Complementation Tests Research Articles

LsKN1 and LsOFP6 synergistically regulate the bolting time by modulating the gibberellin pathway in lettuce.

Bolting time is an important agronomic trait in lettuce (Lactuca sativa) production. Premature bolting significantly reduces crop quality and marketability. Here, we report map-based cloning and characterization of a LsKN1 gene that controls bolting in lettuce. A segregating population was developed by crossing a crisphead-type cultivar with a stem-type cultivar to genetically map and clone the LsKN1 gene. In the late-bolting parent (crisphead), the LsKN1 was activated by a CACTA-like transposon which was inserted into the first exon of LsKN1. Complementation test, overexpression, and CRISPR/cas9 knockout showed that the activated LsKN1 allele (LsKN1TP) delays bolting in lettuce. ChIP-seq and phytohormone analysis demonstrated that LsKN1 regulates gibberellin (GA) biosynthesis and response. LsKN1TP binds to the promoter of the LsGA20ox1 and LsRGA1, and down- and upregulates their expression, respectively. Furthermore, LsRGA1 interacts with LsKN1TP to enhance the repression of GA biosynthesis. LsOFP6 acts as a safeguard, interacting with LsKN1TP to prevent excessive inhibition of GA biosynthesis and response during the vegetative-to-reproductive phase transition. The LsKN1-LsOFP6 module orchestrates the GA pathway to regulate bolting time in lettuce, which provides insight into the bolting development in lettuce and offers valuable genetic resources for breeding lettuce varieties resistant to premature bolting.

A mitochondrial metalloprotease FtsH4 is required for symbiotic nitrogen fixation in Lotus japonicus nodules

Symbiotic nitrogen fixation is a highly coordinated process involving legume plants and nitrogen-fixing bacteria known as rhizobia. In this study, we investigated a novel Fix− mutant of the model legume Lotus japonicus that develops root nodules with endosymbiotic rhizobia but fails in nitrogen fixation. Map-based cloning identified the causal gene encoding the filamentation temperature-sensitive H (FtsH) protein, designated as LjFtsH4. The LjFtsH4 gene was expressed in all plant organs without increased levels during nodulation. Subcellular localization revealed that LjFtsH4, fused with a fluorescent protein, localized in mitochondria. The Ljftsh4 mutant nodules showed signs of premature senescence, including symbiosome membrane collapse and bacteroid disintegration. Additionally, nodule cells of Ljftsh4 mutant displayed mitochondria with indistinct crista structures. Grafting and complementation tests confirmed that the Fix− phenotype was determined by the root genotype, and that protease activity of LjFtsH4 was essential for nodule nitrogen fixation. Furthermore, the ATP content in Ljftsh4 mutant roots and nodules was lower than in the wild-type, suggesting reduced mitochondrial function. These findings underscore the critical role of LjFtsH4 in effective symbiotic nitrogen fixation in root nodules.

A single-nucleotide insertion in Rxp confers durable resistance to bacterial pustule in soybean.

The soybean Rxp gene, encoding a bHLH transcription factor and an ACT-like domain, has an rxp allele producing a truncated protein that confers resistance to pustule-causing Xanthomonas axonopodis pv. glycines. In soybean, bacterial pustules caused by Xanthomonas axonopodis pv. glycines lead to premature defoliation and decreased yield in warm, wet climates. In the USA, approximately 70years ago, bacterial pustules were eliminated by introducing a recessive resistance allele, rxp, of the Rxp gene, representing the first example of successful soybean breeding for durable disease resistance in North America. In this study, we isolated this historical Rxp gene from resistant soybean varieties using positional cloning. The 1.06Mb region where Rxp was reported to reside was narrowed down to an 11.1kb region containing a single gene, Glyma.17g090500. The resistance allele, rxp, contains a T insertion. A complementation test of the Rxp allele in resistant plants confirmed the identification of the Rxp gene. The product of the susceptible wild-type allele, Rxp, is presumed to be a basic helix-loop-helix (bHLH) transcription factor with an aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain. This gene was mainly expressed in extended leaves, and its homologs were identified to be distributed in angiosperms. A total of six alleles were obtained: four from spontaneous variation, including the wild-type and three mutant alleles that encoded truncated proteins, and two from ethyl methanesulfonate mutants, including an allele that encoded a truncated protein and a missense allele. By evaluating the resistance of these six alleles, we found that the loss of function of RXP decreased the bacterial pustule lesions. This study provides important insights into the soybean rxp allele, which confers durable resistance to bacterial pustules.

