A genome-wide survey of copy number variations reveals an asymmetric evolution of duplicated genes in rice.

BACKGROUND: Copy number variations (CNVs) are an important type of structural variations in the genome that usually affect gene expression levels by gene dosage effect. Understanding CNVs as part of genome evolution may provide insights into the genetic basis of important agricultural traits and contribute to the crop breeding in the future. While available methods to detect CNVs utilizing next-generation sequencing technology have helped shed light on prevalence and effects of CNVs, the complexity of crop genomes poses a major challenge and requires development of additional tools. RESULTS: Here, we generated genomic and transcriptomic data of 93 rice (Oryza sativa L.) accessions and developed a comprehensive pipeline to call CNVs in this large-scale dataset. We analyzed the correlation between CNVs and gene expression levels and found that approximately 13% of the identified genes showed a significant correlation between their expression levels and copy numbers. Further analysis showed that about 36% of duplicate pairs were involved in pseudogenetic events while only 5% of them showed functional differentiation. Moreover, the offspring copy mainly contributed to the expression levels and seemed more likely to become a pseudogene, whereas the parent copy tended to maintain the function of ancestral gene. CONCLUSION: We provide a high-accuracy CNV dataset that will contribute to functional genomics studies and molecular breeding in rice. We also showed that gene dosage effect of CNVs in rice is not exponential or linear. Our work demonstrates that the evolution of duplicated genes is asymmetric in both expression levels and gene fates, shedding a new insight into the evolution of duplicated genes.

Developing disease-resistant thermosensitive male sterile rice by multiplex gene editing.

High-quality and disease-resistant male sterile lines have great potential for applications in hybrid rice breeding. We introduced specific mutations into the TMS5, Pi21, and Xa13 genes in Pinzhan intermediate breeding material using the CRISPR/Cas9 multiplex genome editing system. We found that the transgene-free homozygous triple tms5/pi21/xa13 mutants obtained in the T1 generation displayed characteristics of thermosensitive genic male sterility (TGMS) with enhanced resistance to rice blast and bacterial blight. Our study provides a convenient and effective way of converting breeding intermediate material into TGMS lines through multiplex gene editing, which could significantly accelerate the breeding of sterile lines.

OsPK2 encodes a plastidic pyruvate kinase involved in rice endosperm starch synthesis, compound granule formation and grain filling.

Starch is the main form of energy storage in higher plants. Although several enzymes and regulators of starch biosynthesis have been defined, the complete molecular machinery remains largely unknown. Screening for irregularities in endosperm formation in rice represents valuable prospect for studying starch synthesis pathway. Here, we identified a novel rice white-core endosperm and defective grain filling mutant, ospk2, which displays significantly lower grain weight, decreased starch content and alteration of starch physicochemical properties when compared to wild-type grains. The normal starch compound granules were drastically reduced and more single granules filled the endosperm cells of ospk2. Meanwhile, the germination rate of ospk2 seeds after 1-year storage was observably reduced compared with wild-type. Map-based cloning of OsPK2 indicated that it encodes a pyruvate kinase (PK, ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40), which catalyses an irreversible step of glycolysis. OsPK2 has a constitutive expression in rice and its protein localizes in chloroplasts. Enzyme assay showed that the protein product from expressed OsPK2 and the crude protein extracted from tissues of wild-type exhibits strong PK activity; however, the mutant presented reduced protein activity. OsPK2 (PKpα1) and three other putative rice plastidic isozymes, PKpα2, PKpß1 and PKpß2, can interact to form heteromer. Moreover, the mutation leads to multiple metabolic disorders. Altogether, these results denote new insights into the role of OsPK2 in plant seed development, especially in starch synthesis, compound granules formation and grain filling, which would be useful for genetic improvement of high yield and rice grain quality.

The Phosphoproteomic Response of Rice Seedlings to Cadmium Stress.

