STN7 and STN8 kinases phosphorylate several proteins of the photosynthetic apparatus in response to changes in environmental conditions. Phosphorylation is linked to the dynamic arrangement of both the 3D structure of the thylakoid membrane and thylakoid protein complexes, thereby controlling excitation energy transfer between photosystems and the repair cycle of photosystem II (PSII). However, mutants with phosphorylation of PSII core proteins show only minor changes compared with the wild-type (WT) under conditions tested so far, and thus the physiological significance of reversible thylakoid protein phosphorylation by STN8 remains largely unresolved. In this study, we examined the impact of STN7 and STN8 on osmotic stress and consequent changes in the structure of the thylakoid membrane in Arabidopsis thaliana using physiological, cellular, and biochemical assays. Our results demonstrate that Arabidopsis plants lacking STN7 or STN8 exhibited slow growth and impaired regulation of excitation energy transfer, as well as low photosynthetic efficiency, excessive accumulation of ROS, and more severe cell death in response to osmotic stress compared with the WT. Clear effects of osmotic stress were observed in both stn7 and stn8 mutants but were more pronounced in the stn7stn8 double mutant. Our data strongly suggest that phosphorylation of the PSII reaction center proteins, regulated by STN8, is important for maintaining the functional balance of the photosynthetic apparatus under osmotic stress-induced changes in the thylakoid membrane and thus protects it from oxidative stress.

Calmodulin-binding protein 60b (CBP60b) is a pivotal transcription factor in the transcriptional reprogramming of immune responses. Although its downstream regulatory genes and associated genetic pathways have been reported, its post-translational regulation and modifications remain unexplored. Here, we report that calcium-dependent protein kinases CPK4/5/6/11 physically interact with CBP60b and phosphorylate its serine 568 residue, thereby enhancing its transcriptional activity. The phosphodeficient variant CBP60b[S568A] compromises immune responses, whereas the phosphomimetic variant CBP60b[S568D] enhances resistance against pathogens, demonstrating that ser568 is essential for the function of CBP60b. Moreover, we observed that the expression of CPK4/5/6/11 is induced by pathogens, while this pathogen-induced expression of CPKs is reduced in the cbp60b mutant. Through experiments including dual luciferase (LUC) reporter assays and chromatin immunoprecipitation (ChIP) assays, we further demonstrated that CPK4, CPK5, and CPK11 are direct transcriptional targets of CBP60b. In conclusion, our results demonstrate for the first time that CBP60b is post-translationally regulated by CPKs via phosphorylation, while the pathogen-induced expression of CPKs is dependent on CBP60b. This mutual regulation establishes a positive feedback loop, which enables plants to mount a robust immune response against pathogens.

Photosynthesis impairment and the accompanying leaf yellowing represent a prominent and physiologically critical symptom of phytoplasma infection. However, the underlying molecular mechanisms remain poorly understood. In this study, we identified the Jujube Witches' Broom pathogenic effector PHYL1JWB, which was previously reported to induce phyllody in jujube, as a key inducer of photosynthesis impairment. Overexpression of PHYL1JWB in sour jujube and Arabidopsis thaliana disrupted photosynthesis by causing severe disorganization of chloroplast ultrastructure. PHYL1JWB directly interacts with the integral thylakoid membrane protein ZjCURT1A and promotes its 26S proteasome-dependent degradation independently of lysine ubiquitination, leading to disruption of thylakoid structure and photosynthetic dysfunction. The conserved two α-helices of PHYL1JWB and the C-terminal segment of ZjCURT1A containing the thylakoid membrane curvature-related CAAD motif are essential for their interaction. Together, these findings reveal that PHYL1JWB impairs photosynthesis by disrupting chloroplast ultrastructural in addition to inducing phyllody, thereby contributing to abnormal plant growth. This work highlights the functional pleiotropy of pathogenic effectors and underscores the need to consider their impacts on multiple physiological processes in the context of phytoplasma pathogenicity.

Chilling stress severely restricts tomato cultivation and productivity, yet the mechanisms by which a single regulator integrates photosynthetic efficiency, developmental progression, and chilling tolerance remain to be elucidated. Here, we characterized SlBBX19, a chilling-inducible B-box transcription factor in tomato (Solanum lycopersicum). SlBBX19 is nucleus-localized, functions as a negative regulator of vegetative growth and photosynthetic capacity under normal conditions, while simultaneously promoting fruit ripening and suppressing inflorescence branching. Under chilling stress, SlBBX19 acts as a negative regulator of chilling tolerance: overexpression exacerbates reactive oxygen species (ROS) accumulation, membrane damage, and suppresses COR pathway activation, whereas knockout lines exhibit enhanced chilling resistance. Transcriptomic and molecular analyses reveal that SlBBX19 directly binds to G-box elements in the promoters of light-harvesting chlorophyll a/b-binding genes SlLhcb2.2 and SlLhca4.1 to repress their transcription. Virus-induced gene silencing of these targets recapitulated the overexpression phenotypes, confirming that SlBBX19 compromises photosynthetic efficiency and chilling tolerance primarily through SlLhc downregulation. Collectively, our findings establish SlBBX19 as a key integrator of development and environmental acclimation, revealing a novel regulatory module that links photosynthetic antenna dynamics to chilling stress adaptation. This work may provide a promising genetic target for engineering chilling-tolerant and physiologically optimized tomato varieties.

