Characterization of pyruvate decarboxylase genes from rice

The pdc1 gene encoding pyruvate decarboxylase has been isolated and sequenced from an IR54 rice genomic library. In contrast to a previously isolated intron-less rice genomic pdc, pRgpdc3, this gene contains five intervening introns in the coding region and corresponds to a cDNA clone, pRcpdc1, isolated from an IR54-cDNA library constructed from anaerobically-induced mRNAs. Comparison of the deduced amino acid sequence of this gene with that of the rice pdc2 and pdc3 showed 88% and 89% similarity, and 78% and 79% identity, respectively. Southern blots indicated that more than three genes constitute the pdc gene family in rice. pdc1 is highly inducible under anaerobic conditions. Rice pdc2 is also inducible by anoxia but to a much lesser extent than pdc1.     

Distribution patterns of 104 kDa stress-associated protein in rice

A 104 kDa protein (SAP 104) accumulates in rice seedlings in response to several abiotic stress conditions and immunological homologues of rice SAP 104 have been detected in several monocot and dicot species, as also Neurospora crassa, a fungus. In this report, we show that the amino acid sequence of a tryptic peptide generated from purified SAP 104 bears significant homology with an ATP-binding domain of the HSP 100 family proteins of Arabidopsis thaliana and Glycine max. It is further shown that differential uninduced and induced (by high-temperature stress) levels of this protein are accumulated in various organs of the mature rice plant grown under field conditions. Significant uninduced levels of this protein were in particular found in developing and mature rice grains. Seeds/grains of several other plant genera (i.e. Triticum aestivum, Zea mays, Brassica juncea) were also found to contain high uninduced levels of SAP 104. Importantly, the levels of uninduced SAP 104 in rice grains were found to decline during the seed germination phase: after two days of germination, this protein was undetectable in tissues representing pooled sample of seeds and just-emerged seedlings. Tissue print-immunoblotting analysis has indicated that in seeds high levels of this protein are specifically present in the embryo portion.     

Micropropagation of tea (Camellia sinensis (L.) O. Kuntze) using Thidiazuron

The effect of thidiazuron (TDZ) on the micropropagation of Camellia sinensis (China hybrid) was compared with that of benzylaminopurine (BAP) using nodal segments from in vitro raised seedlings. Extremely low concentrations of TDZ (1pM–100nM) alone were effective in inducing shoot bud proliferation and maintaining high rates of shoot multiplication on hormone-free media. On the other hand, higher concentrations of BAP (1–10M) and its continued presence were required to initiate and sustain shoot proliferation. While wider ranges of BAP combined favourably with auxins like NAA or IBA, only specific combinations of TDZ and NAA were effective for shoot proliferation. TDZ treated explants yielded healthy shoots, with sturdy leaves, even during the initial stages of growth, whereas, the effect of BAP was cumulative over subcultures in attaining a high proliferative rate.     

Gibberellic acid and kinetin partially reverse the effect of water stress on germination and seedling growth in chickpea

The percent germination and seedling growth of chickpea (Cicer arietinum L. cv. PBG-1) decreased with increasing concentrations of exogenous polyethylene glycol 6000 (PEG). With 15% PEG in the growth medium germination was only 33% while with 10% PEG it was 58% as compared to 93% in control. Addition of gibberellic acid (GA₃) and kinetin to medium containing 10% PEG increased the germination and seedling growth and the effect was maximum with 6 µM GA₃ which was a better inducer of growth and germination under reduced water potential than kinetin. However, indole acetic acid (IAA) inhibited germination and growth of stressed seedlings. The activity of amylase in cotyledons under stress was significantly increased with GA₃ while kinetin and IAA were less effective. Gibberellic acid also enhanced the mobilization of starch from cotyledons of stressed seedlings resulting in low starch levels in cotyledons compared with stressed seedlings.     

