Xingming Lian

Genome-wide association analyses provide genetic and biochemical insights into natural variation in rice metabolism

Plant metabolites are important to world food security in terms of maintaining sustainable yield and providing food with enriched phytonutrients. Here we report comprehensive profiling of 840 metabolites and a further metabolic genome-wide association study based on ∼6.4 million SNPs obtained from 529 diverse accessions of Oryza sativa. We identified hundreds of common variants influencing numerous secondary metabolites with large effects at high resolution. We observed substantial heterogeneity in the natural variation of metabolites and their underlying genetic architectures among different subspecies of rice. Data mining identified 36 candidate genes modulating levels of metabolites that are of potential physiological and nutritional importance. As a proof of concept, we functionally identified or annotated five candidate genes influencing metabolic traits. Our study provides insights into the genetic and biochemical bases of rice metabolome variation and can be used as a powerful complementary tool to classical phenotypic trait mapping for rice improvement.

Combining high-throughput phenotyping and genome-wide association studies to reveal natural genetic variation in rice

Even as the study of plant genomics rapidly develops through the use of high-throughput sequencing techniques, traditional plant phenotyping lags far behind. Here we develop a high-throughput rice phenotyping facility (HRPF) to monitor 13 traditional agronomic traits and 2 newly defined traits during the rice growth period. Using genome-wide association studies (GWAS) of the 15 traits, we identify 141 associated loci, 25 of which contain known genes such as the Green Revolution semi-dwarf gene, SD1. Based on a performance evaluation of the HRPF and GWAS results, we demonstrate that high-throughput phenotyping has the potential to replace traditional phenotyping techniques and can provide valuable gene identification information. The combination of the multifunctional phenotyping tools HRPF and GWAS provides deep insights into the genetic architecture of important traits.

OsSPX1 suppresses the function of OsPHR2 in the regulation of expression of OsPT2 and phosphate homeostasis in shoots of rice.

Phosphate (Pi) homeostasis in plants is required for plant growth and development, and is achieved by the coordination of Pi acquisition, translocation from roots to shoots, and remobilization within plants. Previous reports have demonstrated that over-expression of OsPHR2 (the homolog of AtPHR1) and knockdown of OsSPX1 result in accumulation of excessive shoot Pi in rice. Here we report that OsPHR2 positively regulates the low-affinity Pi transporter gene OsPT2 by physical interaction and upstream regulation of OsPHO2 in roots. OsPT2 is responsible for most of the OsPHR2-mediated accumulation of excess shoot Pi. OsSPX1 suppresses the regulation on expression of OsPT2 by OsPHR2 and the accumulation of excess shoot Pi, but it does not suppress induction of OsPT2 or the accumulation of excessive shoot Pi in the Ospho2 mutant. Our data also show that OsSPX1 is a negative regulator of OsPHR2 and is involved in the feedback of Pi-signaling network in roots that is defined by OsPHR2 and OsPHO2. This finding provides new insight into the regulatory mechanism of Pi uptake, translocation, allocation and homeostasis in plants.

Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice.

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme in nitrogen metabolism; it catalyzes the critical incorporation of inorganic ammonium into glutamine. Two full-length cDNAs that encode the rice (Oryza sativa) cytosolic glutamine synthetase1 genes (OsGS1;1 and OsGS1;2) were isolated from a Minghui 63 normalized cDNA library, and glnA encoding GS in Escherichia coli was isolated by PCR amplification. Transformants for GS gene (GS1;1, GS1;2, and glnA) in rice were produced by an Agrobacterium tumefaciens-mediated transformation method, and transcripts of GS gene accumulated at higher levels in the primary transgenic plants. Our results indicated an increased metabolic level in GS-overexpressed plants, which showed higher total GS activities and soluble protein concentrations in leaves and higher total amino acids and total nitrogen content in the whole plant. Decreases in both grain yield production and total amino acids were observed in seeds of GS-overexpressed plants compared with wild-type plants. In addition, GS1;2-overexpressed plants exhibited resistance to Basta selection and higher sensitivity to salt, drought, and cold stress conditions, whereas the other two types of GS-overexpressed plants failed to show any significant changes for these stress conditions compared with wild-type plants.

