Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus

Studies of maize starch branching enzyme mutants suggest that the amylose extender high amylose starch phenotype is a consequence of the lack of expression of the predominant starch branching enzyme II isoform expressed in the endosperm, SBEIIb. However, in wheat, the ratio of SBEIIb and SBEIIa expression are inversely related to the expression levels observed in maize and rice. Analysis of RNA at 15 days post anthesis suggests that there are about 4-fold more RNA for SBE IIa than for SBE IIb. The genes for SBE IIa and SBE IIb from wheat are distinguished in the size of the first three exons, allowing isoform-specific antibodies to be produced. These antibodies were used to demonstrate that in the soluble fraction, the amount of SBE IIa protein is two to three fold higher than SBIIb, whereas in the starch granule, there is two to three fold more SBE IIb protein amount than SBE IIa. In a further difference to maize and rice, the genes for SBE IIa and SBE IIb are both located on the long arm of chromosome 2 in wheat, in a position not expected from rice–maize–wheat synteny.     

Molecular analysis of the gene encoding a rice starch branching enzyme

Summary The sequence of a rice gene encoding a starch branching enzyme (sbe1) shows extreme divergence from that of the rice gene, that is homologous to bacterial glycogen branching enzyme (sbe2). sbe1 is expressed abundantly and specifically in developing seeds and maximally in the middle stages of seed development. This expression pattern completely coincides with that of the waxy gene, which encodes a granule-bound starch synthase. Three G-box motifs and consensus promoter sequences are present in the 5 flanking region of sbe1. It encodes a putative transit peptide, which is required for transport into the amyloplast. A 2.2 kb intron (intron 2) precedes the border between the regions encoding the transit peptide and the mature protein, and contains a high G/C content with several repeated sequences in its 5 half. Although only a single copy of sbe1 is present in the rice genome, Southern analysis using intron 2 as a probe indicates the presence of several homologous sequences in the rice genome, suggesting that this large intron and also the transit peptide coding region may be acquired from another portion of the genome by duplication and insertion of the sequence into the gene.     

Changes in structure of starch and enzyme activities affected by sugary mutations in developing rice endosperm. Possible role of starch debranching enzyme (R-enzyme) in amylopectin biosynthesis

In maturing endosperms of a variety of sugary mutants of rice, phytoglycogen-like polysaccharides with highly branched a -glucans were accumulated instead of amylopectin. while the amylose content greatly decreased. Measurement of activities per endosperm of the 10 major enzymes involved in starch and sucrose metabolism revealed that the activity of starch debranching enzyme (R-enzyme) was specifically reduced in the sugary mutants. The activity of starch branching enzyme I (Q-enzyme I) was also significantly decreased, but less so than the R-enzyme, in the mutants, suggesting some coordination of the expression of the genes coding for R-enzyme and Q-enzyme I. Western blot analysis showed that the sugary mutations of rice resulted in a decrease in the amount of R-enzyme protein, but not in major modification of the enzyme. These findings strongly suggest that R-enzyme plays a critical role in determining the amylopectin fine structure, since at the extremely low level of R-enzyme activity as compared with Q-enzyme activity, as found in sugary mutants, the rice endosperm produced phytoglycogen. We hypothesize that balance of activities or interaction between Q-enzyme and R-enzyme may be responsible for the fine structure of a -polyglucans in plant tissues.     

The structure of starch can be manipulated by changing the expression levels of starch branching enzyme IIb in rice endosperm

When the starch branching enzyme IIb (BEIIb) gene was introduced into a BEIIb-defective mutant, the resulting transgenic rice plants showed a wide range of BEIIb activity and the fine structure of their amylopectins showed considerable variation despite having the two other BE isoforms, BEI and BEIIa, in their endosperm at the same levels as in the wild-type. The properties of the starch granules, such as their gelatinization behaviour, morphology and X-ray diffraction pattern, also changed dramatically depending on the level of BEIIb activity, even when this was either slightly lower or higher than that of the wild-type. The over-expression of BEIIb resulted in the accumulation of excessive branched, water-soluble polysaccharides instead of amylopectin. These results imply that the manipulation of BEIIb activity is an effective strategy for the generation of novel starches for use in foodstuffs and industrial applications.     

