Gursharn S. Randhawa

Mapping of Quantitative Trait Loci for Grain Iron and Zinc Concentration in Diploid A Genome Wheat

Micronutrients, especially iron (Fe) and zinc (Zn), are deficient in the diets of people in underdeveloped countries. Biofortification of food crops is the best approach for alleviating the micronutrient deficiencies. Identification of germplasm with high grain Fe and Zn and understanding the genetic basis of their accumulation are the prerequisites for manipulation of these micronutrients. Some wild relatives of wheat were found to have higher grain Fe and Zn concentrations compared with the cultivated bread wheat germplasm. One accession of Triticum boeoticum (pau5088) that had relatively higher grain Fe and Zn was crossed with Triticum monococcum (pau14087), and a recombinant inbred line (RIL) population generated from this cross was grown at 2 locations over 2 years. The grains of the RIL population were evaluated for Fe and Zn concentration using atomic absorption spectrophotometer. The grain Fe and Zn concentrations in the RIL population ranged from 17.8 to 69.7 and 19.9 to 64.2 mg/kg, respectively. A linkage map available for the population was used for mapping quantitative trait loci (QTL) for grain Fe and Zn accumulation. The QTL analysis led to identification of 2 QTL for grain Fe on chromosomes 2A and 7A and 1 QTL for grain Zn on chromosome 7A. The grain Fe QTL were mapped in marker interval Xwmc382-Xbarc124 and Xgwm473-Xbarc29, respectively, each explaining 12.6% and 11.7% of the total phenotypic variation and were designated as QFe.pau-2A and QFe.pau-7A. The QTL for grain Zn, which mapped in marker interval Xcfd31-Xcfa2049, was designated as QZn.pau-7A and explained 18.8% of the total phenotypic variation.

Plant Cell Wall Matrix Polysaccharide Biosynthesis

The wall of an expanding plant cell consists primarily of cellulose microfibrils embedded in a matrix of hemicellulosic and pectic polysaccharides along with small amounts of structural and enzymatic proteins. Matrix polysaccharides are synthesized in the Golgi and exported to the cell wall by exocytosis, where they intercalate among cellulose microfibrils, which are made at the plasma membrane and directly deposited into the cell wall. Involvement of Golgi glucan synthesis in auxin-induced cell expansion has long been recognized; however, only recently have the genes corresponding to glucan synthases been identified. Biochemical purification was unsuccessful because of the labile nature and very low abundance of these enzymes. Mutational genetics also proved fruitless. Expression of candidate genes identified through gene expression profiling or comparative genomics in heterologous systems followed by functional characterization has been relatively successful. Several genes from the cellulose synthase-like (Csl) family have been found to be involved in the synthesis of various hemicellulosic glycans. The usefulness of this approach, however, is limited to those enzymes that probably do not form complexes consisting of unrelated proteins. Nonconventional approaches will continue to incrementally unravel the mechanisms of Golgi polysaccharide biosynthesis.

Ischemic insult induced apoptotic changes in PC12 cells: Protection by trans resveratrol

In this study, we determined the protective potential of trans resveratrol against oxygen-glucose deprivation (OGD) induced reactive oxygen species mediated apoptotic damages in PC12 cells. In vitro model of ischemic cerebral stroke was created by keeping cells in an OGD condition for 6h followed by 24h reoxygenation. Cells received biologically safe doses (5, 10, and 25 μM) of trans resveratrol in the following schedules for 24h prior to OGD; during 6h of OGD; for 24h post OGD and whole treatment group which starts from 24h before OGD and lasted to 24h post OGD. Anti-ischemic potential of trans resveratrol was assessed by measuring the regulation of lipid peroxidation, reactive oxygen species production, glutathione content, and expression (mRNA and protein) of apoptotic markers such as Bax, Bcl(2) and Caspase-3. Hypoxia inducible factor-1α (HIF-1α) was also assessed to correlate the changes with ischemic injuries. Significant (P<0.05) restoration in lipid peroxidation, reactive oxygen species, and glutathione content were observed following the treatment of trans resveratrol in cells receiving OGD and re-oxygenation. Changes induced by trans resveratrol could be correlated well with alterations in the expression of Bax, Bcl(2), Caspase-3 and HIF-1α. These results indicate that trans resveratrol administration attenuates free radical formation and mitochondria mediated apoptosis perhaps by regulating the expressions of Bax, Bcl(2,) and Caspase-3 in PC12 cells receiving OGD and re-oxygenation insult.

