Lysine is the first limiting essential amino acid in rice. We previously constructed a series of transgenic rice lines to enhance lysine biosynthesis (35S), down-regulate its catabolism (Ri), or simultaneously achieve both metabolic effects (35R). In this study, nine transgenic lines, three from each group, were selected for both field and animal feeding trials. The results showed that the transgene(s) caused no obvious effects on field performance and main agronomic traits. Mature seeds of transgenic line 35R-17 contained 48–60-fold more free lysine than in wild type and had slightly lower apparent amylose content and softer gel consistency. Moreover, a 35-day feeding experiment showed that the body weight gain, food efficiency, and protein efficiency ratio of rats fed the 35R-17 transgenic rice diet were improved when compared with those fed wild-type rice diet. These data will be useful for further evaluation and potential commercialization of 35R high-lysine transgenic rice.Keywords: feeding trial; field performance; free lysine; physicochemical quality; transgenic rice;

OsGBSSI, encoded by the Waxy (Wx) gene, is the key enzyme in the synthesis of amylose chains. Transgenic rice lines with various GBSSI activities were previously developed via site-directed mutagenesis of the Wx gene in the glutinous cultivar Guanglingxiangnuo (GLXN). In this study, grain morphology, molecular structure, and physicochemical properties were investigated in four transgenic lines with modified OsGBSSI activity and differences in amylose content. A milky opaque appearance was observed in low- and non-amylose rice grains due to air spaces in the starch granules. Gel permeation chromatography (GPC) and high-performance anion-exchange chromatography (HPAEC) analyses showed that although OsGBSSI can synthesize intermediate and extra-long amylopectin chains, it is mainly responsible for the longer amylose chains. Amylose content was positively correlated with trough viscosity, final viscosity, setback viscosity, pasting time, pasting temperature, and gelatinization temperature and negatively with gel consistency, breakdown viscosity, gelatinization enthalpy, and crystallinity. Overall, the findings suggest that OsGBSSI may be also involved in amylopectin biosynthesis, in turn affecting grain appearance, thermal and pasting properties, and the crystalline structure of starches in the rice endosperm.Keywords: amylose; crystallinity; Oryza sativa L.; OsGBSSI; physicochemical properties; starch fine structure;

•Kernel morphology and starch properties of cooked high-amylose rice were studied.•Starches with different shapes had different properties during cooking process.•The elongated and interior hollow starches kept their intact status during cooking.•The molecular weight distribution of starch in cooked kernel did not change.•Starch in cooked kernel was not wholly disrupted and highly resistant to digestion.Rice is principally consumed as cooked kernels. A high-amylose rice line, TRS, has a potential to positively impact human health. Though the properties of starch isolated from TRS kernels have been studied, the properties of cooked kernel remain to be resolved. In this study, the changes in kernel morphology and starch properties of brown TRS were investigated during cooking in boiling water. TRS kernel, which had polygonal, aggregate, elongated, and interior hollow starches from the inner to the outer of endosperm, showed different gelatinization properties during cooking. The polygonal starch was gelatinized most completely and fastest, the aggregate starch was partly gelatinized, and the elongated and interior hollow starches kept almost their intact status. The molecular weight distribution of starch in kernel did not show significant change, but starch crystalline structure was not wholly disrupted during the cooking process. The starch in cooked kernel had a very high resistance to digestion.Download high-res image (261KB)Download full-size image

•Subchronic feeding study of different doses of high-free-lysine (HFL) transgenic rice in Sprague-Dawley rats.•No diet-related difference in physiological and biochemical parameters between groups fed transgenic rice diets (T) and non-transgenic (N) or control diets.•No adverse effect in growing rats given a diet containing up to 70% HFL transgenic rice.Lysine is considered to be the first essential amino acid in rice. An elite High-Free-Lysine transgenic line HFL1 was previously produced by metabolic engineering to regulate lysine metabolism. In this study, a 90-day toxicology experiment was undertaken to investigate the potential health effect of feeding different doses of HFL1 rice to Sprague-Dawley rats. During the trial, body weight gain, food consumption and food efficiency were recorded, and no adverse effect was observed in rats fed transgenic (T) rice diets compared with non-transgenic (N) or control diets. At both midterm and final assessments, hematological parameters and serum chemistry were measured, and organ weights and histopathology were examined at the end of the trial. There was no diet-related difference in most hematological or serum chemistry parameters or organ weights between rats fed the T diets and those fed the N or control diets. Some parameters were found to differ between T groups and their corresponding N and/or control groups, but no adverse histological effect was observed. Taken together, the data from the current trial demonstrates that high lysine transgenic rice led to no adverse effect in Sprague-Dawley rats given a diet containing up to 70% HFL1 rice in 90 days.

