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.     

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.     

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.     

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.     

Transgenic rice plants: Characterization of two generations of seed progeny

Transgenic rice plants have been regenerated from kanamycin-resistant callus of Oryza sativa (cv. Taipei 309) derived from protoplasts electroporated with pCaMVNEO carrying the neomycin phosphotransferase II ( nptII ) gene. Of 6 randomly selected plants, all contained the nptll gene, but only 2 plants expressed NPTII activity. The transgenic plants were significantly shorter, produced fewer tillers, took longer to flower and had reduced fertility compared to non-transformed protoplastderived plants. Fifty-six seeds collected from one transgenic plant expressing NPTII activity germinated on medium containing kanamycin sulphate to give 16 green, first seed generation (R₁) plants. The latter could be divided into 3 groups: (i) Plants which set seed, had normal floret morphology and produced a total of 76 seeds; (ii) Plants which flowered, but which failed to set seed; (iii) Plants which failed to flower, were shorter and had significantly fewer tillers than plants of groups (i) and (ii). The nptII gene was present in all transgenic R₁ plants, but only 8 plants expressed the gene. Phenotypic characteristics, observed in transgenic R₁ plants were also seen in the transforned R₂ plants. These included reduced stature, a longer vegetative phase and reduced fertility compared to non-transformed plants.     

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.     

Transient expression of electroporated gene in leaf protoplasts of indica rice and influence of template topology and vector sequences

Electroporation was used for gene delivery and evaluation of various parameters affecting transient expression of a gene for β-glucuronidase ( gus ) in leaf protoplasts of Oryza sativa var. Basmati-370. Transient expression was found to be dependent on voltage, capacitance, amount of plasmid and carrier DNA as well as period of culture. Maximum GUS activity was obtained when a 150 ms pulse at 300 V cm^-1 and 200 μF was applied to the protoplasts (l–2×10⁶ml⁻¹) in an electroporation buffer containing 20 μg of plasmid and 30 μg of calf thymus DNA, assayed 48 h after electroporation. DNA topology did not influence expression of the gene as both linear and supercoiled templates resulted in similar activities, but a 4-fold decrease in expression was observed if the gene was excised, reflecting the positive influence of vector sequences on gene expression.     

Influence of supplemental UV-B radiation on photosynthetic characteristics of rice plants

In a field experiment with rice (Oryza sativa L. cv. Saket 4) grown under ambient and supplemental ultraviolet-B (UV-B) radiation at 20 % ozone depletion, differences in gas exchange, concentrations of photosynthetic pigments, anthocyanins and flavonoids, biomass accumulation, catalase and peroxidase activities, and contents of ascorbic acid and phenol were determined. Decline in photosynthesis was associated with reductions in stomatal conductance and concentrations of photosynthetic pigments. Enhanced UV-B radiation (eUV-B) increased the contents of flavonoid and phenolic compounds in leaves. Peroxidase activity increased and catalase activity was always lower at eUV-B. The total plant biomass decreased at eUV-B.     

Regulation of gene expression by endogenous ABA in tomato plants

In response to water deficit, endogenous abscisic acid (ABA) accumulates in plants. This ABA serves as a signal for a multitude of processes, including regulation of gene expression. ABA accumulated in response to water deficit signals cellular as well as whole plant responses playing a role in the pattern of gene expression throughout the plant. Although the function of genes regulated by ABA during stress are currently poorly understood, a number of these genes may permit the plant to adapt to environmental stress.     

Relationships of differential gene expression in leaves with heterosis and heterozygosity in a rice diallel cross

Using differential display analysis, we assessed the patterns of differential gene expression in hybrids relative to their parents in a diallel cross involving 8 elite rice lines. The analysis revealed several patterns of differential expression including: (1) bands present in one parent and F₁ but absent in the other parent, (2) bands observed in both parents but not in the F₁, (3) bands occurring in only one parent but not in the F₁ or the other parent, and, (4) bands detected only in the F₁ but in neither of the parents. Relationships between differential gene expression and heterosis and marker heterozygosity were evaluated using data for RFLPs, SSRs and a number of agronomic characters. The analysis showed that there was very little correlation between patterns of differential expression and the F₁ means for all six agronomic traits. Differentially expressed fragments that occurred only in one parent but not in the other parent or in F₁ in each of the respective crosses were positively correlated with heterosis and heterozygosity. And conversely, fragments that were detected in F₁s but in neither of the respective parents were negatively correlated with heterosis and heterozygosity. The remaining patterns of differential expression were not correlated with heterosis or heterozygosity. The relationships between the patterns of differential expression and heterosis observed in this study were not consistent with expectations based on dominance or overdominance hypotheses.     

Origins and population genetics of weedy red rice in the USA

Weedy red rice (Oryza sativa spontonea) is a persistent and problematic weed of rice culture worldwide. A major hypothesis for the mechanism of production of this weed in South and Southeast Asia is hybridization between cultivated rice (Oryza sativa) and wild rice (Oryza rufipogon). However, weedy red rice can often be found outside the range of O. rufipogon leaving questions on the origin and process behind weedy rice infestations. In the USA, weedy red rice was first documented as early as 1846 and has continued to affect rice production areas. In this study, we attempt to identify the origin and population structure of weedy red rice sampled from the USA using both DNA sequence data from a neutral nuclear locus as well as microsatellite genotype data. Results suggest that two major accessions of weedy rice exist, strawhull and blackhull, and these forms may both hybridize with the cultivated rice of the USA, O. sativa japonica. Using population assignment of multilocus genotype signatures with principal component analysis and structure, an Asian origin is supported for US weedy rice. Additionally, hybridization between strawhull and blackhull varieties was inferred and may present the opportunity for the production of new weedy forms in the future.     

Characterisation of the PsbX protein from Photosystem II and light regulation of its gene expression in higher plants

The location and expression of the previously uncharacterised photosystem II subunit PsbX have been analysed in higher plants. We show that this protein is a component of photosystem II (PSII) core particles but absent from light-harvesting complexes or PSII reaction centres. PsbX is, however, localised to the near vicinity of the reaction centre because it can be cross-linked to cytochrome b559, which is known to be associated with the D1/D2 dimer. We also show that the expression of this protein is tightly regulated by light, since neither protein nor mRNA is found in dark-grown plants.