Outcomes from the International Society of Nephrology Hemolytic Uremic Syndromes International Forum

The haemolytic uraemic syndromes (HUS) are a heterogeneous group of conditions only some of which are mediated by complement (CaHUS). We report the outcome of the 2023 International Society of Nephrology HUS International Forum where a global panel of experts considered the current state of the art, identified areas of uncertainty, and proposed optimal solutions. Areas of uncertainty and areas for future research included: the nomenclature of HUS; novel complement testing strategies; identification of biomarkers; genetic predisposition to aHUS; optimal dosing and withdrawal strategies for C5 inhibitors; treatment of kidney transplantation recipients; disparity of access to treatment; and the next generation of complement inhibitors in CaHUS. The current rationale for optimal patient management is described.

Challenges in Diagnosis and Management of SLE in Africa: An Online Survey.

We surveyed African physicians about challenges in diagnosis and management of systemic lupus erythematosus (SLE). We used a cross-sectional, online questionnaire-based survey of African specialist physicians on availability of laboratory tests, medications, and specialized services for the diagnosis of SLE. Our 226 respondents from 31 countries were dermatologists (38%), rheumatologists (28%), and nephrologists (23%), the majority practicing at university/state-funded hospitals (80.8%), but over half of patients (59.6%) were self-funded for laboratory tests and medications. Antinuclear antibody (ANA), antiphospholipid antibody, and complement tests were available to 79.4%, 67.6%, and 62.3% of respondents, respectively, but fewer in the East and West African regions. Median turnaround time for the ANA test was within two weeks but more than four weeks for 5.6% of respondents, and longer in West Africa compared with other regions (P = 0.0002). Availability of urine protein-to-creatinine test, skin and renal histopathology was 82%, 82.5%, and 76.2%, respectively. Median turnaround times were within one to two weeks, but more than four weeks for 13.8% of respondents for skin histology results and usually within four weeks but more than four weeks for 24.5% of respondents for renal histology. Glucocorticoids and antimalarials were readily available across all regions, with variable availability of immunosuppressants from 93.7% for methotrexate to 65% for calcineurin inhibitors and only 58.4% for the biologic rituximab. Intensive care units/high care facilities, hemodialysis, and renal transplantation were available to 69.8%, 91.9%, and 56.5% of respondents, respectively. Variable availability of laboratory tests, medications, and supportive services coupled with cost constraints are major impediments to early diagnosis and optimal management of SLE in most of Africa and are likely factors contributing to underreporting and poor prognosis of SLE in Africa.

Genome-Wide Isolation of VIN Gene Family and Functional Identification of HpVIN4 in Red Pitaya (Hylocereus polyrhizus)

Soluble sugars, including glucose, fructose and sucrose, are the most important determinants that affect the flavor and quality of red pitaya (Hylocereus polyrhizus) fruit. Vacuolar invertase (VIN), which catalyzes sucrose hydrolysis into glucose and fructose, is a key type of enzyme responsible for soluble sugar metabolism in plant growth and development. Herein, we conducted genome-wide identification, gene expression analysis, subcellular localization and an enzymatic properties assay for the VIN-encoding genes from red pitaya. During red pitaya fruit development towards ripening, the enzymatic activities of VIN showed an up-regulated trend towards ripening. In total, four isoforms (HpVIN1–4) of the VIN-encoding gene were identified from the pitaya genome. Sequence alignment results revealed that the HpVIN1, HpVIN3 and HpVIN4 proteins contained essential motifs for targeting the vacuole and conserved motifs or residues responsible for sucrose binding and hydrolysis. Gene expression pattern analyses revealed that the level of HpVIN4 was obviously increasing during fruit development and acted as the most abundant VIN isoform towards ripening. Subcellular localization detection via transient expression in Arabidopsis thaliana mesophyll protoplasts revealed that the HpVIN4 protein was localized in the vacuole. Growth complementation tests of heterologous expression in the invertase-deficient baker’s yeast strain suggested that the HpVIN4 protein had a sucrose hydrolysis activity and could restore the yeast growth in vivo. The identification of enzymatic properties in vitro demonstrated that the HpVIN4 protein could degrade sucrose into glucose and fructose with an optimum pH of 4.0. Specifically, the HpVIN4 protein had an estimated Km value of 5.15 ± 1.03 mmol·L−1 for sucrose hydrolysis. Ultimately, this study provides a comprehensive understanding of the potential roles of VINs during fruit development and towards ripening and provides functional gene resources for regulating soluble sugar accumulation in red pitaya fruit.

