Qiaoquan Liu

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.

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.

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.

OsbZIP58, a basic leucine zipper transcription factor, regulates starch biosynthesis in rice endosperm

Starch composition and the amount in endosperm, both of which contribute dramatically to seed yield, cooking quality, and taste in cereals, are determined by a series of complex biochemical reactions. However, the mechanism regulating starch biosynthesis in cereal seeds is not well understood. This study showed that OsbZIP58, a bZIP transcription factor, is a key transcriptional regulator controlling starch synthesis in rice endosperm. OsbZIP58 was expressed mainly in endosperm during active starch synthesis. osbzip58 null mutants displayed abnormal seed morphology with altered starch accumulation in the white belly region and decreased amounts of total starch and amylose. Moreover, osbzip58 had a higher proportion of short chains and a lower proportion of intermediate chains of amylopectin. Furthermore, OsbZIP58 was shown to bind directly to the promoters of six starch-synthesizing genes, OsAGPL3, Wx, OsSSIIa, SBE1, OsBEIIb, and ISA2, and to regulate their expression. These findings indicate that OsbZIP58 functions as a key regulator of starch synthesis in rice seeds and provide new insights into seed quality control.

C-Type Starch from High-Amylose Rice Resistant Starch Granules Modified by Antisense RNA Inhibition of Starch Branching Enzyme

High-amylose starch is a source of resistant starch (RS) which has a great benefit on human health. A transgenic rice line (TRS) enriched amylose and RS had been developed by antisense RNA inhibition of starch branching enzymes. In this study, the native starch granules were isolated from TRS grains as well as the wild type, and their crystalline type was carefully investigated before and after acid hydrolysis. In high-amylose TRS rice, the C-type starch, which might result from the combination of both A-type and B-type starch, was observed and subsequently confirmed by multiple physical techniques, including X-ray powder diffraction, solid-state nuclear magnetic resonance, and Fourier transform infrared. Moreover, the change of starch crystalline structure from C- to B-type during acid hydrolysis was also observed in this RS-rich rice. These data could add to our understanding of not only the polymorph structure of cereal starch but also why high-amylose starch is more resistant to digestion.

Stable Inheritance of the Antisense Waxy Gene in Transgenic Rice with Reduced Amylose Level and Improved Quality

Amylose content in rice endosperm is a key determinant of eating and cooking quality. In the present study, a chimeric antisense construct, which contained a 756-bp antisense Waxy (Wx) gene DNA fragment from rice and the gusA coding sequence, both fused to the 3.1-kb rice Wx promoter, was efficiently introduced into several elite rice cultivars, both of japonica and indica type, via Agrobacterium. More than 200 independent transgenic lines were produced and integration transgene was confirmed by PCR and Southern blotting. Northern blot analysis suggested that the antisense Wx transcript interacted with both the endogenous Wx mature mRNA and unspliced transcripts, but only interaction with the mature mRNA resulted in reduced amylose synthesis. Analysis of GUS activity showed that the gusA fusion gene driven by the rice Wx promoter expressed highly in the endosperm of the transgenic rice plants. Varying degrees of reduction in amylose content, up to 96%, were found in seeds derived from these transformants. Consistently, opaque white seeds, similar to glutinous rice, were observed in several transgenic lines of japonica rice. In transgenic lines derived from indica rice, which usually has a high amylose level, significant reduction of amylose content was also found in the endosperm, but the levels of reduction were lower than those of japonica rice. Genetic analysis demonstrated that transgenes and improved amylose content were stably inherited (up to ninth generation) in these transgenic lines. Several elite transgenic lines with improved amylose level and quality have been selected for field evaluation.

