Yunlu Tian

Association mapping and genetic dissection of nitrogen use efficiency-related traits in rice (Oryza sativa L.)

The increases in the usage of nitrogen fertilizer result in deleterious impacts on the environment; thus, there is an urgent need to improve nitrogen use efficiency (NUE) in crops including rice (Oryza sativa L.). Attentions have focused on quantitative trait loci (QTL) mapping of NUE-related traits using single experimental population, but to date, very few studies have taken advantage of association mapping to examine hundreds of lines for identifying potentially novel QTLs in rice. Here, we conducted association analysis on NUE-related traits using a population containing 184 varieties, which were genotyped with 157 genome-wide simple sequence repeat (SSR) markers. We detected eight statistically significant marker loci associating with NUE-related traits, of which two QTLs at RM5639 and RM3628 harbored known NUE-related genes GS1;2 and AspAt3, respectively. At a novel NUE-related locus RM5748, we developed Kompetitive Allele Specific PCR (KASP) single nucleotide polymorphism (SNP) markers and searched for putative NUE-related genes which are close to the associated SNP marker. Based on a transcriptional map of N stress responses constructed by our lab, we evaluated expressions of the NUE-related genes in this region and validated their effect on NUE. Meanwhile, we analyzed NUE-related alleles of the eight loci that could be utilized in marker-assisted selection. Moreover, we estimated breeding values of all the varieties through genomic prediction approach that could be beneficial for rice NUE enhancement.

TSV, a putative plastidic oxidoreductase, protects rice chloroplasts from cold stress during development by interacting with plastidic thioredoxin Z

Rice is vulnerable to cold stress. Seedlings are very sensitive to cold stress and this harms global rice production. The effects of cold on chloroplast development are well known, but little is known about the underlying molecular mechanisms. Here, we isolated a temperature-sensitive virescent (tsv) mutant that is extremely sensitive to cold stress. It displayed defective chloroplasts, decreased chlorophyll and zero survivorship under cold stress. We isolated and identified TSV by map-based cloning and rescue experiments, combined with genetic, cytological and molecular biological analyses. We found that TSV, a putative plastidic oxidoreductase, is a new type of virescent protein. A mutation in tsv causes premature termination of the gene product. The activity of plastid-encoded RNA polymerase (PEP) and the expression of genes participating in chlorophyll synthesis were severely reduced in the tsv mutant under cold stress, but not at normal temperatures. TSV expression was induced by low temperatures. Strikingly, TSV interacted with OsTrxZ (a subunit of PEP in chloroplasts) and enhanced OsTrxZ stability under low temperatures. We demonstrated that TSV protects rice chloroplasts from cold stress by interacting with OsTrxZ. These results provide novel insights into ways in which rice chloroplast development and chlorophyll synthesis are protected by TSV under cold stress.

OsPPR6, a pentatricopeptide repeat protein involved in editing and splicing chloroplast RNA, is required for chloroplast biogenesis in rice

Key messageOsPPR6, a pentatricopeptide repeat protein involved in editing and splicing chloroplast RNA, is required for chloroplast biogenesis in rice.AbstractThe chloroplast has its own genetic material and genetic system, but it is also regulated by nuclear-encoded genes. However, little is known about nuclear-plastid regulatory mechanisms underlying early chloroplast biogenesis in rice. In this study, we isolated and characterized a mutant, osppr6, that showed early chloroplast developmental defects leading to albino leaves and seedling death. We found that the osppr6 mutant failed to form thylakoid membranes. Using map-based cloning and complementation tests, we determined that OsPPR6 encoded a new Pentatricopeptide Repeat (PPR) protein localized in plastids. In the osppr6 mutants, mRNA levels of plastidic genes transcribed by the plastid-encoded RNA polymerase decreased, while those of genes transcribed by the nuclear-encoded RNA polymerase increased. Western blot analyses validated these expression results. We further investigated plastidic RNA editing and splicing in the osppr6 mutants and found that the ndhB transcript was mis-edited and the ycf3 transcript was mis-spliced. Therefore, we demonstrate that OsPPR6, a PPR protein, regulates early chloroplast biogenesis and participates in editing of ndhB and splicing of ycf3 transcripts in rice.

