Publications

Small interfering RNAs (siRNAs) are responsible for establishing and maintaining DNA methylation through the RNA-directed DNA methylation (RdDM) pathway in plants. Although siRNA biogenesis is well known, it is relatively unclear about how the process is regulated. By a forward genetic screen in Arabidopsis thaliana, we identified a mutant defective in NOT1 and demonstrated that NOT1 is required for transcriptional silencing at RdDM target genomic loci. We demonstrated that NOT1 is required for Pol IV-dependent siRNA accumulation and DNA methylation at a subset of RdDM target genomic loci. Furthermore, we revealed that NOT1 is a constituent of a multi-subunit CCR4-NOT deadenylase complex by immunoprecipitation combined with mass spectrometry and demonstrated that the CCR4-NOT components can function as a whole to mediate chromatin silencing. Therefore, our work establishes that the CCR4-NOT complex regulates the biogenesis of Pol IV-dependent siRNAs, and hence facilitates DNA methylation and transcriptional silencing in Arabidopsis.

Abstract Abscisic acid (ABA) is an important phytohormone regulating plant growth, development, and stress responses. It has an essential role in multiple physiological processes of plants, such as stomatal closure, cuticular wax accumulation, leaf senescence, bud dormancy, seed germination, osmotic regulation, and growth inhibition among many others. Abscisic acid controls downstream responses to abiotic and biotic environmental changes through both transcriptional and posttranscriptional mechanisms. During the past 20 years, ABA biosynthesis and many of its signaling pathways have been well characterized. Here we review the dynamics of ABA metabolic pools and signaling that affects many of its physiological functions.

Cold stress affects crop growth and productivity worldwide. Understanding the genetic basis of cold tolerance in germplasms is critical for crop improvement. Plants can coordinate environmental stimuli of light and temperature to regulate cold tolerance. However, it remains unknown which gene in germplasms could have such function. Here, we utilized genome-wide association study (GWAS) to investigate the cold tolerance of wild and cultivated tomato accessions and discovered that increased cold tolerance is accompanied with tomato domestication. We further identified a 27-bp InDel in the promoter of the CONSTANS-like transcription factor (TF) SlBBX31 is significantly linked with cold tolerance. Coincidentally, a key regulator of light signalling, SlHY5, can directly bind to the SlBBX31 promoter to activate SlBBX31 transcription while the 27-bp InDel can prevent S1HY5 from transactivating SlBBX31. Parallel to these findings, we observed that the loss of function of SlBBX31 results in impaired tomato cold tolerance. SlBBX31 can also modulate the cold-induced expression of several ERF TFs including CBF2 and DREBs. Therefore, our study has uncovered that SlBBX31 is possibly selected during tomato domestication for cold tolerance regulation, providing valuable insights for the development of hardy tomato varieties.

SummaryHyperosmotic stress caused by drought and salinity is a significant environmental factor that limits plant growth and agricultural productivity. Plants respond to osmotic stress by activating Ca2+ signaling, accumulating the stress hormone abscisic acid (ABA), reprogramming gene expression and altering growth. Despite intensive efforts, no global regulators of all of these responses have been identified. Here, we show that the Ca2+-responsive phospholipid binding BONZAI proteins are critical for Ca2+ signaling, ABA accumulation, gene expression reprogramming and plant growth under osmotic stress by antagonizing plant immune responses mediated by intracellular immune receptor NLRs. Using a Ca2+-imaging-based forward genetic screen, we found that the Arabidopsis osmo1/bon1 mutant plants display a reduced cytosolic Ca2+ signal in response to hyperosmotic stress. The bon1/bon2/bon3 triple mutants are impaired in osmotic stress induction of gene regulation and ABA accumulation. Importantly, the bon mutants are hypersensitive to osmotic stress inhibition of plant growth. These defects were suppressed by mutations in the immunity regulator SNC1 or PAD4. Our findings suggest that that BON proteins function as global regulators of plant osmotic stress responses by repressing immune signaling.

In plants, establishment of de novo DNA methylation is regulated by the RNA-directed DNA methylation (RdDM) pathway. RdDM machinery is known to concentrate in the Cajal body, but the biological significance of this localization has remained elusive. Here, we show that the antiviral methylation of the Tomato yellow leaf curl virus (TYLCV) genome requires the Cajal body in Nicotiana benthamiana cells. Methylation of the viral genome is countered by a virus-encoded protein, V2, which interacts with the central RdDM component AGO4, interfering with its binding to the viral DNA; Cajal body localization of the V2-AGO4 interaction is necessary for the viral protein to exert this function. Taken together, our results draw a long sought-after functional connection between RdDM, the Cajal body, and antiviral DNA methylation, paving the way for a deeper understanding of DNA methylation and antiviral defences in plants.