Linkage mapping of starch branching enzyme III in rice (Oryza sativa L.) and prediction of location of orthologous genes in other grasses

 The chromosomal position of Starch Branching Enzyme III (SBEIII) was determined via linkage to RFLP markers on an existing molecular map of rice (Oryza sativa L.). A cDNA of 890 bp was generated using specific PCR primers designed from available SBEIII sequence data and used as a probe in Southern analysis. The SBEIII cDNA hybridized to multiple restriction fragments, but these fragments mapped to a single locus on rice chromosome 2, flanked by CDO718 and RG157. The detection of a multiple-copy hybridization pattern suggested the possibility of a tandemly duplicated gene at this locus. The map location of orthologous SBE genes in maize, wheat, and oat were predicted based on previously published genetic studies and comparative maps of the grass family. Received : 5 August 1996 / Accepted : 13 September 1996     

Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms

The dynamic changes of the activities of enzymes involving in starch biosynthesis, including ADP-glucose pyrophosphorylase (AGPase), soluble starch synthases (SSS), starch branching enzyme (SBE) and starch debranching enzymes (DBE) were studied, and changes of fine structure of amy- lopectin were characterized by isoamylase treatment during rice grain development, using trans anti-waxy gene rice plants. The relationships between the activities of those key enzymes were also analyzed. The amylose synthesis was significantly inhibited in transgenic Wanjing 9522, but the total starch content and final grain weight were less affected as compared with those of non-transgenic Wanjing 9522 rice cultivar. Analyses on the changes of activities of enzymes involving in starch bio- synthesis showed that different enzyme activities were expressed differently during rice endosperm development. Soluble starch synthase is relatively highly expressed in earlier stage of endosperm de- velopment, whilst maximal expression of granule-bound starch synthase (GBSS) occurred in mid-stage of endosperm development. No obvious differences in changes of the activities of AGPase and SBE between two rice cultivars investigated, except the DBEs. Distribution patterns of branches of amy- lopectin changed continually during the development of rice grains and varied between two rice culti- vars. It was suggested that amylopectin synthesis be prior to the synthesis of amylose and different enzymes have different roles in controlling syntheses of branches of amylopectin.     

Inheritance of two endosperm protein loci in rice (Oryza sativa L.)

Summary Previous studies indicated two types of phenotypic protein markers as two minor bands of SDS-PAGE for rice storage protein. A variant derived from a Pakistani variety, Dular, was found to show a mobility variant with Band 11, a relatively faster-moving band as compared to Band 10, while most of the other cultivated rices exhibited Band 10 at a molecular weight of around 100–110 K. Band 11 was also observed in several wild rice species. How this variant occurred is not known. Another marker is characterized by the presence of either Band 56 (slower-migrating band) or Band 57 (faster-migrating band) in most cultivars at a molecular weight of about 28–27 K. Most indica varieties developed in Taiwan have Band 57 and japonica varieties have Band 56. Genetic analysis of F₁, F₂ and F₃ seeds from interstrain crosses indicated that Band 10 versus Band 11 and Band 56 versus Band 57 are due to codominant alleles at two loci. Tests of independent inheritance between these two loci (Band 10/11 versus Band 56/57) indicated that there is no linkage between them. Both of these two protein loci encode for endosperm proteins and mostly belong to the minor polypeptide subunits of the glutelin fraction of rice seed proteins. Studies on reciprocal crosses indicate dosage effects as exhibited in band patterns. Variations in band intensity were frequently observed when the maternal genotype was different.     

