Mechanistic Association of Quantitative Trait Locus with Malate Secretion in Lentil (Lens culinaris Medikus) Seedlings under Aluminium Stress.

Aluminium (Al) toxicity acts as a major delimiting factor in the productivity of many crops including lentil. To alleviate its effect, plants have evolved with Al exclusion and inclusion mechanisms. The former involves the exudation of organic acid to restrict the entry of Al3+ to the root cells while latter involves detoxification of entered Al3+ by organic acids. Al-induced secretion of organic acids from roots is a well-documented mechanism that chelates and neutralizes Al3+ toxicity. In this study, F6 recombinant inbred lines (RILs) derived from a cross between L-7903 (Al-resistant) and BM-4 (Al-sensitive) were phenotyped to assess variation in secretion levels of malate and was combined with genotypic data obtained from 10 Al-resistance linked simple sequence repeat (SSRs) markers. A major quantitative trait loci (QTL) was mapped for malate (qAlt_ma) secretion with a logarithm of odd (LOD) value of 7.7 and phenotypic variation of 60.2%.Validated SSRs associated with this major QTL will be useful in marker assisted selection programmes for improving Al resistance in lentil.

Morpho-physiological characterization coupled with expressional accord of exclusion mechanism in wild and cultivated lentil under aluminum stress.

Aluminum stress deteriorates lentil production under acidic soils. Enhanced insight into Al tolerance traits is needed to improve its productivity. Therefore, Al-resistant (L-4602, PAL-8) and Al-sensitive (BM-4, EC-223229) cultivars along with a resistant wild (ILWL-15) were characterized for morpho-physiological traits viz. seedling root architecture (SRA), Al accumulation, and localization via fluorescent and non-fluorescent staining under control and Al-treated conditions. Also, antioxidant activities and organic acid secretion were quantified, and expressions of 10 associated genes were analyzed. Roots of Al-resistant cultivars and wild genotype showed higher root growth, antioxidant enzyme activities, and organic acid secretion than Al-sensitive ones. Among these traits, higher organic acid secretion was influenced by enhanced expression of genes, especially-aluminum sensitive-3 (ALS 3), aluminum-activated malate transporter (ALMT), multidrug and toxic compound extrusion (MATE), citrate synthase (CS), and phospho enol pyruvate carboxylase (PEPC)-which helped in reducing Al and callose accumulation. These genes were located on lentil chromosomes via sequence alignment with lentil draft genome. A strong link between morpho-physiological variation and organic acid secretion was noted which reinforced the prominence of exclusion mechanism. It was complemented by enhanced antioxidant activities at seedling stage which mitigated Al stress effects on SRA. Wild outperformed over cultivars indicating its impregnable evolution which can be exploited to better understand tolerance mechanisms. Al-resistant cultivars had significantly higher seed yield than Al-sensitive and national checks on Al-toxic fields, confirming-tolerance is sustained till reproductive stage in lentil. This study elucidated role of gene families in eliminating Al toxicity that will assist breeders to formulate strategies for developing Al-resistant cultivars.

Molecular mapping of aluminium resistance loci based on root re-growth and Al-induced fluorescent signals (callose accumulation) in lentil (Lens culinaris Medikus).

Development of aluminium (Al) resistant genotypes through molecular breeding is a major approach for increasing seed yield under acidic conditions. There are no available reports on mapping of Al resistance loci and molecular breeding for Al resistant varieties in lentil. The present study reports a major quantitative trait loci (QTL) for Al resistance using simple sequence repeat (SSR) markers in F2 and F3 mapping populations derived from contrasting parents. Phenotypic response to Al was measured on the bases of root re-growth (RRG), fluorescent signals (callose accumulation) and Al contents in hydroponic assay. After screening 495 SSR markers to search polymorphism between two contrasting parents, 73 polymorphic markers were used for bulk segregation analysis. Two major QTLs were identified using seven trait linked markers, one each for fluorescent signals and RRG mapped on linkage group (LG) 1 under Al stress conditions in F2 mapping population of cross BM-4 × L-4602. One major QTL (qAlt_fs) was localised between PLC_88 and PBA_LC_373, covering 25.9 cM with adjacent marker PLC_88 at a distance of 0.4 cM. Another major QTL (qAlt_rrg) for RRG was in the marker interval of PBA_LC_1247 and PLC_51, covering a distance of 45.7 cM with nearest marker PBA_LC_1247 at a distance of 21.2 cM. Similarly, in F3 families of BM-4 × L-4602 and BM-4 × L-7903, LG-1 was extended to 285.9 and 216.4 cM respectively, having four newly developed genic-SSR markers. These QTLs had a logarithm of odd (LOD) value of 140.5 and 28.8 along with phenotypic variation of 52% and 11% for fluorescent signals and RRG respectively, whereas, qAlt_rrg had LOD of 36 and phenotypic variance of 25% in F3 population of BM-4 × L-4602. Two major QTLs identified in the present study can be further dissected for candidate gene discovery and development of molecular markers for breeding improved varieties with high Al resistance.

