Jian-Zhong Huang

Workable male sterility systems for hybrid rice: Genetics, biochemistry, molecular biology, and utilization

The exploitation of male sterility systems has enabled the commercialization of heterosis in rice, with greatly increased yield and total production of this major staple food crop. Hybrid rice, which was adopted in the 1970s, now covers nearly 13.6 million hectares each year in China alone. Various types of cytoplasmic male sterility (CMS) and environment-conditioned genic male sterility (EGMS) systems have been applied in hybrid rice production. In this paper, recent advances in genetics, biochemistry, and molecular biology are reviewed with an emphasis on major male sterility systems in rice: five CMS systems, i.e., BT-, HL-, WA-, LD- and CW- CMS, and two EGMS systems, i.e., photoperiod- and temperature-sensitive genic male sterility (P/TGMS). The interaction of chimeric mitochondrial genes with nuclear genes causes CMS, which may be restored by restorer of fertility (Rf) genes. The PGMS, on the other hand, is conditioned by a non-coding RNA gene. A survey of the various CMS and EGMS lines used in hybrid rice production over the past three decades shows that the two-line system utilizing EGMS lines is playing a steadily larger role and TGMS lines predominate the current two-line system for hybrid rice production. The findings and experience gained during development and application of, and research on male sterility in rice not only advanced our understanding but also shed light on applications to other crops.

Disruption of OsSULTR3;3 reduces phytate and phosphorus concentrations and alters the metabolite profile in rice grains.

Two low phytic acid (lpa) mutants have been developed previously with the aim to improve the nutritional value of rice (Oryza sativa) grains. In the present study, the impacts of lpa mutations on grain composition and underlying molecular mechanisms were investigated. Comparative compositional analyses and metabolite profiling demonstrated that concentrations of both phytic acid (PA) and total phosphorus (P) were significantly reduced in lpa brown rice, accompanied by changes in other metabolites and increased concentrations of nutritionally relevant compounds. The lpa mutations modified the expression of a number of genes involved in PA metabolism, as well as in sulfate and phosphate homeostasis and metabolism. Map-based cloning and complementation identified the underlying lpa gene to be OsSULTR3;3. The promoter of OsSULTR3;3 is highly active in the vascular bundles of leaves, stems and seeds, and its protein is localized in the endoplasmic reticulum. No activity of OsSULTR3;3 was revealed for the transport of phosphate, sulfate, inositol or inositol 1,4,5 triphosphate by heterologous expression in either yeast or Xenopus oocytes. The findings reveal that OsSULTR3;3 plays an important role in grain metabolism, pointing to a new route to generate value-added grains in rice and other cereal crops.

Expression of cytochrome P450 CYP81A6 in rice: tissue specificity, protein subcellular localization, and response to herbicide application

