Dibyendu N. Sengupta

Amelioration of salinity stress by exogenously applied spermidine or spermine in three varieties of indica rice differing in their level of salt tolerance

We present here the comparative protective potentiality of exogenously applied polyamines (PAs), namely spermidine (Spd) and spermine (Spm), in mitigating NaCl toxicity and inducing short-term salinity tolerance in three indica rice varieties, namely M-1-48 (salt-sensitive), Nonabokra (salt-tolerant) and Gobindobhog (highly sensitive). The retardation in root length or shoot length and toxic Na(+) accumulation or K(+) loss, the considerable increment in malondialdehyde/H(2)O(2) accumulation or lipoxygenase activity, all of which were particularly noteworthy in M-1-48 and Gobindobhog during salinity stress, was appreciably reduced by co-treatment with Spd or Spm. Both the PAs also inhibited the extent of salt-induced protein carbonylation in all the varieties and enhanced protease activity, especially in Gobindobhog. The prevention of chlorophyll degradation was better with Spd in Nonabokra and Gobindobhog. While the salt-induced increase in anthocyanin or reducing sugar level was further prompted by Spd or Spm in all the varieties, the proline content was elevated by Spd particularly in Gobindobhog. During salinity stress, both the PAs were effective in lowering the putrescine accumulation in M-1-48 and Gobindobhog, and strikingly increasing the Spm level in all the varieties, the highest being in Gobindobhog. In addition, they enhanced the activity of peroxidases and compensated for the decreased catalase activity in all the varieties. Thus the two PAs could recuperate all the three varieties from salt-induced damages to different degrees. The salt injuries, encountered in M-1-48 and Gobindobhog, both of which showed greater susceptibility to salinity stress, were more pronouncedly alleviated and counteracted by the PAs, than the salt-tolerant Nonabokra. The reversal of inhibitory effect of salinity stress was conferred by preventing growth inhibition or various forms of cellular damages, maintaining proper K(+)/Na(+) balance or triggering the level of osmolytes and activity of antioxidant enzymes. Our communication offers a referenced evidence for an understanding of the mechanism by which higher PAs relieve the damages particularly in salt-sensitive rice varieties.

Differential antioxidative responses of indica rice cultivars to drought stress.

The present study investigated the linkages between drought stress, oxidative damages and variations in antioxidants in the three rice varieties IR-29 (salt-sensitive), Pokkali (salt-tolerant) and aromatic Pusa Basmati (PB), to elucidate the antioxidative protective mechanism governing differential drought tolerance. Water deficit, induced by 20% (w/v) polyethylene glycol (PEG-6000), provoked severe damages in IR-29 and PB in the form of huge chlorophyll degradation and elevated H2O2, malondialdehyde and lipoxygenase (LOX, EC 1.13.11.12) levels as compared to Pokkali. The protein oxidation was more conspicuous in IR-29. Increment in antioxidants, particularly flavonoids and phenolics was several folds higher over control in Pokkali, while much lesser in IR-29 and PB. The activity of catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) were decreased in IR-29 and PB, but unaltered in Pokkali. However, marked drought-induced increase in guaiacol peroxidase (GPX, EC 1.11.1.7) activity was noted in both IR-29 and PB. Induction in radical scavenging activity, being the maximum in IR-29, and increased reducing power ability in all the cultivars, accompanied with drought stress, were observed as a defense mechanism. The novelty of our work is that it showed the aromatic rice PB behaving more closely to IR-29 in greater susceptibility to dehydration stress, while the salt-tolerant Pokkali also showed effective drought tolerance properties.

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.

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.

Characterization of transcriptional profiles of MA-ACS1 and MA-ACO1 genes in response to ethylene, auxin, wounding, cold and different photoperiods during ripening in banana fruit

The ripening-specific genes MA-ACS1 (Musa acuminata ACC synthase1) and MA-ACO1 (M. acuminata ACC oxidase 1) are regulated in response to a wide variety of factors. Here, we have studied the differential transcript accumulation pattern and protein levels of MA-ACS1 and MA-ACO1 genes in response to ethylene, auxin, wounding and low temperature in preclimacteric banana fruit. We have shown that exogenous application of ethylene and auxin induced the expression of MA-ACS1, while MA-ACO1 showed marginal expression following ethylene treatment in preclimacteric stage. Auxin did not induce MA-ACO1 expression. Thus, auxin-treated banana fruits showed lower ethylene production rate as compared to ethylene-treated fruits. Conversely, wounding and cold treatment down-regulated the expression of both the genes and thus inhibited ethylene production. Furthermore, we have detected a GCC-box putative ethylene-responsive element (ERE)- and an auxin-responsive element (ARE)-specific DNA-binding activity in the banana pulp and studied the ethylene and auxin responsive characteristics of the GCC-box and ARE (TGTCTC) containing synthetic promoter fragments. In addition, we have detected an enhanced ethylene production rate and expression level of MA-ACS1 and MA-ACO1 genes along with a strong GCC-box-specific DNA-binding activity following exposure to constant dark period for 8d at the preclimacteric stage. Together, our study provides interesting information about the regulation of expression of MA-ACS1 and MA-ACO1 genes in response to various factors during ripening in banana fruit, which may have physiological relevance concerning ethylene biosynthesis during post-harvest conditions.