Temperature during the growing season is a critical factor affecting grain quality. High temperatures at grain filling affect kernel development, resulting in reduced yield, increased chalkiness, reduced amylose content, and poor milling quality. Here, we investigated the grain quality and starch structure of two japonica rice cultivars with good sensory properties grown at different temperatures during the filling stage under natural field conditions. Compared to those grown under normal conditions, rice grains grown under hot conditions showed significantly reduced eating and cooking qualities, including a higher percentage of grains with chalkiness, lower protein and amylose contents, and higher pasting properties. Under hot conditions, rice starch contained reduced long-chain amylose (MW 107.1 to 107.4) and significantly fewer short-chain amylopectin (DP 5–12) but more intermediate- (DP 13–34) and long- (DP 45–60) chain amylopectin than under normal conditions, as well as higher crystallinity and gelatinization properties.

The plastid ribosome is essential for chloroplast biogenesis as well as seedling formation. As the plastid ribosome closely resembles the prokaryotic 70S ribosome, many plastid ribosomal proteins (PRPs) have been identified in higher plants. However, their assembly in the chloroplast ribosome in rice remains unclear. In the present study, we identified a novel rice mutant, albino lethal 1 (al1), from a chromosome segment substitution line population. The al1 mutant displayed an albino phenotype at the seedling stage and did not survive past the three-leaf stage. No other apparent differences in plant morphology were observed in the al1 mutant. The albino phenotype of the al1 mutant was associated with decreased chlorophyll content and abnormal chloroplast morphology. Using fine mapping, AL1 was shown to encode the PRPL12, a protein localized in the chloroplasts of rice, and a spontaneous single-nucleotide mutation (C/T), resulting in a residue substitution from leucine in AL1 to phenylalanine in al1, was found to be responsible for the early seedling lethality. This point mutation is located at the L10 interface feature of the L12/AL1 protein. Yeast two-hybrid analysis showed that there was no physical interaction between al1 and PRPL10. In addition, the mutation had little effect on the transcript abundance of al1, but had a remarkable effect on the protein abundance of al1 and transcript abundance of chloroplast biogenesis-related and photosynthesis-related genes. These results provide a first glimpse into the molecular details of L12’s function in rice.

•High-amylose TRS starch had higher resistance to alkali treatment than TQ starch.•Alkali treatment had no significant effect on the crystalline structure of starch.•Alkali treatment increased the hydrolysis of starch by HCl and amylolytic enzymes.•The 0.4% NaOH treatment affected morphology and SAXS peak intensity of starch.•TRS starch treated with 0.4% NaOH had lower AC, and higher To and Tp.Native starches were isolated from mature grains of high-amylose transgenic rice TRS and its wild-type rice TQ and treated with 0.1% and 0.4% NaOH for 7 and 14 days at 35 °C. Alkali-treated starches were characterised for structural and functional properties using various physical methods. The 0.1% NaOH treatment had no significant effect on structural and functional properties of starches except that it markedly increased the hydrolysis of starch by amylolytic enzymes. The 0.4% NaOH treatment resulted in some changes in structural and functional properties of starches. The alkali treatment affected granule morphology and decreased the electron density between crystalline and amorphous lamellae of starch. The effect of alkali on the crystalline structure including long- and short-range ordered structure was not pronounced. Compared with control starch, alkali-treated TRS starches had lower amylose content, higher onset and peak gelatinisation temperatures, and faster hydrolysis of starch by HCl and amylolytic enzymes.

High-amylose cereal starches usually have heterogeneous starch granules in morphological structure. In the present study, the polygonal, aggregate, elongated, and hollow starch granules were separated from different regions of the kernels of high-amylose rice, and their structures were investigated. The results showed that the polygonal starch granules had low amylose content and high short branch-chain and branching degree of amylopectin, and exhibited A-type crystallinity. The aggregate starch granules had high long branch-chain of amylopectin, relative crystallinity, and double helix content, and exhibited C-type crystallinity. The elongated starch granules had high amylose content and low branching degree of amylopectin and relative crystallinity, and exhibited C-type crystallinity. The hollow starch granules had very high amylose content, proportion of amorphous conformation, and amylose–lipid complex, and very low branch-chain of amylopectin, branching degree of amylopectin, and double helix content, and exhibited no crystallinity. The different structures of heterogeneous starch granules from high-amylose rice resulted in significantly different thermal properties.