Despite the economic importance of the rubber tree (Hevea brasiliensis) and its sensitivity to cold stress, the molecular mechanisms that govern its cold response are still largely unknown. Here, we identified H. brasiliensis CATALASE2 (HbCAT2) as a factor associated with cold tolerance in this species, likely through reducing excess H2O2. Cold stress significantly induced HbCAT2 transcription and catalase activity in H. brasiliensis, and overexpression of HbCAT2 in both H. brasiliensis and Arabidopsis enhanced cold tolerance and reduced H2O2 accumulation. Furthermore, we found that HbCBF1, a key transcription factor in plant cold signaling, could directly bind to and activate HbCAT2, and overexpression of HbCAT2 was able to alleviate the cold hypersensitivity of the Arabidopsis cbf123 triple mutant. Additionally, the protein kinase HbSnRK2.6A interacted with and phosphorylated HbCAT2 to stimulate its catalase activity. Genetic evidence showed that HbSnRK2.6A-mediated cold tolerance is associated with CAT2 function. Together, our findings are consistent with a dual regulatory module in which HbCBF1-mediated HbCAT2 transcription and HbSnRK2.6A-mediated phosphorylation of HbCAT2 may contribute to enhancing cold tolerance by preventing H2O2 overaccumulation in H. brasiliensis.

The JAGGED transcription factor family regulates lateral organ development across angiosperms. In soybean (Glycine max Merr.), a D9H mutation in the EAR repression motif of GmJAG1 causes a narrow-leaflet phenotype and explains over 70% of phenotypic variance in leaf shape. Because this mutation does not affect the zinc finger DNA-binding domain, both alleles bind identical targets but differ in repressor recruitment. Previous studies mapped GmJAG1 binding sites, but the functional targets controlling leaf morphology are uncharacterized. Here, we used comparative transcriptomics across four soybean genotypes with contrasting leaf shape, spanning a developmental time series from shoot apex to mature leaf, and identified 1567 putative candidate target genes. GmJAG1 expression was confined to the shoot apex, yet 99.1% of candidate targets maintained differential expression throughout development. We found that neither Kip-Related Protein (KRP) cell cycle inhibitors nor Cyclin-Dependent Kinases (CDKs) showed differential expression despite binding evidence in Arabidopsis. However, D-type cyclins were upregulated in narrow-leaf genotypes suggesting that soybean GmJAG1 acts through cyclin-mediated rather than KRP-mediated cell cycle regulation described in Arabidopsis- a divergence in regulatory logic between the two species. Pathway analysis revealed enrichment of auxin (1.8-fold, P = 0.02) and salicylic acid (fourfold, P = 0.016) genes among JAG1D9H targets. Filtering by differential expression, binding data, phenotype correlation, and co-expression network membership identified 79 high-confidence targets, including orthologs of NPH3 (phototropin-mediated leaf flattening), MIK2 (cell wall integrity sensing), RD22 (ABA-responsive stress signaling), and SCL23 (GRAS transcription factor in bundle sheath development). These candidates provide targets for functional validation and breeding in legumes.

Leaf senescence, the final stage of leaf development, is regulated by complex interplays of intrinsic genetic programs and environmental cues. Throughout their lifetimes, all living organisms encounter various endogenous and environmental challenges, many of which can cause potentially fatal DNA damage. Among these, DNA-protein crosslinks (DPCs) are particularly deleterious, as they obstruct essential processes such as replication and transcription, thereby compromising genome integrity and ultimately leading to premature aging across species. However, the biological significance of DPCs and their repair mechanisms in leaf senescence remains unexplored. Here, we demonstrate that cis-platin (cis-Pt), a potent DPC inducer, accelerates leaf senescence in Arabidopsis. We reveal that Arabidopsis DPC repair factor WSS1A, a WLM/Spr-T metalloprotease, plays a negative role in leaf senescence induced by cis-Pt treatment, darkness, and leaf age. WSS1A forms nuclear condensates via liquid-liquid phase separation both in vitro and in vivo, which is cooperatively driven by its N-terminal segment and intrinsically disordered region. Mechanistically, WSS1A non-covalently interacts with SMALL UBIQUITIN MODIFIER 3 (SUMO3) through its SUMO-interacting motif and is also covalently SUMOylated by SUMO3. Genetic analysis further reveals that WSS1A and SUMO3 act in the same pathway to control cis-Pt-induced leaf senescence. Together, this study establishes a conceptual framework connecting DPC repair and SUMO3-dependent regulation of WSS1A in modulating leaf senescence in Arabidopsis.