The environmental damage caused by cadmium (Cd) pollution is of increasing concern in China. While the overall plant response to Cd has been investigated in some depth, the contribution (if any) of protein phosphorylation to the detoxification of Cd and the expression of tolerance is uncertain. Here, the molecular basis of the plant response has been explored in hydroponically raised rice seedlings exposed to 10 µΜ and 100 µΜ Cd2+ stress. An analysis of the seedlings' quantitative phosphoproteome identified 2454 phosphosites, associated with 1244 proteins. A total of 482 of these proteins became differentially phosphorylated as a result of exposure to Cd stress; the number of proteins affected in this way was six times greater in the 100 µΜ Cd2+ treatment than in the 10 µΜ treatment. A functional analysis of the differentially phosphorylated proteins implied that a significant number was involved in signaling, in stress tolerance and in the neutralization of reactive oxygen species, while there was also a marked representation of transcription factors.

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) regulates carbon metabolism during grain filling in rice.

KEY MESSAGE: Decreased PFPase activity in rice perturbs the equilibration of carbon metabolism during grain filling but has no visible phenotypic effects during the vegetative and reproductive growth stages. Starch is a primary energy reserve for various metabolic processes in plant. Despite much advance has been achieved in pathways involved in starch biosynthesis, information was still lacked for precise regulation related to carbon metabolism during seed filling in rice (Oryza sativa). The objective of this study was to identify and characterize new gene associated with carbon metabolism during grain filling. By screening our chemical mutant pool, two allelic mutants exhibiting floury endosperm were isolated. No visible phenotypic defects were observed during both the vegetative and reproductive growth stages, except for the floury-like endosperm of grains with significantly reduced kernel thickness, 1000-grain weight and total starch content. Map-based cloning revealed that the mutant phenotypes were controlled by a gene encoding pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) ß subunit (PFPß), which catalyzes reversible interconversion between fructose-6-phosphate and fructose-1, 6-bisphosphate. The identity of PFP ß was further confirmed by a genetic complementation test. Subcellular analysis demonstrated that PFPß was localized in cytoplasm. Quantitative PCR and histochemical staining indicated PFP ß was ubiquitously expressed in various tissues. Furthermore, we found PFP ß could express in both the early and late phases of starch accumulation during grain filling and decreased activity of PFP ß in pfp mutants resulted in compromised carbon metabolism with increased soluble sugar contents and unfavorable starch biosynthesis. Our results highlight PFPß functions in modulating carbon metabolism during grain filling stage.

[Mapping of QTL associated with rice cooking quality and candidate gene analysis].

Excavating the quantitative trait locus (QTL) associated with rice cooking quality, analyzing candidate genes, and improving cooking quality-associated traits of rice varieties by genetic breeding can effectively improve the taste of rice. In this study, we used the indica rice HZ, the japonica rice Nekken2 and 120 recombinant inbred lines (RILs) populations constructed from them as experimental materials to measure the gelatinization temperature (GT), gel consistency (GC) and amylose content (AC) of rice at the maturity stage. We combined the high-density genetic map for QTL mapping. A total of 26 QTLs associated with rice cooking quality (1 QTL associated with GT, 13 QTLs associated with GC, and 12 QTLs associated with AC) were detected, among which the highest likelihood of odd (LOD) value reached 30.24. The expression levels of candidate genes in the localization interval were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and it was found that the expression levels of six genes were significantly different from that in parents. It was speculated that the high expression of LOC_Os04g20270 and LOC_Os11g40100 may greatly increase the GC of rice, while the high expression of LOC_Os01g04920 and LOC_Os02g17500 and the low expression of LOC_Os03g02650 and LOC_Os05g25840 may reduce the AC. The results lay a molecular foundation for the cultivation of new high-quality rice varieties, and provide important genetic resources for revealing the molecular regulation mechanism of rice cooking quality.

OsLESV and OsESV1 promote transitory and storage starch biosynthesis to determine rice grain quality and yield.

