Arjula R. Reddy

Differential sensitivity of rice pathogens to growth inhibition by flavonoids

Differential sensitivity of the major pathogens of rice, Xanthomonas oryzae pv. oryzae, Pyricularia oryzae and Rhizoctonia solani to inhibition by certain flavonoids was tested using paper disc/liquid culture and spore germination assays. Naringenin, the first intermediate of the flavonoid pathway, displayed growth inhibition of Xanthomonas strains and spore germination of P. oryzae. On the other hand, no such inhibition was found with Rhizoctonia solani. Crude extracts of leaf and pericarp tissues of a fully purple pigmented rice cultivar, Purpleputtu, also showed growth inhibition of Xanthomonas.

DNA helix destabilization by proline and betaine: possible role in the salinity tolerance process

Evidence is provided for the ability of proline, a salinity induced osmoprotectant, to destabilize the double helix and lower the T m of DNA in a concentration dependent manner. At the reported salinity‐adaptive bio‐accumulation of 1 M and above, proline could considerably decrease the T m and partially counteract the effect of sodium chloride and spermidine on DNA stability. On the contrary, several other amino acids tested did not show any such destabilizing effect on DNA helix. Enhanced susceptibility to S1 nuclease and insensitivity to DNase I in presence of increasing proline concentrations have further suggested a clear destabilization of the double helix. Such an effect is somewhat reminiscent of the interaction between betaine, another salinity induced osmolyte, and DNA resulting in decreased T m values. These interactions may be significant in view of the abundance of such osmolytes in cells under salinity stress‐adapted conditions, with many a bacterial mutant accumulating them exhibiting improved tolerance to salinity.

Ultraviolet-B-Responsive Anthocyanin Production in a Rice Cultivar Is Associated with a Specific Phase of Phenylalanine Ammonia Lyase Biosynthesis

Seedlings of 17 rice (Oryza sativa L.) cultivars were classified on the basis of anthocyanin pigmentation into three groups: an acyanic group with 9 cultivars, a moderately cyanic group with 5 cultivars, and a cyanic group with 3 cultivars. Seedlings of the cyanic group were deep purple in color, possessing copious amounts of anthocyanin in shoots. Sunlight (SL)-mediated anthocyanin and phenylalanine ammonia lyase (PAL) induction in a cyanic cultivar, purple puttu, was compared with an acyanic cultivar, black puttu. A brief exposure of dark-grown purple puttu seedlings to SL induced anthocyanin formation during a subsequent dark period with a peak at 24 h. The magnitude of SL-mediated anthocyanin induction is age dependent, the 4-d-old seedlings being the most responsive to SL. The anthocyanin induction in purple puttu seedlings is mediated exclusively by the ultraviolet-B (UV-B) component of SL. The SL-triggered anthocyanin induction was reduced by about 30% by a terminal far-red light pulse and was restored by a red light pulse, indicating the role of phytochrome in modulation of anthocyanin level. The SL-mediated induction of PAL showed two peaks, one at 4 h and the other at 12 h. Whereas the first PAL peak (4 h) was induced by phytochrome and was seen in both cultivars, the second PAL peak (12 h) was inducible by UV-B only in the cyanic purple puttu cultivar.

Isolation and characterization of expressed sequence tags (ESTs) from subtracted cDNA libraries of Pennisetum glaucum seedlings

Pearl millet (Pennisetum glaucum), used as forage and grain crop is a stress tolerant species. Here we identify differentially regulated transcripts in response to abiotic (salinity, drought and cold) stresses from subtracted cDNA libraries by single-pass sequencing of cDNA clones. A total of 2,494 EST sequences were clustered and assembled into a collection of 1,850 unique sequences with 224 contigs and 1,626 singleton sequences. By sequence comparisons the putative functions of many ESTs could be assigned. Genes with stress related functions include those involved in cellular defense against abiotic stresses and transcripts for proteins involved in stress response signaling and transcription in addition to ESTs encoding unknown functions. These provide new candidate genes for investigation to elucidate their role in abiotic stress. The relative mRNA abundance of 38 selected genes, quantified using real time quantitative RT-PCR, demonstrated the existence of a complex gene regulatory network that differentially modulates gene expression in a kinetics-specific manner in response to different abiotic stresses. Notably, housekeeping and non-target genes were effectively reduced in these subtracted cDNA libraries constructed. These EST sequences are a rich source of stress-related genes and reveal a major part of the stress-response transcriptome that will provide the foundation for further studies into understanding Pennisetum’s adaptability to harsh environmental conditions.