Because plants cannot move, they have evolved complex sensing and response systems to cope with the physical environment. Adverse environmental conditions, such as those causing abiotic stress, often cause significant losses in crop productivity and quality. Because of a paucity of well-defined visible phenotypes, conventional genetic screens have not been very successful in isolating abiotic stress signal transduction mutants of plants. Here, we describe a reporter gene-based strategy to screen for mutants affected in abiotic stress-regulated gene transcription. Our genetic screen uses the firefly luciferase reporter gene driven by the cold, drought, salt, and abscisic acid (ABA)-responsive RD29A promoter (RD29A::LUC). Arabidopsis plants transformed with the RD29A::LUC reporter emit bioluminescence in response to cold, drought, salt, or ABA treatment. After mutagenesis of these plants with ethyl methanesulfonate (EMS), mutants can be screened from the M2 population by monitoring the level of stress-inducible bioluminescence with a high-throughput, low-light imaging system. This protocol describes in detail the procedures for this luciferase reporter-based genetic screen for Arabidopsis mutants defective in abiotic stress signaling.

Summary CRISPR /Cas9 and TALEN are currently the two systems of choice for genome editing. We have studied the efficiency of the TALEN system in rice as well as the nature and inheritability of TALEN ‐induced mutations and found important features of this technology. The N287C230 TALEN backbone resulted in low mutation rates (0–6.6%), but truncations in its C‐terminal domain dramatically increased efficiency to 25%. In most transgenic T0 plants, TALEN produced a single prevalent mutation accompanied by a variety of low‐frequency mutations. For each independent T0 plant, the prevalent mutation was present in most tissues within a single tiller as well as in all tillers examined, suggesting that TALEN ‐induced mutations occurred very early in the development of the shoot apical meristem. Multigenerational analysis showed that TALEN ‐induced mutations were stably transmitted to the T1 and T2 populations in a normal Mendelian fashion. In our study, the vast majority of TALEN ‐induced mutations (~81%) affected multiple bases and ~70% of them were deletions. Our results contrast with published reports for the CRISPR /Cas9 system in rice, in which the predominant mutations affected single bases and deletions accounted for only 3.3% of the overall mutations.

The SOS3 (for SALT OVERLY SENSITIVE3) calcium binding protein and SOS2 protein kinase are required for sodium and potassium ion homeostasis and salt tolerance in Arabidopsis. We have shown previously that SOS3 interacts with and activates the SOS2 protein kinase. We report here the identification of a SOS3 binding motif in SOS2 that also serves as the kinase autoinhibitory domain. Yeast two-hybrid assays as well as in vitro binding assays revealed a 21–amino acid motif in the regulatory domain of SOS2 that is necessary and sufficient for interaction with SOS3. Database searches revealed a large family of SOS2-like protein kinases containing such a SOS3 binding motif. Using a yeast two-hybrid system, we show that these SOS2-like kinases interact with members of the SOS3 family of calcium binding proteins. Two-hybrid assays also revealed interaction between the N-terminal kinase domain and the C-terminal regulatory domain within SOS2, suggesting that the regulatory domain may inhibit kinase activity by blocking substrate access to the catalytic site. Removal of the regulatory domain of SOS2, including the SOS3 binding motif, resulted in constitutive activation of the protein kinase, indicating that the SOS3 binding motif can serve as a kinase autoinhibitory domain. Constitutively active SOS2 that is SOS3 independent also was produced by changing Thr168 to Asp in the activation loop of the SOS2 kinase domain. Combining the Thr168-to-Asp mutation with the autoinhibitory domain deletion created a superactive SOS2 kinase. These results provide insights into regulation of the kinase activities of SOS2 and the SOS2 family of protein kinases.

