Glucose-induced delay of seed germination in rice is mediated by the suppression of ABA catabolism rather than an enhancement of ABA biosynthesis.
Both glucose and ABA play crucial roles in the regulation of seed germination and post-germination development. In Arabidopsis thaliana, up-regulation of ABA biosynthesis is suggested as one of the possible mechanisms mediating the glucose-induced delay in seed germination. Since the endogenous ABA level is controlled by the equilibrium between ABA biosynthesis and catabolism, we investigated how this equilibrium is related to the regulation of seed germination by glucose in rice. When ABA biosynthesis was inhibited by nordihydroguaiaretic acid (NDGA), an inhibitor of the ABA anabolic enzyme 9-cis-epoxycarotenoid dioxygenase (NCED), rice seed germination showed no response. In contrast, inhibition of ABA catabolism by diniconazole significantly arrested seed germination, suggesting that the regulation of ABA catabolism plays a major role. Further experiments indicated that the expression of OsABA8ox3, a key gene in ABA catabolism and encoding ABA 8'-hydroxylase in rice, was significantly increased during the first 6 h of imbibition, which was consistent with the decline of ABA content in the imbibed seeds. Expression of OsABA8ox genes, especially OsABA8ox2 and OsABA8ox3, was sensitively suppressed in the presence of exogenously supplied glucose. In contrast, the expression profiles of OsNCED genes that control the limiting step of ABA biosynthesis showed no significant changes in response to low levels of glucose. Our results demonstrated that the glucose-induced delay of seed germination is a result of the suppression of ABA catabolism rather than any enhancement of ABA biosynthesis during rice seed germination.
Characterization and expression profiles of miRNAs in rice seeds
Small RNAs (sRNAs) are common and effective modulators of gene expression in eukaryotic organisms. To characterize the sRNAs expressed during rice seed development, massively parallel signature sequencing (MPSS) was performed, resulting in the obtainment of 797 399 22-nt sequence signatures, of which 111 161 are distinct ones. Analysis on the distributions of sRNAs on chromosomes showed that most sRNAs originate from interspersed repeats that mainly consist of transposable elements, suggesting the major function of sRNAs in rice seeds is transposon silencing. Through integrative analysis, 26 novel miRNAs and 12 miRNA candidates were identified. Further analysis on the expression profiles of the known and novel miRNAs through hybridizing the generated chips revealed that most miRNAs were expressed preferentially in one or two rice tissues. Detailed comparison of the expression patterns of miRNAs and corresponding target genes revealed the negative correlation between them, while few of them are positively correlated. In addition, differential accumulations of miRNAs and corresponding miRNA*s suggest the functions of miRNA*s other than being passenger strands of mature miRNAs, and in regulating the miRNA functions.
Physiological and biochemical aspects of pre-sowing seed treatments in fine rice (Oryza sativa L.)
The effects of pre-sowing seed treatments on the germination and emergence of fine rice and on reducing, nonreducing and total sugars and α-amylase activity were studied. Fine rice seeds were either soaked in tap water by a traditional method, hardened for 18 or 24 h (two cycles) or osmoconditioned (-1.1 MPa KNO3) for 24 or 48 h. Seed hardening (24 h) and the traditional soaking treatments resulted in a higher germination percentage, germination index and energy of germination and lower mean germination time and mean emergence time. Performance was slightly better in seeds subjected to hardening for 24 h than the traditional soaking because of lower T50 and non-reducing sugars and higher reducing and total sugars and α-amylase activity. In seeds subjected to osmoconditioning for 48 h seed performance was impaired possibly because of KNO3 toxicity.
Identification of rice seed varieties using neural network.
A digital image analysis algorithm based color and morphological features was developed to identify the six varieties (ey7954, syz3, xs11, xy5968, xy9308, z903) rice seeds which are widely planted in Zhejiang Province. Seven color and fourteen morphological features were used for discriminant analysis. Two hundred and forty kernels used as the training data set and sixty kernels as the test data set in the neural network used to identify rice seed varieties. When the model was tested on the test data set, the identification accuracies were 90.00%, 88.00%, 95.00%, 82.00%, 74.00%, 80.00% for ey7954, syz3, xs11, xy5968, xy9308, z903 respectively.
