Qingmei Guan

Heat stress induction of miR398 triggers a regulatory loop that is critical for thermotolerance in Arabidopsis

microRNAs (miRNAs) play important roles in plant growth and development. Previous studies have shown that down-regulation of miR398 in response to oxidative stress permits up-regulation of one of its target genes, CSD2 (copper/zinc superoxide dismutase), and thereby helps plants to cope with oxidative stress. We report here that heat stress rapidly induces miR398 and reduces transcripts of its target genes CSD1, CSD2 and CCS (a gene encoding a copper chaperone for both CSD1 and CSD2). Transgenic plants expressing miR398-resistant forms of CSD1, CSD2 and CCS under the control of their native promoters are more sensitive to heat stress (as indicated by increased damage at the whole-plant level and to flowers) than transgenic plants expressing normal coding sequences of CSD1, CSD2 or CCS under the control of their native promoters. In contrast, csd1, csd2 and ccs mutant plants are more heat-tolerant (as indicated by less damage to flowers) than the wild-type. Expression of genes encoding heat stress transcription factors (HSF genes) and heat shock proteins (HSP genes) is reduced in heat-sensitive transgenic plants expressing miR398-resistant forms of CSD1, CSD2 or CCS but is enhanced in the heat-tolerant csd1, csd2 and ccs plants. Chromatin immunoprecipitation assays revealed that HSFA1b and HSFA7b are the two HSFs responsible for heat induction of miR398. Together, our results suggest that plants use a previously unrecognized strategy to achieve thermotolerance, especially for the protection of reproductive tissues. This strategy involves the down-regulation of CSD genes and their copper chaperone CCS through heat-inducible miR398.

A DEAD Box RNA Helicase Is Critical for Pre-mRNA Splicing, Cold-Responsive Gene Regulation, and Cold Tolerance in Arabidopsis

This article characterizes RCF1, a cold-inducible DEAD box RNA helicase that is important for pre-mRNA splicing of genes. RCF1 regulates the expression of cold-regulated genes and is vital for cold tolerance in plants. Cold stress resulting from chilling and freezing temperatures substantially reduces crop production worldwide. To identify genes critical for cold tolerance in plants, we screened Arabidopsis thaliana mutants for deregulated expression of a firefly luciferase reporter gene under the control of the C-REPEAT BINDING FACTOR2 (CBF2) promoter (CBF2:LUC). A regulator of CBF gene expression1 (rcf1-1) mutant that is hypersensitive to cold stress was chosen for in-depth characterization. RCF1 encodes a cold-inducible DEAD (Asp-Glu-Ala-Asp) box RNA helicase. Unlike a previously reported DEAD box RNA helicase (LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENES4 [LOS4]) that regulates mRNA export, RCF1 does not play a role in mRNA export. Instead, RCF1 functions to maintain proper splicing of pre-mRNAs; many cold-responsive genes are mis-spliced in rcf1-1 mutant plants under cold stress. Functional characterization of four genes (PSEUDO-RESPONSE REGULATOR5 [PRR5], SHAGGY-LIKE SERINE/THREONINE KINASE12 [SK12], MYB FAMILY TRANSCRIPTION FACTOR CIRCADIAN1 [CIR1], and SPFH/PHB DOMAIN-CONTAINING MEMBRANE-ASSOCIATED PROTEIN [SPFH]) that are mis-spliced in rcf1-1 revealed that these genes are cold-inducible positive (CIR1 and SPFH) and negative (PRR5 and SK12) regulators of cold-responsive genes and cold tolerance. Together, our results suggest that the cold-inducible RNA helicase RCF1 is essential for pre-mRNA splicing and is important for cold-responsive gene regulation and cold tolerance in plants.

The Protein Phosphatase RCF2 and Its Interacting Partner NAC019 Are Critical for Heat Stress–Responsive Gene Regulation and Thermotolerance in Arabidopsis

