Publications

DNA methylation is an important epigenetic mark. In plants, de novo DNA methylation occurs mainly through the RNA-directed DNA methylation (RdDM) pathway. Researchers have previously inferred that a flowering regulator, MULTICOPY SUPPRESSOR OF IRA1 4 (MSI4)/FVE, is involved in non-CG methylation at several RdDM targets, suggesting a role of FVE in RdDM. However, whether and how FVE affects RdDM genome-wide is not known. Here, we report that FVE is required for DNA methylation at thousands of RdDM target regions. In addition, dysfunction of FVE significantly reduces 24-nucleotide siRNA accumulation that is dependent on factors downstream in the RdDM pathway. By using chromatin immunoprecipitation and sequencing (ChIP-seq), we show that FVE directly binds to FVE-dependent 24-nucleotide siRNA cluster regions. Our results also indicate that FVE may function in RdDM by physically interacting with RDM15, a downstream factor in the RdDM pathway. Our study has therefore revealed that FVE, by associating with RDM15, directly regulates DNA methylation and siRNA accumulation at a subset of RdDM targets.

Abstract The extensive phenotypic diversity within natural populations of Arabidopsis is associated with differences in gene expression. Transcript levels can be considered as inheritable quantitative traits, and used to map expression quantitative trait loci (eQTL) in genome‐wide association studies (GWASs). In order to identify putative genetic determinants for variations in gene expression, we used publicly available genomic and transcript variation data from 665 Arabidopsis accessions and applied the single nucleotide polymorphism‐set (Sequence) Kernel Association Test (SKAT) method for the identification of eQTL. Moreover, we used the penalized orthogonal‐components regression (POCRE) method to increase the power of statistical tests. Then, gene annotations were used as test units to identify genes that are associated with natural variations in transcript accumulation, which correspond to candidate regulators, some of which may have a broad impact on gene expression. Besides increasing the chances to identify real associations, the analysis using POCRE and SKAT significantly reduced the computational cost required to analyze large datasets. As a proof of concept, we used this approach to identify eQTL that represent novel candidate regulators of immune responses. The versatility of this approach allows its application to any process that is subjected to natural variation among Arabidopsis accessions.

ABSTRACT Unlike pathogens that trigger plant defense responses, commensal or beneficial microbes are compatible with plants and do not elicit a defense response. An assumption underlying the compatibility is that plants are inert in mounting a defense response to non-pathogenic microbial factors. However, the mechanisms underlying this inertness in defense are unknown. Here a forward genetic screen led to the isolation of an Arabidopsis mutant displaying a new type of immunity which we named as latent defense response (LDR) to a beneficial rhizobacterium. The mutant, known as gp1 for Growth-Promotion 1 , is impaired in rhizobacteria-induced plant growth-promotion due to disrupted oleic acid homeostasis and consequent activation of defense responses. Several bacterial volatile compounds trigger LDR in gp1 but not wild type plants. GP1 dysfunction strongly represses colonization of the beneficial rhizobacterium and alters root-associated microbiota. Our findings reveal a hidden layer of plant defense, LDR, which is suppressed by GP1 to allow mutualistic association between plants and beneficial rhizobacteria. One Sentence Summary A hidden layer of host immunity against non-pathogenic microbes leads to plant incompatibility with beneficial rhizobacteria.

SOS2 (salt overly sensitive 2) is a serine/threonine protein kinase required for salt tolerance in Arabidopsis thaliana . In this study, we identified the protein phosphatase 2C ABI2 (abscisic acid-insensitive 2) as a SOS2-interacting protein. Deletion analysis led to the discovery of a novel protein domain of 37 amino acid residues, designated as the protein phosphatase interaction (PPI) motif, of SOS2 that is necessary and sufficient for interaction with ABI2. The PPI motif is conserved in protein kinases of the SOS2 family (i.e., protein kinase S, PKS) and in the DNA damage repair and replication block checkpoint kinase, Chk1, from various organisms including humans. Mutations in the conserved amino acid residues in the PPI motif abolish the interaction of SOS2 with ABI2. We also identified a protein kinase interaction domain in ABI2 and examined the interaction specificity between PKS and the ABI phosphatases. We found that some PKSs interact strongly with ABI2 whereas others interact preferentially with ABI1. The interaction between SOS2 and ABI2 was disrupted by the abi2-1 mutation, which causes increased tolerance to salt shock and abscisic acid insensitivity in plants. Our results establish the PPI motif and the protein kinase interaction domain as novel protein interaction domains that mediate the binding between the SOS2 family of protein kinases and the ABI1/2 family of protein phosphatases.

Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants.

DNA methylation is an important epigenetic mark involved in many biological processes. The genome of the climacteric tomato fruit undergoes a global loss of DNA methylation due to active DNA demethylation during the ripening process. It is unclear whether the ripening of other fruits is also associated with global DNA demethylation. We characterized the single-base resolution DNA methylomes of sweet orange fruits. Compared with immature orange fruits, ripe orange fruits gained DNA methylation at over 30,000 genomic regions and lost DNA methylation at about 1,000 genomic regions, suggesting a global increase in DNA methylation during orange fruit ripening. This increase in DNA methylation was correlated with decreased expression of DNA demethylase genes. The application of a DNA methylation inhibitor interfered with ripening, indicating that the DNA hypermethylation is critical for the proper ripening of orange fruits. We found that ripening-associated DNA hypermethylation was associated with the repression of several hundred genes, such as photosynthesis genes, and with the activation of hundreds of genes, including genes involved in abscisic acid responses. Our results suggest important roles of DNA methylation in orange fruit ripening.

Cold temperatures trigger the expression of the CBF family of transcription factors, which in turn activate many downstream genes that confer freezing tolerance to plants. It has been shown previously that the cold regulation of CBF3 involves an upstream bHLH-type transcription factor, ICE1. ICE1 binds to the Myc recognition sequences in the CBF3 promoter. Apart from Myc recognition sequences, CBF promoters also have Myb recognition sequences. We report here that the Arabidopsis MYB15 is involved in cold-regulation of CBF genes and in the development of freezing tolerance. The MYB15 gene transcript is up-regulated by cold stress. The MYB15 protein interacts with ICE1 and binds to Myb recognition sequences in the promoters of CBF genes. Overexpression of MYB15 results in reduced expression of CBF genes whereas its loss-of-function leads to increased expression of CBF genes in the cold. The myb15 mutant plants show increased tolerance to freezing stress whereas its overexpression reduces freezing tolerance. Our results suggest that MYB15 is part of a complex network of transcription factors controlling the expression of CBFs and other genes in response to cold stress.

Summary Two allelic Arabidopsis mutants, leaf wilting 2‐1 and leaf wilting 2‐2 ( lew2‐1 and lew2‐2 ), were isolated in a screen for plants with altered drought stress responses. The mutants were more tolerant to drought stress as well as to NaCl, mannitol and other osmotic stresses. lew2 mutant plants accumulated more abscisic acid (ABA), proline and soluble sugars than the wild type. The expression of a stress‐inducible marker gene RD29A , a proline synthesis‐related gene P5CS (pyrroline‐5‐carboxylate synthase) and an ABA synthesis‐related gene SDR1 (alcohol dehydrogenase/reductase) was higher in lew2 than in the wild type. Map‐based cloning revealed that the lew2 mutants are new alleles of the AtCesA8/IRX1 gene which encodes a subunit of a cellulose synthesis complex. Our results suggest that cellulose synthesis is important for drought and osmotic stress responses including drought induction of gene expression.

It has been reported that double-stranded break (DSB)-induced small RNAs (diRNAs) are generated via the RNA-directed DNA methylation pathway and function in DSB repair in Arabidposis. However, important questions remain regarding the biogenesis and function of diRNAs. Here, we used CRISPR/Cas9- or TALEN-triggered DSBs to characterize diRNAs in Arabidopsis and rice. We found that 21-nt diRNAs were generated from a 35S promoter::GU-US reporter transgene targeted by CRISPR/Cas9. Unexpectedly, Pol II transcription of the transgene was required for efficient diRNA production and the level of diRNA accumulation correlated with the expression level of the transgene. diRNAs were not detected from CRISPR/Cas9- or TALEN-induced DSBs within the examined endogenous genes in Arabidopsis or rice. We also found that DCL4 and RDR6 that are known to be involved in posttranscriptional gene silencing were required to generate diRNAs. Our results suggest that DSBs are necessary but not sufficient for efficient diRNA generation and a high level of diRNAs is not necessary for DSB repair.

