Shujun Ou

OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice

Significance Premature leaf senescence is known to decrease rice yield severely, but the molecular mechanism underlying this relationship remains largely unknown. Similarly, although abscisic acid (ABA)-induced leaf senescence has long been observed, the mechanism of this pathway has yet to be determined. In this study we identified and characterized a dominant premature leaf senescence mutant, prematurely senile 1 (ps1-D). The data demonstrated both that PS1/Oryza sativa NAC (no apical meristem, Arabidopsis ATAF1/2, and cup-shaped cotyledon2)-like, activated by apetala3/pistillata (OsNAP) is an ideal marker of natural senescence onset and that it functions as an important link between ABA and leaf senescence in rice. Furthermore, reduced OsNAP expression led to extended grain filling and an improved seed-setting rate, which significantly enhanced the grain yield. Thus, fine-tuning OsNAP expression should be a means of improving rice yield. It has long been established that premature leaf senescence negatively impacts the yield stability of rice, but the underlying molecular mechanism driving this relationship remains largely unknown. Here, we identified a dominant premature leaf senescence mutant, prematurely senile 1 (ps1-D). PS1 encodes a plant-specific NAC (no apical meristem, Arabidopsis ATAF1/2, and cup-shaped cotyledon2) transcriptional activator, Oryza sativa NAC-like, activated by apetala3/pistillata (OsNAP). Overexpression of OsNAP significantly promoted senescence, whereas knockdown of OsNAP produced a marked delay of senescence, confirming the role of this gene in the development of rice senescence. OsNAP expression was tightly linked with the onset of leaf senescence in an age-dependent manner. Similarly, ChIP-PCR and yeast one-hybrid assays demonstrated that OsNAP positively regulates leaf senescence by directly targeting genes related to chlorophyll degradation and nutrient transport and other genes associated with senescence, suggesting that OsNAP is an ideal marker of senescence onset in rice. Further analysis determined that OsNAP is induced specifically by abscisic acid (ABA), whereas its expression is repressed in both aba1 and aba2, two ABA biosynthetic mutants. Moreover, ABA content is reduced significantly in ps1-D mutants, indicating a feedback repression of OsNAP on ABA biosynthesis. Our data suggest that OsNAP serves as an important link between ABA and leaf senescence. Additionally, reduced OsNAP expression leads to delayed leaf senescence and an extended grain-filling period, resulting in a 6.3% and 10.3% increase in the grain yield of two independent representative RNAi lines, respectively. Thus, fine-tuning OsNAP expression should be a useful strategy for improving rice yield in the future.

Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies

Asian cultivated rice (Oryza sativa L.) consists of two main subspecies, indica and japonica. Indica has higher nitrate-absorption activity than japonica, but the molecular mechanisms underlying that activity remain elusive. Here we show that variation in a nitrate-transporter gene, NRT1.1B (OsNPF6.5), may contribute to this divergence in nitrate use. Phylogenetic analysis revealed that NRT1.1B diverges between indica and japonica. NRT1.1B-indica variation was associated with enhanced nitrate uptake and root-to-shoot transport and upregulated expression of nitrate-responsive genes. The selection signature of NRT1.1B-indica suggests that nitrate-use divergence occurred during rice domestication. Notably, field tests with near-isogenic and transgenic lines confirmed that the japonica variety carrying the NRT1.1B-indica allele had significantly improved grain yield and nitrogen-use efficiency (NUE) compared to the variety without that allele. Our results show that variation in NRT1.1B largely explains nitrate-use divergence between indica and japonica and that NRT1.1B-indica can potentially improve the NUE of japonica.

OsbZIP71, a bZIP transcription factor, confers salinity and drought tolerance in rice

