A natural gene drive system confers reproductive isolation in rice.

Hybrid sterility restricts the utilization of superior heterosis of indica-japonica inter-subspecific hybrids. In this study, we report the identification of RHS12, a major locus controlling male gamete sterility in indica-japonica hybrid rice. We show that RHS12 consists of two genes (iORF3/DUYAO and iORF4/JIEYAO) that confer preferential transmission of the RHS12-i type male gamete into the progeny, thereby forming a natural gene drive. DUYAO encodes a mitochondrion-targeted protein that interacts with OsCOX11 to trigger cytotoxicity and cell death, whereas JIEYAO encodes a protein that reroutes DUYAO to the autophagosome for degradation via direct physical interaction, thereby detoxifying DUYAO. Evolutionary trajectory analysis reveals that this system likely formed de novo in the AA genome Oryza clade and contributed to reproductive isolation (RI) between different lineages of rice. Our combined results provide mechanistic insights into the genetic basis of RI as well as insights for strategic designs of hybrid rice breeding.

Rice LIKE EARLY STARVATION1 cooperates with FLOURY ENDOSPERM6 to modulate starch biosynthesis and endosperm development.

In cereal grains, starch is synthesized by the concerted actions of multiple enzymes on the surface of starch granules within the amyloplast. However, little is known about how starch-synthesizing enzymes access starch granules, especially for amylopectin biosynthesis. Here, we show that the rice (Oryza sativa) floury endosperm9 (flo9) mutant is defective in amylopectin biosynthesis, leading to grains exhibiting a floury endosperm with a hollow core. Molecular cloning revealed that FLO9 encodes a plant-specific protein homologous to Arabidopsis (Arabidopsis thaliana) LIKE EARLY STARVATION1 (LESV). Unlike Arabidopsis LESV, which is involved in starch metabolism in leaves, OsLESV is required for starch granule initiation in the endosperm. OsLESV can directly bind to starch by its C-terminal tryptophan (Trp)-rich region. Cellular and biochemical evidence suggests that OsLESV interacts with the starch-binding protein FLO6, and loss-of-function mutations of either gene impair ISOAMYLASE1 (ISA1) targeting to starch granules. Genetically, OsLESV acts synergistically with FLO6 to regulate starch biosynthesis and endosperm development. Together, our results identify OsLESV-FLO6 as a non-enzymatic molecular module responsible for ISA1 localization on starch granules, and present a target gene for use in biotechnology to control starch content and composition in rice endosperm.

A CYP78As-small grain4-coat protein complex â ¡ pathway promotes grain size in rice.

CYP78A, a cytochrome P450 subfamily that includes rice (Oryza sativa L.) BIG GRAIN2 (BG2, CYP78A13) and Arabidopsis thaliana KLUH (KLU, CYP78A5), generate an unknown mobile growth signal (referred to as a CYP78A-derived signal) that increases grain (seed) size. However, the mechanism by which the CYP78A pathway increases grain size remains elusive. Here, we characterized a rice small grain mutant, small grain4 (smg4), with smaller grains than its wild type due to restricted cell expansion and cell proliferation in spikelet hulls. SMG4 encodes a multidrug and toxic compound extrusion (MATE) transporter. Loss of function of SMG4 causes smaller grains while overexpressing SMG4 results in larger grains. SMG4 is mainly localized to endoplasmic reticulum (ER) exit sites (ERESs) and partially localized to the ER and Golgi. Biochemically, SMG4 interacts with coat protein complex Ⅱ (COPⅡ) components (Sar1, Sec23, and Sec24) and CYP78As (BG2, GRAIN LENGTH 3.2 [GL3.2], and BG2-LIKE 1 [BG2L1]). Genetically, SMG4 acts, at least in part, in a common pathway with Sar1 and CYP78As to regulate grain size. In summary, our findings reveal a CYP78As-SMG4-COPⅡ regulatory pathway for grain size in rice, thus providing new insights into the molecular and genetic regulatory mechanism of grain size.

Functional Diversification of Sec13 Isoforms for Storage Protein Trafficking in Rice Endosperm Cells.