The cytochrome P450 gene CYP81A6 confers tolerance to bentazon and metsulfuron-methyl, two selective herbicides widely used for weed control in rice and wheat fields. Knockout mutants of CYP81A6 are highly susceptible to both herbicides. The present study aimed to characterize the CYP81A6 expression in rice. Quantitative real-time polymerase chain reaction (PCR) analyses demonstrated that foliar treatment of bentazon (500 mg/L) greatly induced expression of CYP81A6 in both wild-type (Jiazhe B) and its knockout mutant (Jiazhe mB): a 10-fold increase at 9 h before returning to basal levels at 24 h in Jiazhe B, while in the mutant the expression level rose to >20-fold at 12 h and maintained at such high level up to 24 h post exposure. In contrast, metsulfuron-methyl (500 mg/L) treatment did not affect the expression of CYP81A6 in Jiazhe B within 80 h; thereafter the expression peaked at 120 h and returned gradually to basal levels by Day 6. We suggest that a metabolite of metsulfuron-methyl, 1H-2,3-benzothiazin-4-(3H)-one-2,2-dioxide, is likely to be responsible for inducing CYP81A6 expression, rather than the metsulfuron-methyl itself. Use of a promoter-GUS reporter construct (CYP81A6Pro::GUS) demonstrated that CYP81A6 was constitutively expressed throughout the plant, with the highest expression in the upper surfaces of leaves. Subcellular localization studies in rice protoplasts showed that CYP81A6 was localized in the endoplasmic reticulum. These observations advance our understanding of CYP81A6 expression in rice, particularly its response to the two herbicides.摘要目的分析CYP81A6基因在苯达松及甲磺隆处理下的诱导表达模式, 解释该基因与两种除草剂代谢相关的可能原因。创新点从两种除草剂降解途径中产生的小分子物质的结构相似性出发, 通过基因诱导表达的特点分析, 解释CYP81A6和两种除草剂降解相关的原因。方法通过实时定量聚合酶链反应(PCR)来分析基因表达的特点; 利用CYP81A6启动子与GUS报告基因构建的载体来分析组织特异性表达; 通过亚细胞定位来确定CYP81A6发挥功能的场所。结论CYP81A6基因受苯达松及甲磺隆诱导, 在不同的时间点开始上调, 说明了甲磺隆的降解中间产物可以诱导这个基因的表达; CYP81A6是组成型表达, 在根、 茎、 叶中均有表达; 亚细胞定位结果证明CYP81A6是一个内质网上的蛋白。

Modified accumulation of selected heavy metals in Bt transgenic rice

Safety assessment of genetically modified crops generally does not take into account the potential hazard of altered patterns of heavy metal accumulation in plants. A pot experiment was conducted under greenhouse conditions to evaluate the impact of heavy metal amendments on the accumulation of Cd, Cu, Pb and Zn in a Bt transgenic rice Ke-Ming-Dao (KMD) and its wild-type Xiushui 11 (Xs11). In control soils, significant difference was only found in contents of Cu (p < 0.01) and Pb (p < 0.05) in straw between KMD and Xs11. At three levels of Cd amendments (5, 10, and 20 mg/kg), the Cd contents in grain and straw of KMD were significantlyhigher than those of Xs11, and all grain Cd contents were significantly higher than the International Criteria (0.2-0.4 mg/kg) as specified by the Codex Alimentarius Commission (CAC). These results implied that it may be unsafe for growing Bt transgenic rice in heavily Cd-polluted areas. No significant difference in Zn was found between the two varieties with the exception of roots at Zn amendment level of 600 mg/kg, while Pb contents in KMD were much higher in the straw at the lead amendment level of 1000 mg/kg and inthe root at 250 mg Pb/kg. Data on the heavy metal accumulation patterns for the genetically modified rice may be used for the selection of growing areas as well as for plant residue management for Bt rice.

Characterization of an RNase Z nonsense mutation identified exclusively in environment-conditioned genic male sterile rice

The two-line hybrid system in rice is becoming more important and employs environment-conditioned genic male sterile (EGMS) lines sensitive to photoperiod (photoperiod-sensitive genic male sterile), temperature [temperature genic male sterile (TGMS)], or a combination of the two (photoperiod temperature genic male sterile). At least 18 EGMS genes have been mapped, and two cloned, but controversies exist. For example, three different genes were reported to underlie the TGMS trait in three independently identified progenitors, Annong S-1, Zhu 1S, and Guangzhan 63S, while another study demonstrated that the TGMS genes in Annong S-1 and Zhu 1S are allelic. In the present study, we confirmed the allelism of the three TGMS genes, which means there is a common TGMS gene(s) in these lines. Knowing there is an association between the mutant allele (RNZm) of a ribonuclease gene (RNZ) with the TGMS trait in Guangzhuan 63S, we then sequenced RNZ for 14 commercial EGMS and 21 non-EGMS lines, and we developed two derived cleaved amplified polymorphic sequence (dCAPS) markers to detect RNZm alleles in 32 EGMS and 310 non-EGMS lines. The analyses showed that the RNZm allele existed exclusively in EGMS lines; all non-EGMS lines contained the functional RNZgc or RNZtc allele. Furthermore, two segregating populations that included 2,429 individuals were developed by crossing Zhu 1S (RNZm) to two non-EGMS lines (both with RNZtc); examination of the segregation of male sterile and fertile plants indicated that the TGMS trait was under the control of a single gene; analysis of the markers revealed the RNZm allele exclusively in TGMS plants and the RNZtc allele only in non-TGMS plants in both populations. The dCAPS markers could therefore help select TGMS progeny in breeding programs, which will save time and labor, and improve breeding efficiency and accuracy.

