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- Author or Editor: Abhaya Dandekar x
Sorbitol is the primary photosynthetic end product in the leaves of many tree fruit species in the Rosaceae family, but its physiological role remains unclear. In this study, we determined the effect of decreased sorbitol synthesis on the antioxidant system that scavenges reactive oxygen species (ROS) in apple leaves. Sorbitol synthesis was decreased in apple leaves by antisense inhibition of aldose-6-phosphate reductase activity. Dehydroascorbate reductase (DHAR), glutathione reductase, and catalase (CAT) activities increased in the leaves of the transgenic plants with decreased sorbitol synthesis, whereas superoxide dismutase, ascorbate peroxidase, NADH dependent and NADPH dependent monodehydroascorbate reductase activity did not show significant changes. Ascorbate and glutathione concentrations were higher in leaves of the transgenic plants compared with the control. The effect of decreased sorbitol synthesis on the antioxidant enzyme activity was dependent on leaf developmental stages. Larger changes in the enzyme activities of CAT, DHAR, and GR were observed in the old leaves than in the young leaves. These results suggest that sorbitol may play a role in ROS scavenging in apple leaves.
The reaction catalyzed by ADP-glucose pyrophosphorylase (AGPase) to form ADP-glucose is a regulatory and rate-limiting step in starch synthesis in plants. In response to decreased sorbitol synthesis, starch synthesis was up-regulated in the transgenic apple plants. In this study, we examined both redox and metabolite regulation of AGPase to understand the mechanism responsible for the up-regulation of starch synthesis. No difference in the monomerization/dimerization of apple leaf AGPase small subunits was observed between the transgenic plants and the untransformed control. NADP-dependent malate dehydrogenase, indicative of chloroplastic redox status, did not show significant change in the transgenic plants either. Determination of key metabolites with nonaqueous fractionation indicated that concentrations of hexose phosphates (mainly glucose-6-phosphate and fructose-6-phosphate) were higher in both the cytosol and chloroplasts of the transgenic plants than in the control, whereas 3-phosphoglycerate (PGA) concentration in the chloroplast was not higher in the transgenic plants. We conclude that accumulation of hexose-phosphates results in a decrease in inorganic phosphate (Pi) concentration and an increase in PGA/Pi ratio in the chloroplast, leading to up-regulation of starch synthesis via activating AGPase.
Considering starch synthesis was enhanced in leaves of transgenic apple trees with decreased sorbitol synthesis, we hypothesized that starch degradation must be up-regulated correspondingly to maintain carbon supply to sink tissues. Compared with the untransformed control, mature leaves of the transgenic plants had a larger drop in starch concentration between dusk and pre-dawn, higher maltose concentration, and higher activities of two key enzymes in starch degradation: -amylase and cytosolic glucosyltransferase during the day and night. 14C-maltose and 14C-glucose were fed to the apple leaves to study the fate of starch breakdown products in the synthesis of sorbitol and sucrose. Under light, a larger proportion of both 14C-maltose and 14C-glucose were converted to sorbitol than to sucrose in the untransformed control, whereas conversion of 14C-maltose and 14C-glucose to sucrose predominated over that to sorbitol in the transgenic apple leaves. The leaf samples fed with 14C-maltose and 14C-glucose in the dark are still being analyzed, but it appears that sucrose is the main product in both the untransformed control and the transgenic plants. These results support the hypothesis that starch degradation is up-regulated in the transgenic plants.
Many tree crops belonging to the Rosaceae family translocate and metabolize sorbitol. We have determined that some species of bacteria belonging to the genus Agrobacterium, Pseudomonas, and Erwinia pathogenic to the Rosaceae demonstrate the ability to metabolize sorbitol while those that were isolated from other hosts could not utilize sorbitol. Employing cellulose acetate electrophoresis (CAE) we have been able to demonstrate the presence of isoenzymes of sorbitol dehydrogenase (SDH) that correlate with the ability to metabolize sorbitol in these organisms. In order to study the properties of SDH in these organisms we carried out a detailed enzymatic analysis of the enzyme from A. tumefaciens. We found that the enzyme displayed activity when mannitol or xylitol were used as substrates, in addition to sorbitol. Michaelis constants (Km) were 32.8 mM, 0.19 mM, and 38.2 mM for sorbitol, mannitol, and xylitol respectively. To further distinguish the reactions with the different substrates the enzymatic extracts were further characterized on CAE using different substrates to visualize patterns of isoenzymes for a particular sugar alcohol. These analyses revealed the presence of unique isoenzymes for SDH. In addition we observed the presence of mannitol dehydrogenase (MDH) representing in most species a non-specific polyol dehydrogenase.
