Molecular analyses of the metallothionein gene family in rice (Oryza sativa L.)

Metallothioneins are a group of low molecular mass and cysteine-rich metal-binding proteins, ubiquitously found in most living organisms. They play an important role in maintaining intracellular metal homeostasis, eliminating metal toxification and protecting against intracellular oxidative damages. Analysis of complete rice genome sequences revealed eleven genes encoding putative metallothionein (OsMT), indicating that OsMTs constitute a small gene family in rice. Expression profiling revealed that each member of the OsMT gene family differs not only in sequence but also in their tissue expression patterns, suggesting that these isoforms may have different functions they perform in specific tissues. On the basis of OsMT structural and phylogenetic analysis, the OsMT family was classified as two classes and class I was subdivided into four types. Additionally, in this paper we also present a complete overview of this family, describing the gene structure, genome localization, upstream regulatory element, and exon/intron organization of each member in order to provide valuable insight into this OsMT gene family.     

rHsp90 gene expression in response to several environmental stresses in rice (Oryza sativa L.).

In this study, the gene for a rice (Oryza sativa L.) 90 kDa heat shock protein (rHsp90, GenBank accession no. AB037681) was identified by screening rice root cDNAs that were up-regulated under carbonate (NaHCO(3)) stress using the method of differential display, and cloned. The open-reading-frame of rHsp90-cDNA was predicted to encode a protein containing 810 amino acids, which showed high similarity to proteins in Hordeum vulgare (accession no. X67960) and Catharathus roseus (accession no. L14594). Further studies showed that rHsp90 mRNA accumulated following exposure to several abiotic stresses, including salts (NaCl, NaHCO(3) and Na(2)CO(3)), desiccation (using polyethylene glycol), high pH (8.0 and 11.0) and high temperature (42 and 50 degrees C). Yeast (Saccharomyces cerevisiae) over-expressing rHsp90 exhibited greater tolerance to NaCl, Na(2)CO(3) and NaHCO(3) and tobacco seedlings over-expressing rHsp90 could tolerate salt concentrations as high as 200 mM NaCl, whereas untransformed control seedlings couldn't. These results suggest that rHsp90 plays an important role in multiple environmental stresses.     

Structure, expression, and functional analysis of the hexokinase gene family in rice (Oryza sativa L.)

Hexokinase (HXK) is a dual-function enzyme that both phosphorylates hexose to form hexose 6−phosphate and plays an important role in sugar sensing and signaling. To investigate the roles of hexokinases in rice growth and development, we analyzed rice sequence databases and isolated ten rice hexokinase cDNAs, OsHXK1 (Oryza sativa Hexokinase 1) through OsHXK10. With the exception of the single-exon gene OsHXK1, the OsHXKs all have a highly conserved genomic structure consisting of nine exons and eight introns. Gene expression profiling revealed that OsHXK2 through OsHXK9 are expressed ubiquitously in various organs, whereas OsHXK10 expression is pollen-specific. Sugars induced the expression of three OsHXKs, OsHXK2, OsHXK5, and OsHXK6, in excised leaves, while suppressing OsHXK7 expression in excised leaves and immature seeds. The hexokinase activity of the OsHXKs was confirmed by functional complementation of the hexokinase-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). OsHXK4 was able to complement this mutant only after the chloroplast-transit peptide was removed. The subcellular localization of OsHXK4 and OsHXK7, observed with green fluorescent protein (GFP) fusion constructs, indicated that OsHXK4 is a plastid-stroma-targeted hexokinase while OsHXK7 localizes to the cytosol.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The phytocyanin gene family in rice (Oryza sativa L.): genome-wide identification, classification and transcriptional analysis

Background

Phytocyanins (PCs) are plant-specific blue copper proteins involved in electron transport, and a large number of known PCs are considered to be chimeric arabinogalactan proteins (AGPs). To date there has not been a genome-wide overview of the OsPC gene family. Therefore, as the first step and a useful strategy to elucidate the functions of OsPCs, there is an urgent need for a thorough genome-wide analysis of this gene family.

Methodology/Principal Findings

In this study, a total of 62 OsPC genes were identified through a comprehensive bioinformatics analysis of the rice (Oryza sativa L.) genome. Based on phylogeny and motif constitution, the family of OsPCs was classified into three subclasses: uclacyanin-like proteins (OsUCLs), stellacyanin-like proteins (OsSCLs) and early nodulin-like proteins (OsENODLs). Structure and glycosylation prediction indicated that 46 OsPCs were glycosylphosphatigylinositol-anchored proteins and 38 OsPCs were chimeric AGPs. Gene duplication analysis revealed that chromosomal segment and tandem duplications contributed almost equally to the expansion of this gene family, and duplication events were mostly happened in the OsUCL subfamily. The expression profiles of OsPC genes were analyzed at different stages of vegetative and reproductive development and under abiotic stresses. It revealed that a large number of OsPC genes were abundantly expressed in the various stages of development. Moreover, 17 genes were regulated under the treatments of abiotic stresses.

