Publications

ABSTRACT Plants have evolved signaling mechanisms that guide growth away from adverse environments that can cause yield losses. Root halotropism is a sodium-specific negative tropism that is crucial for surviving and thriving under high salinity. Although root halotropism was discovered some years ago, the underlying molecular and cellular mechanisms remain unknown. Here, we show that abscisic acid (ABA)-mediated root twisting determines halotropism in Arabidopsis . An ABA-activated SnRK2 protein kinase (SnRK2.6) phosphorylates the microtubule-associated protein SP2L at Ser406, which induces a change in the anisotropic cell expansion at the root transition zone that located between the apical meristem and basal elongation zone, and is required for root twisting during halotropsim. Salt stress triggers SP2L-mediated cortical microtubule reorientation in cells at the transition zone, which guides cellulose microfibril patterns. Our findings outline the cellular and molecular mechanisms of root halotropism and indicate that anisotropic cell expansion through MT-reorientation and microfibril deposition have a central role in mediating tropic responses.

Lysine-ε-acetylation (Kac) is a post-translational modification (PTM) that is critical for metabolic regulation and cell signaling in mammals. However, its prevalence and importance in plants remain to be determined. Employing high-resolution tandem mass spectrometry, we analyzed protein lysine acetylation in five representative Arabidopsis organs with 2 ~ 3 biological replicates per organ. A total of 2887 Kac proteins and 5929 Kac sites were identified. This comprehensive catalog allows us to analyze proteome-wide features of lysine acetylation. We found that Kac proteins tend to be more uniformly expressed in different organs, and the acetylation status exhibits little correlation with the gene expression level, indicating that acetylation is unlikely caused by stochastic processes. Kac preferentially targets evolutionarily conserved proteins and lysine residues, but only a small percentage of Kac proteins are orthologous between rat and Arabidopsis. A large portion of Kac proteins overlap with proteins modified by other PTMs including ubiquitination, SUMOylation and phosphorylation. Although acetylation, ubiquitination and SUMOylation all modify lysine residues, our analyses show that they rarely target the same sites. In addition, we found that "reader" proteins for acetylation and phosphorylation, i.e., bromodomain-containing proteins and GRF (General Regulatory Factor)/14-3-3 proteins, are intensively modified by the two PTMs, suggesting that they are main crosstalk nodes between acetylation and phosphorylation signaling. Analyses of GRF6/14-3-3λ reveal that the Kac level of GRF6 is decreased under alkaline stress, suggesting that acetylation represses plant alkaline response. Indeed, K56ac of GRF6 inhibits its binding to and subsequent activation of the plasma membrane H+-ATPase AHA2, leading to hypersensitivity to alkaline stress. These results provide valuable resources for protein acetylation studies in plants and reveal that protein acetylation suppresses phosphorylation output by acetylating GRF/14-3-3 proteins.

Multicellular organisms can be regenerated from totipotent differentiated somatic cell or nuclear founders [1-3]. Organisms regenerated from clonally related isogenic founders might a priori have been expected to be phenotypically invariant. However, clonal regenerant animals display variant phenotypes caused by defective epigenetic reprogramming of gene expression [2], and clonal regenerant plants exhibit poorly understood heritable phenotypic ("somaclonal") variation [4-7]. Here we show that somaclonal variation in regenerant Arabidopsis lineages is associated with genome-wide elevation in DNA sequence mutation rate. We also show that regenerant mutations comprise a distinctive molecular spectrum of base substitutions, insertions, and deletions that probably results from decreased DNA repair fidelity. Finally, we show that while regenerant base substitutions are a likely major genetic cause of the somaclonal variation of regenerant Arabidopsis lineages, transposon movement is unlikely to contribute substantially to that variation. We conclude that the phenotypic variation of regenerant plants, unlike that of regenerant animals, is substantially due to DNA sequence mutation.

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.

DNA methylation, a conserved epigenetic mark, is critical for tuning temporal and spatial gene expression. The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci. However, how ROS1 is recruited to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor that is required to prevent gene silencing and DNA hypermethylation. AGDP3 binds to H3K9me2 marks in its target DNA via its AGD12 cassette. Analysis of the crystal structure of the AGD12 cassette of AGDP3 in complex with an H3K9me2 peptide revealed that dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. A histidine residue located in the methyllysine binding aromatic cage provides AGDP3 with pH-dependent H3K9me2 binding capacity. Our results uncover a molecular mechanism for the regulation of DNA demethylation by the gene silencing mark H3K9me2.

Abstract Drought is one of the most important environmental constraints limiting plant growth and agricultural productivity. To understand the underlying mechanism of drought tolerance and to identify genes for improving this important trait, we conducted a gain-of-function genetic screen for improved drought tolerance in Arabidopsis thaliana. One mutant with improved drought tolerance was isolated and designated as enhanced drought tolerance1. The mutant has a more extensive root system than the wild type, with deeper roots and more lateral roots, and shows a reduced leaf stomatal density. The mutant had higher levels of abscisic acid and Pro than the wild type and demonstrated an increased resistance to oxidative stress and high levels of superoxide dismutase. Molecular genetic analysis and recapitulation experiments showed that the enhanced drought tolerance is caused by the activated expression of a T-DNA tagged gene that encodes a putative homeodomain-START transcription factor. Moreover, overexpressing the cDNA of the transcription factor in transgenic tobacco also conferred drought tolerance associated with improved root architecture and reduced leaf stomatal density. Therefore, we have revealed functions of the homeodomain-START factor that were gained upon altering its expression pattern by activation tagging and provide a key regulator that may be used to improve drought tolerance in plants.

Additional file 5: Table S4. Distribution of the indicated TF targets in each hub.