Rice Seed Cultivar Identification Using Near-Infrared Hyperspectral Imaging and Multivariate Data Analysis
A near-infrared (NIR) hyperspectral imaging system was developed in this study. NIR hyperspectral imaging combined with multivariate data analysis was applied to identify rice seed cultivars. Spectral data was exacted from hyperspectral images. Along with Partial Least Squares Discriminant Analysis (PLS-DA), Soft Independent Modeling of Class Analogy (SIMCA), K-Nearest Neighbor Algorithm (KNN) and Support Vector Machine (SVM), a novel machine learning algorithm called Random Forest (RF) was applied in this study. Spectra from 1,039 nm to 1,612 nm were used as full spectra to build classification models. PLS-DA and KNN models obtained over 80% classification accuracy, and SIMCA, SVM and RF models obtained 100% classification accuracy in both the calibration and prediction set. Twelve optimal wavelengths were selected by weighted regression coefficients of the PLS-DA model. Based on optimal wavelengths, PLS-DA, KNN, SVM and RF models were built. All optimal wavelengths-based models (except PLS-DA) produced classification rates over 80%. The performances of full spectra-based models were better than optimal wavelengths-based models. The overall results indicated that hyperspectral imaging could be used for rice seed cultivar identification, and RF is an effective classification technique.
Effects of different forms of antimony on rice during the period of germination and growth and antimony concentration in rice tissue
Seed germination and pot experiments for rice were conducted to investigate the effects of Sb(III) and Sb(V) on the growth and yield of rice, and the antimony concentration in rice tissue. Antimony was applied either as antimony potassium tartrate (III) or as potassium antimonate (V) in the experiments. The result show that Sb(III) and Sb(V) can affect the growth of root and rice sprout, and reduce the transformation ratio of dry matter during the germination period of rice seed, also, it can affect the activity of α-amylase and the growth of rice. A reduction in yield and an increase of antimony in rice were significantly related to Sb application rates to soils. The results also suggest that not only the application rate of Sb, but also the chemical form of Sb form should be considered while assessing the effect of Sb on plant and the danger of Sb contamination of soils as well as of food.
Protective roles of nitric oxide on seed germination and seedling growth of rice (Oryza sativa L.) under cadmium stress.
Nitric oxide (NO) is a bioactive molecule in plants which mediates a variety of physiological processes and responses to biotic and abiotic stresses including heavy metals. In the present study, the effects of exogenous NO donor sodium nitroprusside (SNP) on rice seed germination and seedlings growth were investigated under Cd stress and a possible mechanism was postulated. The results indicated that 100μM Cd significantly decreased rice seed germination index, vigor index, root and shoot lengths as well as fresh weight compared to control. Exogenous SNP dose-dependently attenuated the inhibition of rice seed germination and thereafter seedling growth caused by Cd. The promoting effect was most pronounced at 30μM SNP. Cd exposure caused oxidative stress by elevating hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents in root and shoot of rice seedlings. 30μM SNP counteracted partly Cd toxicity by reducing the H2O2 and MDA contents of Cd-exposed seedlings. Meanwhile, application of SNP markedly stimulated the activities of superoxide dismutases (SOD), ascorbate peroxidases (APX), guaiacol peroxidase (POD) and catalases (CAT) compared with Cd treatment alone, thereby indicating the enhancement of the antioxidative capacity in the root and shoot under Cd stress. In addition, addition of 30μM SNP increased accumulation of proline in both root and shoot. The Cd accumulation in seedlings was significant reduced by SNP, implicating that the protective role of SNP was responsible for preventing Cd accumulation. However, the effects of SNP were reverted by addition of cPTIO, a NO scavenger, suggesting the protective roles of SNP might be related to the induction of NO. Furthermore, K3Fe(CN)6 and [Formula: see text] / [Formula: see text] had no similar roles as SNP. Based on these results, it can be concluded that SNP exerted an advantageous effect on alleviating the inhibitory effect of Cd on rice seed germination and seedling growth, which might interact with NO.
