DNA uptake by imbibition and expression of a foreign gene in rice

Uptake of DNA by imbibition of dry and viable rice ( Oryza sativa L.) embryos from a DNA solution and expression of a foreign gene were detected using two different vectors contaíning gusA (β-glucuronidase) and hpt (hygromycin phosphotransferase) as reporter genes. The frequency of transient expression of gusA and hpt genes using the CaMV35S promoter was about 30 to 50%. The main sites of gusA gene expression were meristems of roots and vascular bundles of leaves. Also, DNA uptake, integration and expression of the hpt gene in selected rice were investigated by various PCR methods and Southern blot analysis of genomic DNA. It was shown that the hygromycin phosphotransferase (HPT) DNA was present in the rice genome in an integrated form and not as a plasmid form.     

The light-dependent regulation of gene expression during plastid development in higher plants

In recent years there has been a considerable increase in our understanding of the manner by which light affects gene expression during chloroplast development. In most systems that have been studied, light acts through sensitive photoreceptor molecules and quantitatively increases or represses the level of expression of specific nuclear-and plastid-encoded genes. Although the mechanisms are obscure, a picture is beginning to emerge in which the coordination of nuclear and plastid gene expression is controlled by regulatory mechanisms originating within their respective subcellular compartments. This review summarizes some of our current knowledge concerning the nature of light-regulated gene expression in higher plants and provides a prospectus for future research in this area.     

Regulation of the photosynthetic apparatus under fluctuating growth light

Safe and efficient conversion of solar energy to metabolic energy by plants is based on tightly inter-regulated transfer of excitation energy, electrons and protons in the photosynthetic machinery according to the availability of light energy, as well as the needs and restrictions of metabolism itself. Plants have mechanisms to enhance the capture of energy when light is limited for growth and development. Also, when energy is in excess, the photosynthetic machinery slows down the electron transfer reactions in order to prevent the production of reactive oxygen species and the consequent damage of the photosynthetic machinery. In this opinion paper, we present a partially hypothetical scheme describing how the photosynthetic machinery controls the flow of energy and electrons in order to enable the maintenance of photosynthetic activity in nature under continual fluctuations in white light intensity. We discuss the roles of light-harvesting II protein phosphorylation, thermal dissipation of excess energy and the control of electron transfer by cytochrome b₆f, and the role of dynamically regulated turnover of photosystem II in the maintenance of the photosynthetic machinery. We present a new hypothesis suggesting that most of the regulation in the thylakoid membrane occurs in order to prevent oxidative damage of photosystem I.     

Efficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in the plants

Summary Rice is one of the most important crops in the world with 35% of the total population (over two billion people) depending on it as their source of food. It is therefore essential to develop efficient methods for the transformation and regeneration of rice plants in order to delineate the exact regulatory sequences responsible for gene expression and to transfer beneficial genes into this plant. Here, for the first time, we present definitive evidence for the regeneration of a large number of transgenic rice plants after introduction of the bacterial -glucuronidase gene into rice protoplasts. The presence of integrated copies of this gene was detected in the genome of transgenic plants by DNA hybridization analysis. Furthermore, under the control of regulatory regions from a maize alcohol dehydrogenase sequence, -glucuronidase gene expression was detected in the roots of transgenic plants. This expression was stimulated up to six fold under anaerobic conditions.     

Silicon partially preserves the photosynthetic performance of rice plants infected by Monographella albescens

Leaf scald, caused by Monographella albescens, is one of the major diseases in rice worldwide. This study investigated the effect of silicon (Si) on the photosynthetic gas exchange parameters [net CO₂ assimilation rate (A), stomatal conductance to water vapour (g_s), transpiration rate (E)] and internal CO₂ concentration (C_i), chlorophyll (Chl) fluorescence a parameters [minimal fluorescence (F₀), maximum fluorescence (F_m), maximum quantum yield of photosystem II (F_v/F_m)], photochemical quenching coefficient (q_p), effective quantum yield of PSII [Y(II)], quantum yield of regulated energy dissipation [Y(NPQ)] and quantum yield dissipation non‐regulated [Y(NO)] and the concentrations of pigments in rice plants grown in nutrient solutions containing either 0 (?Si) or 2 mM Si (+Si) and non‐inoculated or inoculated with M. albescens. Leaf scald severity decreased with higher foliar Si concentration. For the inoculated +Si plants, A, g_s and E were significantly higher in comparison with the inoculated ?Si plants, in which C_i was significantly increased. Similarly, the concentrations of Chl_a, Chl_b, total Chl_a+b and carotenoids were higher for the +Si plants in comparison with the ?Si plants. Changes in the images of Chl a fluorescence were first observed precisely on the ?Si plants leaves in comparison with the +Si plants. A decrease of q_P and Y(II) in inoculated ?Si plants, in comparison with the inoculated +Si plants, was accompanied by an increase in Y(NPQ) and Y(NO). Notably, the extent of the leaf areas was much more evident for Y(II) and q_P in comparison with F₀, F_m and F_v/F_m, suggesting that Y(II) and q_P were good predictors in detecting the early effects of leaf scald on the leaf photosynthesis. For the +Si non‐inoculated plants, changes in Y(II) were associated with alterations in both Y(NPQ) and Y(NO) compared with non‐inoculated ?Si plants. In conclusion, the photosynthetic performance (as demonstrated by the gas exchange and Chl a fluorescence parameters) and the pigment pools of rice plants infected with M. albescens were preserved by Si supply and, therefore, provided an increase in rice resistance against leaf scald.     

