regulated by the expression of MAPKKK4. Further investigation identified BnaMAPKKK4 elicited ROS through the downstream MPK3. These results indicate that BnaMAPKKK4 and its downstream components function in the ROS-induced cell death.

Identification, expression and interaction analyses of calcium-dependent protein kinase (CPK) genes in canola (Brassica napus L.).

BACKGROUND: Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a well-known ubiquitous intracellular secondary messenger in plants. Calcium-dependent protein kinases (CPKs) are Ser/Thr protein kinases found only in plants and some protozoans. CPKs are Ca2+ sensors that have both Ca2+ sensing function and kinase activity within a single protein and play crucial roles in plant development and responses to various environmental stresses. RESULTS: In this study, we mined the available expressed sequence tags (ESTs) of B. napus and identified a total of 25 CPK genes, among which cDNA sequences of 23 genes were successfully cloned from a double haploid cultivar of canola. Phylogenetic analysis demonstrated that they could be clustered into four subgroups. The subcellular localization of five selected BnaCPKs was determined using green fluorescence protein (GFP) as the reporter. Furthermore, the expression levels of 21 BnaCPK genes in response to salt, drought, cold, heat, abscisic acid (ABA), low potassium (LK) and oxidative stress were studied by quantitative RT-PCR and were found to respond to multiple stimuli, suggesting that canola CPKs may be convergence points of different signaling pathways. We also identified and cloned five and eight Clade A basic leucine zipper (bZIP) and protein phosphatase type 2C (PP2C) genes from canola and, using yeast two-hybrid and bimolecular fluorescence complementation (BiFC), determined the interaction between individual BnaCPKs and BnabZIPs or BnaPP2Cs (Clade A). We identified novel, interesting interaction partners for some of the BnaCPK proteins. CONCLUSION: We present the sequences and characterization of CPK gene family members in canola for the first time. This work provides a foundation for further crop improvement and improved understanding of signal transduction in plants.

Arabidopsis CIPK14 positively regulates glucose response.

Calcium is a ubiquitous intracellular secondary messenger in plants. Calcineurin B-like proteins (CBLs), which contain four Ca(2+)-binding EF hand motifs, are Ca(2+) sensors and regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs). Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in glucose signaling. In the present study, we identified CIPK14 gene from Arabidopsis that play a role in glucose signaling. The subcellular localization of CIPK14 was determined using green fluorescence protein (GFP) as the reporter. Furthermore, the expression levels of CIPK14 in response to salt, drought, cold, heat, ABA, methyl viologen (MV) and glucose treatments were examined by quantitative RT-PCR and it was found to respond to multiple stimuli, suggesting that CIPK14 may be a point of convergence for several different signaling pathways. Moreover, knock-out mutation of CIPK14 rendered it more sensitive to glucose treatment. Yeast two-hybrid assay demonstrated that CIPK14 interacted with three CBLs and also with two key kinases, sucrose non-fermenting 1-related kinase (SnRK) 1.1 and SnRK1.2 implicated in glucose signaling. This is the first report to demonstrate that CIPK also plays a role in glucose signaling.

Identification and characterization of CBL and CIPK gene families in canola (Brassica napus L.).

BACKGROUND: Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a ubiquitous intracellular secondary messenger in plants. Calcineurin B-like proteins (CBLs) are Ca2+ sensors and regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs). Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in canola. RESULTS: In the present study, we identified seven CBL and 23 CIPK genes from canola by database mining and cloning of cDNA sequences of six CBLs and 17 CIPKs. Phylogenetic analysis of CBL and CIPK gene families across a variety of species suggested genome duplication and diversification. The subcellular localization of three BnaCBLs and two BnaCIPKs were determined using green fluorescence protein (GFP) as the reporter. We also demonstrated interactions between six BnaCBLs and 17 BnaCIPKs using yeast two-hybrid assay, and a subset of interactions were further confirmed by bimolecular fluorescence complementation (BiFC). Furthermore, the expression levels of six selected BnaCBL and 12 BnaCIPK genes in response to salt, drought, cold, heat, ABA, methyl viologen (MV) and low potassium were examined by quantitative RT-PCR and these CBL or CIPK genes were found to respond to multiple stimuli, suggesting that the canola CBL-CIPK network may be a point of convergence for several different signaling pathways. We also performed a comparison of interaction patterns and expression profiles of CBL and CIPK in Arabidospsis, canola and rice, to examine the differences between orthologs, highlighting the importance of studying CBL-CIPK in canola as a prerequisite for improvement of this crop. CONCLUSIONS: Our findings indicate that CBL and CIPK family members may form a dynamic complex to respond to different abiotic or hormone signaling. Our comparative analyses of the CBL-CIPK network between canola, Arabidopsis and rice highlight functional differences and the necessity to study CBL-CIPK gene functions in canola. Our data constitute a valuable resource for CBL and CPK genomics.