Variety Identification of Single Rice Seed Using Hyperspectral Imaging Combined with Convolutional Neural Network
The feasibility of using hyperspectral imaging with convolutional neural network (CNN) to identify rice seed varieties was studied. Hyperspectral images of 4 rice seed varieties at two different spectral ranges (380–1030 nm and 874–1734 nm) were acquired. The spectral data at the ranges of 441–948 nm (Spectral range 1) and 975–1646 nm (Spectral range 2) were extracted. K nearest neighbors (KNN), support vector machine (SVM) and CNN models were built using different number of training samples (100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 and 3000). KNN, SVM and CNN models in the Spectral range 2 performed slightly better than those in the Spectral range 1. The model performances improved with the increase in the number of training samples. The improvements were not significant when the number of training samples was large. CNN model performed better than the corresponding KNN and SVM models in most cases, which indicated the effectiveness of using CNN to analyze spectral data. The results of this study showed that CNN could be adopted in spectral data analysis with promising results. More varieties of rice need to be studied in future research to extend the use of CNNs in spectral data analysis.
Global Proteome Analyses of Lysine Acetylation and Succinylation Reveal the Widespread Involvement of both Modification in Metabolism in the Embryo of Germinating Rice Seed.
Regulation of rice seed germination has been shown to mainly occur at post-transcriptional levels, of which the changes on proteome status is a major one. Lysine acetylation and succinylation are two prevalent protein post-translational modifications (PTMs) involved in multiple biological processes, especially for metabolism regulation. To investigate the potential mechanism controlling metabolism regulation in rice seed germination, we performed the lysine acetylation and succinylation analyses simultaneously. Using high-accuracy nano-LC-MS/MS in combination with the enrichment of lysine acetylated or succinylated peptides from digested embryonic proteins of 24 h after imbibition (HAI) rice seed, a total of 699 acetylated sites from 389 proteins and 665 succinylated sites from 261 proteins were identified. Among these modified lysine sites, 133 sites on 78 proteins were commonly modified by two PTMs. The overlapped PTM sites were more likely to be in polar acidic/basic amino acid regions and exposed on the protein surface. Both of the acetylated and succinylated proteins cover nearly all aspects of cellular functions. Ribosome complex and glycolysis/gluconeogenesis-related proteins were significantly enriched in both acetylated and succinylated protein profiles through KEGG enrichment and protein-protein interaction network analyses. The acetyl-CoA and succinyl-CoA metabolism-related enzymes were found to be extensively modified by both modifications, implying the functional interaction between the two PTMs. This study provides a rich resource to examine the modulation of the two PTMs on the metabolism pathway and other biological processes in germinating rice seed.
Prevention of allergic asthma by vaccination with transgenic rice seed expressing mite allergen: induction of allergen-specific oral tolerance without bystander suppression.
This study tested the feasibility of oral immunotherapy for bronchial asthma using a newly developed subunit vaccine in which a fragment (p45-145) of mite allergen (Der p 1) containing immunodominant human and mouse T cell epitopes was encapsulated in endoplasmic reticulum-derived protein bodies of transgenic (Tg) rice seed. Allergen-specific serum immunoglobulin responses, T cell proliferation, Th1/Th2 cytokine production, airway inflammatory cell infiltration, bronchial hyper-responsiveness (BHR) and lung histology were investigated in allergen-immunized and -challenged mice. Prophylactic oral vaccination with the Tg rice seeds clearly reduced the serum levels of allergen-specific IgE and IgG. Allergen-induced CD4(+) T cell proliferation and production of Th2 cytokines in vitro, infiltration of eosinophils, neutrophils and mononuclear cells into the airways and BHR were also inhibited by oral vaccination. The effects of the vaccine were antigen-specific immune response because the levels of specific IgE and IgG in mice immunized with Der f 2 or ovalbumin were not significantly suppressed by oral vaccination with the Der p 1 expressing Tg rice. Thus, the vaccine does not induce nonspecific bystander suppression, which has been a problem with many oral tolerance regimens. These results suggest that our novel vaccine strategy is a promising approach for allergen-specific oral immunotherapy against allergic diseases including bronchial asthma.
Nondestructive determination of transgenic Bacillus thuringiensis rice seeds (Oryza sativa L.) using multispectral imaging and chemometric methods.
Crop-to-crop transgene flow may affect the seed purity of non-transgenic rice varieties, resulting in unwanted biosafety consequences. The feasibility of a rapid and nondestructive determination of transgenic rice seeds from its non-transgenic counterparts was examined by using multispectral imaging system combined with chemometric data analysis. Principal component analysis (PCA), partial least squares discriminant analysis (PLSDA), least squares-support vector machines (LS-SVM), and PCA-back propagation neural network (PCA-BPNN) methods were applied to classify rice seeds according to their genetic origins. The results demonstrated that clear differences between non-transgenic and transgenic rice seeds could be easily visualized with the nondestructive determination method developed through this study and an excellent classification (up to 100% with LS-SVM model) can be achieved. It is concluded that multispectral imaging together with chemometric data analysis is a promising technique to identify transgenic rice seeds with high efficiency, providing bright prospects for future applications.