The WRKY transcription factor OsWRKY78 regulates stem elongation and seed development in rice

WRKY proteins are a large super family of transcriptional regulators primarily involved in various plant physiological programs. In present study, the expression profile and putative function of the WRKY transcriptional factor, WRKY78, in rice were identified. Real-time RT-PCR analysis showed that OsWRKY78 transcript was most abundant in elongating stems though its expression was detected in all the tested organs. The expression profiles were further confirmed by using promoter-GUS analysis in transgenic rice. OsWRKY78::GFP fusion gene transient expression analysis demonstrated that OsWRKY78 targeted to the nuclei of onion epidermal cell. Furthermore, OsWRKY78 RNAi and overexpression transgenic rice lines were generated. Transgenic plants with OsWRKY78 overexpression exhibited a phenotype identical to the wild type, whereas inhibition of OsWRKY78 expression resulted in a semi-dwarf and small kernel phenotype due to reduced cell length in transgenic plants. In addition, a T-DNA insertion mutant line oswrky78 was identified and a phenotype similar to that of RNAi plants was also observed. Grain quality analysis data showed no significant differences, with the exception of minor changes in endosperm starch crystal structure in RNAi plants. Taken together, these results suggest that OsWRKY78 may acts as a stem elongation and seed development regulator in rice.

Granule structure and distribution of allomorphs in C-type high-amylose rice starch granule modified by antisense RNA inhibition of starch branching enzyme.

C-type starch, which is a combination of both A-type and B-type crystal starch, is usually found in legumes and rhizomes. We have developed a high-amylose transgenic line of rice (TRS) by antisense RNA inhibition of starch branching enzymes. The starch in the endosperm of this TRS was identified as typical C-type crystalline starch, but its fine granular structure and allomorph distribution remained unclear. In this study, we conducted morphological and spectroscopic studies on this TRS starch during acid hydrolysis to determine the distribution of A- and B-type allomorphs. The morphology of starch granules after various durations of acid hydrolysis was compared by optical microscopy, scanning electron microscopy, and transmission electron microscopy. The results showed that amorphous regions were located at the center part of TRS starch subgranules. During acid hydrolysis, starch was degraded from the interior of the subgranule to the outer surface, while the peripheral part of the subgranules and the surrounding band of the starch granule were highly resistant to acid hydrolysis. The spectroscopic changes detected by X-ray powder diffraction, 13C cross-polarization magic-angle spinning NMR, and attenuated total reflectance Fourier transform infrared showed that the A-type allomorph was hydrolyzed more rapidly than the B-type, and that the X-ray diffraction profile gradually changed from a native C-type to a CB-type with increasing hydrolysis time. Our results showed that, in TRS starch, the A-type allomorph was located around the amorphous region, and was surrounded by the B-type allomorph located in the peripheral region of the subgranules and the surrounding band of the starch granule. Thus, the positions of A- and B-type allomorphs in the TRS C-type starch granule differ markedly from those in C-type legume and rhizome starch.

A 90-day toxicology study of high-amylose transgenic rice grain in Sprague-Dawley rats.

A transgenic rice line (TRS) with high amylose level has been developed by antisense RNA inhibition of starch branching enzymes. Compositional analysis of TRS demonstrated that the content of resistant starch (RS) was significantly higher compared to conventional non-transgenic rice. High level of RS is an important raw material in food industry and has various physiological effects for human health. In order to provide the reliable theory basis for field release of TRS rice, we evaluated the potential health effects of long-term consumption of the TRS. The 90-day toxicology feeding experiment was conducted in Sprague-Dawley rats fed with diets containing 70% of either TRS rice flour, its near-isogenic rice flour or the control diet. The clinical performance variables (body weight, body weight gain and food consumption) were measured and pathological responses (hematological parameters and serum chemistry at the midterm and the completion of the experiment, urinalysis profile and serum sex hormone response at the completion of the experiment) were performed. Besides, clinical signs, relative organ weights and microscopic observations were also compared between TRS group and its near-isogenic rice group. The combined data indicates that high-amylose TRS grain is as safe as the conventional non-transgenic rice for rat consumption.