Submergence tolerance of rice – A new glasshouse method for the experimental submergence of plants

A new method is described for evaluation of submergence tolerance of rice ( Oryza sativa L.) plants. Responses of a range of cultivars corresponded with known differences in field performance. The method 1) allows fast and effective determination of submergence tolerance, 2) allows screening of many plants in a small glasshouse area, 3) provides for recovery of superior plants for seed collection, 4) allows manipulation of many environmental variables to mimic the natural submergence environment, and 5) uses simple, inexpensive, readily available equipment. Physiological studies performed with this method gave results similar to those obtained from field studies and showed that submergence tolerance increased in older plants; it decreased with increasing depth, increasing temperature and with high or low light levels. The system is ideal for the rapid evaluation of rice germplasm under controlled conditions and physiological studies on the mechanism of rice submergence tolerance.     

A rice promoter containing both novel positive and negative cis-elements for regulation of green tissue-specific gene expression in transgenic plants

The tissue-specific expression of transgenes is essential in plant breeding programmes to avoid the fitness costs caused by constitutive expression of a target gene. However, knowledge on the molecular mechanisms of tissue-specific gene expression and practicable tissue-specific promoters is limited. In this study, we identified the cis -acting elements of a tissue-specific promoter from rice, P_D54O , and tested the application of original and modified P_D54O and its cis -elements in the regulation of gene expression. P_D54O is a green tissue-specific promoter. Five novel tissue-specific cis -elements (LPSE1, LPSE2, LPSRE1, LPSRE2, PSE1) were characterized from P_D54O . LPSE1 activated gene expression in leaf and young panicle. LPSRE2 suppressed gene expression in leaf, root, young panicle and stem, and PSE1 suppressed gene expression in young panicle and stem. LPSRE1 and LPSE2 had dual roles in the regulation of tissue-specific gene expression; both functioned as activators in leaf, but LPSRE1 acted as a repressor in stem and LPSE2 as a repressor in young panicle and root. Transgenic rice plants carrying cry1Ac encoding Bacillus thuringiensis endotoxin, regulated by P_D54O , were resistant to leaf-folders, with no Cry1Ac protein found in endosperm or embryo. A reporter gene regulated by a series of truncated P_D54O showed various tissue-specific expression patterns. Different fragments of P_D54O fused with the constitutive cauliflower mosaic virus 35S promoter suppressed 35S -regulated gene expression in various tissues. P_D54O , truncated P_D54O and the tissue-specific cis -elements provide useful tools for the regulation of tissue-specific gene expression in rice breeding programmes.     

Transient expression of gus gene in intact seed embryos of Indica rice after electroporation-mediated gene delivery

Summary Two-day-old germinating intact seed embryos of Oryza sativa variety Basmati 370 were electroporated with a view to examine suitability of this system for gene delivery. The experiments were done with a plasmid having gus gene under the control of CaMV 35S promoter. Spectrofluorophotometric GUS assay revealed high activity of the introduced gene when embryos were given three electrical pulses at 1600 V cm^-1 and 100 F capacitance with a pulse length of 75 ms. Additionally, histochemical localization of GUS activity in seedlings and various organs such as leaves, coleoptiles and roots was also done. Expression of GUS activity was studied up to 15 days and found to be organ-specific, thereby showing that embryos can indeed serve as efficient recipient system. Use of cycloheximide revealed that GUS activity appears as a result of early protein synthesis after electroporation and is substantially stable in vivo.     

