Vidhu A. Sane

Mango - Postharvest Biology and Biotechnology

Mango is one of the choicest fruits in the world and popular due to its delicate taste, pleasant aroma and nutritional value. Mango is indigenous to north-east India and north Burma, but now grown in over 90 countries. In the past two decades, mango production has increased appreciably with international trade jumping approximately four-fold valued close to US

Activation of ethylene-responsive p-hydroxyphenylpyruvate dioxygenase leads to increased tocopherol levels during ripening in mango

950 million. Mango belongs to the category of climacteric fruits and its ripening is initiated and proceeded by a burst in ethylene production and a dramatic rise in the rate of respiration. Although there are a few hundred mango cultivars grown in the Indian subcontinent and other parts of the world, the most popular cultivars are generally highly perishable and ripen within 7 to 9 days of harvest at ambient temperature. Currently, the export potential and international trade of mango is limited due to several factors such as its perishable nature, disease and pest infestation, and susceptibility of certain premium cultivars to chilling injury when stored at low temperatures. Efforts are ongoing to develop technologies for improved storage and packaging, and overcome limitations encountered during storage and transit. Controlled atmosphere (CA) and hypobaric storage of mango are powerful means to overcome its perishable nature. The composition of CA varies among cultivars to ensure its original taste, flavor and aroma. Edible coating on the fruit skin may further cut down the rate of deterioration. Recently, significant advances have been made in understanding ripening characteristics of mango at the molecular level. Candidate genes related to ethylene biosynthesis and signalling, cell wall modification, aroma production and stress response have been cloned and characterized for future use in mango improvement. Efforts are also being made to establish a suitable transformation and plant regeneration system so that transgenic mango with added value and increased shelf life for long distance transportation could be developed.

Over-expression of JcDGAT1 from Jatropha curcas increases seed oil levels and alters oil quality in transgenic Arabidopsis thaliana

Mango is characterized by high tocopherol and carotenoid content during ripening. From a cDNA screen of differentially expressing genes during mango ripening, a full-length p-hydroxyphenylpyruvate dioxygenase (MiHPPD) gene homologue was isolated that encodes a key enzyme in the biosynthesis of tocopherols. The gene encoded a 432-amino-acid protein. Transcript analysis during different stages of ripening revealed that the gene is ripening related and rapidly induced by ethylene. The increase in MiHPPD transcript accumulation was followed by an increase in tocopherol levels during ripening. The ripening-related increase in MiHPPD expression was also seen in response to abscisic acid and to alesser extent to indole-3-acetic acid. The expression of MiHPPD was not restricted to fruits but was also seen in other tissues such as leaves particularly during senescence. The strong ethylene induction of MiHPPD was also seen in young leaves indicating that ethylene induction of MiHPPD is tissue independent. Promoter analysis of MiHPPD gene in tomato discs and leaves of stable transgenic lines of Arabidopsis showed that the cis elements for ripening-related, ethylene-responsive, and senescence-related expression resided within the 1590 nt region upstream of the ATG codon. Functionality of the gene was demonstrated by the ability of the expressed protein in bacteria to convert p-hydroxyphenylpyruvate to homogentisate. These results provide the first evidence for HPPD expression during ripening of a climacteric fruit.

Differential expression of the mango alcohol dehydrogenase gene family during ripening.

The increasing consumption of fossil fuels and petroleum products is leading to their rapid depletion and is a matter of concern around the globe. Substitutes of fossil fuels are required to sustain the pace of economic development. In this context, oil from the non food crops (biofuel) has shown potential to substitute fossil fuels. Jatropha curcas is an excellent shrub spread and naturalized across the globe. Its oil contains a high percentage of unsaturated fatty acids (about 78-84% of total fatty acid content) making the oil suitable for biodiesel production. Despite its high oil content, it has been poorly studied in terms of important enzymes/genes responsible for oil biosynthesis. Here, we describe the isolation of the full length cDNA clone of JcDGAT1, a key enzyme involved in oil biosynthesis, from J. curcas seeds and manipulation of oil content and composition in transgenic Arabidopsis plants by its expression. Transcript analysis of JcDGAT1 reveals a gradual increase from early seed development to its maturation. Homozygous transgenic Arabidopsis lines expressing JcDGAT1 both under CaMV35S promoter and a seed specific promoter show an enhanced level of total oil content (up by 30-41%) in seeds but do not show any phenotypic differences. In addition, our studies also show alterations in the oil composition through JcDGAT1 expression. While the levels of saturated FAs such as palmitate and stearate in the oil do not change, there is significant reproducible decrease in the levels of oleic acid and a concomitant increase in levels of linolenic acid both under the CaMV35S promoter as well as the seed specific promoter. Our studies thus confirm that DGAT is involved in flux control in oil biosynthesis and show that JcDGAT1 could be used specifically to manipulate and improve oil content and composition in plants.

Isolation of High Quality RNA from Oilseeds of Jatropha curcas

Alcohol dehydrogenases play an important role during fruit ripening and aroma production. Three full-length cDNAs (MiAdh1, 2 and 3) encoding alcohol dehydrogenases were obtained from mango fruit pulp using RT-PCR approaches. All three members displayed strong homology in the coding region when compared at the protein and nucleotide levels, however showed variations in untranslated regions. Expression patterns of these ADHs were different during fruit development and ripening. MiADH1 and MiADH2 transcripts accumulated at the onset of ripening in mango fruit whereas MiADH3 accumulated during early development of fruit. Expression analysis also indicated that mango ADHs were responsive to ethylene but regulated differently by ABA. MiADH1 was induced by ABA treatment whereas MiADH2 transcript was negatively regulated by ABA. MiADH3 did not respond to ABA in ripening fruit. Differences in substrate specificity for NADH and NADPH were also observed between the three enzymes. Total ADH enzyme activity correlated positively with increased transcript levels at the initiation of ripening.

