Pravendra Nath

A simple procedure for the isolation of high quality RNA from ripening banana fruit

Isolation of high quality RNA from ripening banana fruit tissue is difficult due to high levels of polysaccharides and other substances that interfere when using conventional procedures for RNA isolation. These substances not only decrease the yield but the quality of RNA is almost unusable. We describe here a simple RNA procedure that effectively removes these contaminating substances without affecting the yield. Following this procedure, we routinely obtained 80–150 μg of total RNA per g fresh tissue. The RNA is of good quality and suitable for northern analysis, RT-PCR and cDNA library construction.

Ripening of fleshy fruit: molecular insight and the role of ethylene.

Development and ripening in fruit is a unique phase in the life cycle of higher plants which encompasses several stages progressively such as fruit development, its maturation, ripening and finally senescence. During ripening phase, several physiological and biochemical changes take place through differential expression of various genes that are developmentally regulated. Expression and/or suppression of these genes contribute to various changes in the fruit that make it visually attractive and edible. However, in fleshy fruit massive losses accrue during post harvest handling of the fruit which may run into billions of dollars worldwide. This encouraged scientists to look for various ways to save these losses. Genetic engineering appears to be the most promising and cost effective means to prevent these losses. Most fleshy fruit ripen in the presence of ethylene and once ripening has been initiated proceeds uncontrollably. Ethylene evokes several responses during ripening through a signaling cascade and thousands of genes participate which not only sets in ripening but also responsible for its spoilage. Slowing down post ripening process in fleshy fruit has been the major focus of ripening-related research. In this review article, various developments that have taken place in the last decade with respect to identifying and altering the function of ripening-related genes have been described. Role of ethylene and ethylene-responsive genes in ripening of fleshy fruit is also included. Taking clues from the studies in tomato as a model fruit, few case studies are reviewed.

Modulation of transcriptome and metabolome of tobacco by Arabidopsis transcription factor, AtMyb12, leads to insect resistance

Flavonoids synthesized by the phenylpropanoid pathway participate in myriad physiological and biochemical processes in plants. Due to the diversity of secondary transformations and the complexity of the regulation of branched pathways, single gene strategies have not been very successful in enhancing the accumulation of targeted molecules. We have expressed an Arabidopsis (Arabidopsis thaliana) transcription factor, AtMYB12, in tobacco (Nicotiana tabacum), which resulted in enhanced expression of genes involved in the phenylpropanoid pathway, leading to severalfold higher accumulation of flavonols. Global gene expression and limited metabolite profiling of leaves in the transgenic lines of tobacco revealed that AtMYB12 regulated a number of pathways, leading to flux availability for the phenylpropanoid pathway in general and flavonol biosynthesis in particular. The tobacco transgenic lines developed resistance against the insect pests Spodoptera litura and Helicoverpa armigera due to enhanced accumulation of rutin. Suppression of flavonol biosynthesis by artificial microRNA reversed insect resistance of the AtMYB12-expressing tobacco plants. Our study suggests that AtMYB12 can be strategically used for developing safer insect pest-resistant transgenic plants.

Expression and activities of ethylene biosynthesis enzymes during ripening of banana fruits and effect of 1-MCP treatment

The respiratory climacteric, ethylene evolution and activities and expression of ethylene biosynthesis enzymes show a characteristic pattern in banana during ethylene induced ripening. A unique biphasic respiratory climacteric with a 10 and 6 fold increment in respiration rates on days 2 and 6 respectively after ethylene treatment is preceded by ethylene evolution on days 1 and 4 with 8.3 and 6.93 fold increments respectively. This represents a unique feature of ripening in banana. While ACC synthase transcript accumulation matched the respiratory climacteric, the ACC synthase activity and ACC oxidase transcript accumulation showed a different pattern. The most significant observation was the effect of 1-MCP on ACC content and in vitro ACC oxidase activity. Though 1-MCP treated fruit did not show any respiratory climacteric or burst in ethylene production, it did not inhibit completely ACC accumulation and in vitro ACC oxidase activity. No transcript accumulation of ACC synthase was observed at any time in 1-MCP treated fruits, whereas a basal level of ACC oxidase transcript was detected throughout. It is concluded that ethylene induced ripening of banana is characteristically different from that of other climacteric fruits and that ethylene biosynthesis may have more than one mechanisms operating during ripening which are tightly controlled at various levels.

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

Functional Analysis of Regulatory Elements in the Gene Promoter for an Abscission-Specific Cellulase from Bean and Isolation, Expression, and Binding Affinity of Three TGA-Type Basic Leucine Zipper Transcription Factors

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.