Coat protein complex II (COPII) vesicles play crucial roles in mediating the endoplasmic reticulum (ER) exit of newly synthesized proteins to the Golgi in eukaryotic cells. However, the molecular functions of COPII components and their functional diversifications in plant seeds remain obscure. Here, we showed that the rice (Oryza sativa) glutelin precursor accumulation12 (gpa12) mutant is defective in storage protein export from the ER, resulting in the formation of aggregated protein bodies. Map-based cloning revealed that GPA12 encodes a COPII outer layer protein, Sec13a, that mainly localizes to endoplasmic reticulum exit sites (ERES) and partially localizes to the Golgi. Biochemical experiments verified that Sec13a physically interacts with Sec31 and Sec16, and mutation in Sec13 compromises its interaction with Sec31 and Sec16, thereby affecting the membrane association of the inner complex components Sar1b and Sec23c. Apart from Sec13a, the rice genome encodes two other Sec13 isoforms, Sec13b and Sec13c. Notably, we observed an abnormal accumulation of globular ER structures in the sec13bc double mutant but not in the single mutants, suggesting a functional redundancy of Sec13b and Sec13c in modulating ER morphology. Taken together, our results substantiated that Sec13a plays an important role in regulating storage protein export from the ER, while Sec13b and Sec13c are required for maintaining ER morphology in rice endosperm cells. Our findings provide insights into the functional diversification of COPII components in plants.

Auxin regulates source-sink carbohydrate partitioning and reproductive organ development in rice.

Carbohydrate partitioning between the source and sink tissues plays an important role in regulating plant growth and development. However, the molecular mechanisms regulating this process remain poorly understood. In this study, we show that elevated auxin levels in the rice dao mutant cause increased accumulation of sucrose in the photosynthetic leaves but reduced sucrose content in the reproductive organs (particularly in the lodicules, anthers, and ovaries), leading to closed spikelets, indehiscent anthers, and parthenocarpic seeds. RNA sequencing analysis revealed that the expression of AUXIN RESPONSE FACTOR 18 (OsARF18) and OsARF2 is significantly up- and down-regulated, respectively, in the lodicule of dao mutant. Overexpression of OsARF18 or knocking out of OsARF2 phenocopies the dao mutant. We demonstrate that OsARF2 regulates the expression of OsSUT1 through direct binding to the sugar-responsive elements (SuREs) in the OsSUT1 promoter and that OsARF18 represses the expression of OsARF2 and OsSUT1 via direct binding to the auxin-responsive element (AuxRE) or SuRE in their promoters, respectively. Furthermore, overexpression of OsSUT1 in the dao and Osarf2 mutant backgrounds could largely rescue the spikelets' opening and seed-setting defects. Collectively, our results reveal an auxin signaling cascade regulating source-sink carbohydrate partitioning and reproductive organ development in rice.

Safety, efficacy, and survival outcomes of immune checkpoint inhibitors rechallenge in patients with cancer: a systematic review and meta-analysis.

BACKGROUNDS: There is little evidence on the safety, efficacy, and survival benefit of restarting immune checkpoint inhibitors (ICI) in patients with cancer after discontinuation due to immune-related adverse events (irAEs) or progressive disease (PD). Here, we performed a meta-analysis to elucidate the possible benefits of ICI rechallenge in patients with cancer. METHODS: Systematic searches were conducted using PubMed, Embase, and Cochrane Library databases. The objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and incidence of irAEs were the outcomes of interest. RESULTS: Thirty-six studies involving 2026 patients were analyzed. ICI rechallenge was associated with a lower incidence of all-grade (OR, 0.05; 95%CI, 0.02-0.13, P < .05) and high-grade irAEs (OR, 0.37; 95%CI, 0.21-0.64, P < .05) when compared with initial ICI treatment. Though no significant difference was observed between rechallenge and initial treatment regarding ORR (OR, 0.69; 95%CI, 0.39-1.20, P = .29) and DCR (OR, 0.85; 95%CI, 0.51-1.40, P = 0.52), patients receiving rechallenge had improved PFS (HR, 0.56; 95%CI, 0.43-0.73, P < .05) and OS (HR, 0.55; 95%CI, 0.43-0.72, P < .05) than those who discontinued ICI therapy permanently. Subgroup analysis revealed that for patients who stopped initial ICI treatment because of irAEs, rechallenge showed similar safety and efficacy with initial treatment, while for patients who discontinued ICI treatment due to PD, rechallenge caused a significant increase in the incidence of high-grade irAEs (OR, 4.97; 95%CI, 1.98-12.5, P < .05) and a decrease in ORR (OR, 0.48; 95%CI, 0.24-0.95, P < .05). CONCLUSION: ICI rechallenge is generally an active and feasible strategy that is associated with relative safety, similar efficacy, and improved survival outcomes. Rechallenge should be considered individually with circumspection, and randomized controlled trials are required to confirm these findings.