Characterization of the ddt1 Mutant in Rice and Its Impact on Plant Height Reduction and Water Use Efficiency.

Rice (Oryza sativa L.), a fundamental global staple, nourishes over half of the world's population. The identification of the ddt1 mutant in rice through EMS mutagenesis of the indica cultivar Shuhui527 revealed a dwarf phenotype, characterized by reduced plant height, smaller grain size, and decreased grain weight. Detailed phenotypic analysis and map-based cloning pinpointed the mutation to a single-base transversion in the LOC_Os03g04680 gene, encoding a cytochrome P450 enzyme, which results in a premature termination of the protein. Functional complementation tests confirmed LOC_Os03g04680 as the DDT1 gene responsible for the observed phenotype. We further demonstrated that the ddt1 mutation leads to significant alterations in gibberellic acid (GA) metabolism and signal transduction, evidenced by the differential expression of key GA-related genes such as OsGA20OX2, OsGA20OX3, and SLR1. The mutant also displayed enhanced drought tolerance, as indicated by higher survival rates, reduced water loss, and rapid stomatal closure under drought conditions. This increased drought resistance was linked to the mutant's improved antioxidant capacity, with elevated activities of antioxidant enzymes and higher expression levels of related genes. Our findings suggest that DDT1 plays a crucial role in regulating both plant height and drought stress responses. The potential for using gene editing of DDT1 to mitigate the dwarf phenotype while retaining improved drought resistance offers promising avenues for rice improvement.

The neurodevelopmental genes alan shepard and Neuroglian contribute to female mate preference in African Drosophila melanogaster.

Mate choice is a key trait that determines fitness for most sexually reproducing organisms, with females often being the choosy sex. Female preference often results in strong selection on male traits that can drive rapid divergence of traits and preferences between lineages, leading to reproductive isolation. Despite this fundamental property of female mate choice, very few loci have been identified that contribute to mate choice and reproductive isolation. We used a combination of population genetics, quantitative complementation tests, and behavioural assays to demonstrate that alan shepard and Neuroglian contribute to female mate choice, and could contribute to partial reproductive isolation between populations of Drosophila melanogaster. Our study is among the first to identify genes that contribute to female mate preference in this historically important system, where female preference is an active premating barrier to reproduction. The identification of loci that are primarily known for their roles in neurodevelopment provides intriguing questions of how female mate preference evolves in populations via changes in sensory system and higher learning brain centres.

The pentatricopeptide repeat protein TCD6 functions RNA editing and cleavage of ndhA and is required for chloroplast development in early rice seedlings

Pentatricopeptide repeat (PPR) proteins compose one of the largest protein families in higher plants and play a role in regulating organellar gene expression. In this study, we discovered that a new rice mutant tcd6 exhibited albino phenotype and aberrant chloroplast before the three-leaf (autotrophic) seedling stage. Through Map-based cloning and complementation tests, it was shown that TCD6 encodes a chloroplast-located PPR protein, with 14 PPR motifs and an atypical DYW-like motif. In addition, the disruption of TCD6 hindered the nuclear-encoded polymerase (NEP)-dependent transcript levels for plastid genes and led to defects in the cleavage and editing of ndhA (encoding NDH subunit) in early tcd6 mutant seedlings. Taken together, our results indicate that TCD6 is indispensable for chloroplast development and involves in RNA editing and cleavage of ndhA during early seedling (autotrophic) growth of rice.

Assessing Transcriptomic Responses to Oxidative Stress: Contrasting Wild-Type Arabidopsis Seedlings with dss1(I) and dss1(V) Gene Knockout Mutants.