WRKY47 transcription factor modulates leaf senescence through regulating PCD-associated genes in Arabidopsis.

Transcription factors play crucial roles in almost all physiological processes including leaf senescence. Cell death is a typical symptom appearing in senescing leaves, which is also classified as developmental programmed cell death (PCD). However, the link between PCD and leaf senescence still remains unclear. Here, we found a WRKY transcription factor WRKY47 positively modulates age-dependent leaf senescence in Arabidopsis (Arabidopsis thaliana). WRKY47 was expressed preferentially in senescing leaves. A subcellular localization assay indicated that WRKY47 was exclusively localized in nuclei. Overexpression of WRKY47 showed precocious leaf senescence, with less chlorophyll content and higher electrolyte leakage, but loss-of-function mutants of WRKY47 delayed this biological process. Through qRT-PCR and dual luciferase reporter assays, we found that WRKY47 could activate the expression of senescence-associated genes (SAGs) and PCD-associated genes to regulate leaf senescence. Furthermore, through electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-qPCR, WRKY47 was found to bind to W-box fragments in promoter regions of BFN1 (Bifunctional Nuclease 1) and MC6 (Metacaspase 6) directly. In general, our research revealed that WRKY47 regulates age-dependent leaf senescence by activating the transcription of two PCD-associated genes.

The interaction and physicochemical properties of the starch-polyphenol complex: Polymeric proanthocyanidins and maize starch with different amylose/amylopectin ratios.

This study investigated the impact of polymeric proanthocyanidins (PPC) on the physicochemical characteristics of maize starch with varying amylose content, and their potential interaction mechanism. PPC with a lower content (1 %) reduced the viscoelasticity of the high amylose maize starch (HAM) system, inhibited amylose rearrangement, and enhanced its fluidity. However, excessive PPC restrained the interaction between PPC and amylose. In contrast to HAM, PPC improved the gelation ability of waxy maize starch (WAM) as PPC concentration was raised. PPC suppressed the recrystallization of starch during storage, and PPC had a superior inhibition influence on the retrogradation of WAM in comparison to HAM. This indicated that amylopectin was more likely to interact with PPC than amylose. Hydrogen bonds were the main driving force between PPC and starch chains, which was clarified by Fourier transform-infrared, nuclear magnetic resonance, X-ray diffraction, iodine bonding reaction, and dynamic light scattering data. Additionally, the mechanism of interaction between PPC and the two starch components may be similar, and variance in physicochemical attributes can be primarily credited to the percentage of amylose to amylopectin in starch.

Identification, cloning and characterization of R2R3-MYB gene family in canola (Brassica napus L.) identify a novel member modulating ROS accumulation and hypersensitive-like cell death.

The R2R3-MYB proteins comprise one of the largest families of transcription factors in plants. Although genome-wide analysis of this family has been carried out in some plant species, little is known about R2R3-MYB genes in canola (Brassica napus L.). In this study, we have identified 76 R2R3-MYB genes in the canola genome through mining of expressed sequence tags (ESTs). The cDNA sequences of 44 MYB genes were successfully cloned. The transcriptional activities of BnaMYB proteins encoded by these genes were assayed in yeast. The subcellular localizations of representative R2R3-MYB proteins were investigated through GFP fusion. Besides, the transcript abundance level analysis during abiotic conditions and ABA treatment identified a group of R2R3-MYB genes that responded to one or more treatments. Furthermore, we identified a previously functionally unknown MYB gene-BnaMYB78, which modulates reactive oxygen species (ROS)-dependent cell death in Nicotiana benthamiana, through regulating the transcription of a few ROS- and defence-related genes. Taken together, this study has provided a solid foundation for understanding the roles and regulatory mechanism of canola R2R3-MYB genes.