Expression of a rice glutelin promoter in transgenic tobacco

A chimeric gene consisting of the 5 flanking sequences of a rice glutelin gene (Gt3) linked to the chloramphenicol acetyltransferase (CAT) coding segment was introduced into tobacco via Agrobacterium tumefaciens-mediated transformation. CAT enzyme activity could be detected in extracts from seeds as early as 8 days after flowering and obtained a maximum level at 16 days after flowering, the onset of overall protein accumulation. Significant expression of CAT activity in non-seed tissues occurred in some, but not all plants, suggesting possible chromosome position effects on non-seed tissue expression. A positive correlation was observed between expression levels in seeds and gene copy numbers.Author for correspondence     

Sequence variation, differential expression and chromosomal location of rice chitinase genes

Rice chitinases are encoded by a small multigene family. To clarify the overall organization of rice chitinase genes, we have isolated and characterized the genes Cht-1, Cht-2 and Cht-3. Although all the three genes encode class I chitinase, the nucleotide sequences of the coding regions of Cht-1 and Cht-3 are very similar (90%), while that of Cht-2 is clearly more divergent (78%). Only Cht-2 has a 130 by intron and encodes a C-terminal peptide sequence similar to that known to function as a vacuolar targeting signal. In 5 flanking regions of Cht-1 and Cht-3, but not of Cht-2, conserved sequences (GGCCGGCYGCCCYAG) were found. Related sequences were found also in the 5 flanking regions of another chitinase gene and a -glucanase gene which has also been reported to be stress-induced in rice. RNA blot hybridization analysis demonstrated that the stress-induced expression patterns of the Cht-1 and Cht-3 genes are similar, but quite different from that of Cht-2. However, all three genes are active in unstressed roots. By restriction fragment length polymorphism (RFLP) linkage analysis, Cht-1 and Cht-3 were mapped onto chromosome 6 and shown to be closely linked (0.8 cM). Cht-2 was mapped onto chromosome 5. All these features suggest that the expression patterns of rice class I chitinase genes may be correlated with their levels of sequence divergence and their chromosomal location.     

Nucleotide sequence of a rice cDNA similar to a maize NADP-dependent malic enzyme

We have isolated a rice cDNA clone that is homologous to the gene for the maize NADP-dependent malic enzyme (EC 1.1.1.40; NADP-ME). The deduced amino acid sequence coded for by the cDNA indicates a high level of homology to chloroplast type NADP-ME, including a transit peptide with pronounced hydrophobic properties at the amino terminus. Northern blot analysis indicates that the expression of this gene is regulated by external stress such as submergence.     

Quantitative nature of the Prolamin-box, ACGT and AACA motifs in a rice glutelin gene promoter: minimal cis-element requirements for endosperm-specific gene expression

The -197 bp promoter of the rice seed storage protein gene, GluB-1, is capable of conferring endosperm-specific gene expression. This proximal 5' flanking region contains four motifs, GCN4, AACA, ACGT and Prolamin-box, which are conserved in many seed storage protein genes. We previously showed that multiple copies of GCN4 conferred endosperm expression pattern when fused to the -46 core promoter of CaMV 35S. In this paper we demonstrate, using a similar approach, that tandem repeated copies of any of the other three motifs are unable to direct expression in seeds as well as other tissues of transgenic rice plants. Mutational analysis of individual motifs in the -197 bp promoter resulted in remarkable reductions in promoter activity. These results indicate that the GCN4 motif acts as an essential element determining endosperm-specific expression and that the AACA, ACGT and Prolamin-box are involved in quantitative regulation of the GluB-1 gene. A set of gain-of-function experiments using transgenic rice showed that either the Prolamin-box or AACA, although often coupled with GCN4 in many genes, is insufficient to form a functional promoter unit with GCN4, whereas a combination of GCN4, AACA and ACGT motifs was found sufficient to confer a detectable level of endosperm expression. Taken together, our results provide direct insight into the importance of combinatorial interplay between cis-elements in regulating the expression of seed storage protein genes.     

