Publications

The <i>Arabidopsis</i> NFYA5 Transcription Factor Is Regulated Transcriptionally and Posttranscriptionally to Promote Drought Resistance

Abstract Nuclear factor Y (NF-Y) is a ubiquitous transcription factor composed of three distinct subunits (NF-YA, NF-YB, and NF-YC). We found that the Arabidopsis thaliana NFYA5 transcript is strongly induced by drought stress in an abscisic acid (ABA)-dependent manner. Promoter:β-glucuronidase analyses showed that NFYA5 was highly expressed in vascular tissues and guard cells and that part of the induction by drought was transcriptional. NFYA5 contains a target site for miR169, which targets mRNAs for cleavage or translational repression. We found that miR169 was downregulated by drought stress through an ABA-dependent pathway. Analysis of the expression of miR169 precursors showed that miR169a and miR169c were substantially downregulated by drought stress. Coexpression of miR169 and NFYA5 suggested that miR169a was more efficient than miR169c at repressing the NFYA5 mRNA level. nfya5 knockout plants and plants overexpressing miR169a showed enhanced leaf water loss and were more sensitive to drought stress than wild-type plants. By contrast, transgenic Arabidopsis plants overexpressing NFYA5 displayed reduced leaf water loss and were more resistant to drought stress than the wild type. Microarray analysis indicated that NFYA5 is crucial for the expression of a number of drought stress–responsive genes. Thus, NFYA5 is important for drought resistance, and its induction by drought stress occurs at both the transcriptional and posttranscriptional levels.

ICE1: a regulator of cold-induced transcriptome and freezing tolerance in<i>Arabidopsis</i>

Cold temperatures trigger the expression of the CBF family of transcription factors, which in turn activate many downstream genes that confer chilling and freezing tolerance to plants. We report here the identification of ICE1 ( i nducer of C BF e xpression 1 ), an upstream transcription factor that regulates the transcription of CBF genes in the cold. An Arabidopsis ice1 mutant was isolated in a screen for mutations that impair cold-induced transcription of a CBF3 promoter-luciferase reporter gene. The ice1 mutation blocks the expression of CBF3 and decreases the expression of many genes downstream of CBFs , which leads to a significant reduction in plant chilling and freezing tolerance. ICE1 encodes a MYC-like bHLH transcriptional activator. ICE1 binds specifically to the MYC recognition sequences in the CBF3 promoter. ICE1 is expressed constitutively, and its overexpression in wild-type plants enhances the expression of the CBF regulon in the cold and improves freezing tolerance of the transgenic plants.

Additional file 7 of 3D organization of regulatory elements for transcriptional regulation in Arabidopsis

Additional file 7: Table S6. List of the long-range chromatin interactions spatially linking SNPs to flowering-time-associated target genes.

Reconstitution in yeast of the <i>Arabidopsis</i> SOS signaling pathway for Na ⁺ homeostasis

The Arabidopsis thaliana SOS1 protein is a putative Na + /H + antiporter that functions in Na + extrusion and is essential for the NaCl tolerance of plants. sos1 mutant plants share phenotypic similarities with mutants lacking the protein kinase SOS2 and the Ca 2+ sensor SOS3. To investigate whether the three SOS proteins function in the same response pathway, we have reconstituted the SOS system in yeast cells. Expression of SOS1 improved the Na + tolerance of yeast mutants lacking endogenous Na + transporters. Coexpression of SOS2 and SOS3 dramatically increased SOS1-dependent Na + tolerance, whereas SOS2 or SOS3 individually had no effect. The SOS2/SOS3 kinase complex promoted the phosphorylation of SOS1. A constitutively active form of SOS2 phosphorylated SOS1 in vitro independently of SOS3, but could not fully substitute for the SOS2/SOS3 kinase complex for activation of SOS1 in vivo . Further, we show that SOS3 recruits SOS2 to the plasma membrane. Although sos1 mutant plants display defective K + uptake at low external concentrations, neither the unmodified nor the SOS2/SOS3-activated SOS1 protein showed K + transport capacity in vivo , suggesting that the role of SOS1 on K + uptake is indirect. Our results provide an example of functional reconstitution of a plant response pathway in a heterologous system and demonstrate that the SOS1 ion transporter, the SOS2 protein kinase, and its associated Ca 2+ sensor SOS3 constitute a functional module. We propose a model in which SOS3 activates and directs SOS2 to the plasma membrane for the stimulatory phosphorylation of the Na + transporter SOS1.

DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV

DNA methylation is an important epigenetic mark in many eukaryotic organisms. De novo DNA methylation in plants can be achieved by the RNA-directed DNA methylation (RdDM) pathway, where the plant-specific DNA-dependent RNA polymerase IV (Pol IV) transcribes target sequences to initiate 24-nt siRNA production and action. The putative DNA binding protein DTF1/SHH1 of Arabidopsis has been shown to associate with Pol IV and is required for 24-nt siRNA accumulation and transcriptional silencing at several RdDM target loci. However, the extent and mechanism of DTF1 function in RdDM is unclear. We show here that DTF1 is necessary for the accumulation of the majority of Pol IV-dependent 24-nt siRNAs. It is also required for a large proportion of Pol IV-dependent de novo DNA methylation. Interestingly, there is a group of RdDM target loci where 24-nt siRNA accumulation but not DNA methylation is dependent on DTF1. DTF1 interacts directly with the chromatin remodeling protein CLASSY 1 (CLSY1), and both DTF1 and CLSY1 are associated in vivo with Pol IV but not Pol V, which functions downstream in the RdDM effector complex. DTF1 and DTF2 (a DTF1-like protein) contain a SAWADEE domain, which was found to bind specifically to histone H3 containing H3K9 methylation. Taken together, our results show that DTF1 is a core component of the RdDM pathway, and suggest that DTF1 interacts with CLSY1 to assist in the recruitment of Pol IV to RdDM target loci where H3K9 methylation may be an important feature. Our results also suggest the involvement of DTF1 in an important negative feedback mechanism for DNA methylation at some RdDM target loci.

General Control Non-derepressible 1 (AtGCN1) Is Important for Flowering Time, Plant Growth, Seed Development, and the Transcription/Translation of Specific Genes in Arabidopsis

We have previously demonstrated that General Control Non-derepressible 1 (AtGCN1) is essential for translation inhibition under cold stress through interacting with GCN2 to phosphorylate eukaryotic translation initiation factor 2 (eIF2). Here, we report that the flower time of the atgcn1 mutant is later than that of the wild type (WT), and some siliques of atgcn1 cannot develop and produce seeds. Total and polysomal RNA of atgcn1-1 and wild type (WT) after cold treatments were sequenced. The sequencing results show that the mutation of atgcn1 selectively alters the expression of genes at both transcriptional and translational levels. The classification of AtGCN1 target genes reveals that AtGCN1 regulated gens are involved in flower development, seed dormancy and seed development, response to osmotic stress, amino acid biosynthesis, photosynthesis, cell wall organization, protein transport and localization, lipid biosynthesis, transcription, macroautophagy, proteolysis and cell death. Further analysis of AtGCN1 regulated genes at translational levels shows that the Kozak sequence and uORFs (upstream open reading frame) of transcripts affect translation selection. These results show that AtGCN1 is required for the expression of selective genes in Arabidopsis.

Gene Targeting by Homology-Directed Repair in Rice Using a Geminivirus-Based CRISPR/Cas9 System

RNA Splicing Factors and RNA-Directed DNA Methylation

RNA-directed histone and/or DNA modification is a conserved mechanism for the establishment of epigenetic marks from yeasts and plants to mammals. The heterochromation formation in yeast is mediated by RNAi-directed silencing mechanism, while the establishment of DNA methylation in plants is through the RNA-directed DNA methylation (RdDM) pathway. Recently, splicing factors are reported to be involved in both RNAi-directed heterochromatin formation in yeast and the RdDM pathway in plants. In yeast, splicing factors may provide a platform for facilitating the siRNA generation through an interaction with RDRC and thereby affect the heterochromatin formation, whereas in plants, various splicing factors seem to act at different steps in the RdDM pathway.