Characterization of Pepstatin-Sensitive Acid Protease in Resting Rice Seeds
Resting rice seeds contained an acid protease whose optimal pH was 3.5 for casein containing 3.6 m urea and 2.5~3.0 for acid denatured haemoglobin. The enzyme was stable in a pH range between 5 and 9. The apparent molecular weight of the enzyme was 60,000~65,000. The enzyme was separated into several fractions by ion-exchange chromatography on DE-32 and by isoelectric focusing. Multiple forms of the enzyme were assumed to be due to the complex formation of the enzyme and inactive proteins. The enzyme activity was completely inhibited by 1 × 10−6 m pepstatin. ID50 value of the enzyme for pepstatin was estimated to be 5 × 10−6 m, which indicates a high affinity of the enzyme for pepstatin. The enzyme is a typical acid protease (carboxyl proteinase) that is sensitive to pepstatin.
Priming Effect of Rice Seeds on Seedling Establishment under Adverse Soil Conditions
An experiment was carried out to find out the priming effects of rice seeds, Oryza sativa L. (cv. Ilpumbyeo) on. the seedling establishment and early emergence under excess soil moisture conditions. Seeds were primed by soaking in -0.6 MPa polyethylene glycol (PEG) solution at 25~circC for 4 days. The primed seeds were sown in soils with various soil moistures (60, 80, 100, 120, and 140% field capacity) at 17 and 25~circC , respectively. Germination and emergence rates, plumule height, and radicle length of primed seeds were higher than those of untreated seeds at any soil moisture and temperature examined. The time from planting to 50% germination (T50 ) of primed seeds was less than that of untreated seeds by 0.9～3.7 days. Germination rate, emergence rate, plumule height, and radicle length were highest at the soil moisture of 80% field capacity among the soil moistures. Priming effects of rice seeds on germination and emergence rates were more prominent under the unfavorable soil moistures (60, 100, 120, and 140% field capacity) than those under the optimum soil moisture condition (80% field capacity). However, priming effects on seedling growth were greater at near optimum soil moisture compared with too lower or higher soil moistures. Therefore, these findings suggest that priming of rice seeds may be a useful way for better seedling establishment under the adverse soil conditions.
Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits.
Flavonoids possess diverse health-promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone-3-hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm-specific GluB-1 promoter or embryo- and aleurone-specific 18-kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB-II-type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes.
Unintended compositional changes in transgenic rice seeds ( Oryza sativa L.) studied by spectral and chromatographic analysis coupled with chemometrics methods.
Unintended compositional changes in transgenic rice seeds were studied by near-infrared reflectance, GC-MS, HPLC, and ICP-AES coupled with chemometrics strategies. Three kinds of transgenic rice with resistance to fungal diseases or insect pests were comparatively studied with the nontransgenic counterparts in terms of key nutrients such as protein, amino acids, fatty acids, vitamins, elements, and antinutrient phytic acid recommended by the Organization for Economic Co-operation and Development (OECD). The compositional profiles were discriminated by chemometrics methods, and the discriminatory compounds were protein, three amino acids, two fatty acids, two vitamins, and several elements. Significance of differences for these compounds was proved by analysis of variance, and the variation extent ranged from 20 to 74% for amino acids, from 19 to 38% for fatty acids, from 25 to 57% for vitamins, from 20 to 50% for elements, and 25% for protein, whereas phytic acid content did not change significantly. The unintended compositional alterations as well as unintended change of physical characteristic in transgenic rice compared with nontransgenic rice might be related to the genetic transformation, the effect of which needs to be elucidated by additional studies.
