Kailash C. Upadhyaya

Calcium signaling : Linking environmental signals to cellular functions

Topics discussed in this review include Ca in signal-response coupling and the specificity of Ca 2+ signals. It is concluded that Ca is the most important signalling molecule in plants. The source of Ca and the type of cell responding to a particular signal confers specificity at the primary level. At other levels, specificity is achieved by the strength of the Ca signal and the specific patterns of Ca change. Another level of control is achieved by changes in activity of various other proteins and factors that become activated or modulated by changes in Ca levels. Ca-dependent kinases and phosphatases belong to this category and are main components of signal response coupling. Changes in the level of other factors in concert with Ca also activate a specific set of proteins, and this could also be involved in contribution to the specificity of signalling. Ca directly affecting the expression of certain genes gives another level of control on specificity. Other components in the regulation of Ca signalling are also discussed.

Interaction of Piriformospora indica with Azotobacter chroococcum.

Microbial communities in rhizosphere interact with each other and form a basis of a cumulative impact on plant growth. Rhizospheric microorganisms like Piriformospora indica and Azotobacter chroococcum are well known for their beneficial interaction with plants. These features make P. indica /A. chroococcum co-inoculation of crops most promising with respect to sustainable agriculture and to understanding the transitions in the evolution of rhizospheric microbiome. Here, we investigated interactions of P. indica with A. chroococcum in culture. Out of five Azotobacter strains tested, WR5 exhibited growth-promoting while strain M4 exerted growth-inhibitory effect on the fungus in axenic culture. Electron microscopy of co-culture indicated an intimate association of the bacterium with the fungus. 2-D gel electrophoresis followed by mass spectrometry of P. indica cellular proteins grown with or without WR5 and M4 showed differential expression of many metabolic proteins like enolase-I, ureaseD, the GTP binding protein YPT1 and the transmembrane protein RTM1. Fungal growth as influenced by bacterial crude metabolites was also monitored. Taken together, the results conform to a model where WR5 and M4 influence the overall growth and physiology of P. indica which may have a bearing on its symbiotic relationship with plants.

Effects of vanadate and insulin on the activities of selected enzymes of amino acid metabolism in alloxan diabetic rat kidney

The effects of insulin and the insulin mimetic agent “vanadate” were studied on the activities of alanine aminotransferase, aspartate aminotransferase, glutamate dehydrogenase and arginase in the cytosolic and the mitochondrial fractions of the kidney in control and alloxan induced diabetic rats. An enhancement in the activities of these enzymes were noted in both the fractions of diabetic kidney. Vanadate treatment (0.6 mg/ml in drinking water) of alloxan induced diabetic rats restored the activities of these enzymes almost completely in the cytosolic and partially in the mitochondrial fractions. Vanadate treatment also normalized hyperglycaemia without altering the depressed levels of insulin secretion in diabetic rats. The effect of insulin treatment was found to be the same as that of vanadate in diabetic rats.

A multiplex RT‐PCR assay for analysis of relative transcript levels of different members of multigene families: Application to Arabidopsis calmodulin gene family

The high degree of conservation of nucleotide sequences among different members of a multigene family poses problems in analysis of expression patterns governed by each member of the gene family. In this report we describe a simple, semi‐quantitative and single tube multiplex RT‐PCR assay for simultaneous and relative expression analysis with an application to all the six members of Arabidopsis calmodulin multigene family. In the multiplex primer set, individual gene specific primers were derived from 3′‐untranslated region of the genes and a single common primer from the conserved exonic region. Transcriptional activation of all the members of the calmodulin gene family in response to touch was monitored. The results demonstrate that two of the genes are not regulated by touch; however, the other four that are induced by touch show a differential response including their kinetics of induction.

CARE1, a TY3-gypsy like LTR-retrotransposon in the food legume chickpea (Cicer arietinum L.).

We report a Ty3-gypsy like retrotransposon CARE1 (Cicer arietinum retro-element 1) in chickpea (Accession no. DQ239702). This 5,920-bp AT-rich (63%) element carries 723-bp 5′-LTR and 897-bp 3′-LTR flanking to an internal region of 4,300-bp. The LTRs of CARE1 show 93.9% nucleotide identity to each other and have 4-bp (ACTA) terminal inverted repeats. A 17-bp potential tRNAmet primer binding site downstream to 5′-LTR and a 13-bp polypurine tract upstream to 3′-LTR have been identified. The order of characteristic domains (Gag-proteinase-reverse transcriptase-RNaseH-integrase) in the deduced amino acid sequence and its phylogenetic analysis with other retrotransposons, places CARE1 in the gypsy group of retrotransposons. Homologues of a number of cis-elements including CCAAT, TATA and GT-1 have been detected in the regulatory region or the 5′-LTR of CARE1. Transgenic tobacco plants containing 5′-LTR:GUS construct showed that its 5′-LTR is inactive in a heterologous system under normal as well as tissue culture conditions. Genomic Southern blot experiments using 5′-LTR of the element as a probe showed that CARE1 or its related elements are present in the genomes of various chickpea accessions from various geographic regions.