Conclusions/Significance

The genome-wide identification and expression analysis of OsPC genes should facilitate research in this gene family and give new insights toward elucidating their functions in higher plants.     

RFLP analysis of rice (Oryza sativa L.) introgression lines

Summary Fifty-two introgression lines (BC₂F₈) from crosses between two Oryza sativa parents and five accessions of O. officinalis were analyzed for the introgression of O. officinalis chromosome segments. DNA from the parents and introgression lines was analyzed with 177 RFLP markers located at approximately 10-cM intervals over the rice chromosomes. Most probe/enzyme combinations detected RFLPs between the parents. Of the 174 informative markers, 28 identified putative O. officinalis introgressed chromosome segments in 1 or more of the introgression lines. Introgressed segments were found on 11 of the 12 rice chromosomes. In most cases of introgression, O. sativa RFLP alleles were replaced by O. officinalis alleles. Introgressed segments were very small in size and similar in plants derived from early and later generations. Some nonconventional recombination mechanism may be involved in the transfer of such small chromosomal segments from O. officinalis chromosomes to those of O. sativa. Some of the introgressed segments show association with genes for brown planthopper (BPH) resistance in some introgressed lines, but not in others. Thus, none of the RFLP markers could be unambiguously associated with BPH resistance.     

Heterosis breeding in rice (Oryza sativa L.)

Summary Studies conducted at the International Rice Research Institute (IRRI) during 1980 and 1981 have shown up to 73% heterosis, 59% heterobeltiosis and 34% standard heterosis for yield in rice. The latter was estimated in comparison to commercial varieties: IR36 and IR42 (yield 4–5 t/ha in wet season trials and 7–8 t/ha in dry season trials). Generally speaking, absolute yield was lower and extent of standard heterosis was higher in wet season than in dry season with some exception. Yields up to 5.9 t/ha (22% standard heterosis) in the wet season and 10.4 t/ha (34% standard heterosis) in the dry season were obtained. Most of the hybrids performed better in some season while some performed better in both seasons. Hybrids showed better lodging resistance although they were 5–10 cm taller. F₁ hybrids had significant positive correlations with the parental traits viz., yield (r = 0.446), tillering (r = 0.746), height (r = 0.810) and flowering (r = 0.843). Selection of parents among elite breeding lines on the basis of their per se yield performance, diverse origin and resistance to insects and diseases should give heterotic combination. Yield advantage of hybrids was due primarily to increase in number of spikelets per unit area even though tiller number was reduced. Grain weight was either the same or slightly higher. High yielding hybrids also showed significant heterosis and heterobeltiosis for total dry matter and harvest index. For commercial utilization of heterosis in rice, effective male sterility and fertility restoration systems are available and up to 45% natural outcrossing on male sterile lines has been observed. Consequently, F₁ rice hybrid have been successfully developed and used in China. Prospects of developing hybrid rice varieties elsewhere appear bright especially in countries that have organized seed production, certification and distribution programs and where hybrid seed can be produced at a reasonable cost.     

Genetic analysis of plant regeneration in rice (Oryza sativa L.)

Summary A diallel study involving reciprocal crosses of four genotypes (IR8, 36, 54, and 64) was carried out to understand the genetic mechanism of plant regeneration from immature embryo-calli in rice. Regeneration frequency (% of calli that produced plants) varied from a high of 86% for IR54 to a low of 0% for IR36, while regenerated plants per embryo numbered from 0 to 7 when these same IR lines and the F₁ hybrids were compared. Combining ability analysis revealed that both nuclear (with both additive and dominant effects) and cytoplasmic genes are important in controlling regeneration in rice. Parental lines and F1 hybrids with high ability to regenerate were identified.     

Genetic analysis of waxy locus in rice (Oryza sativa L.)

Summary Inheritance of waxy locus was studied in crosses of a waxy variety with four non-waxy parents having high-, intermediate-, low- or very low-amylose content. The analysis for amylose content was done on a single grain basis in parents, F₁, F₂, B₁F₁, and B₂F₁ seeds. The waxy parent lacking synthesis of amylose content was found to differ from the ones having high-, intermediate-, low- or very low-amylose content by one gene with major effect. Dosage effects for amylose content were observed to have great influence on segregation pattern and efficiency of selection. Selection efficiency for amylose content can be enhanced by selecting for endosperm appearance in early segregating generations.     