Structural relationship among the members of a multigene family coding for the sweet potato tuberous root storage protein

Sporamin, the major soluble protein of the sweet potato tuberous root, is coded for by a multigene family. Fourty-nine essentially full-length sporamin cDNAs isolated from tuberous root cDNA library have been classified by cross hybridization, restriction endonuclease cleavage pattern and ribonuclease cleavage mapping. All the cDNAs fall into one of the two distinct homology groups, subfamilies A and B, which correspond to the polypeptide classes sporamin A and B, respectively. At least 5 different sequences are detected in both of the 22 sporamin A and 27 sporamin B cDNAs. Comparison of the nucleotide sequences of the coding region of three each of sporamin A and B subfamily members, four from cDNAs and two from genomic clones, indicates that intra-subfamily homologies (94 to 98%) are much higher than inter-subfamily homologies (82 to 84%), and there are deletions or insertions of one or two codons at three locations which characterize each subfamily. Large portions of base substitutions in the coding region accompany amino acid substitutions. In contrast to the coding region, most of the structural differences among the members in the 5 and 3 noncoding regions are deletions or insertions.     

The expression of iron homeostasis-related genes during rice germination

To characterize Fe homeostasis during the early stages of seed germination, a microarray analysis was performed. mRNAs extracted from fully mature seeds or seeds harvested 1–3 days after sowing were hybridized to a rice microarray containing approximately 22,000 cDNA oligo probes. Many Fe deficiency-inducible genes were strongly expressed throughout early seed germination. These results suggest that the demand for Fe is extremely high during germination. Under Fe-deficient conditions, rice produces and secretes a metal-cation chelator called deoxymugineic acid (DMA) to acquire Fe from the soil. In addition, DMA and its intermediate nicotianamine (NA) are thought to be involved in long distance Fe transport in rice. Using promoter-β-glucuronidase (GUS) analysis, we investigated the expression patterns during seed germination of the Fe deficiency-inducible genes OsNAS1, OsNAS2, OsNAS3, OsNAAT1, and OsDMAS1, which encode enzymes that participate in the biosynthesis of DMA, and the transporter genes OsYSL2 and OsIRT1, which are involved in Fe transport. All of these genes were expressed in germinating seeds prior to protrusion of the radicle. These results suggest that DMA and NA are produced and involved in Fe transport during germination. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A rice (Oryza sativa L.) mutant having a low content of glutelin and a high content of prolamine

Among the mutant lines of rice that have been selected for morphological characters, one line, NM67, was found to have a low content of glutelin and a higher content of prolamine in its seed protein than other Japanese cultivars. This mutant is a semi-dwarf and partially sterile line, and its leaves turn yellow before heading. Genetic analysis after backcross to the original cultivar, Nihonmasari, revealed the following: (1) the character of low glutelin content was always accompanied by the character of high prolamine content; (2) the low glutelin (and high prolamine) character seemed to be manifested by a single dominant gene; and (3) semi-dwarfness, low fertility and early yellowing leaf of the mutant, which might also be pleiotropy, were controlled by a single recessive gene independent of the gene for protein content. The protein character of NM67 was genetically separated from semi-dwarfness and low fertility, and a new line having low glutelin content and high prolamine content with normal morphological characters comparable to those of the original cultivar was obtained from the progenies of the cross. The possible use of this line as a low protein rice cultivar is discussed.     

Molecular cloning, expression and characterization of a glycosyltransferase from rice

Secondary plant metabolites undergo several modification reactions, including glycosylation. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, we cloned one of the glycosyltransferases from rice, RUGT-5 resulting in 40–42% sequence homology with UGTs from other plants. RUGT-5 was functionally expressed as a glutathione S-transferase fusion protein in Escherichia coli and was then purified. Eight different flavonoids were used as tentative substrates. HPLC profiling of reaction products displayed at least two peaks. Glycosylation positions were located at the hydroxyl groups at C-3, C-7 or C-4′ flavonoid positions. The most efficient substrate was kaempferol, followed by apigenin, genistein and luteolin, in that order. According to in vitro results and the composition of rice flavonoids the in vivo substrate of RUGT-5 was predicted to be kaempferol or apigenin. To our knowledge, this is the first time that the function of a rice UGT has been characterized.     

