Developing Rice Grains Research Articles

Comparison of amylopectin structure and activities of key starch synthesis enzymes in the grains of rice single-segment substitution lines with different Wx alleles

The Wx gene is the major gene controlling amylose synthesis in rice endosperm. So far, the Wx gene can be mainly classified into five common alleles, wx, Wx t, Wx g1, Wx g2, and Wx g3, according to their amylose phenotypes. Besides, the Wx allelic variation was also found to play an important role in regulating other physicochemical properties of rice starch. However, the specific physiological and starch structural mechanisms are not clear yet. With a set of single-segment substitution lines harboring five different Wx alleles, the genotype differences in amylopectin structure and dynamic changes of activities of key starch synthesis enzymes were investigated. The distinct Wx genotype difference in chain length distribution of amylopectin was confirmed by size exclusion chromatography. There were clear allelic differences in activities of starch synthesis enzymes during grain filling. The Wx allelic variation had the highest impact on the activities of starch branching enzyme, followed by pullulanase, and soluble starch synthase, while no influence on isoamylase activity. Differences in structural features of amylopectin and mean enzyme activities during grain filling period correlated well with most of the variations in starch property traits. These findings indicate that the Wx allelic variation may affect starch structure and physicochemical properties through selectively influencing activities of starch synthesis enzymes in developing rice grains. The present results will contribute to our understanding of the regulation of starch biosynthesis in rice endosperm.

Identification and expression analysis of microRNAs at the grain filling stage in rice(Oryza sativa L.)via deep sequencing.

MicroRNAs (miRNAs) have been shown to play crucial roles in the regulation of plant development. In this study, high-throughput RNA-sequencing technology was used to identify novel miRNAs, and to reveal miRNAs expression patterns at different developmental stages during rice (Oryza sativa L.) grain filling. A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain. The expression profiles of these identified miRNAs were analyzed and the results showed that 161 known miRNAs were differentially expressed during grain development, a high proportion of which were up-regulated from 5 to 7 days after fertilization. In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally. Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation. In this study, differences were revealed in the composition and expression profiles of miRNAs among individual developmental stages during the rice grain filling process, and miRNA editing events were also observed, analyzed and validated during this process. The results provide novel insight into the dynamic profiles of miRNAs in developing rice grain and contribute to the understanding of the regulatory roles of miRNAs in grain filling.

A large increase in IAA during development of rice grains correlates with the expression of tryptophan aminotransferase OsTAR1 and a grain‐specific YUCCA

The indole-3-acetic acid (IAA) content of developing grains of Oryza sativa subsp. japonica was measured by combined liquid chromatography, tandem mass spectrometry in multiple-reaction-monitoring mode. The increase from 50 ng g(-1) fresh weight to 2.9 µg g(-1) fresh weight from 1 to 14 days after pollination was much larger than that previously reported by enzyme-linked immunoassay methods. The largest increase in IAA content coincided with the start of the major starch deposition phase of grain-fill. The increase in IAA content was strongly correlated with the expression of putative IAA biosynthesis genes, OsYUC9, OsYUC11 and OsTAR1, measured by quantitative reverse transcriptase polymerase chain reaction. These results confirm the importance of the tryptophan aminotransferase/YUCCA pathway in this system. All three genes were expressed in endosperm; expression of OsYUC11 appeared to be confined to endosperm tissue. Phylogenetic analysis indicated that OsYUC11 and AtYUC10 belong to a separate clade of YUCCAs, which do not have orthologues outside the Angiosperms. This clade may have evolved with a specific role in endosperm. Expression of tryptophan decarboxylase in developing rice grains did not correlate with IAA levels, indicating that tryptamine is unlikely to be important for IAA synthesis in this system. In light of these observations, we hypothesize that IAA production in developing rice grains is controlled via expression of OsTAR1, OsYUC9, OsYUC11 and that IAA may be important during starch deposition in addition to its previously suggested role early in grain development.