FLOURY ENDOSPERM8 , encoding the UDP-glucose pyrophosphorylase 1, affects the synthesis and structure of starch in rice endosperm

Cereal opaque-kernel mutants are ideal genetic materials for studying the mechanism of starch biosynthesis and amyloplast development. Here we isolated and identified two allelic floury endosperm 8 (flo8) mutants of rice, named flo8-1 and flo8-2. In the flo8 mutant, the starch content was decreased and the normal physicochemical features of starch were altered. Map-based cloning and subsequent DNA sequencing analysis revealed a single nucleotide substitution and an 8-bp insertion occurred in UDP-glucose pyrophosphorylase 1 (Ugp1) gene in flo8-1 and flo8-2, respectively. Complementation of the flo8-1 mutant restored normal seed appearance by expressing full length coding sequence of Ugp1. RT-qPCR analysis revealed that Ugp1 was ubiquitously expressed. Mutation caused the decreased UGPase activity and affected the expression of most of genes associated with starch biosynthesis. Meanwhile, western blot and enzyme activity analyses showed the comparability of protein levels and enzyme activity of most tested starch biosynthesis related genes. Our results demonstrate that Ugp1 plays an important role for starch biosynthesis in rice endosperm.

Genetic dissection of leaf-related traits using 156 chromosomal segment substitution lines

A two-line super-hybrid rice (Oryza sativa L.) variety [Liangyoupei9 (LYP9)] demonstrated superiority over its both parents, viz. elite inbred lines 93-11 and Pei-ai64S (PA64S), as well as other conventional hybrids, and had long been exploited in China. However, the genetic basis of its leaf-related traits, supposed to be an important component for yield potential, remains elusive. Here, initially a set of chromosome segment substitution lines (CSSLs) was constructed, in which the genome of Pei-ai64S has been introgressed into the background of 93-11. This set was developed by marker aided selection, based on 123 polymorphic SSR markers. The introgressed chromosomal segments presented in the 156 CSSLs covered 96.46% of Pei-ai64S genome. Afterwards, the CSSLs were deployed to assess the genetic basis of leaf size (length and width) and chlorophyll content of top three leaves across five different environments. The CSSLs showed transgressive segregation for all of the traits, and significant correlations were detected among most of the traits. A total of 27 quantitative trait loci (QTL) were identified on ten chromosomes, and three QTL cluster affecting related traits were found on chromosome 3, 6, and 8, respectively. Remarkably, two key QTLs, qALW3-1 and qALW3-2, both controlling the antepenultimate leaf width, were identified in all five environments, and their effect were further validated by CSSLs harboring the two QTL alleles. Our results indicate that developing CSSLs is a powerful tool for genetic dissection of quantitative traits. Meanwhile, the QTLs controlling leaf-related traits uncovered here provide useful information for marker-assisted selection in improving the performance of leaf morphology and photosynthetic ability.

A selfish genetic element confers non-Mendelian inheritance in rice

Sterility in rice via toxin and antidote Crossing wild and domestic rice often results in hybrid sterility. Such genetic barriers can prevent the movement of potentially beneficial genes from wild rice into domestic varieties. To understand the barriers preventing gene flow, Yu et al. mapped a quantitative trait locus (QTL) that determines sterility between wild-type and domestic rice. This QTL encodes two open reading frames (ORFs) that are both expressed during gametogenesis. The ORFs encode a toxin, which affects the development of pollen, and an antidote, which is required for pollen viability. Thus, selfish genetic elements can underlie evolutionary strategies that facilitate reproductive isolation. Science, this issue p. 1130 A toxin-antidote system with a role in postzygotic reproductive isolation of different wild and cultivated rice species is described. Selfish genetic elements are pervasive in eukaryote genomes, but their role remains controversial. We show that qHMS7, a major quantitative genetic locus for hybrid male sterility between wild rice (Oryza meridionalis) and Asian cultivated rice (O. sativa), contains two tightly linked genes [Open Reading Frame 2 (ORF2) and ORF3]. ORF2 encodes a toxic genetic element that aborts pollen in a sporophytic manner, whereas ORF3 encodes an antidote that protects pollen in a gametophytic manner. Pollens lacking ORF3 are selectively eliminated, leading to segregation distortion in the progeny. Analysis of the genetic sequence suggests that ORF3 arose first, followed by gradual functionalization of ORF2. Furthermore, this toxin-antidote system may have promoted the differentiation and/or maintained the genome stability of wild and cultivated rice.