Effect of aluminium on metabolism of starch and sugars in growing rice seedlings

The effect of increasing concentrations of Al₂(SO₄)₃ in situ on the content of starch, sugars and activity behaviour of enzymes related to their metabolism were studied in growing seedlings of two rice cvs. Malviya-36 and Pant-12 in sand cultures. Al₂(SO₄)₃ levels of 80 and 160 μM in the growth medium caused an increase in the contents of starch, total sugars as well as reducing sugars in roots as well as shoots of the rice seedlings during a 5–20 days growth period. The activities of the enzymes of starch hydrolysis α-amylase, β-amylase and starch phosphorylase declined in Al-exposed seedlings, whereas the activities of sucrose hydrolyzing enzymes sucrose synthase and acid invertase increased in the seedlings due to Al³⁺ treatment. The enzyme of sucrose synthesis, sucrose phosphate synthase showed decreased activity in Al³⁺ treated seedlings compared to controls. Results suggest that Al³⁺ toxicity in rice seedlings impairs the metabolism of starch and sugars and favours the accumulation of hexoses by enhancing the activities of sucrose hydrolyzing enzymes.     

The expression of the rice (Oryza sativa L.) homologue of Snm1 is induced by DNA damages

We isolated and characterized the rice homologue of the DNA repair gene Snm1 (OsSnm1). The length of the cDNA was 1862bp; the open reading frame encoded a predicted product of 485 amino acid residues with a molecular mass of 53.2kDa. The OsSnm1 protein contained the conserved beta-lactamase domain in its internal region. OsSnm1 was expressed in all rice organs. The expression was induced by MMS, H(2)O(2), and mitomycin C, but not by UV. Transient expression of an OsSnm1/GFP fusion protein in onion epidermal cells revealed the localization of OsSnm1 to the nucleus. These results suggest that OsSnm1 is involved not only in the repair of DNA interstrand crosslinks, but also in various other DNA repair pathways.     

Drought-induced male sterility in rice: Changes in carbohydrate levels and enzyme activities associated with the inhibition of starch accumulation in pollen

Male reproductive development of rice (Oryza sativa L.) is very sensitive to drought. A brief, transitory episode of water stress during meiosis in pollen mother cells of rice grown under controlled environmental conditions induced pollen sterility. Anthers containing sterile pollen were smaller, thinner, and often deformed compared to normal anthers of well-watered plants. Only about 20% of the fully developed florets in stressed plants produced grains, compared to 90% in well-watered controls. Water stress treatments after meiosis were progressively less damaging. Levels of starch and sugars and activities of key enzymes involved in sucrose cleavage and starch synthesis were analyzed in anthers collected at various developmental stages from plants briefly stressed during meiosis and then re-watered. Normal starch accumulation during pollen development was strongly inhibited in stress-affected anthers. During the period of stress, both reducing and non-reducing sugars accumulated in anthers. After the relief of stress, reducing sugar levels fell somewhat below those in controls, but levels of non-reducing sugars remained higher than in controls. Activities of acid invertase and soluble starch synthase in stressed anthers were lower than in controls at comparable stages throughout development, during as well as after stress. Stress had no immediate effect on ADP-glucose pyrophosphorylase activity, but had an inhibitory aftereffect throughout post-stress development. Sucrose synthase activity, which was, relatively speaking, much lower than acid invertase activity, was only slightly suppressed by stress. The results show that it is unlikely that pollen sterility, or the attendant inhibition of starch accumulation, in water-stressed rice plants are caused by carbohydrate starvation per se. Instead, an impairment of enzymes of sugar metabolism and starch synthesis may be among the potential causes of this failure.     

Introduction of sense and antisense cDNA for branching enzyme in the amylose-free potato mutant leads to physico-chemical changes in the starch

One isoform of the branching enzyme (BE; EC 2.4.1.18) of potato (Solarium tuberosum L.) is known and catalyses the formation of α-1,6 bonds in a glucan chain, resulting in the branched starch component amylopectin. Constructs containing the antisense or sense-orientated distal 1.5-kb part of a cDNA for potato BE were used to transform the amylose-free (amf) mutant of potato, the starch of which stains red with iodine. The expression of the endogenous BE gene was inhibited either largely or fully as judged by the decrease or absence of the BE mRNA and protein. This resulted in a low percentage of starch granules with a small blue core and large red outer layer. There was no effect on the amylose content, degree of branching or λ_max of the iodine-stained starch. However, when the physico-chemical properties of the different starch suspensions were assessed, differences were observed, which although small indicated that starch in the transformants was different from that of theamf mutant.     