Development of transgenic rice plants expressing maize anthocyanin genes and increased blast resistance

The functional association of flavonoids with plant stress responses, though widely reported in the literature, remains to be documented in rice. Towards this end we chose a transgenic approach with well characterized regulatory and structural genes from maize involved in flavonoid biosynthesis. Activation of anthocyanin pathway in rice was investigated with the maize genes. Production of purple anthocyanin pigments were observed in transformed Tp309 (a japonica rice variety) calluses upon the introduction of the maize regulatory genes C1 (coloured-1), R (red) and the structural gene C2 (coloured-2, encoding chalcone synthase). In addition, stable transgenic plants carrying the maize C2 gene under the control of the maize Ubiquitin promoter were generated. A localized appearance of purple/red pigment in the leaf blade and leaf sheath of R0C2 transgenic seedlings was observed. Such a patchy pattern of the transgene expression appears to be conditioned by the genetic background of Tp309, which is homozygous for dominant color inhibitor gene(s) whose presence was unravelled by appropriate genetic crosses. Southern blot analysis of the transgenic plants demonstrated that c2 cDNA was integrated into the genome. Western blot analysis of these primary transgenics revealed the CHS protein while it was not detected in the control untransformed Tp3O9, suggesting that Tp309 might have a mutation at the corresponding C2 locus or that the expression of this gene is suppressed in Tp309. Further analysis of C2 transgenics revealed CHS protein only in three out of sixteen plants that were western-positive in the R0 generation, suggesting gene silencing. Preliminary screening of these R1 plants against the rice blast fungus Magnaporthe grisea revealed an increase in resistance.

Aldose reductase in rice (Oryza sativa L.): stress response and developmental specificity

Aldose reductase (AR) protein and enzyme (alditol: NAD (P)(+) 1-oxidoreductase, EC 1.1.1.21) activity have been identified in mature seeds of indica rice cultivars. The protein begins to accumulate 15 days after pollination, reaches a peak at seed maturity and disappears upon imbibition. Furthermore, AR is induced in vegetative tissues in response to exogenous ABA application and other stress conditions, such as PEG mediated water stress and salinity. Increase in AR protein levels upon stress are in close agreement with a similar increase in enzyme activity. Varietal differences in AR levels have been demonstrated. Interestingly, all tested tolerant cultivars (as denoted by breeders) accumulate AR in vegetative tisssue in response to ABA application, while the sensitive line, Hamsa, does not do this under similar stress conditions, suggesting that AR may be associated with stress tolerance. Furthermore, AR protein has been identified in mature seeds of some selected cereals indicating the conserved nature of AR across grasses.

Chalcone synthase in rice (Oryza sativa L.): Detection of the CHS protein in seedlings and molecular mapping of the chs locus

The chalcone synthase is a key enzyme that catalyses the first dedicated reaction of the flavonoid pathway in higher plants. The chs gene and its protein product in rice has been investigated. The presence of a chalcone synthase (CHS) protein in rice seedlings and its developmental stage-specific expression has been demonstrated by western analysis. The chalcone synthase of rice was found to be immunologically similar to that of maize. A rice cDNA clone, Os-chs cDNA, encoding chalcone synthase, isolated from a leaf cDNA library of an indica rice variety Purpleputtu has been mapped to the centromeric region of chromosome 11 of rice. It was mapped between RFLP markers RG2 and RG103. RG2 is the nearest RFLP marker located at a genetic distance of 3.3 cM. Some segments of chromosome 11 of rice including chs locus are conserved on chromosome 4 of maize. The markers, including chs locus on chromosome 11 of rice are located, though not in the same order, on chromosome 4 of maize. Genetic analysis of purple pigmentation in two rice lines, Abhaya and Shyamala, used in the present mapping studies, indicated the involvement of three genes, one of which has been identified as a dominant inhibitor of leaf pigmentation. The Os-chs cDNA shows extensive sequence homology, both for DNA and protein (deduced), to that of maize, barley and also to different monocots and dicots.