QTLs for low nitrogen tolerance at seedling stage identified using a recombinant inbred line population derived from an elite rice hybrid.

Tolerance to low nitrogen conditions is a highly desired characteristic for sustainable crop production. In this study, we analyzed the genetic components associated with low N tolerance in rice at seedling stage, including main effects, epistatic effects of the quantitative trait locus (QTLs), and QTL by environment interactions (QEs), using a population of 239 recombinant inbred lines (RILs) from a cross between Zhenshan 97 and Minghui 63, the parents of an elite hybrid. A genetic linkage map with 253 DNA maker loci was constructed. Seedlings of RILs were cultivated in low N and normal N solutions. Root, shoot and plant weight in the two N treatments were measured and the relative weight of the two treatments for each trait was considered as measurements for low N tolerance. Four to eight QTLs with main effects were detected for each of the nine traits. Very few QTLs were detected in both low and normal N conditions, and most QTLs for the relative measurements were different from those for traits under the two N treatments, indicating very little commonality in the genetic basis of the traits and their relative performance under low and normal N conditions. A total of 103 digenic interactions were detected for the nine traits. While the epistatic effects collectively accounted for large proportions of the variation for several traits, the effects of QEs appeared to be trivial. It was concluded that low N tolerance of rice seedling had complex genetic basis that requires extensive studies for full characterization.

Overexpression of a pH-sensitive nitrate transporter in rice increases crop yields

Significance Significant progress has been made in our understanding of plant adaptive responses to maintain cellular pH under varied N supply forms. Rice is a plant adapted to grow in waterlogged or dryland environments, in contrast to other crops, such as wheat, soybean, and maize. The nitrate transporter OsNRT2.3b provides a molecular mechanism explaining plant adaptation to the ammonium-nitrate supply shift between the waterlogged and drained soil environments. The sensing of cytosolic pH by OsNRT2.3b can function to improve rice nitrogen use efficiency and pH balance, providing an explanation for plant adaptation to changes in the form of N supply. Cellular pH homeostasis is fundamental for life, and all cells adapt to maintain this balance. In plants, the chemical form of nitrogen supply, nitrate and ammonium, is one of the cellular pH dominators. We report that the rice nitrate transporter OsNRT2.3 is transcribed into two spliced isoforms with a natural variation in expression ratio. One splice form, OsNRT2.3b is located on the plasma membrane, is expressed mainly in the phloem, and has a regulatory motif on the cytosolic side that acts to switch nitrate transport activity on or off by a pH-sensing mechanism. High OsNRT2.3b expression in rice enhances the pH-buffering capacity of the plant, increasing N, Fe, and P uptake. In field trials, increased expression of OsNRT2.3b improved grain yield and nitrogen use efficiency (NUE) by 40%. These results indicate that pH sensing by the rice nitrate transporter OsNRT2.3b is important for plant adaption to varied N supply forms and can provide a target for improving NUE.

Transcriptome response to nitrogen starvation in rice

Nitrogen is an essential mineral nutrient required for plant growth and development. Insufficient nitrogen (N) supply triggers extensive physiological and biochemical changes in plants. In this study, we used Affymetrix GeneChip rice genome arrays to analyse the dynamics of rice transcriptome under N starvation. N starvation induced or suppressed transcription of 3518 genes, representing 10.88% of the genome. These changes, mostly transient, affected various cellular metabolic pathways, including stress response, primary and secondary metabolism, molecular transport, regulatory process and organismal development. 462 or 13.1% transcripts for N starvation expressed similarly in root and shoot. Comparative analysis between rice and Arabidopsis identified 73 orthologous groups that responded to N starvation, demonstrated the existence of conserved N stress coupling mechanism among plants. Additional analysis of transcription profiles of microRNAs revealed differential expression of miR399 and miR530 under N starvation, suggesting their potential roles in plant nutrient homeostasis.