Oxidative stress represents a critical facet of the array of abiotic stresses affecting crop growth and yield. In this paper, we investigated the potential differences in the functions of two highly homologous Arabidopsis DSS1 proteins in terms of maintaining genome integrity and response to oxidative stress. In the context of homologous recombination (HR), it was shown that overexpressing AtDSS1(I) using a functional complementation test increases the resistance of the Δdss1 mutant of Ustilago maydis to genotoxic agents. This indicates its conserved role in DNA repair via HR. To investigate the global transcriptome changes occurring in dss1 plant mutant lines, gene expression analysis was conducted using Illumina RNA sequencing technology. Individual RNA libraries were constructed from three total RNA samples isolated from dss1(I), dss1(V), and wild-type (WT) plants under hydrogen peroxide-induced stress. RNA-Seq data analysis and real-time PCR identification revealed major changes in gene expression between mutant lines and WT, while the dss1(I) and dss1(V) mutant lines exhibited analogous transcription profiles. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed significantly enriched metabolic pathways. Notably, genes associated with HR were upregulated in dss1 mutants compared to the WT. Otherwise, genes of the metabolic pathway responsible for the synthesis of secondary metabolites were downregulated in both dss1 mutant lines. These findings highlight the importance of understanding the molecular mechanisms of plant responses to oxidative stress.

Genetic bias in repeated evolution of pigment loss in cave populations of the Asellus aquaticus species complex.

Similar phenotypes can evolve repeatedly under the same evolutionary pressures. A compelling example is the evolution of pigment loss and eye loss in cave-dwelling animals. While specific genomic regions or genes associated with these phenotypes have been identified in model species, it remains uncertain whether a bias towards particular genetic mechanisms exists. An isopod crustacean, Asellus aquaticus, is an ideal model organism to investigate this phenomenon. It inhabits surface freshwaters throughout Europe but has colonized groundwater on multiple independent occasions and evolved several cave populations with distinct ecomorphology. Previous studies have demonstrated that three different cave populations utilized common genetic regions, potentially the same genes, in the evolution of pigment and eye loss. Expanding on this, we conducted analysis on two additional cave populations, distinct either phylogenetically or biogeographically from those previously examined. We generated F2 hybrids from cave × surface crosses and tested phenotype-genotype associations, as well as conducted complementation tests by crossing individuals from different cave populations. Our findings revealed that pigment loss and orange eye pigment in additional cave populations were associated with the same genomic regions as observed in the three previously tested cave populations. Moreover, the lack of complementation across all cross combinations suggests that the same gene likely drives pigment loss. These results substantiate a genetic bias in the recurrent evolution of pigment loss in this model system. Future investigations should focus on the cause behind this bias, possibly arising from allele recruitment from ancestral surface populations' genetic variation or advantageous allele effects via pleiotropy.

Mechanism Analysis of OsZF8-Mediated Regulation of Rice Resistance to Sheath Blight.

Transcription factors are key molecules involved in transcriptional and post-transcriptional regulation in plants and play an important regulatory role in resisting biological stress. In this study, we identified a regulatory factor, OsZF8, mediating rice response to Rhizoctonia solani (R. solani) AG1-IA infection. The expression of OsZF8 affects R. solani rice infection. OsZF8 knockout and overexpressed rice plants were constructed, and the phenotypes of mutant and wild-type (WT) plants showed that OsZF8 negatively regulated rice resistance to rice sheath blight. However, it was speculated that OsZF8 plays a regulatory role at the protein level. The interacting protein PRB1 of OsZF8 was screened using the yeast two-hybrid and bimolecular fluorescence complementation test. The results showed that OsZF8 effectively inhibited PRB1-induced cell death in tobacco cells, and molecular docking results showed that PRB1 had a strong binding effect with OsZF8. Further, the binding ability of OsZF8-PRB1 to ergosterol was significantly reduced when compared with the PRB1 protein. These findings provide new insights into elucidating the mechanism of rice resistance to rice sheath blight.

