The OsNAC24-OsNAP protein complex activates OsGBSSI and OsSBEI expression to fine-tune starch biosynthesis in rice endosperm.

Starch accounts for up to 90% of the dry weight of rice endosperm and is a key determinant of grain quality. Although starch biosynthesis enzymes have been comprehensively studied, transcriptional regulation of starch-synthesis enzyme-coding genes (SECGs) is largely unknown. In this study, we explored the role of a NAC transcription factor, OsNAC24, in regulating starch biosynthesis in rice. OsNAC24 is highly expressed in developing endosperm. The endosperm of osnac24 mutants is normal in appearance as is starch granule morphology, while total starch content, amylose content, chain length distribution of amylopectin and the physicochemical properties of the starch are changed. In addition, the expression of several SECGs was altered in osnac24 mutant plants. OsNAC24 is a transcriptional activator that targets the promoters of six SECGs; OsGBSSI, OsSBEI, OsAGPS2, OsSSI, OsSSIIIa and OsSSIVb. Since both the mRNA and protein abundances of OsGBSSI and OsSBEI were decreased in the mutants, OsNAC24 functions to regulate starch synthesis mainly through OsGBSSI and OsSBEI. Furthermore, OsNAC24 binds to the newly identified motifs TTGACAA, AGAAGA and ACAAGA as well as the core NAC-binding motif CACG. Another NAC family member, OsNAP, interacts with OsNAC24 and coactivates target gene expression. Loss-of-function of OsNAP led to altered expression in all tested SECGs and reduced the starch content. These results demonstrate that the OsNAC24-OsNAP complex plays key roles in fine-tuning starch synthesis in rice endosperm and further suggest that manipulating the OsNAC24-OsNAP complex regulatory network could be a potential strategy for breeding rice cultivars with improved cooking and eating quality.

OsNAC129 Regulates Seed Development and Plant Growth and Participates in the Brassinosteroid Signaling Pathway.

Grain size and the endosperm starch content determine grain yield and quality in rice. Although these yield components have been intensively studied, their regulatory mechanisms are still largely unknown. In this study, we show that loss-of-function of OsNAC129, a member of the NAC transcription factor gene family that has its highest expression in the immature seed, greatly increased grain length, grain weight, apparent amylose content (AAC), and plant height. Overexpression of OsNAC129 had the opposite effect, significantly decreasing grain width, grain weight, AAC, and plant height. Cytological observation of the outer epidermal cells of the lemma using a scanning electron microscope (SEM) revealed that increased grain length in the osnac129 mutant was due to increased cell length compared with wild-type (WT) plants. The expression of OsPGL1 and OsPGL2, two positive grain-size regulators that control cell elongation, was consistently upregulated in osnac129 mutant plants but downregulated in OsNAC129 overexpression plants. Furthermore, we also found that several starch synthase-encoding genes, including OsGBSSI, were upregulated in the osnac129 mutant and downregulated in the overexpression plants compared with WT plants, implying a negative regulatory role for OsNAC129 both in grain size and starch biosynthesis. Additionally, we found that the expression of OsNAC129 was induced exclusively by abscisic acid (ABA) in seedlings, but OsNAC129-overexpressing plants displayed reduced sensitivity to exogenous brassinolide (BR). Therefore, the results of our study demonstrate that OsNAC129 negatively regulates seed development and plant growth, and further suggest that OsNAC129 participates in the BR signaling pathway.

High-resolution quantitative trait locus mapping for rice grain quality traits using genotyping by sequencing.

Rice is a major food crop that sustains approximately half of the world population. Recent worldwide improvements in the standard of living have increased the demand for high-quality rice. Accurate identification of quantitative trait loci (QTLs) for rice grain quality traits will facilitate rice quality breeding and improvement. In the present study, we performed high-resolution QTL mapping for rice grain quality traits using a genotyping-by-sequencing approach. An F2 population derived from a cross between an elite japonica variety, Koshihikari, and an indica variety, Nona Bokra, was used to construct a high-density genetic map. A total of 3,830 single nucleotide polymorphism markers were mapped to 12 linkage groups spanning a total length of 2,456.4 cM, with an average genetic distance of 0.82 cM. Seven grain quality traits-the percentage of whole grain, percentage of head rice, percentage of area of head rice, transparency, percentage of chalky rice, percentage of chalkiness area, and degree of chalkiness-of the F2 population were investigated. In total, 15 QTLs with logarithm of the odds (LOD) scores >4 were identified, which mapped to chromosomes 6, 7, and 9. These loci include four QTLs for transparency, four for percentage of chalky rice, four for percentage of chalkiness area, and three for degree of chalkiness, accounting for 0.01%-61.64% of the total phenotypic variation. Of these QTLs, only one overlapped with previously reported QTLs, and the others were novel. By comparing the major QTL regions in the rice genome, several key candidate genes reported to play crucial roles in grain quality traits were identified. These findings will expedite the fine mapping of these QTLs and QTL pyramiding, which will facilitate the genetic improvement of rice grain quality.