Phylogenetic Analysis of Nuclear-Encoded RNA Maturases.

Posttranscriptional processes, such as splicing, play a crucial role in gene expression and are prevalent not only in nuclear genes but also in plant mitochondria where splicing of group II introns is catalyzed by a class of proteins termed maturases. In plant mitochondria, there are 22 mitochondrial group II introns. matR, nMAT1, nMAT2, nMAT3, and nMAT4 proteins have been shown to be required for efficient splicing of several group II introns in Arabidopsis thaliana. Nuclear maturases (nMATs) are necessary for splicing of mitochondrial genes, leading to normal oxidative phosphorylation. Sequence analysis through phylogenetic tree (including bootstrapping) revealed high homology with maturase sequences of A thaliana and other plants. This study shows the phylogenetic relationship of nMAT proteins between A thaliana and other nonredundant plant species taken from BLASTP analysis.

Transcription Factor Information System (TFIS): A Tool for Detection of Transcription Factor Binding Sites.

Transcription factors are trans-acting proteins that interact with specific nucleotide sequences known as transcription factor binding site (TFBS), and these interactions are implicated in regulation of the gene expression. Regulation of transcriptional activation of a gene often involves multiple interactions of transcription factors with various sequence elements. Identification of these sequence elements is the first step in understanding the underlying molecular mechanism(s) that regulate the gene expression. For in silico identification of these sequence elements, we have developed an online computational tool named transcription factor information system (TFIS) for detecting TFBS for the first time using a collection of JAVA programs and is mainly based on TFBS detection using position weight matrix (PWM). The database used for obtaining position frequency matrices (PFM) is JASPAR and HOCOMOCO, which is an open-access database of transcription factor binding profiles. Pseudo-counts are used while converting PFM to PWM, and TFBS detection is carried out on the basis of percent score taken as threshold value. TFIS is equipped with advanced features such as direct sequence retrieving from NCBI database using gene identification number and accession number, detecting binding site for common TF in a batch of gene sequences, and TFBS detection after generating PWM from known raw binding sequences in addition to general detection methods. TFIS can detect the presence of potential TFBSs in both the directions at the same time. This feature increases its efficiency. And the results for this dual detection are presented in different colors specific to the orientation of the binding site. Results obtained by the TFIS are more detailed and specific to the detected TFs as integration of more informative links from various related web servers are added in the result pages like Gene Ontology, PAZAR database and Transcription Factor Encyclopedia in addition to NCBI and UniProt. Common TFs like SP1, AP1 and NF-KB of the Amyloid beta precursor gene is easily detected using TFIS along with multiple binding sites. In another scenario of embryonic developmental process, TFs of the FOX family (FOXL1 and FOXC1) were also identified. TFIS is platform-independent which is publicly available along with its support and documentation at http://tfistool.appspot.com and http://www.bioinfoplus.com/tfis/ . TFIS is licensed under the GNU General Public License, version 3 (GPL-3.0).

Isolation and characterization of stress induced Ty1-copia like retrotransposable elements in chickpea (Cicer arietinum L.).