We discovered four QTLs that maintain proper rice amylose content at high temperature by increasing the splicing efficiency ofWxgene.Amylose content mainly controlled by Wx gene is a key physicochemical property for eating and cooking quality in rice. During the grain filling stage, high temperature can harm rice grain quality by significantly reducing the amylose content in many rice varieties. Here, we provide genetic evidences between Wx gene expression and rice amylose content at high temperature, and identified several quantitative trait loci (QTLs) in this pathway. We performed a genome-wide survey on a set of chromosome segment substitution lines (CSSLs) which carried chromosomal segments from the heat resistant indica 9311 in the heat-sensitive japonica Nipponbare background. Four QTLs, qHAC4, qHAC8a, qHAC8b and qHAC10, which can reduce the deleterious effects of amylose content at high temperature, were identified and mapped to chromosome 4, 8, 8 and 10, respectively. The major QTL qHAC8a, with the highest LOD score of 6.196, was physically mapped to a small chromosome segment (~300 kb). The CSSLs carrying the qHAC8a, qHAC8b and/or qHAC4 from 9311 have the high pre-mRNA splicing efficiency of Wx gene and likely lead to stable amylose content at high temperature. Thus, increasing pre-mRNA processing efficiency of Wx gene could be an important regulation mechanism for maintaining stable amylose content in rice seeds at high temperature. In addition, our results provide a theoretical basis for breeding heat-stable grain in rice.

High-amylose starches are attracting considerable attention because of their potential health benefits and industrial uses. Enzyme hydrolysis of starch is involved in many biological and industrial processes. In this paper, starches were isolated from high-amylose transgenic rice (TRS) and its wild type rice, Te-qing (TQ). The morphological and structural changes of starch residues following Aspergillus niger amyloglucosidase (AAG) hydrolysis were investigated. AAG hydrolysed TQ starch from the granule surface, and TRS starch from the granule interior. During AAG hydrolysis, the content of amorphous structure increased, the contents of ordered structure and single helix decreased, and gelatinisation enthalpy decreased in TQ and TRS starch residues. The A-type polymorph of TRS C-type starch was hydrolysed faster than the B-type polymorph. The short-range ordered structure and B-type polymorph in the peripheral region of the subgranule and the surrounding band of TRS starch increased the resistance of TRS starch to AAG hydrolysis.Highlights► High-amylose rice TRS starch was more resistant to AAG hydrolysis than TQ starch. ► Crystalline structure was hydrolysed faster than the amorphous structure. ► The AAG hydrolysis pattern of TRS starch was different from that of TQ starch. ► A-type polymorph of TRS C-type starch was hydrolysed faster than B-type polymorph. ► The external region with B-type crystal increased resistance of TRS starch to AAG.

Heading date is a major determinant of adaptability and yield potential in rice (Oryza sativa L.) and is influenced by photoperiod. Among chromosome segment substitution lines, the introgression line C63 contains a segment of the short arm of chromosome 6 from indica Qingluzhan 11 in the japonica Nipponbare background and exhibits a delayed heading date under both long day (LD) and short day (SD) natural field conditions. This study demonstrates that the late heading date of the C63 line is controlled by a single recessive gene, Heading date from Qingluzhan 11 (Hd-q). Hd-q was mapped to a region of less than 43.7 kb. Complementation testing revealed that Ef7 (LOC_Os06g05060), a homolog of ArabidopsisELF3, is the candidate gene, while Hd-q is a new allele of Ef7. Sequence alignment revealed at least five Ef7 alleles among 11 rice cultivars based on polymorphism in the coding region. Unlike other alleles, Hd-q has a single nucleotide polymorphism (T/A) in exon 2, which leads to premature termination of translation. In addition to delayed heading date, Hd-q has pleiotropic effects on major agronomic characteristics, which were determined by comparing the near-isogenic line, NIL (Hd-q), with its recurrent parent Nipponbare. The Hd-q allele improved grain yield under both LD and SD conditions and in different geographical regions. Finally, a dCAPS (derived cleaved amplified polymorphic sequence) marker was developed based on the T/A polymorphism, and will be useful for introgression of the Hd-q allele via marker-assisted selection. The Hd-q allele is a useful target for the improvement of rice adaptation and production, especially at low latitudes.