Purification and characterization of the mitochondrial F1 ATPase from rice

High quality RNA with good yield is a prerequisite for carrying out several molecular biology studies. Recalcitrant tissues such as oilseeds pose several problems while isolating good quality RNA. We have standardized a fast and simple protocol for RNA isolation from the seeds of Jatropha curcas, which gives good quality RNA without compromising for the yield. By including pre wash of seed powder with acetone and removal of polysaccharides through selective precipitation, we have been regularly isolating good quality total RNA in the range of 300–450 μg g−1 depending upon tissue type. The RNA isolated by this procedure is devoid of any contaminating DNA. The RNA preparations have been subjected to cDNA synthesis and PCR, and found suitable for these studies. This method also works satisfactorily with groundnut and mustard seeds.

Purification and characterization of the mitochondrial F1 atpase from SORGHUM

Abstract The mitochondrial F 1 ATPase from rice has been purified to homogeneity. One of the characteristic features of this ATPase is that it consists of six subunits of sizes 55 kDa (α + β), 36 kDa (γ), 26.2 kDa (δ′), 22 kDa (δ) and 11 kDa (ϵ) unlike other monocot F 1 ATPases. It exhibits typical non-linear kinetics for ATP and can be stimulated by oxyanions like bicarbonate and sulphite. High concentrations of sulphite (> 10 mM) markedly inhibit the ATPase. Compared to maize F 1 ATPase, it has a relatively lower GTPase activity and completely lacks Ca 2+ ATPase activity. However, in spite of the differences in the kinetic properties of the F 1 ATPases from rice and maize, no significant differences in the amino acid sequence of α and β subunits of rice and maize are observed except for three amino acid changes.

Heterologous expression of two GPATs from Jatropha curcas alters seed oil levels in transgenic Arabidopsis thaliana

Abstract Purification and kinetic analysis of the sorghum mitochondrial F 1 ATPase was attempted in order to understand the behaviour of plant monocot F 1 ATPases. The purified F 1 ATPase consisted of 6 subunits viz. α, β, γ, δ′, δ and e unlike other monocot F 1 ATPases reported so far. The additional subunit designated as δ′ is larger in size than the δ subunit, unlike in other dicot ATPase preparations. The enzyme has a low specific activity and is markedly affected by anions like bicarbonate and sulphite and cations like Mg 2+ and Mn 2+ . It shows no Ca 2+ dependent ATPase activity and has a very low GTPase activity, compared with other plant ATPases. Comparison of the sorghum ATPase with ATPases from other members of Gramineae reveals differences in their characteristics within the same family.

Polygalacturonase (PG) gene expression in Musa acuminata cultivars from Kerala

Oils and fats are stored in endosperm during seed development in the form of triacylglycerols. Three acyltransferases: glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidyl acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT) are involved in the storage lipid biosynthesis and catalyze the stepwise acylation of glycerol backbone. In this study two members of GPAT gene family (JcGPAT1 and JcGPAT2) from Jatropha seeds were identified and characterized. Sequence analysis suggested that JcGPAT1 and JcGPAT2 are homologous to Arabidopsis acyltransferase-1 (ATS1) and AtGPAT9 respectively. The sub-cellular localization studies of these two GPATs showed that JcGPAT1 localizes into plastid whereas JcGPAT2 localizes in to endoplasmic reticulum. JcGPAT1 and JcGPAT2 expressed throughout the seed development with higher expression in fully matured seed compared to immature seed. The transcript levels of JcGPAT2 were higher in comparison to JcGPAT1 in different developmental stages of seed. Over-expression of JcGPAT1 and JcGPAT2 under constitutive and seed specific promoters in Arabidopsis thaliana increased total oil content. Transgenic seeds of JcGPAT2-OE lines accumulated 43-60% more oil than control seeds whereas seeds of Arabidopsis lines over-expressing plastidial GPAT lead to only 13-20% increase in oil content. Functional characterization of GPAT homologues of Jatropha in Arabidopsis suggested that these are involved in oil biosynthesis but might have specific roles in Jatropha.

Tocopherol levels in different mango varieties correlate with MiHPPD expression and its over-expression elevates tocopherols in transgenic Arabidopsis and tomato

Banana cannot be preserved for a long time after harvesting due to a short shelf life. Fruit softening is associated with textural changes due to disassembly of the primary cell wall and modification of the structure and composition of various polysaccharides. Cell wall degradation is caused by the action of various cell wall hydrolase enzymes. Polygalacturonase (PG) is the key enzyme involved in this process. The ripening period is different in cultivars maintained under domesticated cultivation in Kerala. PG activity was profiled in eight Musa acuminata cultivars from Kerala and expression analysis of the PG gene was accomplished using semi-quantitative RT-PCR. Maximum PG activity was observed in Palayankodan and minimum activity was observed in Kadali. Gene expression analysis showed variation between ethylene treated fruits and controls in Palayankodan, whereas the expression patterns in Kadali were similar. The fruit softening process is cultivar specific.