Comparative analysis of some biochemical responses of three indica rice varieties during polyethylene glycol-mediated water stress exhibits distinct varietal differences

Extensive investigation into plant response and adaptation to diverse osmotic stresses like high salt/dehydration/low temperature, involving a broad spectrum of cellular physiological and biochemical changes, is essential to unravel intrinsic mechanism to mitigate against such stresses. In our previous communications, we conducted biochemical analyses of indica rice varieties, subjected to exogenous salt/abscisic acid-mediated oxidative stress. The aim of this study was to compare differential biochemical responses of the salt-sensitive (IR-29), salt-tolerant (Pokkali) and aromatic (Pusa Basmati or PB) rice varieties during polyethylene glycol (PEG)-induced dehydration stress. The greater susceptibility of IR-29 and PB, to water scarcity, was reflected by the higher toxic Na+ and putrescine accumulation, considerable decrease in (reduced/oxidized) glutathione, maximal increment in protease activity and greater downregulation of nitrate reductase activity. On the other hand, Pokkali appeared to suffer lesser damages as evidenced from much lower endogenous Na+ but higher K+, Ca2+ and Mg2+ accumulation, registering the highest levels of osmolytes like glycinebetaine and higher polyamines (spermidine and spermine) accounting to improved relative water content, higher (reduced/oxidized) glutathione, maximal induction of the enzyme phenylalanine ammonia-lyase and practically unhindered nitrate reductase activity, following PEG treatment. The highest induction of sugars and proline in IR-29 and PB probably played the osmoprotective/antioxidative functions, enabling to a certain extent to heighten their lipoxygenase inhibition or H2O2 scavenging potential, more than Pokkali, to ward off oxidative damages and sustain survival under critical dehydrated situations. Thus, the salt-tolerant Pokkali also showed prominent dehydration-tolerance properties, whereas the aromatic rice PB, almost identical in their biochemical responses to IR-29, showed greater sensitivity to PEG-mediated water deficit.

Overexpression of Rab16A gene in indica rice variety for generating enhanced salt tolerance

We report here the overexpression of Rab16A full length gene (promoter + ORF), from the salt-tolerant indica rice Pokkali, in the salt-susceptible indica rice variety Khitish, via particle bombardment. Molecular analysis of the transgenics revealed stable integration of the transgene upto T2 generation. High level of expression of the transgene (driven by its own stress-inducible promoter), as well as the protein, was detectable in the leaves under simulated salinity stress (250 mM NaCl, 24 h); the expression level being higher than wild type (WT) plants. The Rab16A transcript also increased gradually with seed maturity, with its maximal accumulation at 30 d after pollination (DAP) i.e., fully matured seeds, explaining the protective role of Rab16A gene during seed maturation. Enhanced tolerance to salinity was observed in the plants transformed with Rab16A. The superior physiological performances of the transgenics under salt treatment were also reflected in lesser shoot or root length inhibition, reduced chlorophyll damages, lesser accumulation of Na+ and reduced loss of K+, increased proline content as compared with the WT plants. All these results indicated that the overproduction of RAB16A protein in the transgenics enable them to display enhanced tolerance to salinity stress with improved physiological traits. Our work demonstrates the profound potential of Group 2 LEA proteins (to which RAB16A belongs to) in conferring stress tolerance in crop plants through their genetic manipulation.