Simplified adenine base editors improve adenine base editing efficiency in rice

Summary Adenine base editors ( ABE s) have been exploited to introduce targeted adenine (A) to guanine (G) base conversions in various plant genomes, including rice, wheat and Arabidopsis . However, the ABE s reported thus far are all quite inefficient at many target sites in rice, which hampers their applications in plant genome engineering and crop breeding. Here, we show that unlike in the mammalian system, a simplified base editor ABE ‐P1S (Adenine Base Editor‐Plant version 1 Simplified) containing the ecTadA*7.10‐ nSpC as9 (D10A) fusion has much higher editing efficiency in rice compared to the widely used ABE ‐P1 consisting of the ecTadA‐ecTadA*7.10‐ nSpC as9 (D10A) fusion. We found that the protein expression level of ABE ‐P1S is higher than that of ABE ‐P1 in rice calli and protoplasts, which may explain the higher editing efficiency of ABE ‐P1S in different rice varieties. Moreover, we demonstrate that the ecTadA*7.10‐ nC as9 fusion can be used to improve the editing efficiency of other ABE s containing SaCas9 or the engineered Sa KKH ‐Cas9 variant. These more efficient ABE s will help advance trait improvements in rice and other crops.

A Rice Kinase-Protein Interaction Map

Abstract Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

Biochemical Characterization of the Arabidopsis Protein Kinase SOS2 That Functions in Salt Tolerance

The Arabidopsis Salt Overly Sensitive 2 (SOS2) gene encodes a serine/threonine (Thr) protein kinase that has been shown to be a critical component of the salt stress signaling pathway. SOS2 contains a sucrose-non-fermenting protein kinase 1/AMP-activated protein kinase-like N-terminal catalytic domain with an activation loop and a unique C-terminal regulatory domain with an FISL motif that binds to the calcium sensor Salt Overly Sensitive 3. In this study, we examined some of the biochemical properties of the SOS2 in vitro. To determine its biochemical properties, we expressed and isolated a number of active and inactive SOS2 mutants as glutathione S-transferase fusion proteins in Escherichia coli. Three constitutively active mutants, SOS2T168D, SOS2T168D Delta F, and SOS2T168D Delta 308, were obtained previously, which contain either the Thr-168 to aspartic acid (Asp) mutation in the activation loop or combine the activation loop mutation with removal of the FISL motif or the entire regulatory domain. These active mutants exhibited a preference for Mn(2+) relative to Mg(2+) and could not use GTP as phosphate donor for either substrate phosphorylation or autophosphorylation. The three enzymes had similar peptide substrate specificity and catalytic efficiency. Salt overly sensitive 3 had little effect on the activity of the activation loop mutant SOS2T168D, either in the presence or absence of calcium. The active mutant SOS2T168D Delta 308 could not transphosphorylate an inactive protein (SOS2K40N), which indicates an intramolecular reaction mechanism of SOS2 autophosphorylation. Interestingly, SOS2 could be activated not only by the Thr-168 to Asp mutation but also by a serine-156 or tyrosine-175 to Asp mutation within the activation loop. Our results provide insights into the regulation and biochemical properties of SOS2 and the SOS2 subfamily of protein kinases.

MAG2 and MAL Regulate Vesicle Trafficking and Auxin Homeostasis With Functional Redundancy

Auxin is a central phytohormone and controls almost all aspects of plant development and stress response. Auxin homeostasis is coordinately regulated by biosynthesis, catabolism, transport, conjugation, and deposition. Endoplasmic reticulum (ER)-localized MAIGO2 (MAG2) complex mediates tethering of arriving vesicles to the ER membrane, and it is crucial for ER export trafficking. Despite important regulatory roles of MAG2 in vesicle trafficking, the mag2 mutant had mild developmental abnormalities. MAG2 has one homolog protein, MAG2-Like (MAL), and the mal-1 mutant also had slight developmental phenotypes. In order to investigate MAG2 and MAL regulatory function in plant development, we generated the mag2-1 mal-1 double mutant. As expected, the double mutant exhibited serious developmental defects and more alteration in stress response compared with single mutants and wild type. Proteomic analysis revealed that signaling, metabolism, and stress response in mag2-1 mal-1 were affected, especially membrane trafficking and auxin biosynthesis, signaling, and transport. Biochemical and cell biological analysis indicated that the mag2-1 mal-1 double mutant had more serious defects in vesicle transport than the mag2-1 and mal-1 single mutants. The auxin distribution and abundance of auxin transporters were altered significantly in the mag2-1 and mal-1 single mutants and mag2-1 mal-1 double mutant. Our findings suggest that MAG2 and MAL regulate plant development and auxin homeostasis by controlling membrane trafficking, with functional redundancy.