Mutants lacking glutelin subunits in rice: mapping and combination of mutated glutelin genes

Nine mutant lines lacking glutelin subunits were selected from M₂ seeds of about 10000 M₁ plants mutagenized with gamma rays or EMS and from 1400 mutant lines selected originally for morphological characters. There were three types of mutants, one line lacking the largest subunit among four minor bands of glutelin acidic subunits (Type 1), five lines lacking the second largest subunit band (Type 2), and three lines lacking the third largest subunit band (Type 3). Mutants lacking the smallest subunit band were not found. Type 1 lacked 2 of the 10 spots of glutelin acidic subunits separated by two-dimensional electrophoresis and 1 of the 11 spots of the 57-kDa glutelin precursor. Type 2 lacked 2 spots of acidic subunits and 1 spot of the 57-kDa glutelin precursor, and had low amounts of 1 of the 8 spots of glutelin basic subunits. Type 3 mutants lacked each of 1 spot of the acidic subunits and glutelin precursor and had low amount of 1 spot of the basic subunits. Genetic analysis of the mutated genes showed that these mutant characters were controlled by single recessive genes named glu-1, glu-2, and glu-3, respectively. Mutated genes of different lines of the same type were found to be at the same locus. RFLP analysis of F₂ plants between the mutant lines and cv `Kasalath' indicated that glu-1 is on chromosome 2, glu-2 on chromosome 10, and glu-3 on chromosome 1. These mutant genes were combined by crossing, and a line lacking the 3 minor bands of the glutelin acidic subunits was developed. However, the total glutelin content of this line was not remarkably reduced, showing a only 13% decrease. Received: 1 April 1996 / Accepted: 14 June 1996     

Extraction and composition of rice endosperm glutelin

“Crude” glutelin was prepared from milled rice (Oryza sativa) flour by sequential extraction of the albumin-globulin fraction with 0.5 M NaCl and prolamin with 70% ethanol-0.6% β-mercaptoethanol. The solvent, 0.5% sodium dodecyl sulphate (SDS)-0.6% β-mercaptoethanol, extracted 91% of the endosperm glutelin without gelatinizing starch granules, whereas chaotropic solvents such as urea and guanidine caused extensive gelatinization. The S-cyanoethyl glutelin (Ce-glutelin) prepared by SDS extraction of the “crude” glutelin (9.5% protein) of IR480-5-9 rice gave three major subunits with MW 38000, 25000 and 16000 in the ratio 2:1:1 as determined by SDS polyacrylamide gel electrophoresis. A similar preparation from “crude” glutelin of a lower protein containing rice had the corresponding subunits in the ratio of 16:3:1. The MW 38000 subunit was unique to glutelin and was not present in C3-albumin-globulin or prolamin; the subunits were only partially purified by SDS Sephadex G-150 gel-filtration. The C3-glutelin was also prepared from a crude glutelin-prolamin preparation from IR480-5-9 by NaOH extractions followed by precipitation at pH 10 and ethanol extraction of the precipitate (C3-glutelin). This preparation had the same three major subunits and in the same ratio as C3-glutelin prepared by the SDS method. The subunits of the former preparation were separated by carboxymethyl Sephadex C-50 chromatography; the MW 38000 subunit eluted between pH 6.2–8.5, the MW 25000 in an impure state at pH values above 9, and the MW 16000 subunit was eluted at pH 8.6—9.2. Amino acid composition of the Ce-glutelin preparations were similar to each other. The MW 38000 and 16000 subunits had lower lysine contents than whole C3-glutelin, whereas the MW 25000 subunit had a higher lysine content.     

Cloning and characterization of three genes encoding Qb-SNARE proteins in rice

Qb-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and function as important components of the vesicle trafficking machinery in eukaryotic cells. Here, we report three novel plant SNARE (NPSN) genes isolated from rice and named OsNPSN11, OsNPSN12 and OsNPSN13. They have about 70% nucleotide identity over their entire coding regions and similar genomic organization with ten exons and nine introns in each gene. Multiple alignment of deduced amino acid sequences indicate that the OsNPSNs proteins are homologous to AtNPSNs from Arabidopsis, containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region. Semi-quantitative RT-PCR assays showed that the OsNPSNs were ubiquitously and differentially expressed in roots, culms, leaves, immature spikes and flowering spikes. The expression of OsNPSNs was significantly activated in rice seedlings treated with H₂O₂, but down-regulated under NaCl and PEG6000 stresses. Transient expression method in onion epidermal cells revealed that OsNPSNs were located in the plasma membrane. Transformed yeast cells with OsNPSNs had better growth rates than empty-vector transformants when cultured on either solid or liquid selective media containing various concentrations of H₂O₂, but more sensitive to NaCl and mannitol stresses. The 35S:OsNPSN11 transgenic tobacco also showed more tolerance to H₂O₂ and sensitivity to NaCl and mannitol than non-transgenic tobacco. These results indicate that OsNPSNs may be involved in different aspects of the signal transduction in plant and yeast responses to abiotic stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sequence and expression analysis of the thioredoxin protein gene family in rice