Fine-tuning rice heading date through multiplex editing of the regulatory regions of key genes by CRISPR-Cas9.

Heading date (or flowering time) is a key agronomic trait that affects seasonal and regional adaption of rice cultivars. An unoptimized heading date can either not achieve a high yield or has a high risk of encountering abiotic stresses. There is a strong demand on the mild to moderate adjusting the heading date in breeding practice. Genome editing is a promising method which allows more precise and faster changing the heading date of rice. However, direct knock out of major genes involved in regulating heading date will not always achieve a new germplasm with expected heading date. It is still challenging to quantitatively adjust the heading date of elite cultivars with best adaption for broader region. In this study, we used a CRISPR-Cas9 based genome editing strategy called high-efficiency multiplex promoter-targeting (HMP) to generate novel alleles at cis-regulatory regions of three major heading date genes: Hd1, Ghd7 and DTH8. We achieved a series of germplasm with quantitative variations of heading date by editing promoter regions and adjusting the expression levels of these genes. We performed field trials to screen for the best adapted lines for different regions. We successfully expanded an elite cultivar Ningjing8 (NJ8) to a higher latitude region by selecting a line with a mild early heading phenotype that escaped from cold stress and achieved high yield potential. Our study demonstrates that HMP is a powerful tool for quantitatively regulating rice heading date and expanding elite cultivars to broader regions.

Prolongation of seed viability and grain quality in rice by editing OsLOX1 using CRISPR/Cas9.

Deterioration of rice (Oryza sativa L.) affects grain quality and seed viability during storage. Lipoxygenase (LOX), a key enzyme in lipid metabolism, directly affects the rate of ageing. Here, we found that knock-out of lipoxygenase gene OsLOX1 by CRISPR/Cas9 delayed loss of seed viability and quality. Transcriptome analysis showed that during storage, OsLOX1 affected transcription of multiple genes, including genes related to lipid metabolism and antioxidant pathways such as phosphatase and acetaldehyde dehydrogenase, which may regulate the seed storability. The genes significantly down- and up-regulated only in Ningjing 4 after NA for 13 months and 3 days of AA suggesting that OsLOX1 likely promoted seed viability in rice by balancing ageing and storage related genes, and regulated the seed storability through the amino acid synthesis and metabolic pathways. Moreover, knock-out of OsLOX1 without CRISPR/Cas9 not only improved the seed viability, but also had little impact on agronomic traits. More importantly, the OsLOX1 knock-out lines were approved in 2019 (Agricultural Foundation of China Report No. 770). Collectively, our study showed that knock-out of OsLOX1 is beneficial for prolongation of seed viability and can be directly applied to agricultural production. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01506-4.

The association between remnant cholesterol and bone mineral density in US adults: the National Health and Nutrition Examination Survey (NHANES) 2013-2018.

BACKGROUND: Previous evidence showed a possible link of dyslipidemia with bone health. Nevertheless, the correlation of remnant cholesterol (RC) with bone mineral density (BMD) has yet to be well investigated. This study investigated the association of RC with total spine BMD in general Americans. METHODS: This study explored the relationship of RC with total spine BMD in subjects aged ≥ 20 years from the National Health and Nutrition Examination Survey (NHANES) 2013-2018. After adjusting for covariates, multivariate linear regression and stratified analyses were conducted to determine the correlation of serum RC with total spine BMD in adult Americans. Restricted cubic spline (RCS) was applied to examine the nonlinear association of serum RC with total spine BMD. RESULTS: This study included 3815 individuals ≥ 20 years old, 1905 (49.93%) of whom were men and 1910 (50.07%) of whom were women. After adjusting for all covariates, the results showed a negative relationship of serum RC with total spine BMD (ß= -0.024, 95% CI: -0.039, -0.010). The interaction tests of age, sex, race, and BMI showed no statistically significant effects on the association. The RCS also indicated a negative linear correlation of serum RC with total spine BMD (nonlinear P = 0.068, overall P < 0.001). Moreover, RC had a stronger effect on total spine BMD than total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). CONCLUSIONS: This study found that serum RC was negatively related to total spine BMD in U.S. adults. These findings emphasized the important role of RC in bone health in American adults.

Neural basis of opioid-induced respiratory depression and its rescue.