Molecular Structure and Physicochemical Properties of Starches from Rice with Different Amylose Contents Resulting from Modification of OsGBSSI Activity.

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.

Modulation of amylose content by structure-based modification of OsGBSS1 activity in rice (Oryza sativa L.).

The rice Waxy (Wx) gene encodes granule-bound starch synthase 1 (EC 2.4.1.242), OsGBSS1, which is responsible for amylose synthesis in rice seed endosperm. In this study, we determined the functional contribution of eight amino acids on the activity of OsGBSS1 by introducing site-directed mutated Wx gene constructs into the wx mutant glutinous rice. The eight amino acid residues are suspected to play roles in OsGBSS1 structure maintenance or function based on homologous enzyme sequence alignment and homology modelling. Both OsGBSS1 activity and amylose content were analysed in homozygous transgenic lines carrying the mutated OsGBSS1 (Wx) genes. Our results indicate that mutations at diverse sites in OsGBSS1 reduces its activity by affecting its starch-binding capacity, its ADP-glucose-binding capability or its protein stability. Our results shed new light on the structural basis of OsGBSS1 activity and the mechanisms of OsGBSS1 activity on amylose synthesis in vivo. This study also demonstrates that it is feasible to finely modulate amylose content in rice grains by modifying the OsGBSS1 activity.

Oligomerization of rice granule-bound starch synthase 1 modulates its activity regulation.

Granule-bound starch synthase 1 (GBSS1) is responsible for amylose synthesis in cereals, and this enzyme is regulated at the transcriptional and post-transcriptional levels. In this study, we show that GBSS1 from Oryza sativa L. (OsGBSS1) can form oligomers in rice endosperm, and oligomerized OsGBSS1 exhibits much higher specific enzymatic activity than the monomer. A monomer-oligomer transition equilibrium for OsGBSS1 occurs in the endosperm during development. Redox potential is a key factor affecting the oligomer percentage as well as the enzymatic activity of OsGBSS1. Adenosine diphosphate glucose, the direct donor of glucose, also impacts OsGBSS1 oligomerization in a concentration-dependent manner. OsGBSS1 oligomerization is influenced by phosphorylation status, which was strongly enhanced by Mitogen-activated protein kinase (MAPK) and ATP treatment and was sharply weakened by protein phosphatase (PPase) treatment. The activity of OsGBSS1 affects the ratio of amylose to amylopectin and therefore the eating quality of rice. Understanding the regulation of OsGBSS1 activity may lead to the improvement of rice eating quality.

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.

OsRRMh, a Spen-like gene, plays an important role during the vegetative to reproductive transition in rice.

OsRRMh, a homologue of OsRRM, encodes a Spen-like protein, and is composed of two N-terminal RNA recognition motifs (RRM) and one C-terminal Spen paralogue and an orthologue C-terminal domain (SPOC). The gene has been found to be constitutively expressed in the root, stem, leaf, spikelet, and immature seed, and alternative splicing patterns were confirmed in different tissues, which may indicate diverse functions for OsRRMh. The OsRRMh dsRNAi lines exhibited late-flowering and a larger panicle phenotype. When full-length OsRRMh and/or its SPOC domain were overexpressed, the fertility rate and number of spikelets per panicle were both markedly reduced. Also, overexpression of OsRRMh in the Arabidopsis fpa mutant did not restore the normal flowering time, and it delayed flowering in Col plants. Therefore, we propose that OsRRMh may confer one of its functions in the vegetative-to-reproductive transition in rice (Oryza sativa L. subsp. japonica cv. Zhonghua No. 11 (ZH11)).

Establishing a gene trap system mediated by T-DNA(GUS) in rice.

Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promoterless beta-glucuronidase (GUS) reporter gene in the T-DNA region. Transformation of these two plasmids into the rice variety Zhonghua 11 (Oryza sativa ssp. japonica cv.), mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 T(0) plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistant (hyg(r)) homozygotes were screened and the copy number of the T-DNA inserts was determined in the GUS-positive transgenic plants. The flanking sequences of the T-DNA were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of T-DNA were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calli induced from the seeds of the T(1) generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. The results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.