Effect of Temperature on Starch Accumulation and Activities of Starch Synthetic Key Enzymes during Grain-Filling Stage of Aromatic Rice

Changes of the content of starch, amylose and the activities of 3 key enzymes involved in starch synthetic metabolism in developing rice grains under four different temperature in phytotrons during grain filling stage of two aromatic rice varieties were studied. The results showed that compared with that under the lower temperature (21°C and 23°C), the content of starch and amylose and the activities of SBE and GBSS under higher temperatures (26°Cand 30°C) were generally higher at the beginning of the grain filling stage and obviously lower during the middle and late grain filling stage, with the exception of the activity SSS which were always higher under lower temperature. The relationship between the changes of activities of SSS, SBE and the variation of starch accumulating rate was very close and so did the relationship between GBSS activity and the amylose content. The lower activities of SSS, SBE and GBSS during the middle and late grain filling stage may be the key physiological factors which lead to the poorer progress quality and appearance quality of aromatic rice under high temperature.

Bioactive compounds in rice during grain development

The bioactive compounds in two developing rice grains, KFSW (a waxy indica red rice) and TK16 (a non-waxy japonica rice), were investigated. The total phenolics and total flavonoids of two developing rice grains were significantly higher than those in mature seeds. The phenolics extract was further fractionated into three fractions, free, soluble-ester and insoluble-bound; ferulic acid was the most abundant compound in each fraction, in both immature grain and mature seed. α-Tocopherol was the most abundant vitamin E homologue, followed by γ-tocotrienol in KFSW and α-tocotrienol in TK16. High contents of free and soluble-ester ferulic acids, soluble dietary fibre, total tocols and oryzanols were found in the 15th and 18th DAA (days after anthesis) grains, which were suitable for dehulling, with a reasonable brown rice yield. Immature rice grains, containing a remarkable amount of bioactive compounds with a high reducing power, have a great potential for applications in nutraceutical foods.

Variability in head shapes in three populations of the Rice Bug Leptocorisa oratorius (Fabricius) (Hemiptera: Alydidae)

The rice bug, Leptocorisa oratorius (Fabricius) is an insect pest of rice that feeds on developing rice grains reducing the yield and quality of rice. Identification of this pest has been vague owing to variability in its external morphological characteristics. It has been referred to as L. acuta (Thunberg) and L. varicornis in some literature. In this study, the shapes of the head capsule were compared in populations of the rice bug using a landmark-based geometric morphometric method, and analysis of relative warp scores. Samples were collected from three different localities of which eighty-three were from Alubijid, Misamis Oriental; nineteen from Maigo, Lanao del Norte; and eighty-two from Buug, Zamboanga Sibugay. Of the 184 individuals, 116 were classified as males and 68 were classified as females. Images of the dissected head capsule was acquired with the use of MacronCam and Leica ES2 microscope. Landmark analyses was done on the x- and y- coordinates of the head outline which totalled to 35 landmark points. The results showed variability within and among populations of the rice bugs in the regions at the labrum, vertex, and the outline of the compound eye insertion.This variability may represent unique genotypes and that this geographic variation may have an important effect on expanding population sizes in following years which should be considered in control methods.

Activity and expression of adenosine diphosphate glucose pyrophosphorylase in developing rice grains: Varietal differences and implications on grain filling

ADP-glucose pyrophosphorylase (AGPase) is a key enzyme in starch biosynthesis in plants. The activity, expression and gene sequence of AGPase were studied in grains of five rice cultivars representing different origins to gain insight into their variations in relation to starch accumulation. The varieties were Jaya, GEB24 (indica), BSI 115 (japonica), Mahsuri (indica-japonica derivative) and CoRH2 (hybrid). The grain weight increased in all of them until 20 dpa, although at varying rates. It was relatively steady in Mahsuri, in others there was a steep increase from 10 to 20 days post-anthesis (dpa). The total AGPase activity increased steadily from 5 to 20 dpa in Jaya and GEB24, but dropped after 15 dpa in BSI 115 and CoRH2 while in Mahsuri, it maintained the 10 dpa low level until 20 dpa. The AGPase cDNAs did not show any sequence differences in the five varieties. The transcripts of both sub units of AGPase accumulated comparably during the initial phase of grain development suggesting their coordinated expression and dropped after 15 dpa in all the varieties. On the other hand, AGPase protein level remained unchanged after reaching the maximum in CoRH2 and continued to increase up to 20 dpa in the other four varieties. Thus the drop in the enzyme activity cannot be attributed to changes in the transcript or protein amounts per se. We infer that the drop in activity, its varying levels and varying duration in the varieties studied is likely due to differential stability of the active enzyme protein during grain development and that the extended duration of enzyme activity may contribute to increased grain weight.