Abiotic stress responses in plants

Nucleocytoplasmic Trafficking of the Arabidopsis WD40 Repeat Protein XIW1 Regulates ABI5 Stability and Abscisic Acid Responses

Arabidopsis AGDP1 links H3K9me2 to DNA methylation in heterochromatin

Abstract Heterochromatin is a tightly packed form of chromatin that is associated with DNA methylation and histone 3 lysine 9 methylation (H3K9me). Here, we identify an H3K9me2-binding protein, Agenet domain (AGD)-containing p1 (AGDP1), in Arabidopsis thaliana . Here we find that AGDP1 can specifically recognize the H3K9me2 mark by its three pairs of tandem AGDs. We determine the crystal structure of the Agenet domain 1 and 2 cassette (AGD12) of Raphanus sativus AGDP1 in complex with an H3K9me2 peptide. In the complex, the histone peptide adopts a unique helical conformation. AGD12 specifically recognizes the H3K4me0 and H3K9me2 marks by hydrogen bonding and hydrophobic interactions. In addition, we find that AGDP1 is required for transcriptional silencing, non-CG DNA methylation, and H3K9 dimethylation at some loci. ChIP-seq data show that AGDP1 preferentially occupies long transposons and is associated with heterochromatin marks. Our findings suggest that, as a heterochromatin-binding protein, AGDP1 links H3K9me2 to DNA methylation in heterochromatin regions.

A domesticated <i>Harbinger</i> transposase forms a complex with HDA6 and promotes histone H3 deacetylation at genes but not TEs in <i>Arabidopsis</i>

In eukaryotes, histone acetylation is a major modification on histone N-terminal tails that is tightly connected to transcriptional activation. HDA6 is a histone deacetylase involved in the transcriptional regulation of genes and transposable elements (TEs) in Arabidopsis thaliana. HDA6 has been shown to participate in several complexes in plants, including a conserved SIN3 complex. Here, we uncover a novel protein complex containing HDA6, several Harbinger transposon-derived proteins (HHP1, SANT1, SANT2, SANT3, and SANT4), and MBD domain-containing proteins (MBD1, MBD2, and MBD4). We show that mutations of all four SANT genes in the sant-null mutant cause increased expression of the flowering repressors FLC, MAF4, and MAF5, resulting in a late flowering phenotype. Transcriptome deep sequencing reveals that while the SANT proteins and HDA6 regulate the expression of largely overlapping sets of genes, TE silencing is unaffected in sant-null mutants. Our global histone H3 acetylation profiling shows that SANT proteins and HDA6 modulate gene expression through deacetylation. Collectively, our findings suggest that Harbinger transposon-derived SANT domain-containing proteins are required for histone deacetylation and flowering time control in plants.

SANT proteins modulate gene expression by coordinating histone H3KAc and Khib levels and regulate plant heat tolerance

Abstract Histone post-translational modifications (PTMs), such as acetylation and recently identified lysine 2-hydroxyisobutyrylation (Khib), act as active epigenomic marks in plants. SANT domain-containing proteins SANT1, SANT2, SANT3, and SANT4 (SANT1/2/3/4), derived from PIF/Harbinger transposases, form a complex with HISTONE DEACETYLASE 6 (HDA6) to regulate gene expression via histone deacetylation. However, whether SANT1/2/3/4 coordinates different types of PTMs to regulate transcription and mediate responses to specific stresses in plants remains unclear. Here, in addition to modulating histone deacetylation, we found that SANT1/2/3/4 proteins acted like HDA6 or HDA9 in regulating the removal of histone Khib in Arabidopsis (Arabidopsis thaliana). Histone H3 lysine acetylation (H3KAc) and histone Khib were coordinated by SANT1/2/3/4 to regulate gene expression, with H3KAc playing a predominant role and Khib acting complementarily to H3KAc. SANT1/2/3/4 mutation significantly increased the expression of heat-inducible genes with concurrent change of H3KAc levels under normal and heat stress conditions, resulting in enhanced thermotolerance. This study revealed the critical roles of Harbinger transposon-derived SANT domain-containing proteins in transcriptional regulation by coordinating different types of histone PTMs and in the regulation of plant thermotolerance by mediating histone acetylation modification.

Targeted, efficient sequence insertion and replacement in rice