Almost all active plant retroelements are known to be induced by various biotic and abiotic stresses. Here, we show the presence of transcriptionally active Ty1-copia like retroelements in chickpea and their induction in response to abiotic stress. Eight Ty1-copia retrotransposon like mRNA sequences were reverse transcribed, amplified, cloned and characterized from stressed plants. These mRNA sequences were not detected in chickpea plants grown under normal conditions. Basing on the similarity analysis, these RT transcript sequences were classified into three families. It is proposed that all sequences except CARE7 might be transcript sequences of functional retrotransposons. The mRNA sequence CARE3 shows 99% nucleotide identity to a genomic Ty1-copia like sequence present in the Genbank with accession no. AJ535883.

Characterization of heterogeneity in Ty1-copia GROUP retrotransposons in chickpea (Cicer arietinum L.).

Retrotransposons represent a major fraction of the plant genome and play a significant role in the molecular evolution through sequence re-organization. In order to access the diversity in Ty1-copia group of retrotransposons in chickpea, reverse transcriptase (RT) conserved domain specific primers were selected to amplify RT conserved sequences. Thirty-six amplified fragments were cloned and characterized. On the basis of deduced amino acid homology among them, these sequences were grouped into five families. These sequence families showed from 34 to 81% inter-family homology at the amino acid level. Although these sequences belong to a highly conserved region no two sequences were identical. The results show that there is a high degree of heterogeneity among the Ty1-copia group of retroelements in chickpea. The genomic Southern hybridization with one of the reverse transcriptase sequences as a probe shows the presence of a large population of the Ty1-copia group of retrotransposons in chickpea.

Panzee, a copia-like retrotransposon from the grain legume, pigeonpea ( Cajanus cajan L.).

We report the isolation and characterization of a copia-like retrotransposon, Panzee, from pigeonpea ( Cajanus cajan). The 4947-bp Panzee element is AT rich (60%) and the integrated element is flanked by a target-site duplication of 5 bp. The structure of Panzee is that of a typical LTR-retrotransposon containing long terminal repeats (LTRs) which flank its internal region. The 5' LTR is 372 bp in length and the 3' LTR is 383 bp long. Both LTRs start with 5'-TG and end with CA-3' and have 4-bp terminal inverted repeats. The internal region between the LTRs contains two priming sites for DNA synthesis: the first, a 12-bp primer binding site complementary to initiator methionyl tRNA, is located adjacent to the 3' end of the 5' LTR and the other, a 12-bp polypurine tract lies just upstream to the 5' end of the 3' LTR. The putative polyprotein shows homology to all the proteins encoded by LTR retrotransposons, i.e. group-associated antigen ( gag), proteinase, endonuclease, reverse transcriptase (RT) and ribonuclease H (RNase H). However, the cloned copy of the element contains four frameshifts and a premature stop codon in its protein-coding domain. Genomic Southern hybridization experiments using probes derived from three different regions of the element show that Panzee or Panzee-related elements are present in high copy numbers in the pigeonpea genome. Analysis of transgenic tobacco plants containing the LTR:GUS construct shows that the 5' LTR of Panzee drives gene expression in this heterologous system in a tissue-specific manner. A phylogenetic tree constructed using reverse transcriptase sequences places Panzee in the copia group of retrotransposons.

Cloning and characterization of a cell cycle-regulated gene encoding topoisomerase I from Nicotiana tabacum that is inducible by light, low temperature and abscisic acid.

We have cloned a full-length 2874-bp cDNA coding for tobacco topoisomerase I, with an ORF of 2559 bp encoding a protein of 852 amino acids with a calculated molecular mass of 95 kDa and an estimated pI of 9.51. The deduced amino acid sequence shows homology to other eukaryotic topoisomerases I. Tobacco topoisomerase I was over-expressed in Escherichia coli, and the purified recombinant protein was found to relax both positively and negatively super-coiled DNA in the absence of the divalent cation Mg(2+)and ATP. These characteristic features indicate that the tobacco enzyme is a type I topoisomerase. The recombinant protein could be phosphorylated at (a) threonine residue(s) by protein kinase C. However, phosphorylation did not cause any change in its enzymatic activity. The genomic organization of the topoisomerase I gene revealed the presence of 8 exons and 7 introns in the region corresponding to the ORF and one intron in the 3' UTR region. Transcript analysis using RT-PCR showed basal constitutive expression in all organs examined, and the gene was expressed at all stages of the cell cycle--but the level of expression increased during the G1-S phase. The transcript level also increased following exposure to light, low-temperature stress and abscisic acid, a stress hormone.