Japanese Sake‐Brewing Rice Cultivars Show High Levels of Globulin‐like Protein and a Chloroplast Stromal HSP70

ABSTRACTWhite‐core is an important trait in the selection of rice (Oryza sativa L. ssp. japonica) for sake (Japanese rice wine) production. The present study was conducted to find differences between the endosperm protein pattern of white‐core–bearing sake‐brewing cultivars and those of normal rice grains. Protein patterns of white‐core sake‐brewing grains were compared with those of normal cultivars by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). Several proteins showing different amounts were detected. These were identified by microsequencing and found to be known carbohydrate‐metabolizing enzymes as well as proteins with no known involvement in carbohydrate metabolism. Two units of globulin‐like protein (GLP) and a chloroplast stromal heat shock protein 70 (ctHSP70) showed the greatest difference in the white‐core group. Analysis of amyloplast proteins showed slightly higher amounts of GLP and ctHSP70 in the amyloplast than in the cytosol. Also, time‐course analysis of developing rice grains indicated that both proteins were elevated during the later stages of grain development. From this we conclude that GLP and ctHSP70‐II are associated with white‐core appearance and thus behave as biochemical markers for this character in sake‐brewing rice cultivars.

Small RNAs Originated from Pseudogenes: cis- or trans-Acting?

Pseudogenes are significant components of eukaryotic genomes, and some have acquired novel regulatory roles. To date, no study has characterized rice pseudogenes systematically or addressed their impact on the structure and function of the rice genome. In this genome-wide study, we have identified 11,956 non-transposon-related rice pseudogenes, most of which are from gene duplications. About 12% of the rice protein-coding genes, half of which are in singleton families, have a pseudogene paralog. Interestingly, we found that 145 of these pseudogenes potentially gave rise to antisense small RNAs after examining ∼1.5 million small RNAs from developing rice grains. The majority (>50%) of these antisense RNAs are 24-nucleotides long, a feature often seen in plant repeat-associated small interfering RNAs (siRNAs) produced by RNA-dependent RNA polymerase (RDR2) and Dicer-like protein 3 (DCL3), suggesting that some pseudogene-derived siRNAs may be implicated in repressing pseudogene transcription (i.e., cis-acting). Multiple lines of evidence, however, indicate that small RNAs from rice pseudogenes might also function as natural antisense siRNAs either by interacting with the complementary sense RNAs from functional parental genes (38 cases) or by forming double-strand RNAs with transcripts of adjacent paralogous pseudogenes (2 cases) (i.e., trans-acting). Further examinations of five additional small RNA libraries revealed that pseudogene-derived antisense siRNAs could be produced in specific rice developmental stages or physiological growth conditions, suggesting their potentially important roles in normal rice development. In summary, our results show that pseudogenes derived from protein-coding genes are prevalent in the rice genome, and a subset of them are strong candidates for producing small RNAs with novel regulatory roles. Our findings suggest that pseudogenes of exapted functions may be a phenomenon ubiquitous in eukaryotic organisms.

Storage product synthesis and accumulation in developing grains of wheat

The time course of synthesis and accumulation of the major storage components in developing grain of wheat cv Hereward has been determined. Gluten proteins were first detected at 10 dpa and accumulated most rapidly between 12 and 35 dpa, with little change after 42 dpa. Differences in the accumulation patterns of two different types of ω-gliadins were observed while the synthesis of the HMW subunits was initiated about 2 days later than that of the other gluten proteins. Although protein accumulation had essentially ceased by 42 dpa, grain desiccation was associated with a dramatic increase in the proportion of large glutenin polymers. The accumulation of starch essentially paralleled that of gluten proteins, reaching 55% of the grain dry weight at maturity. This was associated with an increase in the amylose content, from about 20 to 26% of the total starch. The expression patterns of transcripts encoding enzymes of the synthesis (ADP glucose pyrophosphorylase, starch synthases), branching and modification of starch were consistent with the pattern of starch accumulation and with the expression patterns reported for orthologous genes in developing rice grain, showing high conservation between species.

