Bharati Ghosh

Expression of arginine decarboxylase in seedlings of indica rice (Oryza sativa L.) cultivars as affected by salinity stress

The effect of salinity stress on the activity of arginine decarboxylase (ADC, EC 4.1.1.19), the first enzyme in biosynthesis of polyamines (PA) from arginine, as well as its transcript level has been compared in salt-sensitive (M-1-48) and salt-tolerant (Pokkali) rice cultivars. Treatment of 72 h grown seedlings either with increasing concentrations of NaCl or with 150 mM NaCl for different time periods, showed a gradual increase of activity in Pokkali. In M-1-48 an immediate increase followed by sharp decrease was observed on prolonged treatment beyond 6 h or above 150 mM NaCl. To generate a DNA probe for ADC, the polymerase chain reaction was used with oat genomic DNA and sequence-specific primers. A region of oat genomic DNA containing a coding sequence for 166 amino acids of the C-terminal part of the ADC enzyme was amplified and called OAD1. Southern analysis of EcoRI- or BamHI-cut genomic DNAs from different cultivars of rice with OAD1 as the probe revealed strong hybridization with one DNA fragment of rice and restriction fragment length polymorphism (RFLP) was noticed. Northern analysis of total RNA of rice with OAD1 as the probe revealed hybridization with a transcript of similar size to the ADC transcript in oat. While in Pokkali, at least a 20-fold accumulation of OAD1 homologous transcript was detected after treatment with 200 mM NaCl, only a seven-fold increase in transcript level was found in M-1-48 after 150 mM NaCl treatment. Results suggest that in the salt-tolerant rice cultivar Pokkali, ADC enzyme activity increases and its transcript also accumulates during the prolonged salinity stress, this mechanism is absent in the salt-sensitive rice cultivar M-1-48 where a prolonged period of salinity stress down-regulates both ADC activity and its transcript level.

Effect of salt stress on polyamine metabolism in Brassica campestris

Abstract Polyamines (PAs) and their major metabolic enzymes were studied under different modes of salinity stress in Brassica . Long stress caused only small changes in PA level, arginine (EC 4.1.1.19) and ornithine decarboxylases (EC 4.1.1.17) and polyamine oxidase (EC 1.5.3.3.) activity. However, short stress increased PA level and enzyme activities. Diamine oxidase (EC 1.4.3.6) activity, unlike other PA metabolic enzymes, increased significantly during long stress.

Expression of abscisic acid-responsive element-binding protein in salt-tolerant indica rice (it Oryza sativa L. cv. Pokkali)

As the products of abiotic stress and ABA inducible genes are predicted to play an important role in the mechanism of salt tolerance, the expression of transcription factor that recognizes abscisic acid-responsive element (ABRE) is likely to be regulated when plants are exposed to abiotic stress. Northern analysis of total RNA from control and salt-treated 10-day-old Pokkali (salt tolerant) rice plants was performed to find out the level of transcripts homologous to wheat cDNA (GC19) for EmBP-1 (bZIP class factor), a transcription factor that recognizes ABRE. Salinity stress (72 h)-induced accumulation of two transcripts, of 2.0 kb (r2.0) and 1.5 kb (r1.5), in roots was detected. Both transcripts were detectable even after 6 h of salt or abscisic acid treatment, whereas sheath and lamina showed constitutive levels of r1.5 transcript. When 32P-labeled DNA containing ABRE was used in a gel mobility shift assay, a low level of complex formation by binding factor was detected from the nuclear extract of lamina of control rice plants. Quantitative enhancement of complex formation was found with the nuclear extract prepared from the lamina of plants treated with 200 mM NaCl for 26 h over control nuclear extract, suggesting a step of regulation of expression of ABRE-binding protein in response to salinity stress. South-western blot analysis of equal amounts of nuclear proteins of lamina showed binding of 32P-labeled ABRE-DNA with two polypeptides (22–28 kDa) present at constitutive levels in control or NaCl-treated plants. Preincubation of the laminar nuclear extract of control plants, with spermidine or proline at 5 mM concentration showed quantitative enhancement of ABRE binding activity. Kinetics of spermidine stimulation showed gradual increase of complex formation from 5 mM concentration. Similarly, addition of GTP to the control nuclear extract also showed quantitative enhancement of complex formation and heparin was found to inhibit GTP activated complex formation by about 25%. Results may suggest the presence of ABRE binding protein in presynthesized and inactive form in control plants and GTP mediated activation is probably one of the way to regulate the expression of ABRE-binding factor.