Effect of phosphorus deficiency on the photosynthetic characteristics of rice plants

The effects of phosphorus deficiency on the photosynthetic characteristics were studied in rice seedlings (Oryza sativa L.) every 8 days after treatment. P deficiency caused a significant reduction in the net photosynthesis rate (P _N) in rice plants. During the first 16 days of P deficiency, the maximum efficiency of PSII photochemistry (F _v/F _m), the effective PSII quantum yield (ϕ_PSII), the electron transport rate (ETR) as well as photochemical quenching (qP) in the P-limited rice plants kept close to the control, but the excitation energy capture efficiency of PSII reaction centers (F′_v/F′_m) was significantly declined in the P-deficient rice leaves. Meanwhile, in the stressed leaves, we also found a significant increase in nonphotochemical quenching (NPQ) as well as in the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX). It was indicated that a series of photoprotective mechanisms had been initiated in rice plants in response to short-term P deficiency. Therefore, PSII functioning was not affected significantly under such stress. As P deficiency continued, the excess excitation energy was accumulated in excess of the capacity of photoprotection systems. When the rice suffered from P deficiency more than 16 days, ϕ_PSII, ETR, and qP were decreased more rapidly than that in the control plants, although NPQ still kept higher in the stressed plants. These results were also consistent with the data on the distribution of excitation energy. The excess energy induced the generation of reactive oxygen species, which might lead to the further damage to PSII functioning. This text was submitted by the authors in English.     

Restoration of gibberellin biosynthesis by 2,6-diisopropylphenoxyacetic acid in uniconazole-treated rice plants

2,6-Diisopropylphenoxyacetic acid (DIPA), a promoter of growth and flowering of Sagittaria species, was found to improve the shoot growth of rice plants treated with uniconazole, an inhibitor of gibberellin (GA) biosynthesis. In a modified micro-drop bioassay using semi-dwarf rice, Oryza sativa L. cv. Tan-ginbozu, in which uniconazole had been incorporated into the agar medium, a significant recovery from growth inhibition was observed for both the 3rd and the 4th leaf sheaths but not for the 2nd sheath. In greenhouse experiments, uniconazole-treated rice plants partially recovered from growth inhibition when DIPA was applied after uniconazole treatment, whereas DIPA applied with, or before, uniconazole treatment did not improve growth. The levels of GA₁ and GA₂₀ in the rice plants treated with uniconazole plus DIPA were almost equal to those of the untreated controls, indicating that the observed growth recovery is attributable to the restoration of GA biosynthesis by DIPA.     

Systemic induction of proteinase-inhibitor-II gene expression in potato plants by wounding

The systemic induction of expression of the gene for proteinase inhibitor II after wounding different parts of potato (Solanum tuberosum L.) plants was analysed at the RNA level. Wounding of either leaves or tubers led to an induction of expression of this gene in non-wounded upper and lower leaves as well as in the upper stem segment, whereas no expression was observed in nonwounded roots or in the lower stem segment. The signal mediating the systemic induction in nonwounded tissue must therefore be able to move both acropetally and basipetally. The systemic wound response is specific for the expression of the proteinase-inhibitor-II gene as no influence was observed for the expression of genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the tuber storage protein patatin which were examined in parallel with the proteinase-inhibitor-II gene.Abbreviation ssRubisco small subunit of ribulose-1,5-bis-phosphate carboxylase     

Changes in growth and photosynthetic capacity of rice with increased UV-B radiation

Sixteen rice ( Oryza sativa L.) cultivars from 7 different geographical regions were grown in greenhouses at the Univ. of Maryland with and without supplemental ultraviolet-B (UV-B) radiation to determine alterations in biomass, morphology and maximum photosynthesis that would be anticipated from potential reductions in the stratospheric ozone column. A wide range of UV-B effects were observed, with the Philippines cultivar Carreon (5993) and the Sri Lankan cultivar Kurkaruppan (15449) showing the greatest decrease and increase, respectively, in total biomass with supplemental UV-B radiation. Approximately one-third of all cultivars tested showed a statistically significant decrease in total biomass with UV-B radiation. For these sensitive cultivars, leaf area and tiller number were also significantly reduced. Photosynthetic capacity as determined by oxygen evolution declined for some cultivars, but the correlation between changes in photosynthesis and biomass with increasing UV-B was equivocal. Results from this experiment indicate that: (1) a number of rice cultivars are sensitive to potential increases in UV-B radiation: and (2) the diversity exhibited by rice in response to increased levels of UV-B suggests that selective breeding might be successfully used to develop UV-B-tolerant rice cultivars.     

