Minghong Gu

Sequence and analysis of rice chromosome 4

Rice is the principal food for over half of the population of the world. With its genome size of 430 megabase pairs (Mb), the cultivated rice species Oryza sativa is a model plant for genome research. Here we report the sequence analysis of chromosome 4 of O. sativa, one of the first two rice chromosomes to be sequenced completely. The finished sequence spans 34.6 Mb and represents 97.3% of the chromosome. In addition, we report the longest known sequence for a plant centromere, a completely sequenced contig of 1.16 Mb corresponding to the centromeric region of chromosome 4. We predict 4,658 protein coding genes and 70 transfer RNA genes. A total of 1,681 predicted genes match available unique rice expressed sequence tags. Transposable elements have a pronounced bias towards the euchromatic regions, indicating a close correlation of their distributions to genes along the chromosome. Comparative genome analysis between cultivated rice subspecies shows that there is an overall syntenic relationship between the chromosomes and divergence at the level of single-nucleotide polymorphisms and insertions and deletions. By contrast, there is little conservation in gene order between rice and Arabidopsis.

Functional Rice Centromeres Are Marked by a Satellite Repeat and a Centromere-Specific Retrotransposon

The centromere of eukaryotic chromosomes is essential for the faithful segregation and inheritance of genetic information. In the majority of eukaryotic species, centromeres are associated with highly repetitive DNA, and as a consequence, the boundary for a functional centromere is difficult to define. In this study, we demonstrate that the centers of rice centromeres are occupied by a 155-bp satellite repeat, CentO, and a centromere-specific retrotransposon, CRR. The CentO satellite is located within the chromosomal regions to which the spindle fibers attach. CentO is quantitatively variable among the 12 rice centromeres, ranging from 65 kb to 2 Mb, and is interrupted irregularly by CRR elements. The break points of 14 rice centromere misdivision events were mapped to the middle of the CentO arrays, suggesting that the CentO satellite is located within the functional domain of rice centromeres. Our results demonstrate that the CentO satellite may be a key DNA element for rice centromere function.

Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities

More than half of the worlds population uses rice as a source of carbon intake every day. Improving grain quality is thus essential to rice consumers. The three main properties that determine rice eating and cooking quality—amylose content, gel consistency, and gelatinization temperature—correlate with one another, but the underlying mechanism of these properties remains unclear. Through an association analysis approach, we found that genes related to starch synthesis cooperate with each other to form a fine regulating network that controls the eating and cooking quality and defines the correlation among these three properties. Genetic transformation results verified the association findings and also suggested the possibility of developing elite cultivars through modification with selected major and/or minor starch synthesis-related genes.

GOLD HULL AND INTERNODE2 Encodes a Primarily Multifunctional Cinnamyl-Alcohol Dehydrogenase in Rice

Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis.

The Central Element Protein ZEP1 of the Synaptonemal Complex Regulates the Number of Crossovers during Meiosis in Rice

This work identifies ZEP1, a rice protein required for the assembly of the synaptonemal complex during meiosis. It finds that, in rice, the number of crossovers between homologous chromosomes is increased in the absence of synapsis, emphasizing the idea that the role of synapsis in crossover formation has diverged among different organisms. ZEP1, a transverse filament (TF) protein, is the rice (Oryza sativa) homolog of Arabidopsis thaliana ZYP1. In the Tos17-insertional zep1 mutants, homologous chromosomes align along the entire length of the chromosome, but the synaptonemal complex is not assembled in early prophase I. Crossovers are well formed, and 12 bivalents could be detected from diakinesis to metaphase I, which leads to equal chromosomal segregation in anaphase I. Moreover, the number of crossovers has a tendency to be increased compared with that in the wild type. These phenomena are different from the TF mutants identified so far in other organisms. Chiasma terminalization of the bivalent, which occurs frequently in the wild type, seldom occurred in zep1. Transmission electron micrographs and immunodetection using an antibody against ZEP1 showed that ZEP1 is the central element of the synaptonemal complex. Although PAIR2 and MER3 were loaded normally in zep1, their dissociation was delayed severely compared with the wild type. In addition, ZEP1 is reloaded onto chromosomes in early microspores as the chromosome decondense, suggesting that ZEP1 might have other biological functions during this process.

Validation of Candidate Reference Genes for the Accurate Normalization of Real-Time Quantitative RT-PCR Data in Rice During Seed Development

Rice seed, a natural storage organ for starch and protein, is also an ideal bioreactor for the production of valuable proteins. Increasingly, studies focused on rice have tried to determine the functions of its genes and also to improve its yield and quality. Real-time RT-PCR is the best available choice at present for gene expression analysis due to its accuracy, sensitivity, and reproducibility. The right choice of reference genes for normalization, however, is a critical precondition for reliable results. In this study, the expression stabilities of nine commonly used housekeeping genes in rice were carefully assessed using the software geNorm. Our results showed that eIF-4a and ACT1 were the most suitable reference genes among almost all the tested samples from two rice varieties, including different temporal and spatial-specific tissues, especially in seeds at different developmental stages. In contrast, 18S and 25S rRNAs, two common reference genes, were found to have the least stable expression. Moreover, it is necessary to use multiple suitable reference genes together for normalization to get a more reliable result in temporal and spatial expression analysis during rice seed development. The validated reference genes were further relied when used to quantify the expression of several genes of interest during rice seed development.