Differential transcriptional regulation of banana sucrose phosphate synthase gene in response to ethylene, auxin, wounding, low temperature and different photoperiods during fruit ripening and functional analysis of banana SPS gene promoter

Sucrose phosphate synthase (SPS) (EC 2.3.1.14) is the key regulatory component in sucrose formation in banana (Musa acuminata subgroup Cavendish, cv Giant governor) fruit during ripening. This report illustrates differential transcriptional responses of banana SPS gene following ethylene, auxin, wounding, low temperature and different photoperiods during ripening in banana fruit. Whereas ethylene strongly stimulated SPS transcript accumulation, auxin and cold treatment only marginally increased the abundance of SPS mRNA level, while wounding negatively regulated SPS gene expression. Conversely, SPS transcript level was distinctly increased by constant exposure to white light. Protein level, enzymatic activity of SPS and sucrose synthesis were substantially increased by ethylene and increased exposure to white light conditions as compared to other treatments. To further study the transcriptional regulation of SPS in banana fruit, the promoter region of SPS gene was cloned and some cis-acting regulatory elements such as a reverse GCC-box ERE, two ARE motifs (TGTCTC), one LTRE (CCGAA), a GAGA-box (GAGA…) and a GATA-box LRE (GATAAG) were identified along with the TATA and CAAT-box. DNA–protein interaction studies using these cis-elements indicated a highly specific cis–trans interaction in the banana nuclear extract. Furthermore, we specifically studied the light responsive characteristics of GATA-box containing synthetic as well as native banana SPS promoter. Transient expression assays using banana SPS promoter have also indicated the functional importance of the SPS promoter in regulating gene expression. Together, these results provide insights into the transcriptional regulation of banana SPS gene in response to phytohormones and other environmental factors during fruit ripening.

Characterization of differential ripening pattern in association with ethylene biosynthesis in the fruits of five naturally occurring banana cultivars and detection of a GCC-box-specific DNA-binding protein.

MA-ACS1 and MA-ACO1 are the two major ripening genes in banana and play crucial role in the regulation of ethylene production during ripening. Here, we report a comparative ripening pattern in five different naturally occurring banana cultivars namely Cavendish (AAA), Rasthali (AAB), Kanthali (AB), Poovan (AAB) and Monthan (ABB), which have distinct genome composition. We found a distinct variation in the climacteric ethylene production and in-vivo ACC oxidase activity level during the ripening stages in the five cultivars. We identified the cDNAs for MA-ACS1 and MA-ACO1 from the five cultivars and studied the transcript accumulation patterns of the two genes, which correlated well with the differential timing in the expression of these two genes during ripening. The GCC-box is one of the ethylene-responsive elements (EREs) found in the promoters of many ethylene-inducible genes. We have identified a GCC-box motif (putative ERE) in the promoters of MA-ACS1 and MA-ACO1 in banana cultivars. DNA–protein interaction studies revealed the presence of a GCC-box-specific DNA-binding activity in the fruit nuclear extract and such DNA-binding activity was enhanced following ethylene treatment. South-Western blotting revealed a 25-kDa nuclear protein that binds specifically to GCC-box DNA in the climacteric banana fruit. Together, these results indicate the probable involvement of the GCC-box motif as the cis-acting ERE in the regulation of MA-ACS1 and MA-ACO1 during ripening in banana fruits via binding of specific ERE-binding protein.

Characterization of an AGAMOUS-like MADS Box Protein, a Probable Constituent of Flowering and Fruit Ripening Regulatory System in Banana

The MADS-box family of genes has been shown to play a significant role in the development of reproductive organs, including dry and fleshy fruits. In this study, the molecular properties of an AGAMOUS like MADS box transcription factor in banana cultivar Giant governor (Musa sp, AAA group, subgroup Cavendish) has been elucidated. We have detected a CArG-box sequence binding AGAMOUS MADS-box protein in banana flower and fruit nuclear extracts in DNA-protein interaction assays. The protein fraction in the DNA-protein complex was analyzed by mass spectrometry and using this information we have obtained the full length cDNA of the corresponding protein. The deduced protein sequence showed ∼95% amino acid sequence homology with MA-MADS5, a MADS-box protein described previously from banana. We have characterized the domains of the identified AGAMOUS MADS-box protein involved in DNA binding and homodimer formation in vitro using full-length and truncated versions of affinity purified recombinant proteins. Furthermore, in order to gain insight about how DNA bending is achieved by this MADS-box factor, we performed circular permutation and phasing analysis using the wild type recombinant protein. The AGAMOUS MADS-box protein identified in this study has been found to predominantly accumulate in the climacteric fruit pulp and also in female flower ovary. In vivo and in vitro assays have revealed specific binding of the identified AGAMOUS MADS-box protein to CArG-box sequence in the promoters of major ripening genes in banana fruit. Overall, the expression patterns of this MADS-box protein in banana female flower ovary and during various phases of fruit ripening along with the interaction of the protein to the CArG-box sequence in the promoters of major ripening genes lead to interesting assumption about the possible involvement of this AGAMOUS MADS-box factor in banana fruit ripening and floral reproductive organ development.