Annotation and BAC/PAC localization of nonredundant ESTs from drought-stressed seedlings of anindica rice

To decipher the genes associated with drought stress response and to identify novel genes in rice, we utilized 1540 high-quality expressed sequence tags (ESTs) for functional annotation and mapping to rice genomic sequences. These ESTs were generated earlier by 3−end single-pass sequencing of 2000 cDNA clones from normalized cDNA libraries constructed from drought-stressed seedlings of anindica rice. A rice UniGene set of 1025 transcripts was constructed from this collection through the BLASTN algorithm. Putative functions of 559 nonredundant ESTs were identified by BLAST similarity search against public databases. Putative functions were assigned at a stringency E value of 10-6 in BLASTN and BLASTX algorithms. To understand the gene structure and function further, we have utilized the publicly available finished and unfinished rice BAC/PAC (BAC, bacterial artificial chromosome; PAC, P1 artificial chromosome) sequences for similarity search using the BLASTN algorithm. Further, 603 nonredundant ESTs have been mapped to BAC/PAC clones. BAC clones were assigned by a homology of above 95% identity along 90% of EST sequence length in the aligned region. In all, 700 ESTs showed rice EST hits in GenBank. Of the 325 novel ESTs, 128 were localized to BAC clones. In addition, 127 ESTs with identified putative functions but with no homology in IRGSP (International Rice Genome Sequencing Program) BAC/PAC sequences were mapped to the Chinese WGS (whole genome shotgun contigs) draft sequence of the rice genome. Functional annotation uncovered about a hundred candidate ESTs associated with abiotic stress in rice andArabidopsis that were previously reported based on microarray analysis and other studies. This study is a major effort in identifying genes associated with drought stress response and will serve as a resource to rice geneticists and molecular biologists.

Flavonoid Biosynthetic Pathway and Cereal Defence Response: An Emerging Trend in Crop Biotechnoloy

The flavonoid pathway, derived from the phenylpropanoid and acetyl CoA and malonyl CoA pathways, gives rise to a diverse array of compounds such as isoflavonoids, anthocyanins, proanthocyanidins, etc. that are attributed with a multitude of biological functions. There is an increasing evidence on the role of flavonoids in defence response in many plant species though the exact mechanism is yet to be defined unequivocally. On the contrary, there is very little information on the role of flavonoids in cereal defence response. A novel strategy to improve disease resistance in cereals is by the engineering of the flavonoid pathway for enhancement of specific flavonoids in selected tissue under pathogen attack. Recent developments in molecular biology and genetics of flavonoid pathway in several plants would allow one to test its potential in improving disease resistance. Further, the powerful molecular techniques and reproducible transformation protocols would enable the manipulation of the flavonoid pathway in cereals. Transferring allied pathways, or a part of pathways to target plants seem to be within the reach of many groups. It is therefore, time for a second look at this secondary pathway particularly in the context of disease resistance phenomenon.

Water stress-responsive 23 kDa polypeptide from rice seedlings is boiling stable and is related to the RAB16 family of proteins

Summary The response of rapidly growing young seedlings of certain indica rice ( Oryza sativa L. Cvs) cultivars to polyethylene glycol (PEG)-mediated water stress and abscisic acid (ABA) treatment was investigated. Several water stress-responsive proteins were identified by SDS-PAGE and one such protein, namely the 23 kDa polypeptide, was purified. Using antibodies raised against it, we proved that this protein was also responsive to air drying (desiccation) of seedlings and ABA treatment but not to NaC1 and cold treatment. Further, Western analysis revealed that the 23 kDa protein is immunologically related to the RAB16 family of proteins. Finally, we demonstrated that the 23 kDa polypeptide is boiling stable. In view of its appearance in response to a variety of water stress conditions in both sensitive and tolerant cultivars, its immunological identity with the RAB16 protein and its novel feature of boiling stability, it is proposed that the 23 kDa protein is associated with a generalized water stress response mechanism and might play a role in water stress tolerance in seedlings.