Deletion in a quantitative trait gene qPE9-1 associated with panicle erectness improves plant architecture during rice domestication.

Rice plant architecture is an important agronomic trait and a major determinant in high productivity. Panicle erectness is the preferred plant architecture in japonica rice, but the molecular mechanism underlying domestication of the erect panicle remains elusive. Here we report the map-based cloning of a major quantitative trait locus, qPE9-1, which plays an integral role in regulation of rice plant architecture including panicle erectness. The R6547 qPE9-1 gene encodes a 426-amino-acid protein, homologous to the keratin-associated protein 5-4 family. The gene is composed of three Von Willebrand factor type C domains, one transmembrane domain, and one 4-disulfide-core domain. Phenotypic comparisons of a set of near-isogenic lines and transgenic lines reveal that the functional allele (qPE9-1) results in drooping panicles, and the loss-of-function mutation (qpe9-1) leads to more erect panicles. In addition, the qPE9-1 locus regulates panicle and grain length, grain weight, and consequently grain yield. We propose that the panicle erectness trait resulted from a natural random loss-of-function mutation for the qPE9-1 gene and has subsequently been the target of artificial selection during japonica rice breeding.

DEP and AFO Regulate Reproductive Habit in Rice

Sexual reproduction is essential for the life cycle of most angiosperms. However, pseudovivipary is an important reproductive strategy in some grasses. In this mode of reproduction, asexual propagules are produced in place of sexual reproductive structures. However, the molecular mechanism of pseudovivipary still remains a mystery. In this work, we found three naturally occurring mutants in rice, namely, phoenix (pho), degenerative palea (dep), and abnormal floral organs (afo). Genetic analysis of them indicated that the stable pseudovivipary mutant pho was a double mutant containing both a Mendelian mutation in DEP and a non-Mendelian mutation in AFO. Further map-based cloning and microarray analysis revealed that dep mutant was caused by a genetic alteration in OsMADS15 while afo was caused by an epigenetic mutation in OsMADS1. Thus, OsMADS1 and OsMADS15 are both required to ensure sexual reproduction in rice and mutations of them lead to the switch of reproductive habit from sexual to asexual in rice. For the first time, our results reveal two regulators for sexual and asexual reproduction modes in flowering plants. In addition, our findings also make it possible to manipulate the reproductive strategy of plants, at least in rice.

Microstructure and ultrastructure of high-amylose rice resistant starch granules modified by antisense RNA inhibition of starch branching enzyme.

A high-amylose transgenic rice line (TRS) modified by antisense RNA inhibition of starch branching enzymes revealed a resistant starch-rich quality. Compound starch granules in whole grains of the regular rice cultivar Teqing (TQ) were readily split during fracturing, whereas the starch granules in TRS were structurally intact and showed large voluminous, non-angular rounded bodies and elongated, filamentous structures tolerant of fracturing. In isolated preparation, TQ starch granules broke up into separate polygonal granules, whereas TRS starch granules kept their intactness. TRS starch granules consisted of packed smaller subgranules, some of which located at the periphery of starch granules were fused to each other with adjacent ones forming a thick band or wall encircling the entire circumference of the granules. TQ starch granules had a high concentration of amylose in the concentric hilum, whereas TRS starch granules showed a relatively even distribution of amylose with intense amylose in both hilum and band.

High‐amylose rice improves indices of animal health in normal and diabetic rats

A high-amylose rice with 64.8% amylose content (AC) was developed by transgenic inhibition of two isoforms of starch branching enzyme (SBE), SBEI and SBEIIb, in an indica rice cultivar. The expression of SBEI and SBEIIb was completely inhibited in the transgenic line, whereas the expression of granule-bound starch synthase was normal. Compared with wild-type rice, drastic reductions in both SBEs in the transgenic rice increased apparent AC in flour from 27.2% to 64.8%, resistant starch (RS) content from 0% to 14.6% and total dietary fibre (TDF) from 6.8% to 15.2%. Elevated AC increased the proportion of long unit chains in amylopectin and increased onset gelatinization temperature and resistance to alkaline digestion; however, kernel weight was decreased. A rat feeding trial indicated that consumption of high-amylose rice decreased body weight gain significantly (P < 0.01); increased faecal mass, faecal moisture and short-chain fatty acids; and lowered the faecal pH. An acute oral rice tolerance test revealed that the high-amylose rice had a positive effect on lowering the blood glucose response in diabetic Zucker fatty rats. This novel rice with its high AC, RS and TDF offers potential benefits for its use in foods and in industrial applications.