Plant-parasitic nematodes deploy secreted effector proteins that reprogram host cellular processes to establish parasitism. Here, we characterized Mi1D08B, a putative dorsal-gland effector from the root-knot nematode Meloidogyne incognita and defined its role in subverting soybean jasmonate-mediated immunity. Mi1D08B is conserved across several Meloidogyne species and localizes to the plant nucleus and cytoplasm. Overexpression of Mi1D08B significantly increased galling and egg production, demonstrating a strong virulence role. A gall-specific yeast two-hybrid screen identified the NINJA-family co-repressor mc410 as a host target of Mi1D08B. Consistent with the functional relevance of this interaction, mc410 promoter activity was detected in galls and giant cells throughout nematode infection. Genetic manipulation of mc410 phenocopied Mi1D08B activity as mc410 overexpression enhanced susceptibility, whereas mc410 silencing reduced nematode infection. Hormone profiling revealed that Mi1D08B overexpression elevated 12-oxo-phytodienoic acid (OPDA) but reduced jasmonic acid (JA) and JA-Ile, consistent with a bottleneck at the peroxisomal OPDA-to-JA conversion. Furthermore, several metabolites associated with this conversion were reduced, indicating that Mi1D08B perturbs metabolic flux through the peroxisomal phase of jasmonate biosynthesis. Gene expression analyses supported this biochemical signature, with upregulation of plastidial OPDA-biosynthetic genes and downregulation of peroxisomal OPDA-reductases, JA-conjugating enzymes, and JA-responsive markers. Together, our data support a model in which Mi1D08B effector interacts with a NINJA co-repressor to suppress JA biosynthesis and signaling, thereby coupling nuclear transcriptional repression to altered metabolism. This mechanism deepens our understanding of nematode manipulation of host hormone networks and highlights the Mi1D08B-mc410 interface and OPDA conversion as promising targets for engineering nematode resistance.

Salinity induces the accumulation of reactive oxygen species (ROS) in plants, thereby regulating plant growth and development, including seed germination. However, the molecular mechanisms underlying the regulation of ROS homeostasis in response to salt stress remain largely unknown. Here, we showed that an leucine-rich-repeat receptor-like kinase, REK1, regulates ROS production by phosphorylating the NADPH oxidase respiratory burst oxidase homolog D (RBOHD) in Arabidopsis in response to salt stress. Salt-induced ROS accumulates extensively in rek1 mutants, causing stress that reduces the seed germination rate of rek1 below that of the wild-type (Col-0), resulting in the salt-sensitive phenotype. Furthermore, REK1 interacts with RBOHD and phosphorylates it at Ser163, thereby regulating its NADPH oxidase activity and influencing ROS production under salt stress. Genetic data revealed that the salt-sensitive phenotype of rek1 is rescued in rek1 rbohd mutants. In summary, our study revealed a salt stress regulation mechanism that modulates ROS production in Arabidopsis through the REK1-RBOHD pathway.

Plants activate osmotic regulation, ion and reactive oxygen species (ROS) homeostasis modulation, and plant hormone abscisic acid (ABA)-mediated stomatal movement regulation for survival under salt stress. However, it is unknown if and how a single molecule coordinates multiple responses. Here we characterized the functions of three closest homologs, cyclic nucleotide-gated channel 14, 15, and 16 (OsCNGC14/15/16), in regulating multiple signaling pathways in response to salt. Mutants of each of these three genes had reduced salt tolerance compared to the wild type. These mutants also had slower stomatal closure, lower Ca[2+] influx, higher Na[+]/K[+] ratio, and ROS accumulation under salt treatment. Conversely, overexpression of OsCNGC16 led to enhanced salt tolerance, increased Ca[2+] influx, lower Na[+]/K[+] ratio, and ROS accumulation in response to salt. OsCNGC14/15/16 also positively regulate shoot growth under ABA treatment. Exogenous ABA treatment rescued the defects in salt tolerance and stomatal closure of the OsCNGC16 mutant. And treatment of ROS scavenger dimethylthiourea (DMTU) partially rescued the salt susceptibility of the OsCNGC16 mutant. In addition, OsCNGC16 can interact with and be phosphorylated by Ca[2+]-dependent protein kinases 5 and 13 (CPK5/13). This study unveils multiple roles of OsCNGC14/15/16 proteins in salt tolerance and identifies common regulators of stomatal closure, Ca[2+] influx, Na[+]/K[+] homeostasis, ROS scavenging, and ABA signaling in response to salt stress.