Opioid-induced respiratory depression (OIRD) causes death following an opioid overdose, yet the neurobiological mechanisms of this process are not well understood. Here, we show that neurons within the lateral parabrachial nucleus that express the µ-opioid receptor (PBL Oprm1 neurons) are involved in OIRD pathogenesis. PBL Oprm1 neuronal activity is tightly correlated with respiratory rate, and this correlation is abolished following morphine injection. Chemogenetic inactivation of PBL Oprm1 neurons mimics OIRD in mice, whereas their chemogenetic activation following morphine injection rescues respiratory rhythms to baseline levels. We identified several excitatory G protein-coupled receptors expressed by PBL Oprm1 neurons and show that agonists for these receptors restore breathing rates in mice experiencing OIRD. Thus, PBL Oprm1 neurons are critical for OIRD pathogenesis, providing a promising therapeutic target for treating OIRD in patients.

Precise Lipidomics Decipher Circulating Ceramide and Sphingomyelin Cycle Associated with the Progression of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a long-term autoimmune condition that causes joint and surrounding tissue inflammation. Lipid mediators are involved in inflammation and deterioration of the joints. Despite attempts to discover effective drug targets to intervene with lipid metabolism in the disease, progress has been limited. In this study, precise lipidomic technology was employed to quantify a broad range of serum ceramides and sphingomyelin (SM) in a large cohort, revealing an association between the accumulation of circulating ceramides and disturbed ceramide/SM cycles during the progression of RA. In our investigation, we discovered that eight ceramides exhibited a positive correlation with the activity of RA, thereby enhancing the accuracy of RA diagnosis, particularly in patients with serum antibody-negative RA. Furthermore, the enzyme SM phosphodiesterase 3 (SMPD3) was found to disrupt the circulating SM cycle and accelerate the progression of RA. The activity of SMPD3 can be inhibited by methotrexate, resulting in decreased metabolic conversion of SM to ceramide. These findings suggest that targeting the SM cycle may provide a new therapeutic option for RA.

Directed evolution rice genes with randomly multiplexed sgRNAs assembly of base editors.

CRISPR-based directed evolution is an effective breeding biotechnology to improve agronomic traits in plants. However, its gene diversification is still limited using individual single guide RNA. We described here a multiplexed orthogonal base editor (MoBE), and a randomly multiplexed sgRNAs assembly strategy to maximize gene diversification. MoBE could induce efficiently orthogonal ABE (<36.6%), CBE (<36.0%), and A&CBE (<37.6%) on different targets, while the sgRNA assembling strategy randomized base editing events on various targets. With respective 130 and 84 targets from each strand of the 34th exon of rice acetyl-coenzyme A carboxylase (OsACC), we observed the target-scaffold combination types up to 27 294 in randomly dual and randomly triple sgRNA libraries. We further performed directed evolution of OsACC using MoBE and randomly dual sgRNA libraries in rice, and obtained single or linked mutations of stronger herbicide resistance. These strategies are useful for in situ directed evolution of functional genes and may accelerate trait improvement in rice.

Hsa_circ_0052611 and mir-767-5p guide the warburg effect, migration, and invasion of BRCA cells through modulating SCAI.

Noncoding RNAs are key regulators in the Warburg Effect, an emerging hallmark of cancer. We intended to investigate the role and mechanism of circular RNA hsa_circ_0052611 (circ_0052611) and microRNA (miR)-767-5p in breast cancer (BRCA) hallmarks, especially the Warburg Effect. Expression of circ_0052611 and SCAI was downregulated, and miR-767-5p was upregulated in human BRCA tissues and cells; moreover, circ_0052611 acted as a miR-767-5p sponge to modulate the expression of miR-767-5p-targeted SCAI. Functionally, re-expressing circ_0052611 suppressed migration, invasion, glucose uptake, lactate production, and extracellular acidification rate (ECAR) in BRCA cells, and promoted apoptotic rate. These effects were accompanied by decreased Vimentin, N-cadherin, Bcl-2, and LDHA, and increased E-cadherin and Bax. Consistently, exhausting miR-767-5p exerted similar effects in BRCA cells. High miR-767-5p could counteract the role of circ_0052611 overexpression, and low SCAI likewise blocked the role of miR-767-5p deletion. In vivo, upregulating circ_0052611 delayed tumor growth of BRCA cells by altering miR-767-5p and SCAI expression. circ_0052611/miR-767-5p/SCAI axis might boycott the malignancy of BRCA cells.

Amino acids-targeted metabolomics reveals novel diagnostic biomarkers for ulcerative colitis and Crohn's disease.