Genetic analysis of salinity tolerance in rice (Oryza sativa L.)

Summary The genetics of salinity tolerance in rice was investigated by a nine-parent complete diallel including reciprocals. Test materials involved susceptible (IR28, IR29, and MI-48), moderately tolerant (IR4595-4-1-13, IR9884-54-3-1E-P1, and IR10206-29-2-1), and tolerant (Nona Bokra, Pokkali, and SR26B) parents. Twoweek-old seedlings were grown in a salinized (EC = 12 dS/m) culture solution for 19 days under controlled conditions in the IRRI phytotron. Typical characteristics of salinity tolerance in rice were found to be Na⁺ exclusion and an increased absorption of K⁺ to maintain a good Na-K balance in the shoot. Genetic component analysis (GCA) revealed that a low Na-K ratio is governed by both additive and dominance gene effects. The trait exhibited overdominance, and two groups of genes were detected. Environmental effects were large, and the heritability of the trait was low. Our findings suggest that when breeding for salt tolerance, selection must be done in a later generation and under controlled conditions in order to minimize environmental effects. Modified bulk and single-seed descent would be the suitable breeding methods. Combining ability analysis revealed that both GCA and specific combining ability (SCA) effects were important in the genetics of salt tolerance. Moderately tolerant parents — e.g., IR4595-4-1-13 and IR9884-54-3-1E-P1 — were the best general combiners. Most of the best combinations had susceptible parents crossed either to moderate or tolerant parents. The presence of reciprocal effects among crosses necessitates the use of susceptible parents as males in hybridization programs. Large heterotic effects suggest the potential of hybrid rice for salt-affected lands.     

Diallel analysis of heading date in rice (Oryza sativa L.)

Summary The genetics of heading date was investigated in an 8×8 diallel set of crosses involving diverse rice cultivars. Wr, Vr graph analysis revealed the presence of a complementary type of non-allelic interaction which apparently affected the position and slope of the regression line such as if there were overdominance. Omission of two interacting parents resulted in a 6×6 subset of diallel crosses from which, as observed in the Wr, Vr graph, the non-allelic interaction had disappeared and the regression line exhibited partial dominance. Estimates of the genetic components of variation were in close conformity with the results obtained from the Wr, Vr graph: the average degree of dominance, as measured by (H₁/D)^1/2, was in overdominance range in the interacting 8×8 set of diallel crosses whereas it was reduced to partial dominance in the non-interacting 6×6 set of crosses. Further analysis by a standardized deviations graph indicated that earliness was controlled, on the average, by an excess of dominant alleles.     

不同环境下水稻谷粒重发育的杂种优势和遗传相关性分析

选用9个不同类型的水稻品种(系),按双列杂交设计(6×5)配成两套亲本和F12个世代的遗传材料,采用数量性状的加性、显性遗传模型、发育遗传模型比较分析了水稻谷粒重发育的杂种优势和遗传相关性。结果表明,早季在谷粒充实前期(1～12d)群体平均优势较小,中后期(13～28d)正向优势趋于明显,在谷粒充实的全过程均表现较小的负向群体超亲优势;晚季则在谷粒充实前中期(1～18d)表现正向群体平均优势。后期(19～28d)优势不明显,在谷粒充实前期(1～12d)表现正向群体超亲优势,之后转为明显的负向群体超亲优势,表明晚季谷粒发育的杂种优势表现较之早季更有利于提高籽粒充实质量,不同发育时期谷粒重之间以及与最终谷粒重之间的遗传相关分析表明,早季以显性相关为主,晚季以加性相关为主,随着发育进程的推进,相关趋于密切。     

Nitrate reductase of rice (Oryza sativa L.) under foot-rot disease

Summary Studies reported here reveal a low nitrate reductase activity in the shoots of MTU 9 rice plants while in roots high enzyme activity has been recorded.Under pathogenesis, a low nitrate reductase activity in the roots and a high enzyme activity in the shoots have been recorded in susceptible rice plants. In the resistant rice plants (GEB 24) no such marked difference in the enzyme activity has been observed.The effect of fusaric and gibberellic acid on this enzyme activity has been studied. In the case of fusaric acid, the nitrate reductase activity in the roots is inversely proportional to the concentration whereas in gibberellic acid, it is directly proportional to the concentration in the shoots.     