Vitamin E comprises a group of eight lipid soluble antioxidant compounds that are an essential part of the human diet. The α-isomers of both tocopherol and tocotrienol are generally considered to have the highest antioxidant activities. γ-tocopherol methyltransferase (γ-TMT) catalyzes the final step in vitamin E biosynthesis, the methylation of γ- and δ-isomers to α- and β-isomers. In present study, the Arabidopsisγ-TMT (AtTMT) cDNA was overexpressed constitutively or in the endosperm of the elite japonica rice cultivar Wuyujing 3 (WY3) by Agrobacterium-mediated transformation. HPLC analysis showed that, in brown rice of the wild type or transgenic controls with empty vector, the α-/γ-tocotrienol ratio was only 0.7, much lower than that for tocopherol (~19.0). In transgenic rice overexpressing AtTMT driven by the constitutive Ubi promoter, most of the γ-isomers were converted to α-isomers, especially the γ- and δ-tocotrienol levels were dramatically decreased. As a result, the α-tocotrienol content was greatly increased in the transgenic seeds. Similarly, over-expression of AtTMT in the endosperm also resulted in an increase in the α-tocotrienol content. The results showed that the α-/γ-tocopherol ratio also increased in the transgenic seeds, but there was no significant effect on α-tocopherol level, which may reflect the fact that γ-tocopherol is present in very small amounts in wild type rice seeds. AtTMT overexpression had no effect on the absolute total content of either tocopherols or tocotrienols. Taken together, these results are the first demonstration that the overexpression of a foreign γ-TMT significantly shift the tocotrienol synthesis in rice, which is one of the world’s most important food crops.

Evaluating exogenous protein expressed in transgenic crops is one of the most effective methods of assessing the safety of transgenic plants. The objective of this study was to assess the food safety of genetically modified (GM) rice containing a lysine-rich fusion protein gene (transgenic GL gene rice) by in vitro digestion and acute toxicity testing of exogenous protein, according to the national standard of the People’s Republic of China. The exogenous protein was rapidly degraded in the simulated gastric and intestinal fluids. In the acute experiment, the exogenous protein was injected into Institute of Cancer Research (ICR) mice via the tail vein at a dose of 438 mg kg−1 body weight. No adverse effects on animal behavior or mortality were observed during the following 15-day period and there were no significant biological changes in body weight, serum biochemistry parameters, relative organ weights or histopathological examinations, compared with the control group. Therefore, exogenous protein in transgenic GL gene rice has a low potential allergenicity or toxicity risk.

Grain shape and size are two key factors that determine rice yield and quality. In the present study, a rice triangular hull mutant (tri1) was obtained from the progeny of japonica rice variety Taipei 309 treated with 60Co γ-rays. Compared to the wild type, the tri1 mutant presents a triangular hull, and exhibits an increase in grain thickness and protein content, but with a slight decrease in plant height and grain weight. Genetic analysis indicated that the mutant phenotype was controlled by a recessive nuclear gene which is stably inherited. Using a map-based cloning strategy, we fine-mapped tri1 to a 47-kb region between the molecular markers CHR0122 and CHR0127 on the long arm of chromosome 1, and showed that it co-segregates with the molecular marker CHR0119. According to the rice genome sequence annotation there are six predicated genes within the mapped region. Sequencing analysis of the mutant and the wild type indicated that there was a deletion of an A nucleotide in exon 3 of the OsMADS32 gene, which could result in a downstream frameshift mutation and premature termination of the predicted polypeptide. Both semi-quantitative and real-time RT-PCR analyses showed that this gene expressed highly in young inflorescences, while expressed at very low levels in other tissues. These results implied that the OsMADS32 gene could be a candidate of TRI1. Taken together, the results of this study lay the foundation for further investigation into the molecular mechanisms regulating rice caryopsis development.

High-amylose cereal starch has a great benefit on human health. Acid modification is very helpful for application of high-amylose starch in food and non-food industries. In this study, the ordered structure of acid-modified high-amylose rice starch was investigated by GPC, HPAEC, 13C CP/MAS NMR and XRD. Acid preferentially degraded the amylose, then A chain and short B chain of amylopectin. Relative double helix content and crystallinity both initially increased sharply and then progressively with acid hydrolysis. The relative crystallinity of starches obtained from 13C CP/MAS NMR was higher than that from XRD. The onset gelatinisation temperature decreased, while the peak and conclusion temperatures increased with increasing hydrolysis time. The endothermic value initially increased and then decreased with acid hydrolysis. The swelling power decreased while solubility increased after acid hydrolysis. These results add to our understanding of the effect of acid hydrolysis on the high-amylose rice starch.Highlights► Acid initially degraded the amylose, then A chain and short B chain of amylopectin. ► Relative double helix content and crystallinity increased with acid hydrolysis. ► The relative crystallinity estimated from 13C CP/MAS NMR was higher than from XRD. ► The To decreased, while the Tp and Tc increased with increasing hydrolysis time. ► The swelling power decreased while solubility increased after acid hydrolysis.