Effects of Nitrogen on Starch Accumulation and Activities of Enzymes Involved in Starch Synthesis in Rice Caryopsis

Starch accumulation is the main event in the developing rice grain, and the activities of key enzymes involved in starch synthesis are the important factors affecting the grain filling. N-fertilizer can regulate the plant growth, flower emergence, grain yield and quality, while the effects of N-fertilizer on starch accumulation and the activities of key enzymes in rice grains are rarely known. In this study, two rice cultivars, Yangdao 6 and Jingdao 941, were used as materials to study the effects of nitrogen on starch accumulation and the activities of related enzymes involved in starch synthesis. The results indicated that increased panicle N-fertilizer could significantly decrease the soluble sugar in the developing caryopsis, while the starch content reduced slightly. The amylose and amylopectin contents in the caryopsis increased synchronously with the raising of grain weight. When the pani-cle N-fertilizer was added, the amylose content in caryopsis as well as the content ratio of amylose to total starch decreased ob-servably. During the development of rice caryopses, the activities of ADP-glucose pyrophosphorylase (AGP), granule-bound starch synthase (GBSS), soluble starch synthase (SSS), and starch branching enzyme (SBE) in endosperm changed as single peak curve. That was, the enzyme activities increased at the early stage of grain filling, till their peak at 10–15 d after flowering, then depressed. Application of nitrogen fertilizer could improve the activities of AGP, SSS, and SBE in rice caryopsis at middle and late filling stages, while decrease GBSS activity at early and middle filling stages. And the effect of the N fertilized at the booting stage was superior to that at the tilling stage.

A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains

Endogenous small RNAs, including microRNAs (miRNAs) and short-interfering RNAs (siRNAs), function as post-transcriptional or transcriptional regulators in plants. miRNA function is essential for normal plant development and therefore is likely to be important in the growth of the rice grain. To investigate the roles of miRNAs in rice grain development, we carried out deep sequencing of the small RNA populations of rice grains at two developmental stages. In a data set of approximately 5.5 million sequences, we found representatives of all 20 conserved plant miRNA families. We used an approach based on the presence of miRNA and miRNA* sequences to identify 39 novel, nonconserved rice miRNA families expressed in grains. Cleavage of predicted target mRNAs was confirmed for a number of the new miRNAs. We identified a putative mirtron, indicating that plants may also use spliced introns as a source of miRNAs. We also identified a miRNA-like long hairpin that generates phased 21 nt small RNAs, strongly expressed in developing grains, and show that these small RNAs act in trans to cleave target mRNAs. Comparison of the population of miRNAs and miRNA-like siRNAs in grains to those in other parts of the rice plant reveals that many are expressed in an organ-specific manner.

Expression of granule‐bound starch synthase in developing rice grain

AbstractThe expression of granule‐bound starch synthase (GBSS) in rice (Oryza sativa) genotype Tainung 67 (200 g kg−1 grain amylose content) and its two NaN3‐induced mutants SA419 and SA418 were examined. G/T polymorphism analysis indicated that SA418 (300 g kg−1 grain amylose content) carried Wxa allele. The insertion of 23 base pairs sequence was found only in SA419 (80 g kg−1 grain amylose content), suggesting that it was a waxy mutant. Microsatellite polymorphisms (CT)n were also detected on the Wx gene encoding GBSS in the tested genotypes. The activities of several key enzymes involving starch biosynthesis in developing grains of field‐grown rice plants were also compared during grain filling period. Significant genotypic differences were only found in the expression of GBSS. The content of amylose in SA418 grain was higher than Tainung 67 and SA419 grains throughout the entire grain filling period, possibly due to its superiority to synthesize amylose through GBSS. The lowest amylose content of SA 419 grain was attributable to its extremely low activity of GBSS in comparison with the two other genotypes. The mutation effects on the expression of GBSS were confirmed by two‐dimensional electrophoresis and mass spectrometry. Copyright © 2007 Society of Chemical Industry