Du1, encoding a novel Prp1 protein, regulates starch biosynthesis through affecting the splicing of Wxb pre-mRNAs in rice (Oryza sativa L.)

Starch is the major component of cereal grains. In rice, starch properties determine the eating and cooking quality. The dull endosperm of rice grains is a classical morphological and agronomical trait that has long been exploited for breeding and genetics study. To understand the molecular mechanism that regulates the starch biosynthesis in rice grains, we characterized a classic rice mutant dull endosperm1 (du1) and isolated Du1 through a map-based cloning approach. Du1, encoding a member of pre-mRNA processing (Prp1) family, is expressed mainly in panicles. Du1 specifically affects the splicing efficiency of Wx(b) and regulates starch biosynthesis by mediating the expression of starch biosynthesis genes. Analysis of du1wx shows that Du1 acts upstream of Wx(b). These results strongly suggest that Du1 may function as a regulator of the starch biosynthesis by affecting the splicing of Wx(b) and the expression of other genes involved in the rice starch biosynthetic pathways.     

Transport of DMAA and MMAA into rice (Oryza sativa L.) roots

Arsenate (As(V)) transport into plant cells has been well studied. A study on rice (Oryza sativa L.) showed that arsenite is transported across the plasma membrane via glycerol transporting channels. Previous studies reported that the dimethylarsinic acid (DMAA) and monomethylarsonic acid (MMAA) uptake in duckweed (Spirodela polyrhiza L.) differed from that of As(V), and was unaffected by phosphate (H₂PO₄). This article reports the transport mechanisms of DMAA and MMAA in rice roots. Linear regression analysis showed that the DMAA and MMAA uptake in rice roots increased significantly (p ≤ 0.0002 and ≤0.0001 for DMAA and MMAA, respectively) with the increase of exposure time. Concentration-dependent influx of DMAA and MMAA showed that the uptake data were well described by Michaelis-Menten kinetics. The MMAA influx was higher than that of DMAA. The DMAA and MMAA uptake in rice roots were decreased significantly (p ≤ 0.0001 and ≤0.0077 for DMAA and MMAA, respectively) with the increase of glycerol concentration indicating that DMAA and MMAA were transported into rice roots using the same mechanisms of glycerol. Glycerol is transported into plant cells by aquaporins, and DMAA and MMAA are transported in a dose-dependent manner of glycerol which reveals that DMAA and MMAA are transported into rice roots through glycerol transporting channels. The DMAA and MMAA concentration in the solution did not affect the inhibition of their uptake rate by glycerol.     

Effect of low temperature on ethanolic fermentation in rice seedlings

Rice seedlings (Oryza sativa L.) were incubated at 5-30 degrees C for 48 h and the effect of temperature on ethanolic fermentation in the seedlings was investigated in terms of low-temperature adaptation. Activities of alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) in roots and shoots of the seedlings were low at temperatures of 20-30 degrees C, whereas temperatures of 5, 7.5 and 10 degrees C significantly increased ADH and PDC activities in the roots and shoots. Temperatures of 5-10 degrees C also increased ethanol concentrations in the roots and shoots. The ethanol concentrations in the roots and shoots at 7.5 degrees C were 16- and 12-times greater than those in the roots and shoots at 25 degrees C, respectively. These results indicate that low temperatures (5-10 degrees C) induced ethanolic fermentation in the roots and shoots of the seedlings. Ethanol is known to prevent lipid degradation in plant membrane, and increased membrane-lipid fluidization. In addition, an ADH inhibitor, 4-methylpyrazole, decreased low-temperature tolerance in roots and shoots of rice seedlings and this reduction in the tolerance was recovered by exogenous applied ethanol. Therefore, production of ethanol by ethanolic fermentation may lead to low-temperature adaptation in rice plants by altering the physical properties of membrane lipids.     

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.