Molecular evolution and functional divergence of HAK potassium transporter gene family in rice （Oryza sativa L.）

The high-affinity K＋ （HAK） transporter gene family is the largest family in plant that functions as potassium transporter and is important for various aspects of plant life. In the present study, we identified 27 members of this family in rice genome. The phylogenetic tree divided the land plant HAK transporter proteins into 6 distinct groups. Although the main characteristic of this family was established before the origin of seed plants, they also showed some differences between the members of non-seed and seed plants. The HAK genes in rice were found to have expanded in lineage-specific manner after the split of monocots and dicots, and both segmental duplication events and tandem duplication events contributed to the expansion of this family. Functional divergence analysis for this family provided statistical evidence for shifted evolutionary rate after gene duplication. Further analysis indicated that both point mutant with positive selection and gene conversion events contributed to the evolution of this family in rice.     

Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles

The coleoptile of rice (Oryza sativa L. cv. Nippon-bare) emerges from an imbibed seed on day 2 after sowing. Then, it matures and senesces rapidly. For analysis of the senescence pattern within individual coleoptiles, we monitored the distribution of chlorophyll (Chl) in entire coleoptiles and in cross-sections of coleoptiles by recording the autofluorescence of Chl. Degradation of Chl was apparent at the tip of the margins of opened-out coleoptiles on day 4, when the overall levels of soluble protein and Chl per coleoptile had reached maximum values. Then, senescence proceeded from the tip to the base and from the inner mesophyll cells towards the outer epidermis, excluding tissues along vascular bundles. Further analysis of cellular senescence using samples embedded in Technovit 7100 resin revealed that the senescence of each green mesophyll cell followed an identical program, which consisted of the following steps: (i) degradation of chloroplast DNA; (ii) condensation of the nucleus, decrease in the size of chloroplasts, degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and chloroplast inner membranes; (iii) disorganization of the nucleus; (iv) complete loss of cellular components, distortion of the cell wall. Although the timing of each step and the rate at which each step was completed differed among cells of different locations within the coleoptile, this sequence was observed in all mesophyll cells in the coleoptile. Received: 31 July 1997 / Accepted: 28 October 1997     

Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles

The cytological sequence of senescence-related changes in coleoptiles of rice (Oryza sativa L. cv. Nippon-bare) was studied using fluorescence and electron microscopy. The coleoptiles reach full size 3 d after sowing, then rapidly senesce and wither completely by day 7. The interveinal region in cross-sections taken 1 mm from the tip of the coleoptile was selected for this analysis. Fluorescence microscopy using samples embedded in Technovit 7100 resin, electron microscopy and immunoelectron microscopy using DNA-specific antibodies were used to elucidate the sequence of senescence-related events. These occur in the following order: (i) degradation of the chloroplast DNA (cpDNA); (ii) condensation of the nucleus in conjunction with a decrease in the size of the dense-chromatin region, shrinkage of the chloroplast, degradation of ribulose-1, 5-bisphosphate carboxylase/oxygenase, dilation of the thylakoid membranes, increase in size and number of osmiophilic globules, condensation of the cytoplasm; (iii) disorganization of the nucleus, degeneration of the tonoplast; (iv) complete loss of the cytoplasmic components, distortion of the cell wall, invasion of microorganisms into the intercellular spaces and ultimately into the cell itself. The mitochondria maintain their ultrastructural integrity and a constant level of mitochondrial DNA throughout senescence. In young mesophyll cells, invagination of the tonoplast into the vacuole frequently occurs. This occasionally includes cytoplasmic material, which is digested in the vacuole as senescence proceeds. Immunoelectron microscopy suggests that cpDNA degradation involves rough digestion first, rather than rapid, direct decomposition of the DNA into nucleotides. The fragmented cpDNA is then dispersed throughout the chloroplast and cytoplasm. Received: 9 April 1998 / Accepted: 11 June 1998     

Potassium nutrition of rice (Oryza sativa L.) varieties under NaCl salinity

A salt-tolerant (Pokkali) and a salt-sensitive (IR28) variety of rice (Oryza sativa L.) were grown in a phytotron to investigate the effect of K (0, 25, 50 and 75 mg K kg^–1 soil) application on their salt tolerance. Potassium application significantly increased potential photosynthetic activity (Rfd value), percentage of filled spikelets, yield and K concentration in straw. At the same time, it also significantly reduced Na and Mg concentrations and consequently improved the K/Na, K/Mg and K/Ca ratios. IR28 responded better to K application than Pokkali. Split application of K failed to exert any beneficial effect over basal application.