Improving Simple Past Tense Mastery Used in Recount Text Through the Use of English Songs at the Eleventh Grade Students of SMA Negeri 2 Sigi

The objective of this research is to prove that the use of English songs can improve simple past tense mastery used in recount text. The researcher used quasi experimental research design. The population of this research is the eleventh grade students of SMA Negeri 2 Sigi, and the samples were the students of XI IPS 2 and XI IPS 4 with the total number of students is 53. The researcher applied purposive random sampling technique. The data were collected through the complement test that consisted of pre-test and post-test. The data were analyzed statistically in order to find out the significant difference of the students’ achievement before and after treatments. The mean score of the pretest of experimental group is 33.78, while the control group is 32.04. The mean score of the posttest of experimental group 74.74, and the control group is 42.27. After analyzing the data t-counted (15.98) is greater than t-table (1.676) by applying 0.05 level of significance and 51 degree of freedom (df). Based on the result of research, it means that the hypothesis is accepted. In conclusion, using English songs can improve students’ simple past tense mastery used in recount text of the eleventh grade at SMA Negeri 2 Sigi

Forward genetics combined with unsupervised classifications identified zebrafish mutants affecting biliary system formation

Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested these 24 mutations affect 24 different genes. We applied unsupervised clustering algorithms to unbiasedly classify the recovered mutants into three classes. Further computational analysis revealed that each of the recovered mutations in these three classes has a unique phenotype on node-subtype composition and distribution within the intrahepatic biliary network. In addition, we found most of the recovered mutations are viable. In those mutant fish, which are already good animal models to study chronic cholestatic liver diseases, the biliary network phenotypes persist into adulthood. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases.

Complementation testing identifies genes mediating effects at quantitative trait loci underlying fear-related behavior

Knowing the genes involved in quantitative traits provides an entry point to understanding the biological bases of behavior, but there are very few examples where the pathway from genetic locus to behavioral change is known. To explore the role of specific genes in fear behavior, we mapped three fear-related traits, tested fourteen genes at six quantitative trait loci (QTLs) by quantitative complementation, and identified six genes. Four genes, Lamp, Ptprd, Nptx2, and Sh3gl, have known roles in synapse function; the fifth, Psip1, was not previously implicated in behavior; and the sixth is a long non-coding RNA, 4933413L06Rik, of unknown function. Variation in transcriptome and epigenetic modalities occurred preferentially in excitatory neurons, suggesting that genetic variation is more permissible in excitatory than inhibitory neuronal circuits. Our results relieve a bottleneck in using genetic mapping of QTLs to uncover biology underlying behavior and prompt a reconsideration of expected relationships between genetic and functional variation.

GhTKPR1_8 functions to inhibit anther dehiscence and reduce pollen viability in cotton.

Sporopollenin, as the main component of the pollen exine, is a highly resistant polymer that provides structural integrity under unfavourable environmental conditions. Tetraketone α-pyrone reductase 1 (TKPR1) is essential for sporopollenin formation, catalyzing the reduction of tetraketone carbonyl to hydroxylated α-pyrone. The functional role of TKPR1 in male sterility has been reported in flowering plants such as maize, rice, and Arabidopsis. However, the molecular cloning and functional characterization of TKPR1 in cotton remain unaddressed. In this study, we identified 68 TKPR1s from four cotton species, categorized into three clades. Transcriptomics and RT-qPCR demonstrated that GhTKPR1_8 exhibited typical expression patterns in the tetrad stage of the anther. GhTKPR1_8 was localized to the endoplasmic reticulum. Moreover, ABORTED MICROSPORES (GhAMS) transcriptionally activated GhTKPR1_8 as indicated by luciferase complementation tests. GhTKPR1_8-knockdown inhibited anther dehiscence and reduced pollen viability in cotton. Additionally, overexpression of GhTKPR1_8 in the attkpr1 mutant restored its male sterile phenotype. This study offers novel insights into the investigation of TKPR1 in cotton while providing genetic resources for studying male sterility.

Receipt of rheumatology care and lupus-specific labs among young adults with systemic lupus erythematosus: A US Medicare retention in care cohort study.