In this paper, endosperm starches were isolated from a high-amylose transgenic rice line (TRS) and its wild type rice Teqing (TQ) kernels at different developmental stages. TQ and TRS starches showed similar amylose contents and shapes at early developmental stage, then the amylose content increased with kernel development. The rate of increase in amylose content was much faster in TRS starches than that in TQ starches. TRS starches showed heterogenous granules at the middle and late developmental stages. TQ starch crystallinity remained A-type, but TRS starch crystallinity changed from A- to C- via CA-type. TRS starches showed higher gelatinization temperatures, lower gelatinization enthalpies, lower swelling powers, and lower hydrolysis rates at middle and late developmental stages compared with TQ starches. The amylose content had a significantly negative correlation with crystallinity, gelatinization enthalpy, swelling power, enzyme digestibility, and acid hydrolysis.Highlights► TQ and TRS starches showed similar amylose contents and shapes at early developmental stage. ► Heterogeneous TRS starches had higher amylose content than homogeneous TQ starches did at middle and late developmental stage. ► TRS starch crystallinity changed from A- to C- via CA-type with kernel development. ► TRS starches showed different crystal, thermal and hydrolytic properties from TQ with amylose content increase. ► The amylose content had negative correlations with crystallinity, gelatinization enthalpy, swelling power, enzyme digestibility, and acid hydrolysis.

High-amylose cereal starch has a great benefit on human health through its resistant starch content. In this paper, starches were isolated from mature grains of high-amylose transgenic rice line (TRS) and its wild-type rice cultivar Te-qing (TQ) and digested in vitro and in vivo. The structural changes of digestive starch residues were characterized using DSC, XRD, 13C CP/MAS NMR, and ATR-FTIR. TQ starch was very susceptible to digestion; its residues following in vitro and in vivo digestion showed similar structural characteristics with TQ control starch, which suggested that both amorphous and crystalline structures were simultaneously digested. Both amorphous and the long-range order structures were also simultaneously hydrolyzed in TRS starch, but the short-range order (double helix) structure in the external region of TRS starch granule increased with increasing digestion time. The A-type polymorph of TRS C-type starch was hydrolyzed more rapidly than the B-type polymorph. These results suggested that B-type crystallinity and short-range order structure in the external region of starch granule made TRS starch resistant to digestion.

A transgenic rice line (TRS) enriched amylose and resistant starch has been developed by antisense RNA inhibition of starch branching enzymes. In this study, gelatinisation and crystalline properties, swelling power, water solubility, morphological and structural changes of starches from TRS and its wild-type Teqing (TQ) were carefully investigated during heating. Compared to TQ, TRS starch showed higher gelatinisation temperatures, lower gelatinisation enthalpy and swelling power. Morphological and structural changes showed that TQ starch drastically swelled after 70 °C, then gradually disrupted with increasing heating temperature. The surrounding band of TRS starch restrained granule swelling, though the subgranules disrupted to form the cavity. The results of spectroscopic analyses indicated that A-type crystalline of TQ changed to amorphous starch after 75 °C, while C-type crystalline of TRS gradually changed to B-type crystalline after 75 °C, then became amorphous starch at 95 °C. These results add to our understanding of the effect of heating on the high-amylose rice starch.Highlights► TRS starch shows higher temperature, lower enthalpy of gelatinisation compared to TQ. ► TRS starch shows lower swelling power and higher solubility at high temperature. ► TQ starch swells and gradually disrupts, and becomes amorphous starch after 75 °C. ► The outer band of TRS starch restrains starch swell and maintains its native shape. ► C-type crystal of TRS gradually changes to B-type, then becomes amorphous starch.

High-amylose cereal starch has a great benefit on human health through its resistant starch (RS) content. Enzyme hydrolysis of native starch is very helpful in understanding the structure of starch granules and utilizing them. In this paper, native starch granules were isolated from a transgenic rice line (TRS) enriched with amylose and RS and hydrolyzed by α-amylase. Structural properties of hydrolyzed TRS starches were studied by X-ray powder diffraction, Fourier transform infrared, and differential scanning calorimetry. The A-type polymorph of TRS C-type starch was hydrolyzed faster than the B-type polymorph, but the crystallinity did not significantly change during enzyme hydrolysis. The degree of order in the external region of starch granule increased with increasing enzyme hydrolysis time. The amylose content decreased at first and then went back up during enzyme hydrolysis. The hydrolyzed starches exhibited increased onset and peak gelatinization temperatures and decreased gelatinization enthalpy on hydrolysis. These results suggested that the B-type polymorph and high amylose that formed the double helices and amylose–lipid complex increased the resistance to BAA hydrolysis. Furthermore, the spectrum results of RS from TRS native starch digested by pancreatic α-amylase and amyloglucosidase also supported the above conclusion.