The gene promoter for a bean abscission cellulase is ethylene-induced in transgenic tomato and shows high sequence conservation with a soybean abscission cellulase

Site-directed mutagenesis was used to identify cis-acting elements that control hormonal and abscission-specific expression of the bean (Phaseolus vulgaris) abscission cellulase (BAC) promoter. Auxin inhibition of BAC promoter expression is at least in part controlled by a negatively regulated element and ethylene induction by a positively regulated element. One of a series of 15 different 10-bp mutations created in a 2.9-kb BAC promoter reduced reporter gene expression by 60%. The native sequence for this 10-bp mutation includes a TGA-type basic leucine zipper (bZIP) motif. Tandem ligation of three 18-bp BAC elements (Z-BAC), which includes the bZIP motif to a minimal −50 35S cauliflower mosaic virus promoter, enhanced expression in abscission zones (AZs) 13-fold over that of the minimal promoter alone. The native forward orientation of the Z-BAC elements was essential for high expression levels. Expression of the Z-BAC minimal construct was 3-fold greater in AZ than stems when compared with the expression levels of an internal control with an enhanced 35S cauliflower mosaic virus promoter. Polymerase chain reaction was used to identify three TGA-type bZIP transcription factors in an AZ cDNA library. One of these factors was of the class I type and two of the class II type. RNA-blot analysis was completed for these genes and electrophoretic mobility shift assays used to confirm their binding to the Z-BAC element. Electrophoretic mobility shift assay-binding affinity was greatest for the class I TGA-type bZIP factor. The results indicate a complex interaction of negative and positive regulating transcription factors that control BAC gene expression.

Petal abscission in rose is associated with the differential expression of two ethylene-responsive xyloglucan endotransglucosylase/hydrolase genes, RbXTH1 and RbXTH2

Bean leaf abscission (organ separation) correlates with thede novo accumulation of a pI 9.5 cellulase and its mRNA. Overlapping genomic clones encoding the bean abscission cellulase (BAC) were isolated and partially sequenced. In addition, a genomic clone for a soybean abscission cellulase (SAC) was identified and the sequence compared to the BAC genomic sequence. Two 5′-upstream regions are particularly well conserved in the two sequences. Of special interest here is the region between −1 and −200 in the BAC promoter which is highly conserved in the SAC gene. Particle gun bombardment with a BAC promoter construct containing 210 bp of BAC sequence 5′ to the transcription start site was sufficient to drive abscission-specific and ethylene and auxin-regulated transient expression in bean. In addition to the transient expression assay, expression was examined in stably transformed tomato. A similar −210 bp BAC promoter construct supported a low level of ethylene-inducible reporter gene expression in tomato leaf abscission zones and adjacent petioles but not in ethylene-treated stem tissue or fruit. Expression from the −210 promoter in tomato abscission zones was inhibited by silver thiosulfate, an ethylene action inhibitor, and was partially inhibited by treatment with auxin.

Biochemical correlates of airway hyperreactivity in guinea pigs: role of lysophosphatidyl choline

Abscission is a process that involves shedding of plant organs from the main plant body. In this study it is shown that the process of petal separation in the fragrant rose, Rosa bourboniana, is accompanied by the expression of two xyloglucan endotransglucosylase/hydrolase genes, RbXTH1 and RbXTH2. The sequences of the two genes show 52% amino acid identity but are conserved at the catalytic site. The genes are up-regulated soon after the initiation of the abscission process and their transcription is associated with the progression of abscission, being faster in ethylene-treated flowers but slower during field abscission. Transcription is ethylene responsive, with the ethylene response being tissue-specific for RbXTH1 but largely tissue-independent for RbXTH2. Expression is correlated with an increase in xyloglucan endotransglucosylase (XET) action in petal abscission zones of both ethylene-treated and field abscising flowers. Proximal promoters of both the genes drive β-glucuronidase expression in an ethylene-responsive and abscission-related manner in agrobacteria-infiltrated rose petals, indicating that cis-elements governing ethylene-responsive and abscission-related expression probably lie within the first 700 nucleotides upstream of the translational initiation codon. The results show that cell wall remodelling of the xyloglucan moieties through the XET action of XTHs may be important for cell separation during abscission.

Transcriptional activation of a 37 kDa ethylene responsive cysteine protease gene, RbCP1, is associated with protein degradation during petal abscission in rose

To elucidate the biochemical basis of airway hyperreactivity, we studied the relationships between in vivo airway sensitivity of guinea pigs to histamine and their tracheal beta-adrenergic binding sites, Ca++- and (Na+-K+)-ATPase activities, and composition of phospholipids. The relationships between tracheal and plasma phospholipids were also examined. beta-Adrenergic receptor binding with 3H-dihydroalprenolol in tracheal tissue showed an inverse relationship with in vivo airway sensitivity to histamine. Among the phospholipids, tracheal phosphatidyl ethanolamine content varied inversely with in vivo airway sensitivity, whereas tracheal and plasma lysophosphatidyl choline contents showed a direct correlation with airway sensitivity. A significant direct correlation was also observed between tracheal and plasma lysophosphatidyl choline levels. Both Ca++-ATPase and (Na+-K+)-ATPase activities increased with increasing airway sensitivity. These enzymes showed inverse correlations with phosphatidyl ethanolamine content and direct correlations with lysophosphatidyl choline content. Our data suggest that increased lysophosphatidyl choline may cause various biochemical changes associated with airway hyperreactivity.