The bZIP transcription factor (TF) family plays an important role in the abscisic acid (ABA) signaling pathway of abiotic stress in plants. We here report the cloning and characterization of OsbZIP71, which encodes a rice bZIP TF. Functional analysis showed that OsbZIP71 is a nuclear-localized protein that specifically binds to the G-box motif, but has no transcriptional activity both in yeast and rice protoplasts. In yeast two-hybrid assays, OsbZIP71 can form both homodimers and heterodimers with Group C members of the bZIP gene family. Expression of OsbZIP71 was strongly induced by drought, polyethylene glycol (PEG), and ABA treatments, but repressed by salt treatment. OsbZIP71 overexpressing (p35S::OsbZIP71) rice significantly improved tolerance to drought, salt and PEG osmotic stresses. In contrast, RNAi knockdown transgenic lines were much more sensitive to salt, PEG osmotic stresses, and also ABA treatment. Inducible expression (RD29A::OsbZIP71) lines were significantly improved their tolerance to PEG osmotic stresses, but hypersensitivity to salt, and insensitivity to ABA. Real-time PCR analysis revealed that the abiotic stress-related genes, OsVHA-B, OsNHX1, COR413-TM1, and OsMyb4, were up-regulated in overexpressing lines, while these same genes were down-regulated in RNAi lines. Chromatin immunoprecipitation analysis confirmed that OsbZIP71 directly binds the promoters of OsNHX1 and COR413-TM1 in vivo. These results suggest that OsbZIP71 may play an important role in ABA-mediated drought and salt tolerance in rice.

Variations in CYP78A13 coding region influence grain size and yield in rice

Grain size is one of the most important determinants of crop yield in cereals. Here, we identified a dominant mutant, big grain2 (bg2-D) from our enhancer-trapping population. Genetic analysis and SiteFinding PCR (polymerase chain reaction) revealed that BG2 encodes a cytochrome P450, OsCYP78A13. Sequence search revealed that CYP78A13 has a paralogue Grain Length 3.2 (GL3.2, LOC_Os03g30420) in rice with distinct expression patterns, analysis of transgenic plants harbouring either CYP78A13 or GL3.2 showed that both can promote grain growth. Sequence polymorphism analysis with 1529 rice varieties showed that the nucleotide diversity at CYP78A13 gene body and the 20 kb flanking region in the indica varieties were markedly higher than those in japonica varieties. Further, comparison of the genomic sequence of CYP78A13 in the japonica cultivar Nipponbare and the indica cultivar 9311 showed that there were three InDels in the promoter region and eight SNPs (single nucleotide polymorphism) in its coding sequence. Detailed examination of the transgenic plants with chimaeric constructs suggested that variation in CYP78A13 coding region is responsible for the variation of grain yield. Taken together, our results suggest that the variations in CYP78A13 in the indica varieties hold potential in rice breeding for application of grain yield improvement.

LTR_retriever: A Highly Accurate and Sensitive Program for Identification of Long Terminal Repeat Retrotransposons

LTR_retriever is an accurate and sensitive program that identifies LTR retrotransposons and generates nonredundant exemplars from DNA sequences for whole-genome annotation and evolutionary studies. Long terminal repeat retrotransposons (LTR-RTs) are prevalent in plant genomes. The identification of LTR-RTs is critical for achieving high-quality gene annotation. Based on the well-conserved structure, multiple programs were developed for the de novo identification of LTR-RTs; however, these programs are associated with low specificity and high false discovery rates. Here, we report LTR_retriever, a multithreading-empowered Perl program that identifies LTR-RTs and generates high-quality LTR libraries from genomic sequences. LTR_retriever demonstrated significant improvements by achieving high levels of sensitivity (91%), specificity (97%), accuracy (96%), and precision (90%) in rice (Oryza sativa). LTR_retriever is also compatible with long sequencing reads. With 40k self-corrected PacBio reads equivalent to 4.5× genome coverage in Arabidopsis (Arabidopsis thaliana), the constructed LTR library showed excellent sensitivity and specificity. In addition to canonical LTR-RTs with 5′-TG…CA-3′ termini, LTR_retriever also identifies noncanonical LTR-RTs (non-TGCA), which have been largely ignored in genome-wide studies. We identified seven types of noncanonical LTRs from 42 out of 50 plant genomes. The majority of noncanonical LTRs are Copia elements, with which the LTR is four times shorter than that of other Copia elements, which may be a result of their target specificity. Strikingly, non-TGCA Copia elements are often located in genic regions and preferentially insert nearby or within genes, indicating their impact on the evolution of genes and their potential as mutagenesis tools.

Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (Fragaria vesca) with chromosome-scale contiguity

Abstract Background Although draft genomes are available for most agronomically important plant species, the majority are incomplete, highly fragmented, and often riddled with assembly and scaffolding errors. These assembly issues hinder advances in tool development for functional genomics and systems biology. Findings Here we utilized a robust, cost-effective approach to produce high-quality reference genomes. We report a near-complete genome of diploid woodland strawberry (Fragaria vesca) using single-molecule real-time sequencing from Pacific Biosciences (PacBio). This assembly has a contig N50 length of ∼7.9 million base pairs (Mb), representing a ∼300-fold improvement of the previous version. The vast majority (>99.8%) of the assembly was anchored to 7 pseudomolecules using 2 sets of optical maps from Bionano Genomics. We obtained ∼24.96 Mb of sequence not present in the previous version of the F. vesca genome and produced an improved annotation that includes 1496 new genes. Comparative syntenic analyses uncovered numerous, large-scale scaffolding errors present in each chromosome in the previously published version of the F. vesca genome. Conclusions Our results highlight the need to improve existing short-read based reference genomes. Furthermore, we demonstrate how genome quality impacts commonly used analyses for addressing both fundamental and applied biological questions.

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice

OsNRT1.1A displays functional divergence with previously reported NRT1.1s in plants and holds great potential in promoting both high yield and early maturation in rice. Nitrogen (N) is a major driving force for crop yield improvement, but application of high levels of N delays flowering, prolonging maturation and thus increasing the risk of yield losses. Therefore, traits that enable utilization of high levels of N without delaying maturation will be highly desirable for crop breeding. Here, we show that OsNRT1.1A (OsNPF6.3), a member of the rice (Oryza sativa) nitrate transporter 1/peptide transporter family, is involved in regulating N utilization and flowering, providing a target to produce high yield and early maturation simultaneously. OsNRT.1A has functionally diverged from previously reported NRT1.1 genes in plants and functions in upregulating the expression of N utilization-related genes not only for nitrate but also for ammonium, as well as flowering-related genes. Relative to the wild type, osnrt1.1a mutants exhibited reduced N utilization and late flowering. By contrast, overexpression of OsNRT1.1A in rice greatly improved N utilization and grain yield, and maturation time was also significantly shortened. These effects were further confirmed in different rice backgrounds and also in Arabidopsis thaliana. Our study paves a path for the use of a single gene to dramatically increase yield and shorten maturation time for crops, outcomes that promise to substantially increase world food security.

Extreme haplotype variation in the desiccation-tolerant clubmoss Selaginella lepidophylla

Plant genome size varies by four orders of magnitude, and most of this variation stems from dynamic changes in repetitive DNA content. Here we report the small 109 Mb genome of Selaginella lepidophylla, a clubmoss with extreme desiccation tolerance. Single-molecule sequencing enables accurate haplotype assembly of a single heterozygous S. lepidophylla plant, revealing extensive structural variation. We observe numerous haplotype-specific deletions consisting of largely repetitive and heavily methylated sequences, with enrichment in young Gypsy LTR retrotransposons. Such elements are active but rapidly deleted, suggesting “bloat and purge” to maintain a small genome size. Unlike all other land plant lineages, Selaginella has no evidence of a whole-genome duplication event in its evolutionary history, but instead shows unique tandem gene duplication patterns reflecting adaptation to extreme drying. Gene expression changes during desiccation in S. lepidophylla mirror patterns observed across angiosperm resurrection plants.Selaginella lepidophylla is a clubmoss with extreme desiccation tolerance. Here, the authors assemble its highly heterozygotic haplotypes and examine gene expression changes during desiccation, which shed light on the mechanisms for maintaining a small genome size and adaptation to extreme drying.

Early selection of bZIP73 facilitated adaptation of japonica rice to cold climates

Cold stress is a major factor limiting production and geographic distribution of rice (Oryza sativa). Although the growth range of japonica subspecies has expanded northward compared to modern wild rice (O. rufipogon), the molecular basis of the adaptation remains unclear. Here we report bZIP73, a bZIP transcription factor-coding gene with only one functional polymorphism (+511 G>A) between the two subspecies japonica and indica, may have facilitated japonica adaptation to cold climates. We show the japonica version of bZIP73 (bZIP73Jap) interacts with bZIP71 and modulates ABA levels and ROS homeostasis. Evolutionary and population genetic analyses suggest bZIP73 has undergone balancing selection; the bZIP73Jap allele has firstly selected from standing variations in wild rice and likely facilitated cold climate adaptation during initial japonica domestication, while the indica allele bZIP73Ind was subsequently selected for reasons that remain unclear. Our findings reveal early selection of bZIP73Jap may have facilitated climate adaptation of primitive rice germplasms.Japonica rice can grow further north than wild or indica rice and is more tolerant of cold climates. Here, the authors show that bZIP73 likely underwent selection in the early phase of rice domestication to facilitate cold tolerance in japonica by modulating ABA and ROS homeostasis.