Chromosome segment duplications are integral in genome evolution by providing a source for the origin of new genes. In the rice genome, besides an ancient polyploidy event known in the rice common ancestor, it had been identified that there was a special segmental duplication involving chromosomes 11 and 12, but the biological role of this duplication remains unknown. In this study, by using a set of chromosome segment substitution lines (CSSLs) and near isogenic lines (NILs) derived from the indica cultivar 9311 and japonica cultivar Nipponbare, a major QTL (qS12) resulting in hybrid male sterility was mapped within ~400 kb region adjacent to the special duplicated segment on the short arm of chromosome 12. Compared to the japonica cultivar Nipponbare, the two sides of the qS12 candidate region were inverted in the indica cultivar 9311. Among 47 of the 111 rice genotypes evaluated by molecular markers, the inverted sides were detected, and found completely homologous to indica cultivar 9311. These results suggested that the two inverted sides protect the sequence in the qS12 regions from recombination. On the short-arm of chromosome 12, two QTLs S-e and S25, in addition to qS12, were previously detected as a distinct segregation distortion and pollen semi-sterility loci. We propose these three hybrid sterility loci are the same locus, and the duplicated segment on chromosome 12 may play a prominent role in diversification, i.e., sub-speciation of cultivated 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.

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.

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.

Cereal starch granules with high-amylose and resistant starch (RS) always show irregular morphology and special crystalline structure, but their formation during grain development is not yet clear. In our previous studies, we had generated a transgenic rice line (TRS) enriched with amylose and RS, which contained semi-compound starch showing a C-type crystalline structure. In this study, the formation of semi-compound C-type starch granule during TRS endosperm development was carefully investigated with light, scanning electron, and transmission electron microscopes and X-ray powder diffraction. The results showed that the TRS starch subgranules, each with a central hilum, were individually initiated in amyloplast and showed an A-type crystal at the early stage of starch granule development, which was similar to that in its wild type. However, with the endosperm development, the amylose content in TRS endosperm starch increased and the B-type starch crystal was deposited in the periphery of subgranules; then, the adjacent subgranules fused together and finally formed a continuous outer layer band surrounding the entire circumference of the starch granule. Accordingly, a mechanistic model for the formation of semi-compound C-type starch granules is proposed.

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.

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.

To produce selectable marker-free (SMF) transgenic rice resistant to chewing insects, the Bacillus thuringiensis cryIA(c) gene (Bt) was introduced into two elite japonica rice varieties by using two Agrobacterium-mediated co-transformation systems. One system is with a single mini-twin T-DNA binary vector in one Agrobacterium strain, which consists of two separate T-DNA regions, one carrying the Bt while the other contains the selectable marker gene, hygromycin resistant gene (HPT). The other system uses two separate binary vectors in two separate Agrobacterium cultures, containing the Bt or HPT gene on individual plasmids. A lot of independent transgenic rice lines harboring both Bt and selectable marker genes were obtained. The results showed that the co-transformation frequency of the Bt gene and HPT gene was much higher by using the mini-twin T-DNA vector system (29.87%) than that by the two separate binary vector systems (4.52%). However, the frequency of the SMF transgenic rice plants obtained from the offspring of co-transgenic plants (21.74%) was lower for the mini-twin T-DNA vector system than that for the latter (50–60%). The data of ELISA implied that the expressed Bt proteins were quantitated as 0.025–0.103% of total leaf soluble proteins in the transgenic plant. Therefore, several elite transgenic rice lines, free of the selectable marker gene, were chosen. The results from both in vitro and in vivo insect bioassays indicated that the SMF transgenic rice was shown to be highly resistant to the striped stem borer and rice leaf folder. Moreover, in a natural field condition without any insecticide applied, all the transgenic rice plants were found to be not injured by the rice leaf folder, whereas the wild types were impaired seriously.