[Preliminary screening of target genes of rice transcription factor OsBP-73].

Previous data showed that a 31-bp (from -840 bp to -810 bp) DNA fragment located at the 5' upstream region of rice waxy gene could interact with nuclear protein extracted from developing endosperm of rice. When this 31 bp DNA sequence was used as a bait to screen a rice cDNA library with a yeast one-hybrid system, three groups of cDNA clones were isolated. One of them is pC73, the correspondent rice gene of pC73 was named as OsBP-73 (Oryza sativa binding protein). A pull-down assay was made to identify the target genes of transcription factor by using genomic DNA and recombinant p73 protein. The cDNA fragment containing DNA-binding domain of OsBP-73 was cloned into expression vector pET28-c(+) (Fig.1) to produce protein p73, fused with a his(6)-tag, from E. coli BL21 (DE3) (Fig.2). The p73 was purified with Ni-NTA under native condition (Fig.3). The target genes of p73 were identified in rice genome-wide by using a pull-down assay, and 22 candidate genes were obtained (Figs.4 and 5, and Table 1). The obtained results show that putative light-repressible receptor protein kinase and GAMYB-binding protein could serve as targets of the OsBP-73, suggesting that OsBP-73 might be involved in light signal transduction.

OsRRM, a Spen-like rice gene expressed specifically in the endosperm.

We used the promoter trap technique to identify a rice plant, named 107#, in which the beta-glucuronidase (GUS) reporter gene was expressed specifically in the endosperm. A single copy of the T-DNA was inserted into the plant genome, and a candidate gene OsRRM was identified by the insertion. The OsRRM promoter directed GUS expression specifically in rice endosperm, analogous to the GUS expression pattern observed in 107#. OsRRM is a single-copy gene in rice and encodes a nuclear protein containing 1005 amino-acid residues with two RNA recognition motifs and one Spen paralog and ortholog C-terminal domain. Western blot analysis confirmed that the OsRRM protein was specifically expressed in rice endosperm. Ectopic expression of OsRRM in transgenic plants led to abnormalities, such as short stature, retarded growth and low fructification rates. Our data, in conjunction with the reported function of Spen genes, implicated OsRRM in the regulation of cell development in rice endosperm.

[Establishment of promoter trapping system mediated by activator/dissociation (beta-glucuronidase) construction in rice].

The coding region of Bar gene, the left border of Ds element, the coding region of GUS gene, the transposase of Ac element, the right border of Ds element and the promoter of Ubi gene were inserted into the T-DNA region of vector pCAMBIA1300 in turn to construct plasmid p13B. The orientations of the ubiquitons' promoter, Ac transposase and Bar are identical but opposite to that of the GUS gene (Fig.1). The plasmid p13B was then introduced into the calli of Oryza sativa subsp. japonica cv. Zhonghua 11 by Agrobacterium tumefaciens-mediated transforming to trap genes in rice. Eighteen independent transgenic lines were obtained and propagated. T(2) generations of 18 independent transgenic lines were screening by herbicide (Basta) (Fig.2) and the herbicide-resistant plants obtained were analyzed by PCR (Fig.3). Ds element transposed in an inheritable manner was found in 37 plants, in which 5 plants showed GUS activity (Fig.4).

A rice quantitative trait locus for salt tolerance encodes a sodium transporter.

Many important agronomic traits in crop plants, including stress tolerance, are complex traits controlled by quantitative trait loci (QTLs). Isolation of these QTLs holds great promise to improve world agriculture but is a challenging task. We previously mapped a rice QTL, SKC1, that maintained K(+) homeostasis in the salt-tolerant variety under salt stress, consistent with the earlier finding that K(+) homeostasis is important in salt tolerance. To understand the molecular basis of this QTL, we isolated the SKC1 gene by map-based cloning and found that it encoded a member of HKT-type transporters. SKC1 is preferentially expressed in the parenchyma cells surrounding the xylem vessels. Voltage-clamp analysis showed that SKC1 protein functions as a Na(+)-selective transporter. Physiological analysis suggested that SKC1 is involved in regulating K(+)/Na(+) homeostasis under salt stress, providing a potential tool for improving salt tolerance in crops.

[Rapidly obtaining the markerless transgenic rice with reduced amylose content by co-transformation and anther culture].