In systemic lupus erythematosus, poor disease outcomes occur in young adults, patients identifying as Black or Hispanic, and socioeconomically disadvantaged patients. These identities and social factors differentially shape care access and quality that contribute to lupus health disparities in the US. Thus, our objective was to measure markers of care access and quality, including rheumatology visits (longitudinal care retention) and lupus-specific serology testing, by race and ethnicity, neighborhood disadvantage, and geographic context. This cohort study used a geo-linked 20% national sample of young adult Medicare beneficiaries (ages 18-35) with lupus-coded encounters and a 1-year assessment period. Retention in lupus care required a rheumatology visit in each 6-month period, and serology testing required ≥1 complement or dsDNA antibody test within the year. Multivariable logistic regression models were fit for visit-based retention and serology testing to determine associations with race and ethnicity, neighborhood disadvantage, and geography. Among 1,036 young adults with lupus, 39% saw a rheumatologist every 6months and 28% had serology testing. White beneficiaries from the least disadvantaged quintile of neighborhoods had higher visit-based retention than other beneficiaries (64% vs 30%-60%). Serology testing decreased with increasing neighborhood disadvantage quintile (aOR 0.80; 95% CI 0.71, 0.90) and in the Midwest (aOR 0.46; 0.30, 0.71). Disparities in care, measured by rheumatology visits and serology testing, exist by neighborhood disadvantage, race and ethnicity, and region among young adults with lupus, despite uniform Medicare coverage. Findings support evaluating lupus care quality measures and their impact on US lupus outcomes.

FLOURY ENDOSPERM24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice.

Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure. Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map-based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant. The flo24 mutant develops more severe floury endosperm when grown under high-temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp-2 to regulate starch biosynthesis and endosperm development in rice. Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice.

Low mutation rate of spontaneous mutants enables detection of causative genes by comparing whole genome sequences.

In the early 1900s, mutation breeding to select varieties with desirable traits using spontaneous mutation was actively conducted around the world, including Japan. In rice, the number of fixed mutations per generation was estimated to be 1.38-2.25. Although this low mutation rate was a major problem for breeding in those days, in the modern era with the development of next-generation sequencing (NGS) technology, it was conversely considered to be an advantage for efficient gene identification. In this paper, we proposed an in silico approach using NGS to compare the whole genome sequence of a spontaneous mutant with that of a closely related strain with a nearly identical genome, to find polymorphisms that differ between them, and to identify the causal gene by predicting the functional variation of the gene caused by the polymorphism. Using this approach, we found four causal genes for the dwarf mutation, the round shape grain mutation and the awnless mutation. Three of these genes were the same as those previously reported, but one was a novel gene involved in awn formation. The novel gene was isolated from Bozu-Aikoku, a mutant of Aikoku with the awnless trait, in which nine polymorphisms were predicted to alter gene function by their whole-genome comparison. Based on the information on gene function and tissue-specific expression patterns of these candidate genes, Os03g0115700/LOC_Os03g02460, annotated as a short-chain dehydrogenase/reductase SDR family protein, is most likely to be involved in the awnless mutation. Indeed, complementation tests by transformation showed that it is involved in awn formation. Thus, this method is an effective way to accelerate genome breeding of various crop species by enabling the identification of useful genes that can be used for crop breeding with minimal effort for NGS analysis.

The plastid-localized lipoamide dehydrogenase 1 is crucial for redox homeostasis, tolerance to arsenic stress and fatty acid biosynthesis in rice.

Soil contamination with arsenic (As) can cause phytotoxicity and reduce crop yield. The mechanisms of As toxicity and tolerance are not fully understood. In this study, we used a forward genetics approach to isolate a rice mutant, ahs1, that exhibits hypersensitivity to both arsenate and arsenite. Through genomic resequencing and complementation tests, we identified OsLPD1 as the causal gene, which encodes a putative lipoamide dehydrogenase. OsLPD1 was expressed in the outer cell layer of roots, root meristem cells, and in the mesophyll and vascular tissues of leaves. Subcellular localization and immunoblot analysis demonstrated that OsLPD1 is localized in the stroma of plastids. In vitro assays showed that OsLPD1 exhibited lipoamide dehydrogenase (LPD) activity, which was strongly inhibited by arsenite, but not by arsenate. The ahs1 and OsLPD1 knockout mutants exhibited significantly reduced NADH/NAD+ and GSH/GSSG ratios, along with increased levels of reactive oxygen species and greater oxidative stress in the roots compared with wild-type (WT) plants under As treatment. Additionally, loss-of-function of OsLPD1 also resulted in decreased fatty acid concentrations in rice grain. Taken together, our finding reveals that OsLPD1 plays an important role for maintaining redox homeostasis, conferring tolerance to arsenic stress, and regulating fatty acid biosynthesis in rice.