Development of Triticum turgidum subsp. durum – Aegilops longissima amphiploids with high iron and zinc content through unreduced gamete formation in F1 hybrids

Four different interspecific hybrids involving three different accessions of Aegilops longissima Schweinf. & Muschl. with high grain iron and zinc content and three Triticum turgidum L. subsp. durum (Desf.) Husn. cultivars with low micronutrient content were made for durum wheat biofortification and investigated for chromosome pairing, fertility, putative amphiploidy, and micronutrient content. The chromosome pairing in the 21-chromosome F1 hybrids (ABSl) consisted of 0-6 rod bivalents and occasionally 1 trivalent. All the F1 hybrids, however, unexpectedly showed partial but variable fertility. The detailed meiotic investigation indicated the simultaneous occurrence of two types of aberrant meiotic divisions, namely first-division restitution and single-division meiosis, leading to regular dyads and unreduced gamete formation and fertility. The F2 seeds, being putative amphiploids (AABBSlSl), had nearly double the chromosome number (40-42) and regular meiosis and fertility. The F1 hybrids were intermediate between the two parents for different morphological traits. The putative amphiploids with bold seed size had higher grain ash content and ash iron and zinc content than durum wheat cultivars, suggesting that Ae. longissima possesses a better genetic system(s) for uptake and seed sequestration of iron and zinc, which could be transferred to elite durum and bread wheat cultivars and exploited.

Development and characterization of Triticum aestivum – Aegilops kotschyi amphiploids with high grain iron and zinc contents

Synthetic amphiploids between Triticum aestivum (AABBDD) landrace Chinese Spring ( Ph I ) and cultivar WL711 with different accessions of Aegilops kotschyi (UUS l S l ) were developed through colchicine treatment of sterile hybrids. The F 1 hybrids and amphiploid plants were intermediate between the parents for plant morphology and spike characteristics. Meiotic metaphase chromosome analysis of the F 1 hybrids (ABDUS l ) showed the expected chromosome number (35) and very little but variable homoeologous chromosome pairing. The amphiploids (AABBDDUUS l S l ), however, had variable frequency of univalents at meiotic metaphase-I. The SDS–PAGE of high molecular weight glutenin subunits of amphiploids along with the parents showed the presence and expression of all the parental genomes in the amphiploids. The amphiploids with seeds as large as that of wheat cultivars had higher grain, flag leaf and grain ash iron and zinc concentrations than the wheat parents and comparable with those of their Ae. kotschyi parents suggest that Ae. kotschyi possesses a distinctive genetic system for the micronutrient uptake, translocation and sequestration than the wheat cultivars. This could, however, be demonstrated unequivocally only with comprehensive data on biomass, grain yield and harvest index of the Aegilops donors and the synthetic amphiploids, which is not feasible due to their shattering and hard threshing. The use of amphiploids for the transfer of high iron and zinc concentrations and development of alien addition and substitution lines in wheat is in progress.

A candidate gene OsAPC6 of anaphase-promoting complex of rice identified through T-DNA insertion

A dwarf mutant (Oryza sativa anaphase-promoting complex 6 (OsAPC6)) of rice cultivar Basmati 370 with 50% reduced plant height as compared to the wild type was isolated by Agrobacterium tumefaciens-mediated transformation using HmRDs cassette. This mutant was found to be insensitive to exogenous gibberellic acid (GA3) application. Homozygous mutant plants showed incomplete penetrance and variable expressivity for plant height and pleiotropic effects including gibberellic acid insensitivity, reduced seed size, panicle length, and female fertility. Single copy insertion of T-DNA and its association with OsAPC6 was confirmed by Southern hybridization, germination on hygromycin, and 3:1 segregation of HPT gene in F2 from OsAPC6 × Basmati 370 cross. The T-DNA flanking region sequenced through thermal asymmetric interlaced polymerase chain reaction showed a single hit on chromosome 3 of japonica rice cultivar Nipponbare in the second exonic region of a gene which encodes for sixth subunit of anaphase-promoting complex/cyclosome. The candidate gene of 8.6-kb length encodes a 728-amino acid protein containing a conserved tetratricopeptide repeat (TPR) domain and has only a paralog, isopenicillin N-synthase family protein on the same chromosome without the TPR domain. There was no expression of the gene in the mutant while in Basmati 370, it was equal in both roots and shoots. The knockout mutant OsAPC6 interferes with the gibberellic acid signaling pathway leading to reduced height and cell size probably through ubiquitin-mediated proteolysis. Further functional validation of the gene through RNAi is in progress.