Inflammatory bowel disease (IBD), which mainly comprises ulcerative colitis (UC) and Crohn's disease (CD), is a common chronic intestinal inflammatory disease that affects the ileum, rectum, and colon. Currently, the diagnosis of IBD is based on clinical history, physical examination and complementary diagnostic tests. It is challenging for physicians to make a definitive diagnosis. This study aimed to analyze the variation in amino acid metabolites in IBD serum and to identify potential predictive biomarkers of IBD diagnosis and progression. Serum samples were collected from 158 UC patients, 130 CD patients and 138 healthy controls (HCs). The 37 amino acids in serum were determined by ultra-high-pressure liquid chromatography coupled to a mass spectrometer. A panel of three-amino-acid metabolites (taurine, homocitrulline and kynurenine) was identified as a specific biomarker panel of IBD. Receiver operating characteristic analysis (ROC) showed that the panel had a sensitivity of 88.4% with a specificity of 84.6% for discriminating CD patients from UC patients. The biomarkers identified are increased in CD compared to UC. Our approach demonstrated a strong relationship between serum amino acid levels and IBD. We successfully identified serum amino acid biomarkers associated with CD and UC. The biomarker panel has potential in clinical practice for IBD diagnosis and will provide new insights into IBD pathogenesis.

An improved oral vaccine with molecular adjuvant ß-defensin protects grouper against nervous necrosis virus infection.

Nervous necrosis virus (NNV) is one of the most important fish viral pathogens infecting more than 120 fish species worldwide. Due to the mass mortality rates often seen among larvae and juveniles, few effective vaccines against NNV were developed up to now. Here, the protective effect of recombinant coat protein (CP) from red-spotted grouper nervous necrosis virus (RGNNV) fused with grouper ß-defensin (DEFB) as an oral vaccine was evaluated using Artemia as a biocarrier delivery system in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀). Feeding with Artemia encapsulated with E. coli expressing control vector (control group), CP, or CP-DEFB showed no obvious side effects on the growth of groupers. ELISA and antibody neutralization assay showed that CP-DEFB oral vaccination group induced higher anti-RGNNV CP specific antibodies and exhibited higher neutralization potency than the CP and control group. Meanwhile, the expression levels of several immune and inflammatory factors in the spleen and kidney after feeding with CP-DEFB were also significantly increased compared with the CP group. Consistently, after challenge with RGNNV, groupers fed CP-DEFB and CP exhibited 100% and 88.23% relative percentage survival (RPS), respectively. Moreover, the lower transcription levels of viral genes and milder pathological changes in CP-DEFB group were detected compared with the CP and control group. Thus, we proposed that grouper ß-defensin functioned as an efficient molecular adjuvant for an improved oral vaccine against nervous necrosis virus infection.

Surface display of major capsid protein on Bacillus subtilis spores against largemouth bass virus (LMBV) for oral administration.

Largemouth bass virus (LMBV) infections resulting in enormous loss are becoming an increasing problem in the largemouth bass industry. Oral vaccination is considered to be an effective and economical measure because of the advantages of non-invasion, no size limitation, lower cost and easily-operated. Based on Bacillus subtilis (B. subtilis) spores, this study successfully constructed the CotC-LMBV recombinant B. subtilis spores and its protective efficacy and immune responses were evaluated. After challenged, the survival rate of largemouth bass orally vaccinated with CotC-LMBV spores was 53.3% and the relative percent survival (RPS) was 45.0% compared to the PBS group. In addition, the specific IgM level in serum in the CotC-LMBV group was significantly higher than in the control groups. In the spleen, the immune-related genes expression detected by quantitative real-time PCR (qRT-PCR) exhibited an increasing trend in different degrees in the CotC-LMBV group, suggesting that innate and adaptive immune responses were activated. This study indicated that oral administration of CotC-LMBV recombinant spores could stimulate an effective immune response and enhance fish immunity against LMBV infection. Therefore, oral vaccination could be an effective approach for the prevention of largemouth bass virus disease.

Oral immunizations with Bacillus subtilis spores displaying VP19 protein provide protection against Singapore grouper iridovirus (SGIV) infection in grouper.