Limiting cytosolic Na+ confers salt tolerance to rice cells in culture: a two-photon microscopy study of SBFI-loaded cells

Rice ( Oryza sativa L.), a staple food in Asia, is very sensitive to soil salinity. However, intraspecific variations exist, with the coastal cultivar Pokkali tolerating even brackish water. This study explores cellular mechanisms that contribute to salt tolerance in rice. It is widely accepted that limiting cytosolic Na⁺ should improve the survival of plants subjected to saline stress. However, an understanding of the mechanisms by which Na⁺ levels are controlled in relatively tolerant cultivars requires monitoring cytosolic Na⁺ non-invasively and in real time, which is technically challenging. We have used two-photon excitation for the ratiometric estimation of cytosolic Na⁺ in cultured cells using sodium-binding benzofuran isophthalate. Pokkali cells maintained low cytosolic Na⁺ (approximately 25 m M ), and a viability of over 85% under high salinity , while Jaya cells were unable to maintain low cytosolic Na⁺ and suffered decreased viability even at moderate saline stress. Here we show that the permeability of the Pokkali plasma membrane to Na⁺ is significantly lower than that of Jaya, to the extent that it is comparable with permeabilities reported for halophytes. Pokkali effectively sequesters Na⁺ in intracellular compartments utilizing a Ca²⁺-regulated transport system(s). Together these cellular mechanisms allow Pokkali to maintain low cytosolic Na⁺ up to a stress of 250 m M NaCl. The findings demonstrate that differences in survival between these contrasting varieties of rice are mainly because of differences in membrane transport mechanisms and thus have significance in crop improvement.     

Biochemical and biophysical characterization of Leishmania donovani gamma-glutamylcysteine synthetase

γ-glutamylcysteine synthetase (Gcs) is a vital enzyme catalyzing the first and rate limiting step in the trypanothione biosynthesis pathway, the ATP-dependent ligation of L-Glutamate and L-Cysteine to form gamma-glutamylcysteine. The Trypanothione biosynthesis pathway is unique metabolic pathway essential for trypanosomatid survival rendering Gcs as a potential drug target. Here we report the cloning, expression, purification and characterization of L. donovani Gcs. Three other constructs of Gcs (GcsN, GcsC and GcsT) were designed on the basis of S. cerevisiae and E. coli Gcs crystal structures. The study shows Gcs possesses ATPase activity even in the absence of substrates L-glutamate and L-Cysteine. Divalent ions however plays an indispensable role in LdGcs ATPase activity. Isothermal titration calorimetry and fluorescence studies illustrates that L. donovani Gcs binds substrate in order ATP >L-glutamate>L-cysteine with Glu 92 and Arg 498 involved in ATP hydrolysis and Glu 92, Glu 55 and Arg 498 involved in glutamate binding. Homology modeling and molecular dynamic simulation studies provided the structural rationale of LdGcs catalytic activity and emphasized on the possibility of involvement of three Mg²⁺ ions along with Glutamates 52, 55, 92, 99, Met 322, Gln 328, Tyr 397, Lys 483, Arg 494 and Arg 498 in the catalytic function of L. donovani Gcs.     

Deciphering the Mechanisms of Endophyte-Mediated Biofortification of Fe and Zn in Wheat

An investigation was carried out to understand the mechanism(s) underlying enhanced Fe or Zn uptake in low Fe–Zn accumulator wheat genotype 4HPYT-414, due to inoculation of siderophore-producing and zinc-solubilizing endophytes—Arthrobacter sulfonivorans DS-68 and Arthrobacter sp. DS-179. Root anatomical features, using transmission electron microscopy (TEM), qualitative and quantitative aspects of production of organic acids and sugars in root exudates, and expression of TaZIP genes were analysed to relate to endophyte-mediated higher concentrations of Fe and Zn in the roots and shoots of wheat plants. TEM studies revealed that the endodermis, cortical region, root hair extension, xylem and xylem vessels, pericycle and vascular bundles were more pronounced and thicker in inoculated treatments, as compared to control. The organic acid profile of root exudates revealed five types of organic acids, with citric acid being predominant. Inoculation of A. sulfonivorans and Arthrobacter sp. brought about 5- and eightfold increases in the amounts of acids, respectively, as compared to control, particularly citric acid, succinic acid and acetic acid. Among the four TaZIP genes targeted, expression was achieved only for TaZIP3 and TaZIP7 genes, which showed 1–2 fold increases in the inoculated treatments. The results clearly indicated that the endophyte-mediated overexpression of TaZIP3 and TaZIP7 genes in roots and shoots, and the observed anatomical and exudate changes were acting synergistically in facilitating higher Fe and Zn translocation in roots and shoots.