Modulation of mRNA levels of liver arginase by insulin and vanadate in experimental diabetes.

This work was carried out to study the modulation of arginase expression in experimental diabetes. Here, we have demonstrated that liver arginase activity and mRNA levels increased significantly in diabetic condition. Insulin treatment reverses the increased activity and mRNA levels nearly to the control values. The reversal effects of vanadate are found to be similar to that of insulin and this observation further reiterates the insulin-like effects of vanadate. ELISA and slot blot assay observations are consistent with biochemical measurements of enzyme activity. These results show an increase in arginase activity and mRNA levels in diabetes and decrease in treated animals may be due to the transcriptional regulation of arginase gene.

Effects of vanadate on expression of liver arginase in experimental diabetic rats.

This work was carried out to study the effects of vanadate on the expression of liver-type arginase in experimentally induced diabetes in the rat. The results showed that the activity and mRNA levels of arginase were increased significantly in the diabetic condition. Vanadate treatment reversed the increased activity and restored mRNA levels of arginase almost to the control values. The reversal effects of vanadate were found to be similar to those of insulin, and this further confirms the insulin-like effects of vanadate. ELISA and slot-blot assay observations were consistent with the biochemical measurements of enzyme activity. The increase in arginase activity and mRNA levels in diabetes and the decrease in insulin- and vanadate-treated animals may be due to the transcriptional regulation of arginase.

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.

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.

Purification and characterization of ribulose-1,5-bisphosphate carboxylase from triticale.

Ribulose-1,5-bisphosphate carboxylase has been isolated from a synthetic cereal triticale and purified using a newly developed rapid procedure involving precipitation with ammonium sulphate (35-55% saturation), DEAE-cellulose (DE-52) chromatography and filtration through Sepharose CL-68. Molecular weights of the enzyme subunits are 15.5 and 52 kDa which corresponds to 540 kDa for the hexadecameric holoenzyme. Isoelectric focussing showed that the enzyme has a pI of 4.2. Various kinetic constants determined under aerobic conditions are: Km (CO2), 118 microM; Km (RuBP), 220 microM (at 20 mM NaHCO3) and Vmax, 690 nmole CO2 fixed/mg enzyme/min.

Structure and organization of a novel calmodulin gene of Arabidopsis thaliana.

The calcium binding protein calmodulin is involved in regulating various cellular and biochemical processes. A gene for calmodulin has been isolated from a genomic library of Arabidopsis thaliana constructed in lambda EMBL-4 using a heterologous cDNA probe from electric eel. The genomic clone was found to contain a complete copy of a distinct calmodulin (CaM) gene. A comparison of the nucleotide sequence with those from the other plant and animal systems reveals that the Arabidopsis CaM gene contains two exons and a single intron of 364 bp. The intron splits the triplet encoding the 25th amino acid of the protein. The Arabidopsis calmodulin protein appears to be distinct in containing a single amino acid substitution, arg to lys at the 126th position from the other known plant and animal calmodulins. The 5'-upstream region of this CaM gene (ACaM4) contains several known sequence-elements, a few of which are known to be implicated in transcriptional activation in response to light, U.V. radiation and heat shock besides the presence of many small multiple repeats. The Southern hybridization analysis and a comparison of nucleotide sequence with those of the other calmodulin genes of Arabidopsis indicate that the calmodulin gene, ACaM4, belongs to a small multigene family consisting of at least four members in the Arabidopsis genome.

Isolation and characterisation of satellite DNA from Brassica juncea (L.) Czern.

Nuclear DNA isolated from hypocotyls (H), proliferating callus (PC) and differentiating callus (DC) of Brassica juncea contains a satellite DNA which can be resolved in actinomycin-D/CsCl gradients. The satellite DNA undergoes changes, when an in vitro culture is raised from hypocotyl tissue and forms a higher percentage of the genome in PC and DC than in mature differentiated tissue (hypocotyl). All the three satellite DNAs are GC-rich compared to main band DNAs. Satellite DNA of H has higher Tm and GC content than that of the PC and DC satellites. A 200 bp basic repeat unit from hypocotyl nuclear DNA has been cloned and characterised.