Thioredoxin (Trx) proteins play important biological functions in cells by changing redox via thioldisulfied exchange. This system is especially widespread in plants. Through database search, we identified 30 potential Trx protein-encoding genes (OsTrx) in rice (Oryza sativa L.). An analysis of the complete set of OsTrx proteins is presented here, including chromosomal location, conserved motifs, domain duplication, and phylogenetic relationships. Our findings suggest that the expansion of the Trx gene family in rice, in large part, occurred due to gene duplication. A comprehensive expression profile of Trx genes family was investigated by analyzing the signal data of this family extracted from the whole genome microarray analysis of Minghui 63 and Zhenshan 97, two indica parents, and their hybrid Shanyou 63, using 27 different tissues representing the entire life cycle of rice. Results revealed specific expression of some members at germination transition as well as the 3-leaf stage during the vegetative growth phase of rice. OsTrx genes were also found to be differentially up- or down-regulated in rice seedlings subjected to treatments of phytohormones and light/dark conditions. The expression levels of the OsTrx genes in the different tissues and under different treatments were also checked by RT-PCR analysis. The identification of OsTrx genes showing differential expression in specific tissues among different genotypes or in response to different environmental cues could provide a new avenue for functional analyses in rice.     

Expression of TERF1 in rice regulates expression of stress-responsive genes and enhances tolerance to drought and high-salinity

Drought and high-salinity are the important constraints that severely affect plant development and crop yield worldwide. It has been established that ethylene response factor (ERF) proteins play important regulatory roles in plant response to abiotic and biotic stresses. Our previous researches have revealed that transgenic tobacco over-expressing TERF1 (encoding a tomato ERF protein) showed enhanced tolerance to abiotic stress. Here, we further investigate the function of TERF1 in transgenic rice. Compared with the wild-type plants, overexpression of TERF1 resulted in an increased tolerance to drought and high-salt in transgenic rice. And the enhanced tolerance may be associated with the accumulation of proline and the decrease of water loss. Furthermore, TERF1 can effectively regulate the expression of stress-related functional genes Lip5, Wcor413-l, OsPrx and OsABA2, as well as regulatory genes OsCDPK7, OsCDPK13 and OsCDPK19 under normal growth conditions. Our analyses of cis-acting elements show that there exist DRE/CRT and/or GCC-box existing in TERF1 targeted gene promoters. Our results revealed that ectopic expression of TERF1 in rice caused a series of molecular and physiological alterations and resulted in the transgenic rice with enhanced tolerance to abiotic stress, indicating that TERF1 might have similar regulatory roles in response to abiotic stress in tobacco and rice. Shumei Gao, Haiwen Zhang and Yun Tian contributed equally to this work.     

Bacterial expression of an active class Ib chitinase from Castanea sativa cotyledons

Ch3, an endochitinase of 32 kDa present in Castanea sativa cotyledons, showed in vitro antifungal properties when assayed against Trichoderma viride. The characterization of a cDNA clone corresponding to this protein indicated that Ch3 is a class Ib endochitinase that is synthesized as a preprotein with a signal sequence preceding the mature polypeptide. Bacterial expression of mature Ch3 fused to the leader peptide of the periplasmic protein ompT resulted in active Ch3 enzyme. A plate assay was adapted for semi-quantitative determination of chitinase activity secreted from cultured bacteria, which should facilitate the identification of mutants with altered capacity to hydrolyse chitin.     

cDNA cloning and gene expression of the major prolamins of rice

A full-length cDNA (pS 18) encoding the 16 kDa rice prolamin composed of 158 amino acids was sequenced. Analysis of N-terminal amino acid sequence of a major rice prolamin indicated that an 18 amino acid signal peptide was removed from 16 kDa precursor prolamin to form the 14 kDa prolamin during seed development. Synthesis of the 16 kDa precursor prolamin began around 8 days after flowering (DAF), increased remarkably at 8–11 DAF and gradually reached maximum levels with the maturation of rice seeds.