Changes in polyamine contents during development and germination of rice seeds

Abstract In rice seed, polyamine concentration on a fresh weight basis increased for 16 days after fertilization, followed by a gradual decline. Arginine decarboxylase activity also followed the same pattern. On germination, the polyamine concentration was greatest after 24 hr and the arginine decarboxylase showed a peak after 48 hr.

Purification and characterization of diamine oxidase from rice embryos

Abstract Diamine oxidase of rice seedlings has been purified 1800-fold to homogeneity. The MW of the enzyme as determined by Sephadex G-100 gel filtration was 12.3 × 10 4 and the enzyme contained two identical subunits each with a MW of 6.12 × 10 4 . The optimal temperature and pH for the enzyme were 30° and 7.5 respectively and the enzyme followed typical Michaelis kinetics with a K m of 10 −5 M. Each enzyme molecule contained four molecules of FAD.

Changes in polyamines and related enzymes with loss of viability in rice seeds

Abstract Putrescine, spermidine and spermine of high vigour, low vigour and non-viable (classes 1, 2 and 3 respectively) seeds of Oryza sativa increased with loss of viability. The largest concentration of spermine was found in non-viable embryos. Spermine was absent in the husks of all the three categories of seeds. Arginine decarboxylase was greatest in high vigoured seeds and its activity gradually declined with loss of viability. However, diamine oxidase and polyamine oxidase activities gradually increased with the loss of viability of the seeds while DNA, RNA and protein contents decreased. The total content of polyamines increased on kinetin treatment but declined on ABA treatment. DNA, RNA and protein followed the same trend as polyamines. The polyamine contents increased by ca 3- and 4-fold, respectively, in high vigoured and low vigoured seeds on 10−4 M kinetin treatment. The activity of ADC followed the same change as that of the polyamines in both cases, but the reverse was observed for the activities of diamine and polyamine oxidases.

Effect of heat stress on ribulose 1,5-bisphosphate carboxylase in rice

Abstract Heat-induced changes of ribulose 1,5-bisphosphate carboxylase (RuBISCO, EC 4.1.1.39) at various times were estimated in tolerant (N 22) and sensitive (IR 8) cultivars of rice (Oryza sativa L.). At a temperature of 40°, specific activity of carboxylase and the titre of RuBISCO holoenzyme were increased or not affected, while at 45°, the specific activity and holoenzyme level were more stable in the tolerant cultivar than in the sensitive one. In both cultivars, a decline in activity and holoenzyme level with time was pronounced at 50°. RLSU was more affected by higher temperatures than RSSU in the tolerant cultivar. However, no such trend was noted in component proteins of the sensitive one.

Changes in polyamine contents during root and nodule growth of Phaseolus mungo

Abstract In Phaseolus mungo seeds, polyamine content increased during early germination, being maximum after 24 hr; and the arginine decarboxylase showed a peak after 18 hr. During nodule initiation and growth two peaks of polyamine contents were noted-the first being 2 weeks after nodule initiation and a second one after 5 weeks. Arginine decarboxylase activity also followed the same pattern. In the roots the polyamine concentration as well as arginine decarboxylase increased up to week 2 after sowing followed by a gradual decrease. Estimation of RNA, DNA and protein contents showed a pattern similar to that of the polyamines.

Ornithine decarboxylase from embryos of jute seeds

Abstract After purification from jute embryos ornithine decarboxylase (EC 4.1.1.17) was separated into two fractions by gel chromatography. Fraction-A was purif

Correlation of endogenous free polyamine levels with root nodule senescence in different genotypes in Vigna mungo L.

Endogenous free polyamines, nitrogenase (EC 1.1.8.6.1, acetylene reduction), and leghaemoglobin (pyridine-hemochrome assay) levels were compared among five genotypes of developing Vigna root nodules grown under field conditions. Nitrogenase activity and leghaemoglobin level attained a peak at the flowering stage and gradually declined thereafter. Individual and total polyamine also followed the same pattern. Ranking on the basis of legume yield and other morphometric attributes was PDU-2 > UH-28 > UH-82 > T-9 > Sardhomash. Except spermine, the levels of putrescine, spermidine, and total polyamine showed significant differences (p<0.05) amongst the genotypes, particularly from flowering to mid-pod development stage. Genotype, development stage, and their interaction between the two had significant (p<0.01) effects on individual as well as total polyamines. Moreover, significant high linear correlations were found between total free polyamine and putrescine with conventional nodule senescence marker like nitrogenase (R2 = 0.94 and R2 = 0.92, respectively). Putrescine had an overall positive correlation with high legume yield. The results strongly suggest a relationship between polyamine and nodule senescence. Endogenous free polyamine and putrescine may be considered as genotypic markers for nodule senescence in field grown V. mungo. It is suggested that the flowering stage is more suitable for selection.