This study employed forward genetic analysis to uncover an important function of a protein phosphatase, RCF2, and its interacting transcription factor, NAC019, in the activation of HSFs and HSPs and thermotolerance. Heat stress is a major environmental constraint for crop production worldwide. To respond to and cope with heat stress, plants synthesize heat shock proteins (HSPs), which are often molecular chaperones and are under the control of heat stress transcription factors (HSFs). Very little is known about the upstream regulators of HSFs. In a forward genetic screen for regulators of C-REPEAT BINDING FACTOR (CBF) gene expression (RCFs), we identified RCF2 and found that it is allelic to CPL1/FIERY2, which encodes a homolog of C-terminal domain phosphatase. Our results also showed that, in addition to being critical for cold stress tolerance, RCF2 is required for heat stress–responsive gene regulation and thermotolerance, because, compared with the wild type, the rcf2-1 mutant is hypersensitive to heat stress and because the reduced thermotolerance is correlated with lower expression of most of the 21 HSFs and some of the HSPs in the mutant plants. We found that RCF2 interacts with the NAC transcription factor NAC019 and that RCF2 dephosphorylates NAC019 in vivo. The nac019 mutant is more sensitive to heat stress than the wild type, and chromatin immunoprecipitation followed by quantitative PCR analysis revealed that NAC019 binds to the promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1. Overexpression of RCF2 or NAC019 in Arabidopsis thaliana increases thermotolerance. Together, our results suggest that, through dephosphorylation of NAC019, RCF2 is an integrator of high-temperature signal transduction and a mechanism for HSF and HSP activation.

A Nuclear Calcium-Sensing Pathway Is Critical for Gene Regulation and Salt Stress Tolerance in Arabidopsis

Salt stress is an important environmental factor that significantly limits crop productivity worldwide. Studies on responses of plants to salt stress in recent years have identified novel signaling pathways and have been at the forefront of plant stress biology and plant biology in general. Thus far, research on salt stress in plants has been focused on cytoplasmic signaling pathways. In this study, we discovered a nuclear calcium-sensing and signaling pathway that is critical for salt stress tolerance in the reference plant Arabidopsis. Through a forward genetic screen, we found a nuclear-localized calcium-binding protein, RSA1 (SHORT ROOT IN SALT MEDIUM 1), which is required for salt tolerance, and identified its interacting partner, RITF1, a bHLH transcription factor. We show that RSA1 and RITF1 regulate the transcription of several genes involved in the detoxification of reactive oxygen species generated by salt stress and that they also regulate the SOS1 gene that encodes a plasma membrane Na+/H+ antiporter essential for salt tolerance. Together, our results suggest the existence of a novel nuclear calcium-sensing and -signaling pathway that is important for gene regulation and salt stress tolerance.

A KH domain-containing putative RNA-binding protein is critical for heat stress-responsive gene regulation and thermotolerance in Arabidopsis.

Heat stress is a severe environmental factor that significantly reduces plant growth and delays development. Heat stress factors (HSFs) are a class of transcription factors that are synthesized rapidly in response to elevations in temperature and are responsible for the transcription of many heat stress-responsive genes including those encoding heat shock proteins (HSPs). There are 21 HSFs in Arabidopsis, and recent studies have established that the HSFA1 family members are master regulators for the remaining HSFs. However, very little is known about upstream molecular factors that control the expression of HSFA1 genes and other HSF genes under heat stress. Through a forward genetic analysis, we identified RCF3, a K homology (KH) domain-containing nuclear-localized putative RNA-binding protein. RCF3 is a negative regulator of most HSFs, including HSFA1a, HSFA1b, and HSFA1d. In contrast, RCF3 positively controls the expression of HSFA1e, HSFA3, HSFA9, HSFB3, and DREB2C. Consistently with the overall increased accumulation of heat-responsive genes, the rcf3 mutant plants are more tolerant than the wild-type to heat stress. Together, our results suggest that a KH domain-containing putative RNA-binding protein RCF3 is an important upstream regulator for heat stress-responsive gene expression and thermotolerance in Arabidopsis.

Regulation of Abiotic Stress Signalling by Arabidopsis C-Terminal Domain Phosphatase-Like 1 Requires Interaction with a K-Homology Domain-Containing Protein

Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD) PHOSPHATASE-LIKE 1 (CPL1) regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds) RNA binding motifs (dsRBMs) at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH) domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.

A Bi-Functional Xyloglucan Galactosyltransferase Is an Indispensable Salt Stress Tolerance Determinant in Arabidopsis

Salinity is an abiotic stress that substantially limits crop production worldwide. To identify salt stress tolerance determinants, we screened for Arabidopsis mutants that are hypersensitive to salt stress and designated these mutants as short root in salt medium (rsa). One of these mutants, rsa3-1, is hypersensitive to NaCl and LiCl but not to CsCl or to general osmotic stress. Reactive oxygen species (ROS) over-accumulate in rsa3-1 plants under salt stress. Gene expression profiling with Affymetrix microarray analysis revealed that RSA3 controls expression of many genes including genes encoding proteins for ROS detoxification under salt stress. Map-based cloning showed that RSA3 encodes a xyloglucan galactosyltransferase, which is allelic to a gene previously named MUR3/KAM1. The RSA3/MUR3/KAM1-encoded xylogluscan galactosyltransferase regulates actin microfilament organization (and thereby contributes to endomembrane distribution) and is also involved in cell wall biosynthesis. In rsa3-1, actin cannot assemble and form bundles as it does in the wild-type but instead aggregates in the cytoplasm. Furthermore, addition of phalloidin, which prevents actin depolymerization, can rescue salt hypersensitivity of rsa3-1. Together, these results suggest that RSA3/MUR3/KAM1 along with other cell wall-associated proteins plays a critical role in salt stress tolerance by maintaining the proper organization of actin microfilaments in order to minimize damage caused by excessive ROS.