Transitory starch is an important carbon source in leaves, and its biosynthesis and metabolism are closely related to grain quality and yield. The molecular mechanisms controlling leaf transitory starch biosynthesis and degradation and their effects on rice (Oryza sativa) quality and yield remain unclear. Here, we show that OsLESV and OsESV1, the rice orthologs of AtLESV and AtESV1, are associated with transitory starch biosynthesis in rice. The total starch and amylose contents in leaves and endosperms are significantly reduced, and the final grain quality and yield are compromised in oslesv and osesv1 single and oslesv esv1 double mutants. Furthermore, we found that OsLESV and OsESV1 bind to starch, and this binding depends on a highly conserved C-terminal tryptophan-rich region that acts as a starch-binding domain. Importantly, OsLESV and OsESV1 also interact with the key enzymes of starch biosynthesis, granule-bound starch synthase I (GBSSI), GBSSII, and pyruvate orthophosphote dikiase (PPDKB), to maintain their protein stability and activity. OsLESV and OsESV1 also facilitate the targeting of GBSSI and GBSSII from plastid stroma to starch granules. Overexpression of GBSSI, GBSSII, and PPDKB can partly rescue the phenotypic defects of the oslesv and osesv1 mutants. Thus, we demonstrate that OsLESV and OsESV1 play a key role in regulating the biosynthesis of both leaf transitory starch and endosperm storage starch in rice. These findings deepen our understanding of the molecular mechanisms underlying transitory starch biosynthesis in rice leaves and reveal how the transitory starch metabolism affects rice grain quality and yield, providing useful information for the genetic improvement of rice grain quality and yield.

The Mining of Genetic Loci and the Analysis of Candidate Genes to Identify the Physical and Chemical Markers of Anti-Senescence in Rice.

Premature senescence is a common occurrence in rice production, and seriously affects rice plants' nutrient utilization and growth. A total of 120 recombinant inbred lines (RILs) were obtained from successive self-crossing of F12 generations derived from Huazhan and Nekken2. The superoxide dismutase (SOD) activity, malondialdehyde (MDA), content and catalase (CAT) activity related to the anti-senescence traits and enzyme activity index of rice were measured for QTL mapping using 4858 SNPs. Thirteen QTLs related to anti-senescence were found, among which the highest LOD score was 5.70. Eighteen anti-senescence-related genes were found in these regions, and ten of them differed significantly between the parents. It was inferred that LOC_Os01g61500, LOC_Os01g61810, and LOC_Os04g40130 became involved in the regulation of the anti-senescence molecular network upon upregulation of their expression levels. The identified anti-senescence-related QTLs and candidate genes provide a genetic basis for further research on the mechanism of the molecular network that regulates premature senescence.

WLP3 Encodes the Ribosomal Protein L18 and Regulates Chloroplast Development in Rice.

Plastid ribosomal proteins play a crucial role in the growth and development of plants, mainly in the gene expression and translation of key genes in chloroplasts. While some information is known about the regulatory processes of plastid ribosomal proteins in various plant species, there is limited knowledge about the underlying mechanisms in rice. In this study, ethyl methanesulfonate (EMS) mutagenesis was used to generate a new mutant called wlp3 (white leaf and panicle3), characterized by white or albino leaves and panicles, which exhibited this phenotype from the second leaf stage until tillering. Furthermore, after a certain period, the newly emerging leaves developed the same phenotype as the rice variety ZH11, while the albino leaves of wlp3 showed an incomplete chloroplast structure and significantly low chlorophyll content. A transition mutation (T to C) at position 380 was identified in the coding region of the LOC_Os03g61260 gene, resulting in the substitution of isoleucine by threonine during translation. WLP3 encodes the ribosomal L18 subunit, which is localized in the chloroplast. Complementation experiments confirmed that LOC_Os03g61260 was responsible for the albino phenotype in rice. WLP3 has high expression in the coleoptile, leaves at the three-leaf stage, and panicles at the heading stage. Compared to the wild-type (WT), wlp3 exhibited reduced chlorophyll synthesis and significantly decreased expression levels of genes associated with plastid development. Yeast two-hybrid (Y2H) analysis revealed that WLP3 interacts with other ribosomal subunits, to influence chloroplast development. These results contribute to a better understanding of the underlying molecular mechanisms of chloroplast development and plastid gene translation.

OsSPL88 Encodes a Cullin Protein that Regulates Rice Growth and Development.