Soybean (Glycine max (L.) Merr.) is among the most important crops in the world for oil and proteins (Zhang et al., 2022). Given that weeds are a major challenge to soybean cultivation, non-transgenic herbicide-tolerant soybean varieties are in great demand, particularly in places where growing transgenic soybean is prohibited. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene editing systems, including base editors built from Cas9 and Cas12 systems, are revolutionary tools in plant breeding, including the breeding for herbicide-tolerant crops (Sun et al., 2016; Wang et al., 2019; Zhang et al., 2023). Although Cas9 base editor systems have been successfully applied in soybean (Huang et al., 2023; Niu et al., 2023), the application of Cas12 base editors in soybean breeding has not been reported. Acetolactate synthase (ALS), a crucial enzyme in the biosynthesis of branched-chain amino acids, is the target of several important herbicides (Powles and Yu, 2010). Variants of ALS that harbours certain point mutations can confer tolerance to a major group of commercial herbicides. A previous study showed that a variant bearing the P197S substitution in AtALS exhibited herbicide resistance in Arabidopsis (Chen et al., 2017). The soybean genome contains four GmALS paralogues (GmALS1-4). Notably, the widely planted non-transgenic herbicide-resistant soybean harbours a single-point mutation P178S in GmALS1, equivalent to P197S in AtALS (Walter et al., 2014). Unfortunately, amino acid substitution mutation in individual ALS genes cannot avoid herbicide injuries (Walter et al., 2014 and this study). Therefore, we explored the use of amino acid substitution mutations in multiple ALS genes to produce soybeans with sufficient herbicide resistance for effective weed control. Among the four GmALS genes, GmALS1 and GmALS3 share 80.6% and 79.0% amino acid sequence identities with AtALS respectively. We selected GmALS1 and GmALS3 as the targets for generation of herbicide-resistant soybean by converting C to T at codons P178 of GmALS1 and P172 of GmALS3 using the BE4max-dCas12-SF01 cytosine base editor system, which was constructed through introducing the E844A mutation in the conserved active site of Cas12-SF01 (Duan et al., 2024) and fusing it to the deaminase Anc689 APOBEC. The cytosine base editor contained a CRISPR RNA (crRNA) transcription box driven by the GmU6 promoter, and the cassette Anc689 APOBEC-dCas12-SF01-UGI (AncBE4max) driven by the SlEF1a promoter (Niu et al., 2023). The crRNA1 and crRNA2 targeted GmALS1 and GmALS3 respectively (Figure 1a). We introduced the base editor into the elite soybean cultivar 'Xudou 18' through Agrobacterium tumefaciens-mediated transformation. The base-editing events at the crRNA target sites showed an overall 2.16% editing efficacy (with 9216 explants, we obtained 416 independent T0 transformants and 9 of them carried gene-editing events (4 for als1, 3 for als3 and 2 for als1/als3, respectively)). Among the plants harbouring C–T substitutions, we successfully obtained ALS1P178S, ALS3P172S and ALS1P178S ALS3P172S mutants (referred to as als1, als3 and als1/als3, respectively), and potential off-target sites were examined and no editing events were detected. We obtained transgene-free stable homozygous als1, als3 and als1/als3 plants in the T1 generation. We evaluated the herbicide resistance of wild-type (WT) and als1/als3 plants grown in a greenhouse by spraying the plants with different concentrations of flucarbazone sodium at the V2 stage. Seven days after the herbicide application, the WT plants were severely damaged while the als1/als3 plants showed no damage (Figure 1c). We repeated the experiment in the following year and included the als1 and als3. The als1 and als3 mutations both conferred some level of herbicide resistance, whereas the als1/als3 showed no symptoms of herbicide damage (Figure 1d). These results showed that als1 and als3 mutations have a synergistic effect that strongly promotes herbicide resistance. We subsequently examined herbicide resistance of the als1/als3 and WT plants grown in the field. The als1/als3 plants showed no symptoms of herbicide damage while the WT plants were severely damaged by the herbicide (Figure 1e). The herbicide-treated als1/als3 plants showed no differences in plant height, internode number, pod number, branching and hundred-grain weight from those of the non-treated WT plants (Figure 1f–k). Furthermore, the productivity of the als1/als3 plants showed no obvious change compared with that of the WT in field tests (4 m2/plot). The yield of the als1/als3 was not reduced under flucarbazone sodium dosages of 280 and 420 mg L−1 compared with 3125 kg/ha for the non-treated WT (Figure 1l). Inappropriate dosages (over 2× (120 mg L−1) flucarbazone sodium) or uneven application of herbicides by farmers in the field could easily exceed the resistance threshold of crops (Walter et al., 2014). Improvement of the herbicide tolerance to meet the requirements of practical breeding applications can overcome these problems. In this study, we successfully established a BE4max editor system that efficiently introduced C-to-T conversions in soybean and generated the als1/als3 mutant, which showed tolerance to 6× (420 mg L−1) flucarbazone sodium herbicide (Figure 1d). The identification of the als1/als3 with 6× flucarbazone sodium herbicide resistance shows the effectiveness of our strategy for breeding herbicide-tolerant soybeans for real-world applications. This work was supported by Bellagen Biotechnology Inc., the National Key Research and Development Program of China (NK2022010301), the Seed-Industrialized Development Program in Shandong Province, China (2021LZGC003), the Key Research & Development Program of Shandong Province, China (2021LZGC012), and the National Natural Science Foundation of China (32270367). The authors have declared no conflict of interest. Q.N., H.X., J.-K. Z. and J.Z. contributed to the conceptualization of the project; Q.N., H.X., X.C., M.S., X.W., S.L., K.P. and Y.Z. performed the research; Q.N., H.X. and J.Z. analysed the data; Q.N., H.X., J.-K. Z. and J.Z. wrote the manuscript. All authors read and approved the final version of the manuscript. Data and materials generated from this work will be avaialble upon request. Dr. Jianhua Zhu is fully responsible for the distributions of all materials associated with this article.

REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylation enzyme that was previously identified during a genetic screen for the silencing of both RD29A-LUC and 35S-NPTII transgenes on a T-DNA construct. Here we performed a genetic screen to identify additional mutants in which the 35S-NPTII transgene is silenced. We identified several alleles of ros1 and of the following components of the RNA-directed DNA methylation (RdDM) pathway: NRPD1 (the largest subunit of polymerase IV), RDR2, NRPE1 (the largest subunit of polymerase V), NRPD2, AGO4, and DMS3. Our results show that the silencing of 35S-NPTII in the RdDM pathway mutants is due to the reduced expression of ROS1 in the mutants. We also identified a putative histone acetyltransferase (ROS4) from the genetic screen. The acetyltransferase contains a PHD-finger domain that binds to unmethylated histone H3K4. The mutation in ROS4 led to reduction of H3K18 and H3K23 acetylation levels. We show that the silencing of 35S-NPTII and some transposable element genes was released by the ddm1 mutation but that this also required ROS4. Our study identifies a unique antisilencing factor, and reveals that the RdDM pathway has an antisilencing function due to its role in maintaining ROS1 expression.

Background The Prognostic Nutritional Index (PNI) has become an important predictive tool for assessing patients’ nutritional status and immune competence. It is widely used in prognostic evaluations for various cancer patients. However, the prognostic relevance of the Prognostic Nutritional Index (PNI) in gastric or gastro-esophageal junction cancer patients (GC/GEJC) undergoing immune checkpoint inhibitors (ICIs) treatment remains unclear. This meta-analysis aimed to determine the prognostic impact of PNI in this specific patient cohort. Methods We conducted a thorough literature search, covering prominent databases such as PubMed, Embase, Web of Science, SpringerLink, and the Cochrane Library. The search spanned from the inception of these databases up to December 5, 2023. Employing the 95% confidence interval and Hazard Ratio (HR), the study systematically evaluated the relationship between PNI and key prognostic indicators, including the objective remission rate (ORR), disease control rate (DCR), overall survival (OS) and progression-free survival (PFS) in GC/GEJC patients undergoing ICI treatment. Results Eight studies comprising 813 eligible patients were selected. With 7 studies consistently demonstrating superior Overall Survival (OS) in the high-Prognostic Nutritional Index (PNI) group compared to their low-PNI counterparts (HR 0.58, 95% CI: 0.47–0.71, P<0.001). Furthermore, the results derived from 6 studies pointed out that the significant correlation between he low-PNI and poorer progression-free survival (PFS) (HR 0.58, 95% CI: 0.47–0.71, P<0.001). Subgroup analyses were performed to validate the robustness of the results. In addition, we conducted a meta-analysis of three studies examining the correlation between PNI and objective response rate/disease control rate (ORR/DCR) and found that the ORR/DCR was significantly superior in the high PNI group (ORR: RR: 1.24, P=0.002; DCR: RR: 1.43, P=0.008). Conclusion This meta-analysis indicates that the low-PNI in GC/GEJC patients undergoing ICI treatment is significantly linked to worse OS and PFS. Therefore, PNI can serve as a prognostic indicator of post-treatment outcomes in patients with GC receiving ICIs. Further prospective studies are required to assess the reliability of these findings. Systematic review registration https://inplasy.com/ , identifier INPLASY202450133.