Approaches to improving the bioavailability and level of iron in rice seeds
Iron deficiency is estimated to affect about 30% of the world population, making iron by far the most deficient nutrient worldwide. Iron supplementation in the form of tablets and food fortification has not been successful in developing countries, and iron deficiency is still the most important deficiency related to malnutrition. Here we present experiments that aim to increase the iron content in rice endosperm and to improve its absorption in the human intestine by means of genetic engineering. We first introduced a ferritin gene from Phaseolus vulgaris into rice grains, increasing their iron content up to twofold. To increase iron bioavailability, we introduced a thermotolerant phytase from Aspergillus fumigatus into the rice endosperm. In addition, as cysteine peptides are considered a major enhancer of iron absorption, we overexpressed the endogenous cysteine-rich metallothionein-like protein. The content of cysteine residues increased about sevenfold and the phytase level in the grains about 130-fold, giving a phytase activity sufficient to completely degrade phytic acid in a simulated digestion experiment. However, the fungal protein did not retain its activity after rice cooking. Therefore a further attempt to specifically reduce the phytic acid in the inner part of the rice endosperm, the tissue eaten after the milling of rice grains, is discussed and biochemical analyses of the transgenic rice seeds are now in progress. Les carences alimentaires en fer touchent 30% de la population mondiale.L'enrichissement en fer de l'endosperme du riz et l'augmentation de l'absorption intestinale du fer sont possibles grâce au genie genetique. Le gene de la ferritine de phaseolus vulgaris est introduit dans les grains de riz, pour multiplier par 2 le contenu en fer. Pour augmenter la biodisponibilite, le gene de la phytase thermotolerente de Aspergillus fumigatus est egalement introduit dans le riz. Une metallothioneine riche en cysteine est surexprimee, son role etant d'ameliorer l'absorption du fer. Apres cuisson, la phytase perd une partie de son activite, il faut donc rechercher d'autres moyens de degrader l'acide phytique, inhibiteur de l'absorption du fer.
Application of terahertz spectroscopy imaging for discrimination of transgenic rice seeds with chemometrics.
Discrimination of genetically modified organisms is increasingly demanded by legislation and consumers worldwide. The feasibility of a non-destructive discrimination of transgenic rice seeds from its non-transgenic counterparts was examined by terahertz spectroscopy imaging system combined with chemometrics. Principal component analysis (PCA), least squares support vector machines (LS-SVM), PCA-back propagation neural network (PCA-BPNN), and random forest (RF) models with the first and second derivative and standard normal variate transformation (SNV) pre-treatments were applied to classify rice seeds based on genotype. The results demonstrated that differences between non-transgenic and transgenic rice seeds did exist, and an excellent classification (accuracy was 96.67% in the prediction set) could be achieved using the RF model combined with the first derivative pre-treatment. The results indicated that THz spectroscopy imaging together with chemometrics would be a promising technique to identify transgenic rice seeds with high efficiency and without any sample preparation.
QTL analysis of seed storability in rice
A double haploid population, which consists of 127 lines derived from anther culture of a typical indica and japonica hybrid 'ZYQ8'/'JX17', was used in this study. Seed storability was investigated by using the storage property measured by the difference of seed germination rates before and after treatment of the rice seeds under 40°C and 95% relative humidity for 10 days in a phytotron. Three QTLs related to rice seed storability were detected on chromosomes 9, 11 and 12, with the LOD scores 2.76, 4.83 and 2.54, respectively, together explaining 35.4% of the genetic variation. The 'JX17' allele at qLS-9 and the 'ZYQ8' alleles at qLS-11 and qLS-12 could enhance the rice seed storability. The effects of the 'ZYQ8' alleles of qLS-11 and qLS-12 were also verified using chromosome segment substitution lines.
Seed longevity of red rice ecotypes buried in soil
Red rice is a troublesome weed in irrigated rice production and is spread through contaminated commercial rice seed and machinery. Seed dormancy is a major trait for red rice. Studies were carried out at two locations to determine red rice seed longevity in the soil of several ecotypes from four US states. Five months after burial near Beaumont, Texas only three ecotypes had viable seed (<1%) when buried at 5 cm, but 9 ecotypes had viable seed after two years when buried at 25 cm. At the thirty-sixth month after burial, ecotypes Arkansas 2, Louisiana 2 and 4, Mississippi 4 and Texas 1 had viable seeds, but at less than 1%. Freshly harvested red rice seeds buried at 12 cm near College Station, TX, survived longer than seeds placed on the soil surface. The percentage of maximum viable seeds was 2% for blackhull type Texas 4, after 17 months. In both studies, commercial rice cultivar seeds were not viable after 5 months, regardless of their position in the soil. Under farming conditions with no fallow land preparations or deep tillage, most red rice seed germinated or was dead after 2 to 3 years, with only minor variation among ecotypes.
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