Alternative splicing (AS) is a common gene regulatory mechanism involving distinct interactions between trans-acting factors and cis-regulatory elements on the precursor messenger RNA (pre-mRNA). In this study, we have functionally characterized the structured motif 45ABC, which is located in the pre-mRNAs of RNA-binding protein (RBP) 45 genes in many plant species. Our data revealed that this element mediates a negative auto- and cross-regulatory feedback loop via AS of the three 45ABC-containing RBP45 genes in Arabidopsis thaliana. We identified a G-rich stretch within the first stem as a potential RBP45 binding site and observed increased RBP45-dependent AS upon structural weakening of this pairing element. The second stem includes the alternative 5' splice site being activated in the presence of RBP45. Based on the known interaction between RBP45 homologs and U1 snRNP components required for 5' splice site recognition, we propose that RBP45 recruitment to stem I of 45ABC may induce usage of the alternative 5' splice site in stem II. The resulting splicing variant is unproductive, thereby diminishing RBP45 expression. Analysing the splicing-regulatory impact of the three At-RBP45 genes in auto- and cross-regulation and a transcriptome-wide manner revealed unequal genetic redundancy with a major role of RBP45B. Furthermore, phenotypical analysis of single- and higher-order rbp45 mutants pointed at these genes' functions in controlling primary root growth and flowering time. Taken together, we demonstrated that both sequence and structural features of 45ABC are critical for proper splicing control, balancing RBP45 expression and functions in plants via a conserved mRNA motif.

Soybean (Glycine max L.) is a major oilseed crop worldwide, and its seed oil quality is largely determined by fatty acid composition. Although the enzymatic framework of plant fatty acid biosynthesis has been extensively characterized, the molecular mechanisms that regulate fatty acid partitioning and seed oil accumulation are not fully understood. In this study, genome-wide association analysis of soybean seed oil content identified GmKASII as a candidate gene underlying natural variation in seed oil accumulation, which was further supported by expression analysis and functional SNP characterization. Genetic manipulation of GmKASII significantly altered soybean seed oil accumulation and fatty acid composition. GmKASII overexpression increased seed oil content and promoted the accumulation of specific C18 fatty acids, including stearic acid (C18:0) and linoleic acid (C18:2), whereas gmkasII knockout reduced seed oil content and resulted in increased palmitic acid (C16:0) and decreased C18:0 levels. Protein-protein interaction assays, including yeast two-hybrid, luciferase complementation, and co-immunoprecipitation analyses, demonstrated that GmKASII physically interacts with acyl-ACP thioesterase GmFATA1B, suggesting a potential functional connection between fatty acid chain elongation and acyl-ACP hydrolysis pathways. Transient expression analysis in tobacco further suggested that co-expression of GmKASII and GmFATA1B influences fatty acid composition in a heterologous system. Collectively, these findings demonstrate that GmKASII plays an important role in regulating plastidial fatty acid flux and seed oil biosynthesis, and reveal a potential regulatory connection between fatty acid elongation and acyl-ACP release pathways in soybean.

Agrobacterium transfers DNA into plant cells, leading to tumors, hairy roots (HR), and natural genetically modified organisms (nGMOs). Transferred DNAs (T-DNAs) from agrobacteria and T-DNA-derived cellular T-DNAs (cT-DNAs) from nGMOs vary considerably and may carry up to 15 different genes. Among these, opine synthase (ops) genes encode the synthesis of opines used as nutrients by the agrobacteria. Earlier studies predicted large numbers of naturally transformed plant species, but only few have been identified and studied so far. We therefore developed a general method to detect cT-DNAs in all publicly available whole genome sequences (WGS) and Sequence Read Archive (SRA) data from land plants. To avoid false positives, we only retained DNA sequences coding for T-DNA proteins. A total of 2614 nGMO species were identified, most are eudicots. However, cT-DNAs were also found in 82 mosses and 75 ferns, showing that Agrobacterium can also generate natural transformants among the early land plants. Analysis of 149 cT-DNA maps revealed different types of T-DNAs. Most notably, these included small T-DNAs (mini T-DNAs) with a single opine synthase gene. Mini T-DNAs are not expected to induce tumors or HRs. The predominance of mini cT-DNAs in mosses and ferns, and the presence of more complex cT-DNAs in spermatophytes, indicate that mini T-DNAs represent the earliest types of T-DNA. Our study also detected unusual T-DNA integration patterns, with multiple copies spread out over several hundreds of kilobases.