Resistance of rice to insect pests mediated by suppression of serotonin biosynthesis

Rice is one of the world’s most important foods, but its production suffers from insect pests, causing losses of billions of dollars, and extensive use of environmentally damaging pesticides for their control1,2. However, the molecular mechanisms of insect resistance remain elusive. Although a few resistance genes for planthopper have been cloned, no rice germplasm is resistant to stem borers. Here, we report that biosynthesis of serotonin, a neurotransmitter in mammals3, is induced by insect infestation in rice, and its suppression confers resistance to planthoppers and stem borers, the two most destructive pests of rice2. Serotonin and salicylic acid derive from chorismate4. In rice, the cytochrome P450 gene CYP71A1 encodes tryptamine 5-hydroxylase, which catalyses conversion of tryptamine to serotonin5. In susceptible wild-type rice, planthopper feeding induces biosynthesis of serotonin and salicylic acid, whereas in mutants with an inactivated CYP71A1 gene, no serotonin is produced, salicylic acid levels are higher and plants are more insect resistant. The addition of serotonin to the resistant rice mutant and other brown planthopper-resistant genotypes results in a loss of insect resistance. Similarly, serotonin supplementation in artificial diet enhances the performance of both insects. These insights demonstrate that regulation of serotonin biosynthesis plays an important role in defence, and may prove valuable for breeding insect-resistant cultivars of rice and other cereal crops.Serotonin, a well-known neurotransmitter in animals, is also produced by plants. In rice, biosynthesis of serotonin through the cytochrome P450 CYP71A1 gene increases the susceptibility of plants by promoting growth of destructive insects.

Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing

Mutation breeding is based on the induction of genetic variations; hence knowledge of the frequency and type of induced mutations is of paramount importance for the design and implementation of a mutation breeding program. Although γ ray irradiation has been widely used since the 1960s in the breeding of about 200 economically important plant species, molecular elucidation of its genetic effects has so far been achieved largely by analysis of target genes or genomic regions. In the present study, the whole genomes of six γ-irradiated M2 rice plants were sequenced; a total of 144–188 million high-quality (Q>20) reads were generated for each M2 plant, resulting in genome coverage of >45 times for each plant. Single base substitution (SBS) and short insertion/deletion (Indel) mutations were detected at the average frequency of 7.5×10−6–9.8×10−6 in the six M2 rice plants (SBS being about 4 times more frequent than Indels). Structural and copy number variations, though less frequent than SBS and Indel, were also identified and validated. The mutations were scattered in all genomic regions across 12 rice chromosomes without apparent hotspots. The present study is the first genome-wide single-nucleotide resolution study on the feature and frequency of γ irradiation-induced mutations in a seed propagated crop; the findings are of practical importance for mutation breeding of rice and other crop species.中文概要目 的研究γ 射线对水稻基因组的诱变效应, 明确其诱发突变的类型、分布和频率。创新点首次针对种子繁殖植物在全基因组范围及单核苷 酸水平上揭示了γ 射线诱发可遗传变异的频率与 特征。方 法利用Illumina Hiseq2000 对三种γ 射线剂量辐照培 育的6 株水稻(日本晴)M2 植株进行基因组重测 序, 生物信息学分析确定单碱基替换(SBS)和 插入缺失(Indel)突变, 以及结构变异和拷贝数 等变异的频率和基因组分布。利用Sanger 测序、 目标片段扩增或定量多聚酶链反应(qPCR)对各 类突变进行验证。综合重测序和验证结果估算诱 发突变频率。结 论结果表明, γ 射线既可以诱发单碱基替换, 也可 以诱发插入缺失突变和结构变异; 水稻M2 代植 株中的平均突变频率达到7.5×10−6∼9.8×10−6; Indel 突变频率约为SBS 变异的1/4, 而结构变异 频率更低; SBS 和Indel 突变随机分布在12 条染 色体上, 无明显的突变热点。