Downregulation of CSD2 by a heat-inducible miR398 is required for thermotolerance in Arabidopsis

MicroRNAs (miRNAs) play important roles in plant growth and development and abiotic stress responses. We report here that heat stress rapidly induces miR398 and reduces transcript of its target gene CSD2. Transgenic plants overexpressing the miR398-resistant form of CSD2 are more sensitive to heat stress than transgenic plants overexpressing normal coding sequence of CSD2. Expression of heat stress transcription factors (HSFs) and heat shock proteins (HSPs) is reduced in the heat-sensitive transgenic plants overexpressing miR398-resistant form of CSD2. Our results suggest that downregulation of CSD2 by the heat-inducible miR398 is required for thermotolerance in Arabidopsis.

Single-base methylome analysis reveals dynamic epigenomic differences associated with water deficit in apple

Summary Cytosine methylation is an essential feature of epigenetic regulation and is involved in various biological processes. Although cytosine methylation has been analysed at the genomic scale for several plant species, there is a general lack of understanding of the dynamics of global and genic DNA methylation in plants growing in environments challenged with biotic and abiotic stresses. In this study, we mapped cytosine methylation at single‐base resolution in the genome of commercial apple (Malus x domestica), and analysed changes in methylation patterns associated with water deficit in representative drought‐sensitive and drought‐tolerant cultivars. We found that the apple genome exhibits ~54%, ~38% and ~8.5% methylation at CG, CHG and CHH sequence contexts, respectively. We additionally documented changes in gene expression associated with water deficit in an attempt to link methylation and gene expression changes. Global methylation and transcription analysis revealed that promoter‐unmethylated genes showed higher expression levels than promoter‐methylated genes. Gene body methylation appears to be positively correlated with gene expression. Water deficit stress was associated with changes in methylation at a multitude of genes, including those encoding transcription factors (TFs) and transposable elements (TEs). These results present a methylome map of the apple genome and reveal widespread DNA methylation alterations in response to water deficit stress. These data will be helpful for understanding potential linkages between DNA methylation and gene expression in plants growing in natural environments and challenged with abiotic and biotic stresses.

Erratum: Regulation of Abiotic Stress Signalling by Arabidopsis C-Terminal Domain Phosphatase-Like 1 Requires Interaction with a K-Homology Domain-Containing Protein (PLoS ONE (2015) 10:10 (e0140735))

Previous data reported in this article were generated using wrong plant materials. In this correction, the plant materials were verified again using PCR, as well as FIT-LUC transgene as phenotypic marker prior to the RT-qPCR analysis. As molecular markers for stress responses, two classes of CUTs (cpl1-UP Transcripts) that represent various osmotic stress (cold, salinity, etc)-regulated (group I) and Fe-deficiency stress-regulated (group II) genes were used [1]. The analysis of the new data indicated that Fe-responsive transcripts regulated were constitutively upregulated in cpl1-6 as reported previously [1]. Similar behavior was observed for LEA and USP, but not other osmotically regulated transcripts tested (Fig 7). In contrast, expression levels of these transcripts in rcf3-2 did not significantly deviate from Col-0 wild type. The gene expression levels in the cpl1-6 rcf3-2 double mutant were similar to the cpl1-6 single mutant. Under Fe deficiency condition, all genotypes produced similar levels of Fe-responsive transcripts. Cold-induction produced two kinds of responses. Transcripts such as COR47, LEA4-5, RD29a, CBF2/3 and COR15 showed strong induction in all genotypes. Only small/moderate induction was observed for LEA18 and USP, for which cpl1-6maintained high constitutive expression levels. Transcript levels in cpl1-6 rcf3-2 were similar to cpl1-6 single mutant. The new results presented here indicate that co-operation of CPL1 and RCF3 is limited in endogenous Fe and osmotic stress signaling, and CPL1 likely plays a predominant role. However, it is likely that the cooperation does exist in certain targets because cpl1 and rcf3mutants have been co-isolated in multiple forward-genetic screening systems based on stress-inducible gene expression phenotypes.