Malate is a central metabolite in plant energy metabolism and biosynthesis and a major carrier of carbon and reducing equivalents between chloroplasts, the cytosol, and mitochondria. Here, we combined reverse genetics with multiple complementary approaches to investigate how disruption of the major mitochondrial malate-converting enzymes affects plant performance under different environmental conditions. We generated two independent mutant lines (mdh1 me1 me2) lacking the predominant mitochondrial malate dehydrogenase isoform MDH1 and both subunits of NAD-dependent malic enzyme (NAD-ME), ME1 and ME2. Growth under contrasting photoperiods and irradiances revealed a conditional phenotype that was most pronounced under short-day/low-light condition. Under this condition, the mutants exhibited impaired growth and photosynthetic performance, accompanied by cytosolic redox imbalance and altered chloroplast ultrastructure. Transfer experiments demonstrated that this phenotype was reversible and determined by the prevailing growth conditions: plants initially grown under short days and low light recovered in newly formed leaves after transfer to long-day/normal-light condition, whereas the reciprocal transfer reproduced the growth impairment in newly formed leaves. Transcriptomic and proteomic analyses revealed dawn-associated perturbations in photosynthetic and redox homeostasis, characterized by reduced investment in chloroplast translation and photosynthetic machinery alongside enhanced proteome maintenance, photoprotection and repair, and iron/ROS homeostasis. Branched-chain amino acids also accumulated in the mutants at the end of the night under low light. Together, these results show that the combined loss of mitochondrial MDH1 and NAD-ME causes a reversible, light-regime-dependent impairment of Arabidopsis growth and photosynthetic performance, highlighting the importance of the simultaneous contribution of both the MDH1 and NAD-ME systems to mitochondrial malate conversion when photosynthetic energy input is limited.
Numerous pathogens invade plants through stomata, and plants, on the other hand, could close the stomata after sensing the pathogen-associated molecular patterns (PAMPs). Subsequently, pathogens could overcome this plant immune response with their phytotoxins and effectors. A previous study showed that Plasmopara viticola, a strictly obligate biotrophic oomycete, could inhibit the Vitis vinifera stomatal closure to promote infection. However, the underlying mechanism stimulating the stomata movement is still poorly understood. Here we demonstrate that PvRxLR24, an effector from P. viticola, functions analogously to a phosphatase by directly inhibiting the kinase activity of OPEN STOMATA 1 (OST1), a central regulator of abscisic acid (ABA) signaling and stomatal closure. This inhibition subsequently abolishes the activation of the slow-type anion channel SLAC1, resulting in a "stomatal lock" open state. Our study demonstrated that OST1 is the direct target of pathogen effectors, shedding light on a new mechanism by which pathogens could manipulate the plant ABA signaling pathways to influence stomatal movement for successful invasion.
Abstract Cellular energy homeostasis relies on the precise exchange of nucleotides across subcellular compartments. The transport of ADP and ATP is essential for maintaining cellular energy balance. Transporters of these molecules are abundant proteins in the mitochondrial membrane, mediating their exchange between the mitochondrial matrix and the cytosol. In this study, we investigated the function of ADP/ATP Carrier 1 (AAC1) in plants to understand whether its regulation and function are similar to those described in animal and yeast systems. Our results revealed that AAC1 specifically transports ADP and ATP, while aac1 mutants displayed lower O2 consumption without evident decreases in cytosolic ATP levels. However, total adenylate profiling revealed altered adenylate balance and reduced adenylate energy charge in aac1 roots, indicating that reduced AAC1 function perturbs energy homeostasis beyond changes in ATP content alone. AAC1 was also required for proper mitochondrial structure, as aac1 mutant mitochondria exhibited a less electron-dense matrix and occasional absence of cristae. Suppressing AAC1 expression in plants altered vegetative and reproductive growth, reducing leaf number and root elongation while increasing silique size and seed weight. These findings provide novel insight into the role of AAC1 in mitochondrial energy dynamics. Biochemical analyses show a high specificity of AAC1 for adenine nucleotides, facilitating ADP/ATP exchange driven by the mitochondrial membrane potential, while excluding monophosphates and divalent ATP complexes. By correlating these biochemical characteristics with impaired mitochondrial respiration, altered adenylate balance, ultrastructural defects, and altered energy allocation, our study highlights the broader physiological impacts of disrupted adenylate transport mediated by AAC1.
Abstract Theanine is a specialized non-protein amino acid that determines the quality of tea (Camellia sinensis), yet the regulatory mechanisms controlling its hydrolysis remain unknown. Here we show that sucrose acts as a signal to modulate theanine accumulation by regulating a transcriptional repressor module that suppresses the theanine hydrolase gene CsPDX2.1. Using a yeast one-hybrid screen, we identified two transcription factors, CsYABBY2 and CsMYB5, that directly bind to distinct cis-elements (TAATGATT and TAACCA) in the CsPDX2.1 promoter. We demonstrate that CsYABBY2 and CsMYB5 interact to form a complex, and that co-expression of both proteins synergistically enhances repression of CsPDX2.1 transcription. Functional analyses via virus-induced gene silencing and transient overexpression confirmed that this module negatively regulates CsPDX2.1 expression and positively controls theanine accumulation. Importantly, exogenous sucrose suppresses CsYABBY2 and CsMYB5 expression, thereby derepressing CsPDX2.1 expression and reducing theanine levels, suggesting that sucrose functions as a regulatory factor affecting this pathway. Our findings reveal a sucrose-responsive (CsYABBY2-CsMYB5-CsPDX2.1) module that mediates theanine accumulation, providing a molecular basis for understanding varietal differences in theanine content.
Abstract Alfalfa (Medicago sativa) as one of the most important leguminous forages globally, plays an irreplaceable role in the development of animal husbandry and ecological conservation. However, its low genetic transformation efficiency severely constrains molecular breeding progress. In this study, the application of elicitor peptides MsPep1 significantly increased the genetic transformation efficiency of alfalfa by 1.61 times and shortened the transformation cycle by 20 days. Using this improved transformation system, we investigated FTO, an m⁶A RNA demethylase gene that regulates plant growth and stress responses. In the greenhouse, overexpression of FTO in alfalfa significantly enhanced plant height, root length, the number of lateral roots, as well as drought and salt tolerance. Under field conditions, it substantially increased fresh weight per plant by 24.98%, relative feed value (RFV) by 10% and hundred-seed weight. Through transcriptome sequencing (RNA-seq), we inferred that the FTO gene promotes alfalfa growth by regulating auxin, optimizes forage quality by enhancing secondary metabolite synthesis and enhances salt-drought tolerance by activating oxidative stress pathways. FTO mediates large amounts of m6A demethylation in plant RNA, altering chromatin composition and transcriptional activation. In summary, these findings provide molecular breeding support for developing novel alfalfa cultivars with high yield, superior quality and strong stress resilience.