Comprehensive Sequence and Whole-Life-Cycle Expression Profile Analysis of the Phosphate Transporter Gene Family in Rice

Plant phosphate transporter (PT) genes comprise a large family with important roles in various physiological and biochemical processes. In this study, a database search yielded 26 potential PT family genes in rice (Oryza sativa). Analysis of these genes led to identification of eight conserved motifs and 5-12 trans-membrane segments, most of them conserved. A total of 237 putative cis elements were found in the 2-kb upstream region of these genes. Of these, a majority were Pi-response and other stress-related cis regulatory elements, such as PHO-like, TATA-box-like, PHR1, or Helix-loop-helix elements, and WRKY1 and ABRE elements, suggesting gene regulation by these signals. Comprehensive expression analysis of these genes was performed using data from microarrays hybridized with RNA from 27 tissues covering the entire lifecycle from three rice genotypes: Minghui 63, Zhenshan 97, and Shanyou 63. Real-time PCR analysis confirmed that three rice PT genes are preferentially expressed in stamen at 1 d before flowering, two in panicle at the heading stage, and two in flag leaf at 14 d after the heading stage. Hormone-treatment experiments revealed differential up-regulation or down-regulation of 11 rice PT genes in seedlings exposed to five hormones, respectively. These results will be useful for elucidating the roles of these genes in the growth, development, and stress response of the rice plant.

Sequence and expression analysis of the thioredoxin protein gene family in rice.

Thioredoxin (Trx) proteins play important biological functions in cells by changing redox via thioldisulfied exchange. This system is especially widespread in plants. Through database search, we identified 30 potential Trx protein-encoding genes (OsTrx) in rice (Oryza sativa L.). An analysis of the complete set of OsTrx proteins is presented here, including chromosomal location, conserved motifs, domain duplication, and phylogenetic relationships. Our findings suggest that the expansion of the Trx gene family in rice, in large part, occurred due to gene duplication. A comprehensive expression profile of Trx genes family was investigated by analyzing the signal data of this family extracted from the whole genome microarray analysis of Minghui 63 and Zhenshan 97, two indica parents, and their hybrid Shanyou 63, using 27 different tissues representing the entire life cycle of rice. Results revealed specific expression of some members at germination transition as well as the 3-leaf stage during the vegetative growth phase of rice. OsTrx genes were also found to be differentially up- or down-regulated in rice seedlings subjected to treatments of phytohormones and light/dark conditions. The expression levels of the OsTrx genes in the different tissues and under different treatments were also checked by RT-PCR analysis. The identification of OsTrx genes showing differential expression in specific tissues among different genotypes or in response to different environmental cues could provide a new avenue for functional analyses in rice.

Sequence and expression analysis of the C3HC4-type RING finger gene family in rice

C3HC4-type RING finger genes comprise a large family in the plant kingdom and play important roles in various physiologic processes of plant life. In this study, we identified 29 C3HC4-type RING finger family genes in rice (Oryza sativa) by database search. Motif analysis revealed the presence of three conserved motifs with unknown functions within the predicted proteins. Promoter analysis found 196 cis-elements in the 2-kb upstream regions of these genes, including a stress-responsive element, a hormone-responsive element, and a light-responsive element. In addition, a comprehensive expression analysis of these genes has been performed using microarray data obtained from 27 tissues or organs of three rice genotypes, Minghui 63, Zhenshan 97, and Shanyou 63. Real-time PCR analysis confirmed that five C3HC4-type RING finger genes are preferentially expressed in reproductive tissues or organs such as stamen, panicle, and endosperm. Expression analysis of C3HC4-type RING finger genes under abiotic stresses suggests that twelve genes are differentially regulated by hormones or stress in rice seedlings. These results would be useful for elucidating their roles in the growth, development, and stress response of the rice plant.