Microbe–Microbe, Microbe–Plant Biocommunication

Quorum Sensing (QS) is a natural phenomenon of communication circuits to regulate a diverse array of physiological activities. QS regulates microbial diversity and ecological balance through various process, i.e., symbiosis, motility, sporulation, virulence, biofilm formation, competence, conjugation, and antibiotic production. There are numerous bacteria, fungi, and plants that have components of a QS system to control a specific phenotype of an organism and to shape the microbial community in the rhizophere. Bacterial intercellular communication is based on the detection of diffusible signal molecules and their transduction mechanisms which convert the information into appropriate molecular–cellular responses. Recent advances in studies of QS systems have brought the understanding that biocommunication and community behavior are crucial parameters for the successful interactions in a mycorrhizosphere. We are only at the beginning to understand how plants control microbial communities in the rhizophere. Interspecies and cross-kingdom communication is implicated in successful symbiotic interactions of a variety of bacteria with fungi and plant systems.

Expression patterns of anArabidopsis phenylalanine ammonia-lyase promoter in transgenic tobacco

We have cloned and characterized one of the genes that encode phenylalanine ammonia-lyase fromArabidopsis thaliana. A 1.8-kb fragment containing the upstream region of this gene,PALI, was transcriptionally fused to the ß-glucuronidase reporter gene, and the construct was introduced into tobacco byAgrobacterium-mediated leaf disc transformation. The distribution of ß-glucuronidase activity in the transgenic plants was analysed histochemically. The spatial pattern of activity showed that thePALI promoter is very active in the vascular tissues. No expression was observed in epidermal cells, trichomes and apical meristems. Activation of thePALI promoter in the early stages of seedling development remained confined to the regions adjacent to the root apical meristems; in later stages of seedling development, the pattern of expression was drastically altered. The results suggest that thePALI promoter is under the control of a complex set of signal transduction pathways which lead to its activation in response to tissue-specific, wounding and developmental cues.

Synthesis of Mitochondrial DNA in Relation to Nuclear Cell Cycle in Germinating Barley Embryos

Summary Mitochondria and nuclei were isolated from excised germinating barley embryos and the incorporation of ( 3 H)-thymidine into the two organelle fractions was measured in order to determine the time of synthesis of mitochondrial DNA (mtDNA) in relation to the synthesis of nuclear DNA (nDNA). The results indicate that in barley seeds, nDNA synthesis begins at the 10th hour after soaking the excised embryos, and continues till 20th hour. The highest synthetic activity is between 13 and 17 hours. The mtDNA synthesis begins after the onset of the first S-phase but most of the mtDNA is synthesized late in the S-phase, between 15 and 17 hours after the initial soaking.

Antisense expression of a gene encoding a calcium-binding protein in transgenic tobacco leads to altered morphology and enhanced chlorophyll

Entamoeba histolytica contains a novel calcium-binding protein like calmodulin, which was discovered earlier, and we have reported the presence of its homologue(s) and a dependent protein kinase in plants. To understand the functions of these in plants, a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP) was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised. These plants showed variation in several phenotypic characters, of which two distinct features, more greenness and leaf thickness, were inherited in subsequent generations. The increase in the level of total chlorophyll in different plants ranged from 60% to 70%. There was no major change in chloroplast structure and in the protein level of D1, D2, LHCP and RuBP carboxylase. These morphological changes were not seen in antisense calmodulin transgenic tobacco plants, nor was the calmodulin level altered in EhCaBP antisense plants.

Cloning and Characterization of a Foldback DNA Element from Antirrhinum majus

A fold back sequence linked to a copy of the transposable element, Tam2, has been Isolated, from a genomic library constructed using partially digested and size-fractionated Antirrhinum DNA Into λ EMBL4 vector, by using Tam2 specific probe. The fold back sequence visualized by heteroduplex analysis reveals a typical stem-loop structure. The foldback element is around 1.2 kb with an average of 227 bp of Inverted termini forming the stem.