Nitro‐blue tetrazolium staining of starch‐synthesizing enzymes in developing rice grains

AbstractAn in situ nitro‐blue tetrazolium (NBT) staining method was used to locate the activities of enzymes involved in starch synthesis in developing grains of field grown rice (Oryza sativa L.) cultivar Tainung 67 and its early ripening mutant SA 419. The results indicated that all the tested enzymes, including sucrose synthase, invertase, hexokinase, ADP‐glucose pyrophosphorylase, UDP‐glucose pyrophosphorylase and starch synthase, were detectable in developing rice endosperms by using NBT staining. The activity of these enzymes was also measurable by using chemical assays. The expression of enzyme activity, as indicated by the formation of blue formazan precipitate, in growing grain of Tainung 67 were visualized in entire endosperm at 11 days after anthesis (DAA). However, NBT staining shifted from central to peripheral region of endosperm after 18 DAA, and the staining disappeared at 25 DAA. Similar staining patterns were also observed in the growing grains of mutant SA 419, but the shift of staining from central to peripheral endosperm occurred at 11 DAA. Electron microscopy examinations showed that the growing patterns of starch granules, sampled from central and peripheral regions of endosperm, varied between two cultivars, with SA 419 growing faster than Tainung 67. Both cultivars showed that the shifts of NBT staining pattern coincided with the changes in the growing pattern of starch granules located in central and peripheral regions of endosperms. The NBT staining results showed that the activities of sucrose to starch conversion enzymes in mutant SA 419 declined and disappeared earlier than its wild‐type Tainung 67 and therefore it ripened earlier than cultivar Tainung 67. Copyright © 2005 Society of Chemical Industry

Profiling the Expression of Genes Controlling Rice Grain Quality

Rice provides a staple source of energy, protein and other nutrients to half of the world population. Over 90 of the rice seeds consists of starch and protein by dry weight. The quantity and property of starch and protein thus play a dominant role in the yield and quality of rice. The amylase content of starch is a determining factor in the eating and cooking quality while the amount and essential amino acids balance of storage proteins affect the nutritional quality of rice. In China, the super-hybrid rice currently under the last phase of development has a 35 yield advantage over the best inbred rice varieties. However, its grain quality needs further improvement. This study reported the expression patterns of 44 genes participating in starch, storage protein, and lysine synthesis in the developing rice grain. Field grown rice cultivar 9311, the paternal line of an elite super-hybrid rice LYP9 with its draft genomic sequence released, was used as plant material. Results revealed diverse yet coordinated expression profiles of the genes involved in the three pathways which lead to the final composition and property of starch, protein and lysine that determine the quality of rice, providing useful information for rice quality improvement.

Changes of Dry Matter Content and Nutrients in Developing Rice Grain

Changes of Dry Matter Content and Nutrients in Developing Rice Grain

Isolation and characterization of two fructokinase cDNA clones from rice

Two cDNA clones, OsFKI and OsFKII, encoding fructokinase (EC 2.7.1.4) were isolated from immature seeds of rice ( Oryza sativa L.) by PCR. OsFKI cDNA encoded a deduced protein of 323 amino acids that was 59–71% identical to previously characterized plant fructokinases. In contrast, OsFKII cDNA encoded a deduced protein of 336 amino acids that shared only 64% amino acid identity with OsFKI. The deduced proteins both possessed an ATP-binding motif and putative substrate recognition site sequences that were previously identified in bacterial fructokinases. Genomic DNA blot analysis also revealed that each fructokinase gene exists as a single copy in the rice genome. The identity of OsFKI and OsFKII as fructokinases was confirmed by the expression of enzyme activity in E. coli. Although both OsFKI and OsFKII utilized fructose as substrate, only OsFKII activity was strongly inhibited at a high fructose concentration. The mRNA corresponding to OsFKII accumulated at high levels in developing rice grains, whereas there were only low levels of OsFKI transcripts in immature seeds. These results indicate that fructokinase in rice endosperm is encoded by two divergent genes, which play different roles in rice grains for starch storage based on their sensitivity to substrate inhibition and level of transcripts in endosperm.