Disease caused by Singapore grouper iridovirus (SGIV) results in major economic losses in the global grouper aquaculture industry. Vaccination is considered to be the most effective way to protect grouper from SGIV. In this study, the spores of Bacillus subtilis (B.subtilis) WB600 were utilized as the vehicle that the VP19 protein was displayed on the spores surface. To further investigate the effect of oral vaccination, the grouper were orally immunized with B.s-CotC-19 spores. After challenged, the survival rate of grouper orally vaccinated with B.s-CotC-19 spores was 34.5% and the relative percent survival (RPS) was 28.7% compared to the PBS group. Moreover, the viral load in the tissues of the B.s-CotC-19 group was significantly lower than that of the PBS group. The histopathological sections of head kidney and liver tissue from the B.s-CotC-19 group showed significantly less histopathology compared to the PBS group. In addition, the specific IgM levels in serum in the B.s-CotC-19 group was higher than those in the PBS group. In the hindgut tissue, the immune-related gene expression detected by quantitative real-time PCR (qRT-PCR) exhibited an increasing trend in different degrees in the B.s-CotC-19 group, suggesting that the innate and adaptive immune responses were activated. These results indicated that the oral administration of recombinant B.subtilis spores was effective for preventing SGIV infection. This study provided a feasible strategy for the controlling of fish virus diseases.

Rapid, sensitive, and high-throughput quantification of broad serological ceramides by using isotope dilution liquid chromatography-negative ion electrospray tandem mass spectrometry.

Ceramides are important intermediates in the metabolism of sphingolipids. High-throughput liquid chromatography-mass spectrometry has been used extensively for monitoring the levels of serological ceramides, but is still limited by inadequate coverage or lack of sensitivity. Herein, a rapid, sensitive, and high-throughput isotope dilution liquid chromatography-negative ion electrospray tandem mass spectrometry (IDLC-nESI-MS/MS) method was developed and verified for accurate quantification of 41 ceramides, involving ceramides with C16-20 sphingosine, dihydro-ceramide, and dehydro-ceramide. This method was validated with excellent linearity (R2 > 0.99) and good recovery in the range of 90-110%. Intra- and inter-day imprecision were below 5.57% and 7.83% respectively. The improved high-throughput quantitative method developed in this study may aid in the accurate characterization of ceramides for understanding ceramide biology and application in disease diagnosis.

Young Leaf White Stripe encodes a P-type PPR protein required for chloroplast development.

Pentatricopeptide repeat (PPR) proteins function in post-transcriptional regulation of organellar gene expression. Although several PPR proteins are known to function in chloroplast development in rice (Oryza sativa), the detailed molecular functions of many PPR proteins remain unclear. Here, we characterized a rice young leaf white stripe (ylws) mutant, which has defective chloroplast development during early seedling growth. Map-based cloning revealed that YLWS encodes a novel P-type chloroplast-targeted PPR protein with 11 PPR motifs. Further expression analyses showed that many nuclear- and plastid-encoded genes in the ylws mutant were significantly changed at the RNA and protein levels. The ylws mutant was impaired in chloroplast ribosome biogenesis and chloroplast development under low-temperature conditions. The ylws mutation causes defects in the splicing of atpF, ndhA, rpl2, and rps12, and editing of ndhA, ndhB, and rps14 transcripts. YLWS directly binds to specific sites in the atpF, ndhA, and rpl2 pre-mRNAs. Our results suggest that YLWS participates in chloroplast RNA group II intron splicing and plays an important role in chloroplast development during early leaf development.

Plastidic pyruvate dehydrogenase complex E1 component subunit Alpha1 is involved in galactolipid biosynthesis required for amyloplast development in rice.

Starch accounts for over 80% of the total dry weight in cereal endosperm and determines the kernel texture and nutritional quality. Amyloplasts, terminally differentiated plastids, are responsible for starch biosynthesis and storage. We screened a series of rice mutants with floury endosperm to clarify the mechanism underlying amyloplast development and starch synthesis. We identified the floury endosperm19 (flo19) mutant which shows opaque of the interior endosperm. Abnormal compound starch grains (SGs) were present in the endosperm cells of the mutant. Molecular cloning revealed that the FLO19 allele encodes a plastid-localized pyruvate dehydrogenase complex E1 component subunit α1 (ptPDC-E1-α1) that is expressed in all rice tissues. In vivo enzyme assays demonstrated that the flo19 mutant showed decreased activity of the plastidic pyruvate dehydrogenase complex. In addition, the amounts of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were much lower in the developing flo19 mutant endosperm, suggesting that FLO19 participates in fatty acid supply for galactolipid biosynthesis in amyloplasts. FLO19 overexpression significantly increased seed size and weight, but did not affect other important agronomic traits, such as panicle length, tiller number and seed setting rate. An analysis of single nucleotide polymorphism data from a panel of rice accessions identified that the pFLO19L haplotype was positively associated with grain length, implying a potential application in rice breeding. In summary, our study demonstrates that FLO19 is involved in galactolipid biosynthesis which is essential for amyloplast development and starch biosynthesis in rice.