Evaluation and identification of wheat-Aegilops addition lines controlling high grain iron and zinc concentration and mugineic acid production

Iron and zinc deficiency affects more than half of the world population due to low inherent micronutrient content of cereals and other staple foods. The micronutrient deficiency is further aggravated by poor availability of these minerals in calcareous soils and their uptake by crop plants. Series of available wheat-Aegilops addition lines were evaluated for identification of alien chromosomes carrying genes for high grain iron and zinc concentrations and release of mugineic acid(s) facilitating micronutrient uptake under their deficient conditions. Addition lines of chromosome 2Sv, 2Uv and 7Uv of Ae. peregrina, 2Sl and 7Sl of Ae. longissima and 2U of Ae. umbellulata were found to carry genes for high grain iron whereas the group 7 chromosomes had genes for higher grain zinc. Higher release of mugineic acid (MA) under iron deficient condition was observed in addition lines of chromosome 2Sv, 2Uv, 4Uv and 7Sv of Ae. peregrina, 2Sl and 6Sl of Ae. longissima and 2U and 5U of Ae. umbellulata. Higher grain and...

Chapter 3 – Guar: An Industrial Crop from Marginal Farms

Guar or cluster bean ( Cyamopsis tetragonoloba [L.] Taub) is a drought tolerant, annual legume grown mainly in semi-arid regions of India and Pakistan on marginal soil. Guar seeds have a large endosperm, which is enriched in galactomannan, a commercial gum with a myriad industrial applications such as paper, textile, oil and gas drilling, pharmaceuticals, food, cosmetics, and explosives. The global demand for guar gum has increased sharply mainly because of fracking, a process involving deep, horizontal drilling for natural gas. Increased demand has translated into improved livelihood of the otherwise marginal farmers in the guar-growing countries. This chapter covers various aspects of the guar crop, which include cultivation, breeding and genetics, galactomannan synthesis and production, and applications of guar gum.

Identification of reference genes for real-time PCR gene expression studies during seed development and under abiotic stresses in Cyamopsis tetragonoloba (L.) Taub.

Guar (Cyamopsis tetragonoloba) is an important industrial crop. The knowledge about genes of guar involved in various processes can help in developing improved varieties of this crop. qRT-PCR is a preferred technique for accurate quantification of gene expression data. This technique requires the use of appropriate reference genes from the crop to be studied. Such genes have not been yet identified in guar. The expression stabilities of the 10 candidate reference genes, viz., CYP, ACT 11, EF-1α, TUA, TUB, ACT 7, UBQ 10, UBC 2, GAPDH and 18S rRNA were evaluated in various tissues of guar under normal and abiotic stress conditions. Four different algorithms, geNorm, NormFinder, BestKeeper and ∆Ct approach, were used to assess the expression stabilities and the results obtained were integrated into comprehensive stability rankings. The most stable reference genes were found to be CYP and ACT 11(tissues), ACT 11, UBC 2 and ACT 7 (seed development), ACT 7 and TUB (drought stress), TUA, UBC 2 and CYP (nitrogen stress), TUA and UBC 2 (cold stress), GAPDH and ACT 7 (heat stress) and GAPDH and EF-1a (salt stress). These results indicated the necessity of identifying a suitable reference gene for each experimental condition. Four selected reference genes were validated by normalizing the expression of CtMT1 gene. To the best of our knowledge this is the first report on the identification of reference genes in guar. These findings are likely to provide a boost to the gene expression studies in this important crop.

Cyamopsis tetragonoloba type 1 metallothionein (CtMT1) gene is upregulated under drought stress and its protein product has an additional C-X-C motif and unique metal binding pattern

Metallothioneins (MTs) are involved in cellular homeostasis of essential metal ions and detoxification of nonessential metal ions. We report here the identification of four MT genes, CtMT1, CtMT2, CtMT3 and CtMT4, encoding CtMT1, CtMT2, CtMT3 and CtMT4 proteins, respectively, from the industrial guar crop. The primary structures of last three proteins were similar to those of respective MT proteins of other plants but the CtMT1 protein primary structure was different from the other plant MT1 proteins in having an additional C-X-C motif. The four MT genes showed tissue specific expression patterns suggesting their specific roles in different tissues. High expression of CtMT1 gene was observed in roots and nodules whereas CtMT2 and CtMT3 genes showed high expression in leaves. The expression of CtMT4 gene was high in seeds. The qRT-PCR studies revealed upregulation in expression of CtMT1 gene under drought stress. Recombinant CtMT1 protein was produced in E. coli Rosetta cells and purified by metal affinity chromatography. The purified protein showed antioxidant property and the order of its metal ion binding affinities was Cu2+ > Zn2+ > Fe2+ > Cd2+. This information about CtMT1 protein is expected to be useful in understanding its role in drought tolerance and other physiological processes of guar.