The plasmid p13W8 carrying antisense fragment of waxy gene and plasmid pCAMBIA1300 containing hpt gene were introduced into rice by Agrobacterium tumefaciens-mediated co-transformation, and 86 transgenic plants were obtained, 32 of them showed positive bands for antisense waxy gene by PCR analysis, the waxy-positive plant frequency is 37.2%. The segregation of antisense fragment of waxy gene and hpt gene was observed by PCR using hpt gene primers and waxy gene primers respectively in 29 T(1) population. One hundred and eighty-three plants containing only the antisense fragment of waxy gene were identified in 1 264 T(1) plants, the waxy-positive plant frequency is 14.4% (Table 1). The amylose content of seeds derived from transgenic plants with only the antisense fragment of waxy gene were determined, varying degrees of reduction in amylose content were found in some plants (Table 2). Four T(1) plants with reduced amylose content were selected through anther culture. Thirty-four anther culture plants seed normally, 23 of them were shown to contain only the antisense fragment of waxy gene (Table 3) by PCR analysis, and the amylose content was reduced to 5%-12% (Table 4). It took only one and half years to obtain the stably inherited markerless transgenic rice with reduced amylose content by co-transformation and anther culture technique.

An interaction between a MYC protein and an EREBP protein is involved in transcriptional regulation of the rice Wx gene.

We previously demonstrated that a 31-bp nucleotide sequence located upstream of the rice Wx gene played an important role in its expression. We further showed that this cis-acting regulator interacts with nuclear proteins extracted from developing rice endosperm. We used the 31-bp sequence as bait in a yeast one-hybrid system to isolate several cDNA clones from a rice cDNA expression library. One of these cDNAs encodes a MYC protein, designated OsBP-5, which is 335 amino acids long and contains a putative basic helix-loop-helix-ZIP DNA-binding domain. This domain exhibits 50% amino acid sequence identity with the R/B proteins that regulate the expression of genes involved in anthocyanin biosynthesis in plants. The results of electrophoretic mobility shift assays (EMSAs) and Southwestern gel blots indicate that this protein binds specifically to the CAACGTG motif within the 31-bp sequence. However, by itself, the OsBP-5 protein is unable to trans-activate a lacZ reporter gene controlled by the 31-bp sequence when tested in a yeast expression system. Interestingly, OsBP-5 can trans-activate this reporter gene when another protein, OsEBP-89, a member of the EREBP family of transcription factors, is present. Furthermore, in vitro pull-down experiments show that a protein isolated from developing rice endosperm interacts with the OsBP-5 protein, and Western blots confirm that the interacting protein is OsEBP-89. The formation of a supershift band in EMSAs also indicates that two proteins interact with each other. Interference of OsBP-5 gene expression by double-stranded RNA reduces the amylose content in mature seed of transgenic rice plants but has no visible effect on their phenotype. These results suggest that the OsBP-5 and OsEBP-89 proteins act synergistically, perhaps as a heterodimer, to regulate the transcription of the rice Wx gene.

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.

[Diagnosis and differential diagnosis of granulocytic sarcomas].

OBJECTIVE: To investigate the diagnosis and differential diagnosis of granulocytic sarcoma (GS). METHODS: The morphological and immunological characteristics of 12 cases of GS were studied. FAB classification was made by peripheral blood, bone marrow picture and bone marrow biopsy assay. RESULTS: All of the 12 cases presented with lymphadenopathy and soft tissue mass. Histologically, the tissue infiltration of GS was composed of blastic cells with round to oval nuclei showing an even, pale chromatin pattern. Some with cleaved or notched nuclei. There were prominent nucleoli and scant cytoplasm in the cells and mitosis was easily found. Immunohistochemically, CD(45) and lysozyme were positive in all of the cases, MPO in 11 (92%), CD(68) in 10 (83%), CD(34) in 5 (42%), and TdT in 2 cases (17%). CD(15) and Mac387 were mainly expressed in mature granulocytes. Examination of bone marrow sections and marrow aspirate smears showed that out of the 11 cases tested 8 were AML-M(2), 2 AML-M(1) and 1 AML-M(0). Only 1 case was nonleukemic, ie. solitary granulocytic sarcoma. CONCLUSION: Granulocytic sarcomas are difficult to identify in routine paraffin-embedded tissue sections and usually misdiagnosed as non-Hodgkin's lymphomas. Immunohistochemistry study with a panel of antibodies in combination with bone marrow and peripheral blood examination are helpful in identification of granulocytic sarcoma.