We identified a leafy head mutant pla1-5 (plastochron 1-5) from the progeny of japonica rice cultivar Taipei 309 treated with 60Co-γ ray irradiation. The pla1-5 mutant has a dwarf phenotype and small leaves. Compared with its wild type, pla1-5 has more leaves and fewer tillers, and it fails to produce normal panicles at the maturity stage. Genetic analysis showed that the pla1-5 phenotype is controlled by a single recessive nuclear gene. Using the map-based cloning strategy, we narrowed down the location of the target gene to a 58-kb region between simple sequence repeat markers CHR1027 and CHR1030 on the long arm of chromosome 10. The target gene cosegregated with molecular markers CHR1028 and CHR1029. There were five predicted genes in the mapped region. The results from sequencing analysis revealed that there was one base deletion in the first exon of LOC_Os10g26340 encoding cytochrome P450 CYP78A11 in the pla1-5 mutant, which might result in a downstream frame shift and premature termination. These results suggest that the P450 CYP78A11 gene is the candidate gene of PLA1-5.

The rapid visco analyser (RVA) profile is an important factor for evaluation of the cooking and eating quality of rice. To improve rice quality, the identification of new quantitative trait loci (QTLs) for RVA profiling is of great significance. We used a japonica rice cultivar Nipponbare as the recipient and indica rice 9311 as the donor to develop a population containing 38 chromosome segment substitution lines (CSSLs) genotyped by a high-throughput re-sequencing strategy. In this study, the population and the parent lines, which contained similar apparent amylose contents, were used to map the QTLs of RVA properties including peak paste viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BKV), setback viscosity (SBV), consistency viscosity (CSV), peak time (PeT) and pasting temperature (PaT). QTL analysis was carried out using one-way analysis of variance and Dunnett's test, and stable QTLs were identified over two years and under two environments. We identified 10 stable QTLs: qPKV2-1, qSBV2-1; qPKV5-1, qHPV5-1, qCPV5-1; qPKV7-1, qHPV7-1, qCPV7-1, qSBV7-1; and qPKV8-1 on chromosomes 2, 5, 7 and 8, respectively, with contributions ranging from −95.6% to 47.1%. Besides, there was pleiotropy in the QTLs on chromosomes 2, 5 and 7.

Development of new technologies for evaluating genetically modified (GM) crops has revealed that there are unintended insertions and expression changes in GM crops. Profiling techniques are non-targeted approaches and are capable of detecting more unintended changes in GM crops. Here, we report the application of a comparative proteomic approach to investigate the protein profile differences between a GM rice line, which has a lysine-rich protein gene, and its non-transgenic parental line. Proteome analysis by two-dimensional gel electrophoresis (2-DE) and mass spectrum analysis of the seeds identified 22 differentially expressed protein spots. Apart from a number of glutelins that were detected as targeted proteins in the GM line, the majority of the other changed proteins were involved in carbohydrate metabolism, protein synthesis and stress responses. These results indicated that the altered proteins were not associated with plant allergens or toxicity.

In rice grains, high amylose content (AC) is correlated with poor grain quality, particularly in indica hybrid rice. To obtain indica hybrid rice with improved cooking and eating qualities, we introduced the antisense Waxy (Wx) gene into 2 elite parental lines of indica hybrid rice by using co-transformation methods. Subsequently, we selected several elite homozygous transgenic lines that did not contain the selectable marker. The expression of the endogenous Wx gene of the selected transgenic lines was significantly downregulated, resulting in low AC in the mature seeds; moreover, the AC in some lines reduced to the level observed in glutinous rice. With the decrease in AC, the gel consistency of the transgenic rice became softer, and the gelatinization temperature tended to be higher than those of the wild types, especially in the case of the Longtefu-derived transformants. We also analyzed the pasting properties of the selected transgenic low-AC lines, and we noted an improvement in the pasting properties of the transgenic rice lines. The results from a field trial indicated that the grain weights of the transgenic lines with lower AC exhibit remarkable reduction compared with those of the wild types.

There are nearly 1000 rice landrace varieties in the Taihu basin, China. To assess the genetic diversity of the rice, 24 intragenic molecular markers (representing 17 starch synthesis-related genes) were investigated in 115 Taihu basin rice landraces and 87 improved cultivars simultaneously. The results show that the average genetic diversity and polymorphism information content values of the landraces were higher than those of improved cultivars. In total, 41 and 39 allele combinations (of the 17 genes) were derived from the landraces and improved cultivars, respectively; only two identical allele combinations were found between the two rice variety sources. Cluster analysis, based on the molecular markers, revealed that the rice varieties could be subdivided into five groups and, within these, the japonica improved rice and japonica landrace rice varieties were in two separate groups. According to the quality reference criteria to classify the rice into grades, some of the landraces were found to perform well, in terms of starch quality. For example, according to NY/T595-2002 criteria from the Ministry of Agriculture of China, 25 and 33 landraces reached grade 1, in terms of their apparent amylose content and gel consistency. The varieties that had outstanding quality could be used as breeding materials for rice quality breeding programs in the future. Our study is useful for future applications, such as genetic diversity studies, the protection of rice variety and improvment of rice quality in breeding programs.