Developmental behavior of gene expression for brown rice thickness under different environments

The dynamic changes of genetic effects, including main effects, and genotype x environment (GE) interaction effects on brown rice thickness (BRT) across environments were investigated by using the developmental genetic models. Seven cytoplasmic male sterile lines of indica rice (Oryza sativa L.) as females and five restoring lines as males were used in a factorial design to produce grains of F(1)s and F(2)s in two environments (years) for developmental genetic analysis. The results indicate that genetic effects, especially GE interaction effects of triploid endosperm genes, cytoplasm genes, and diploid maternal plant genes were important to the performance of BRT at various filling stages of rice. The BRT was genetically controlled by the net genetic effects of genes expressed at the early and late filling stages (1-7 days and 15-21 days after flowering, respectively). The differences in net genetic effects under different environments for endosperm, cytoplasm, and maternal plant genes were found, and the net GE interaction effects were more important to BRT at the early filling and mature stages of rice. Some net genetic effects, especially for net cytoplasm effects spasmodically expressed, were detected among filling stages. Higher additive and cytoplasm main effects, along with their interaction effects, were found, which would be useful for selection for BRT in breeding programs. The predicated genetic effects at different filling stages show that the parents of V20 and Xieqingzao were better than others for improving BRT of progenies.     

Structure, expression pattern and chromosomal localization of the rice Osgrp-2 gene

The plant cell walls comprise various enzymes and several kinds of structural proteins. In addition to the structural roles, the structural cell wall proteins also function in altering the physi-cal properties of cell walls as cells grow, divide and differentiate, and in repairing of cell walls after infection or wounding[1,2]. Plant structural cell wall proteins may be divided into four main classes: extensins, proline-rich proteins (PRPs), arabinogalactan proteins (AGPs) and glycine-rich p…     

Compatibility of glycinebetaine in rice plants: evaluation using transgenic rice plants with a gene for peroxisomal betaine aldehyde dehydrogenase from barley

Glycinebetaine is synthesized in plants by the two‐step oxidation of choline, with betaine aldehyde as the intermediate. The reactions are catalyzed by choline mono‐oxygenase and betaine aldehyde dehydrogenase. Rice plants, which do not accumulate glycinebetaine, possess a gene encoding betaine aldehyde dehydrogenase, whose activity is detectable at low levels. To evaluate the compatibility in rice of glycinebetaine on growth and tolerance to salt, cold and heat, we produced transgenic rice plants by introduction of a cDNA for betaine aldehyde dehydrogenase of barley, which is localized in peroxisomes unlike the chloroplast‐specific localization of betaine aldehyde dehydrogenase in spinach and sugar beet. The transgenic rice plants converted high levels of exogenously applied betaine aldehyde (up to 10 mol m^–3) to glycinebetaine more efficiently than did wild‐type plants. The elevated level of glycinebetaine in transgenic plants conferred significant tolerance to salt, cold and heat stress. However, very high levels of glycinebetaine, resulting from conversion of applied betaine aldehyde to glycinebetaine or from exogenous application, inhibited increases in length of rice plants but not increases in dry weight. Our results suggested that the benefits of accumulation of glycinebetaine by rice plants might be considerable under high light conditions.     

Early and specific gene expression triggered by rice resistance gene Pi33 in response to infection by ACE1 avirulent blast fungus

* Our view of genes involved in rice disease resistance is far from complete. Here we used a gene-for-gene relationship corresponding to the interaction between atypical avirulence gene ACE1 from Magnaporthe grisea and rice resistance gene Pi33 to better characterize early rice defence responses induced during such interaction. * Rice genes differentially expressed during early stages of Pi33/ACE1 interaction were identified using DNA chip-based differential hybridization and QRT-PCR survey of the expression of known and putative regulators of disease resistance. * One hundred genes were identified as induced or repressed during rice defence response, 80% of which are novel, including resistance gene analogues. Pi33/ACE1 interaction also triggered the up-regulation of classical PR defence genes and a massive down-regulation of chlorophyll a/b binding genes. Most of these differentially expressed genes were induced or repressed earlier in Pi33/ACE1 interaction than in the gene-for-gene interaction involving Nipponbare resistant cultivar. * Besides demonstrating that an ACE1/Pi33 interaction induced classical and specific expression patterns, this work provides a list of new genes likely to be involved in rice disease resistance.