FLOURY SHRUNKEN ENDOSPERM1 Connects Phospholipid Metabolism and Amyloplast Development in Rice

The FLOURY SHRUNKEN ENDOSPERM1 gene, encoding a phospholipase-like protein, plays an important role in phospholipid metabolism and amyloplast development in rice endosperm. Starch synthesized and stored in amyloplasts serves as the major energy storage molecule in cereal endosperm. To elucidate the molecular mechanisms underlying amyloplast development and starch synthesis, we isolated a series of floury endosperm mutants in rice (Oryza sativa). We identified the rice mutant floury shrunken endosperm1 (fse1), which exhibited obvious defects in the development of compound starch grains, decreased starch content, and altered starch physicochemical features. Map-based cloning showed that FSE1 encodes a phospholipase-like protein homologous to phosphatidic acid-preferring phospholipase A1. FSE1 was expressed ubiquitously with abundant levels observed in developing seeds and roots. FSE1 was localized to both the cytosol and intracellular membranes. Lipid profiling indicated that total extra-plastidic lipids and phosphatidic acid were increased in fse1 plants, suggesting that FSE1 may exhibit in vivo phospholipase A1 activity on phosphatidylcholine, phosphatidylinositol, phosphatidyl-Ser, phosphatidylethanolamine, and, in particular, phosphatidic acid. Additionally, the total galactolipid content in developing fse1 endosperm was significantly reduced, which may cause abnormal amyloplast development. Our results identify FSE1 as a phospholipase-like protein that controls the synthesis of galactolipids in rice endosperm and provide a novel connection between lipid metabolism and starch synthesis in rice grains during endosperm development.

OsNDUFA9 encoding a mitochondrial complex I subunit is essential for embryo development and starch synthesis in rice

Key messageLoss of function of a mitochondrial complex I subunit (OsNDUFA9) causes abnormal embryo development and affects starch synthesis by altering the expression of starch synthesis-related genes and proteins.AbstractProton-pumping NADH: ubiquinone oxidoreductase (also called complex I) is thought to be the largest and most complicated enzyme of the mitochondrial respiratory chain. Mutations of complex I subunits have been revealed to link with a number of growth inhibitions in plants. However, the function of complex I subunits in rice remains unclear. Here, we isolated a rice floury endosperm mutant (named flo13) that was embryonic lethal and failed to germinate. Semi-thin sectioning analysis showed that compound starch grain development in the mutant was greatly impaired, leading to significantly compromised starch biosynthesis and decreased 1000-grain weight relative to the wild type. Map-based cloning revealed that FLO13 encodes an accessory subunit of complex I protein (designated as OsNDUFA9). A single nucleotide substitution (G18A) occurred in the first exon of OsNDUFA9, introducing a premature stop codon in the flo13 mutant gene. OsNDUFA9 was ubiquitously expressed in various tissues and the OsNDUFA9 protein was localized to the mitochondria. Quantitative RT-PCR and protein blotting indicated loss of function of OsNDUFA9 altered gene expression and protein accumulation associated with respiratory electron chain complex in the mitochondria. Moreover, transmission electron microscopic analysis showed that the mutant lacked obvious mitochondrial cristae structure in the mitochondria of endosperm cell. Our results demonstrate that the OsNDUFA9 subunit of complex I is essential for embryo development and starch synthesis in rice endosperm.

Corrigendum: WSL5, a pentatricopeptide repeat protein, is essential for chloroplast biogenesis in rice under cold stress

Xi Liu, Jie Lan, Yunshuai Huang, Penghui Cao, Chunlei Zhou, Yaken Ren, Niqing He, Shijia Liu, Yunlu Tian, Thanhliem Nguyen, Ling Jiang* and Jianmin Wan* 1 State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China 2 National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China

WSL5, a Pentatricopeptide Repeat Protein, is Essential for Chloroplast Biogenesis in Rice Under Cold Stress.

WSL5 is crucial for the splicing of the chloroplast genes rpl2 and rps12 under cold stress, and affects the retrograde signaling from plastids to the nucleus.