High-resolution melting-based TILLING of γ ray-induced mutations in rice

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M2 plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M2 rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.中文概要目的建立适用于筛选伽马射线诱发突变的、基于高分辨率熔解曲线(high-resolution melting,HRM)技术的高通量定向诱导基因组局部突变技术 (Targeting Induced Local Lesions IN Genomes, TILLING)体系。创新点建立起了基于HRM技术、适用于伽玛射线诱发的小片段插入/缺失突变的高通量TILLING体系(HRM-TILLING)。方法通过不同野生型/突变型比例混池DNA的HRM分析,确定HRM检测不同类型插入/缺失突变的能力,确定M2植株突变检测的适宜混池比例,并用一个伽玛诱变M2 群体(n=4560)筛选OsLCT1和SPDT两个基因的突变体,确定实际效果。结论以4 株M2植株混样,采用HRM可以有效检出突变。建立的基于HRM的TILLING体系适用于伽玛射线诱发突变的高通量筛选。

The xantha Marker Trait Is Associated with Altered Tetrapyrrole Biosynthesis and Deregulated Transcription of PhANGs in Rice

The xantha marker trait, which is controlled by a down-regulating epi-mutation of OsGUN4, has been applied to the production of hybrid rice. However, the molecular basis for the ability of xantha mutants to attain high photosynthetic capacity even with decreased chlorophyll contents has not been characterized. In the present study, we observed that the total chlorophyll content of the xantha mutant was only 27.2% of that of the wild-type (WT) plants. However, the xantha mutant still accumulated 59.9% of the WT δ-aminolevulinic acid content, 72.8% of the WT Mg-protoporphyrin IX content, and 63.0% of the WT protochlorophyllide a content. Additionally, the protoporphyrin IX and heme contents in the mutant increased to 155.0 and 160.0%, respectively, of the WT levels. A search for homologs resulted in the identification of 124 rice genes involved in tetrapyrrole biosynthesis and photosynthesis. With the exception of OsGUN4, OsHO-1, and OsHO-2, the expression levels of the genes involved in tetrapyrrole biosynthesis were significantly higher in the xantha mutant than in the WT plants, as were all 72 photosynthesis-associated nuclear genes. In contrast, there were no differences between the xantha mutant and WT plants regarding the expression of all 22 photosynthesis-associated chloroplast genes. Furthermore, the abundance of 1O2 and the expression levels of 1O2-related genes were lower in the xantha mutant than in the WT plants, indicating 1O2-mediated retrograde signaling was repressed in the mutant plants. These results suggested that the abundance of protoporphyrin IX used for chlorophyll synthesis decreased in the mutant, which ultimately decreased the amount of chlorophyll in the xantha mutant. Additionally, the up-regulated expression of photosynthesis-associated nuclear genes enabled the mutant to attain a high photosynthetic capacity. Our findings confirm that OsGUN4 plays an important role in tetrapyrrole biosynthesis and photosynthesis in rice. GUN4, chlorophyll synthesis pathways, and photosynthetic activities are highly conserved in plants and hence, novel traits (e.g., xantha marker trait) may be generated in other cereal crops by modifying the GUN4 gene.