Eating and cooking qualities (ECQs) of rice are important attributes due to its major influence on consumer acceptability. To better understand the molecular mechanism of the variation in ECQs, we investigated and compared the expressions among different alleles of the Waxy (Wx) gene and its effect on ECQs in specialty rice cultivars. The results showed that the accumulation of amylose was positively and significantly correlated to the level of mature Wx mRNA and granule-bound starch synthase I (GBSS I) in developing rice grain at 12 days after flowering. The amount of GBSS I and its activity together are the main factors controlling amylose synthesis. Differences in ECQs among five Wx allele types were investigated in samples from 15 rice varieties. The apparent amylose content (AAC) and gel consistency (GC) were similar in each type of Wx allele. The AAC followed the order, Wxa type>Wxin type>Wxb type>Wxmg type>wx. Contrary to this, the GC showed an opposite trend compared to AAC. There was a wide variation in rapid visco analyzer (RVA) profile among five Wx allele types, while varieties sharing a specified Wx allele had basically the similar RVA profile, although there was a slight difference in some RVA parameters, peak, hot paste and cool paste viscosities.

Rice kernels of a transgenic high-amylose rice line and its wild type were examined by a high-resolution X-ray microtomography (XMT) and an environmental scanning electron microscope. Two-dimensional (2-D) cross-sectional images and 3-D objects from XMT were reconstructed and analyzed to elucidate their structural features. The lack of two isoforms of starch branching enzyme, termed SBEI and SBEIIb in high-amylose rice (HAR), resulted in a distinctly different grain inner density than wild-type rice (WTR). HAR had smaller, elongated starch granules with air spaces inside the kernels resulting in an opaque grain, whereas WTR had a tight endosperm with little air space and polygonal starch granules. XMT allowed a full 3-D characterization of the rice kernel structure and revealed that air space distribution was not uniform in the HAR kernel.Highlights► Rice with greater than 50% amylose content was developed. ► Ultrastructure of rice kernels was studied by X-ray microtomography. ► Images from X-ray microtomography were compared with scanning electron microscopy. ► High-amylose rice had more air spaces and gave an opaque appearance. ► Air space distribution was not uniform in the high-amylose rice kernel.

To study the efficiency of generating selectable marker-free (SMF) transgenic rice, two transformation methods were employed for four rice varieties (Wuxiangjing 9, Longtefu, Xieqingzao and Zhenshan 97). One method is by using a single twin T-DNA binary vector pYH592 in one Agrobacterium strain, which is composed of two separate T-DNA regions (one carrying an antisense Wx gene and the other carrying a HPT gene). The other one, named as two-strain/two-vector system, is by using two separate binary vectors in two separate Agrobacterium cultures. The results indicated that the average co-transformation frequencies of the antisense Wx gene and the HPT gene were 10.1% and 45.0%, respectively, for the four varieties. And the SMF transgenic plants selected from the offsprings of co-transformants were 55.6% and 60.0% in the two-strain/two-vector and twin T-DNA vector binary systems, respectively.

Chromosome segment substitution lines (CSSLs) are useful for the precise mapping of quantitative trait loci (QTLs) and dissection of the genetic basis of complex traits. In this study, two whole-genome sequenced rice cultivars, the japonica Nipponbare and indica 9311 were used as recipient and donor, respectively. A population with 57 CSSLs was developed after crossing and back-crossing assisted by molecular markers, and genotypes were identified using a high-throughput resequencing strategy. Detailed graphical genotypes of 38 lines were constructed based on resequencing data. These CSSLs had a total of 95 substituted segments derived from indica 9311, with an average of about 2.5 segments per CSSL and eight segments per chromosome, and covered about 87.4% of the rice whole genome. A multiple linear regression QTL analysis mapped four QTLs for 1000-grain weight. The largest-effect QTL was located in a region on chromosome 5 that contained a cloned major QTL GW5/qSW5 for grain size in rice. These CSSLs with a background of Nipponbare may provide powerful tools for future whole-genome discovery and functional study of essential genes/QTLs in rice, and offer ideal materials and foundations for japonica breeding.