Phosphatidic acid (PtdOH) is emerging as an important signalling lipid in abiotic stress responses in plants. The effect of cold stress was monitored using 32P-labelled seedlings and leaf discs of Arabidopsis thaliana. Low, non-freezing temperatures were found to trigger a very rapid 32P-PtdOH increase, peaking within 2 and 5 min, respectively. In principle, PtdOH can be generated through three different pathways, i.e. i) via de novo phospholipid biosynthesis (through acylation of lyso-PtdOH), ii) via phospholipase D hydrolysis of structural phospholipids or iii) via phosphorylation of diacylglycerol (DAG) by DAG kinase (DGK). Using a differential 32P-labelling protocol and a PLD-transphosphatidylation assay, evidence is provided that the rapid 32P-PtdOH response was primarily generated through DGK. A simultaneous decrease in the levels of 32P-PtdInsP, correlating in time, temperature dependency and magnitude with the increase in 32P-PtdOH, suggested that a PtdInsP-hydrolyzing PLC generated the DAG in this reaction. Testing T-DNA insertion lines available for the seven DGK genes, revealed no clear changes in 32P-PtdOH responses, suggesting functional redundancy. Similarly, known cold-stress mutants were analyzed to investigate whether the PtdOH response acted downstream of the respective gene products. The hos1, los1 and fry1 mutants were found to exhibit normal PtdOH responses. Slight changes were found for ice1, snow1, and the overexpression line Super-ICE1, however, this was not cold-specific and likely due to pleiotropic effects. A tentative model illustrating direct cold effects on phospholipid metabolism is presented.

Abstract In plants, RNA-directed DNA methylation (RdDM) is a well-known de novo DNA methylation pathway that involves two plant-specific RNA polymerases, Pol IV and Pol V. In this study, we discovered and characterized an RdDM factor, RDM15. Through DNA methylome and genome-wide siRNA analyses, we show that RDM15 is required for RdDM-dependent DNA methylation and siRNA accumulation at a subset of RdDM target loci. We show that RDM15 contributes to Pol V-dependent downstream siRNA accumulation and interacts with NRPE3B, a subunit specific to Pol V. We also show that the C-terminal tudor domain of RDM15 specifically recognizes the histone 3 lysine 4 monomethylation (H3K4me1) mark. Structure analysis of RDM15 in complex with the H3K4me1 peptide showed that the RDM15 tudor domain specifically recognizes the monomethyllysine through an aromatic cage and a specific hydrogen bonding network; this chemical feature-based recognition mechanism differs from all previously reported monomethyllysine recognition mechanisms. RDM15 and H3K4me1 have similar genome-wide distribution patterns at RDM15-dependent RdDM target loci, establishing a link between H3K4me1 and RDM15-mediated RdDM in vivo. In summary, we have identified and characterized a histone H3K4me1-specific binding protein as an RdDM component, and structural analysis of RDM15 revealed a chemical feature-based lower methyllysine recognition mechanism.

Salt stress is a major environmental factor influencing plant growth and development. To identify salt tolerance determinants, a genetic screen for salt overly sensitive (sos) mutants was performed in Arabidopsis. We present here the characterization of sos4 mutants and the positional cloning of the SOS4 gene. sos4 mutant plants are hypersensitive to Na+, K+, and Li+ ions. Under NaCl stress, sos4 plants accumulate more Na+ and retain less K+ compared with wild-type plants. SOS4 encodes a pyridoxal kinase that is involved in the biosynthesis of pyridoxal-5-phosphate, an active form of vitamin B6. The expression of SOS4 cDNAs complements an Escherichia coli mutant defective in pyridoxal kinase. Supplementation of pyridoxine but not pyridoxal in the growth medium can partially rescue the sos4 defect in salt tolerance. SOS4 is expressed ubiquitously in all plant tissues. As a result of alternative splicing, two transcripts are derived from the SOS4 gene, the relative abundance of which is modulated by development and environmental stresses. Besides being essential cofactors for numerous enzymes, as shown by pharmacological studies in animal cells, pyridoxal-5-phosphate and its derivatives are also ligands for P2X receptor ion channels. Our results demonstrate that pyridoxal kinase is a novel salt tolerance determinant important for the regulation of Na+ and K+ homeostasis in plants. We propose that pyridoxal-5-phosphate regulates Na+ and K+ homeostasis by modulating the activities of ion transporters.

Summary The abiotic stresses of drought, salinity and freezing are linked by the fact that they all decrease the availability of water to plant cells. This decreased availability of water is quantified as a decrease in water potential. Plants resist low water potential and related stresses by modifying water uptake and loss to avoid low water potential, accumulating solutes and modifying the properties of cell walls to avoid the dehydration induced by low water potential and using protective proteins and mechanisms to tolerate reduced water content by preventing or repairing cell damage. Salt stress also alters plant ion homeostasis, and under many conditions this may be the predominant factor affecting plant performance. Our emphasis is on experiments that quantify resistance to realistic and reproducible low water potential (drought), salt and freezing stresses while being suitable for genetic studies where a large number of lines must be analyzed. Detailed protocols for the use of polyethylene glycol‐infused agar plates to impose low water potential stress, assay of salt tolerance based on root elongation, quantification of freezing tolerance and the use of electrolyte leakage experiments to quantify cellular damage induced by freezing and low water potential are also presented.