Effect of Seawater on Photosynthesis and Dry Matter Accumulation in Developing Rice Grains

To understand the physiology of rice under seawater salinity, potted rice plants were irrigated with different concentrations of Japan seawater (electrical conductivity 0.9, 5.7, 11.5, or 21.5 mS cm-1) from 10 d after transplanting (DAT) to 35 DAT, and from 75 to 100 DAT. Seawater salinity decreased the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate, leaf water and osmotic potentials, and relative water content, and increased leaf temperature. The contents of chlorophylls, carotenoids, and total sugars significantly decreased in the leaves but content of non-reducing sugars decreased only slightly. With increasing salinity the Na+ concentration increased, while Ca2+, Mn2+, and K+ concentrations decreased. Salinity decreased the contents of sugars and proteins, dry mass, and rate of dry mater accumulation in developing grains.

Causes of Sterility in Seed Set of Rice under Salinity Stress

The effects of salinity at 50 mM NaCl on floral characteristics, yield components, and biochemical and physiological attributes of the sensitive rice variety IR‐28 were studied under controlled conditions to determine the causes of sterility in seed set under salinity stress. The results showed significant decreases in panicle weight, panicle length, primary branches/panicle, filled seeds/panicle, unfilled seeds/panicle, filled seeds/plant, unfilled seeds/plant, total seeds/panicle, total seed weight/panicle, 1000‐seed weight and total seed weight/plant. The sodium content in different leaves and floral parts increased significantly. In contrast, the potassium content was decreased significantly in leaves and floral parts. A reduction in chlorophyll a and b was also noted in different parts of the leaves. Inhibition of transpiration and photosynthesis was observed in flag leaves at the grain‐filling stage. Soluble carbohydrates in different leaves were reduced significantly in salinized plants but their content in different floral parts was increased, with the exception of primary and secondary branch spikelets. Under salinity stress, total protein concentration in flag, second and third leaves were higher than in control plants. The viability of rice pollen was reduced significantly in salinized plants. It was further observed that starch synthetase activity (α1–4‐glucan glucosyle transferases) in developing rice grains was inhibited very significantly under salinity stress. From these results, it is inferred that sterility and significant reductions in seed set in rice were not merely due to reduction or inhibition of different biochemical constituents and physiological functions, but were mainly due to limitation of soluble carbohydrate translocation in primary and secondary spikelets, accumulation of more sodium and less potassium in all the floral parts, and highly significant inhibition of specific activity of starch synthetase in developing rice grains, thus resulting in failure of seed set.

Cellular localisation and function of a sucrose transporter OsSUT1 in developing rice grains

We previously reported the cloning and tissue-specific expression of a gene encoding a sucrose/proton symporter in rice (Oryza sativa L.), OsSUT1 (Hirose et al. 1997, Plant Cell Physiology38, 1389–1396). This gene is expressed at high levels in the filling grain, leaf sheath and stem. Expression in these tissues occurred only after heading i.e. during the development of the reproductive structure as a major sink. In this paper, we report localisation of the transcript and protein to specific cells in the filling rice grain by in situ hybridisation with a probe from theOsSUT1 cDNA, and immunolocalisation of OsSUT1 and proton-pumping ATPase (H+-ATPase). An OsSUT1 cDNA probe recognises a transcript of approximately 2.4 kb, and the SUT1 antibody recognises a protein of approximately 55 kDa in total membrane protein extracts from the filling grain, leaf sheath and stem. In the developing grain, OsSUT1 is expressed at low levels before heading, with expression reaching a peak approximately ten days after emergence of the panicle from the sheath. Transcript is then present throughout seed development, with expression falling substantially after about 25 days post-heading. Both transcript and protein are localised to the aleurone cells of the developing grain, and are also detected in the maternal tissue, particularly the nucellus, vascular parenchyma tissue and the nucellar projection. Tissue slices from filling rice grain showed high rates of sucrose uptake that were inhibited by pCMBS. The role of OsSUT1 in sucrose transport to the filling grain endosperm is discussed.