Plant lesion mimics refer to necrotic spots spontaneously produced by the plant without mechanical damage, pathogen invasion, and adversity stress. Here, we isolated and characterized two rice (Oryza sativa L) mutants, namely, spl88-1 (spotted leaf88-1) and spl88-2 (spotted leaf88-2), which were identified from an ethyl methanesulfonate-mutagenized japonica cultivar Xiushui 11 population. Physiological and biochemical experiments indicated that more ROS accumulated in spl88-1 and spl88-2 than in wild type. spl88-1 and spl88-2 displayed spontaneous cell death and enhanced their resistance to bacterial blight by affecting the expression of defense-related genes. We isolated SPL88 by map-based cloning, which encoded a highly conserved Cullin protein. A single base deletion was detected in spl88-1 and spl88-2, in which the 132nd base C of SPL88-1 and the 381th base T of SPL88-2 were deleted, causing premature termination of protein translation. SPL88 was expressed in root, stem, leaf, leaf sheath, and panicle. The Cullin protein was localized in the cytoplasm and nucleus. The aforementioned results indicate that SPL88 regulates the growth and development of rice by affecting the expression of defense-related genes.

A Strigolactone Biosynthesis Gene Contributed to the Green Revolution in Rice.

Plant architecture is a complex agronomic trait and a major factor of crop yield, which is affected by several important hormones. Strigolactones (SLs) are identified as a new class hormoneinhibiting branching in many plant species and have been shown to be involved in various developmental processes. Genetical and chemical modulation of the SL pathway is recognized as a promising approach to modify plant architecture. However, whether and how the genes involved in the SL pathway could be utilized in breeding still remain elusive. Here, we demonstrate that a partial loss-of-function allele of the SL biosynthesis gene, HIGH TILLERING AND DWARF 1/DWARF17 (HTD1/D17), which encodes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), increases tiller number and improves grain yield in rice. We found that the HTD1 gene had been widely utilized and co-selected with Semidwarf 1 (SD1), both contributing to the improvement of plant architecture in modern rice varieties since the Green Revolution in the 1960s. Understanding how phytohormone pathway genes regulate plant architecture and how they have been utilized and selected in breeding will lay the foundation for developing the rational approaches toward improving crop yield.

PE-1, Encoding Heme Oxygenase 1, Impacts Heading Date and Chloroplast Development in Rice ( Oryza sativa L.).

The duration of the rice growth phase has always been an important target trait. The identification of mutations in rice that alter these processes and result in a shorter growth phase could have potential benefits for crop production. In this study, we isolated an early aging rice mutant, pe-1, with light green leaves, using γ-mutated indica rice cultivar and subsequent screening methods, which is known as the phytochrome synthesis factor Se5 that controls rice flowering. The pe-1 plant is accompanied by a decreased chlorophyll content, an enhanced photosynthesis, and a decreased pollen fertility. PE-1, a close homologue of HY1, is localized in the chloroplast. Expression pattern analysis indicated that PE-1 was mainly expressed in roots, stems, leaves, leaf sheaths, and young panicles. The knockout of PE-1 using the CRISPR/Cas9 system decreased the chlorophyll content and downregulated the expression of PE-1-related genes. Furthermore, the chloroplasts of pe-1 were filled with many large-sized starch grains, and the number of osmiophilic granules (a chloroplast lipid reservoir) was significantly decreased. Altogether, our findings suggest that PE-1 functions as a master regulator to mediate in chlorophyll biosynthesis and photosynthetic pathways.

OsBT1 encodes an ADP-glucose transporter involved in starch synthesis and compound granule formation in rice endosperm.

Starch is the main storage carbohydrate in higher plants. Although several enzymes and regulators for starch biosynthesis have been characterized, a complete regulatory network for starch synthesis in cereal seeds remains elusive. Here, we report the identification and characterization of the rice Brittle1 (OsBT1) gene, which is expressed specifically in the developing endosperm. The osbt1 mutant showed a white-core endosperm and a significantly lower grain weight than the wild-type. The formation and development of compound starch granules in osbt1 was obviously defective: the amyloplast was disintegrated at early developmental stages and the starch granules were disperse and not compound in the endosperm cells in the centre region of osbt1 seeds. The total starch content and amylose content was decreased and the physicochemical properties of starch were altered. Moreover, the degree of polymerization (DP) of amylopectin in osbt1 was remarkably different from that of wild-type. Map-based cloning of OsBT1 indicated that it encodes a putatively ADP-glucose transporter. OsBT1 coded protein localizes in the amyloplast envelope membrane. Furthermore, the expression of starch synthesis related genes was also altered in the osbt1 mutant. These findings indicate that OsBT1 plays an important role in starch synthesis and the formation of compound starch granules.