To understand the role of ethylene in overall flavor of apple fruits, ethylene production, and action were reduced using apple trees lines transformed for suppressing activity of ACC-synthase or ACC-oxidase enzymes, and 1-methylcyclopropene (1-MCP), an ethylene action inhibitor. A major reduction in ethylene biosynthesis and respiration rates was measured in fruits from these treatments. As expected, we found differential levels of dependence of flavor components on ethylene biosynthesis and action. Regarding aroma production, an ethyleneassociated event, headspace analysis showed a reduction in ester production in the ethylene-suppressed lines and in the apples treated with 1.0 μL·L-1 1-MCP for 20 hours at 20 °C. However, no major differences were observed in concentrations of alcohol and aldehyde volatiles. Other flavor metabolites that showed an ethylene-dependent pattern were organic acids and sugars. Malic acid degradation was significantly reduced under ethylene suppressed conditions, showing a recovery after exposing the fruit to ethylene. Sucrose and fructose concentrations were influenced by suppression or enhancement of ethylene. Total phenolics and individual phenolics showed an ethylene-dependent behavior only when ethylene biosynthesis was reduced, but not when ethylene action was affected. These results suggest that the regulatory mechanisms of aroma biosynthesis in apple are under partial ethylene regulation. Therefore, we are using the ethylene suppressed apple fruits study the channeling and regulation of other metabolic pathways that lead to the manifestation of a complex trait like fruit quality.
Abstract
A system was developed to transform walnut cultivars using the natural gene transfer system of Agrobacterium tumefaciens. We report the infection of English walnut (Juglans regia L.), northern California black walnut (Juglans hindsii), and their F1 hybrid ‘Paradox’ with A. tumefaciens carrying various recombinant derivatives of the tumor-inducing (Ti) plasmids, pTiA6 and pTiB6S3. The three walnut species, each represented by a single micropropagated clone, were found to be equally susceptible to Agrobacterium-induced tumor formation in vitro. Stable lines were established from tumors induced on each clone, and, unlike normal stem callus, these tumor cells grew rapidly in culture media without exogenous plant hormones. High-voltage paper electrophoretic analysis revealed the presence of opines in the walnut tumor tissue. The presence of a foreign gene was demonstrated by expression of a chimeric bacterial gene that encodes resistance to the antibiotic kanamycin, and also by the presence of foreign DNA sequences in genomic DNA isolated from tumors.
Several strains of Agrobacterium tumefaciens and A. rhizogenes were shown to form tumors on runners of the diploid strawberry species Fragaria vesca L. Tumors, weighing from 0.1 to 8.3 mg, appeared from 2 to 4.5 weeks after infection. The majority of tumors tested for opine synthesis by high-voltage paper electrophoresis analysis showed positive results. These results demonstrate that diploid strawberry plants are susceptible to infection with Agrobacterium and that there are differences in the relative virulence of Agrobacterium strains.
Japanese persimmon (Diospyros kaki L. `Jiro') was transformed using a disarmed strain of Agrobacterium tumefaciens, EHA101, carrying the binary plasmid vector, pDU92.710. The T-DNA region of pDU92.710 contained the kanamycin resistance gene (nptII), the β-glucuronidase gene (uidA), and a synthetic reconstruct of cryIA(c) encoding the insecticidal crystal protein fragment of Bacillus thuringiensis subsp. kurstaki HD-73. Leaf discs made from leaves of shoot cultures were cocultivated with Agrobacterium and cultured on a callus-induction medium containing kanamycin and cefotaxime. Among 720 infected leaf discs, 17 putative transformed callus lines showing kanamycin resistance were obtained after 8 weeks of culture. When these were cultured on a regeneration medium containing kanamycin, 15 formed adventitious buds. Of the 15 shoot lines, 11 grew well on a shoot-proliferation medium containing kanamycin, while 4 lines did not grow well. Of the 11 shoot lines, 10 showed GUS activities by fluorometric assay and were subjected to polymerase chain reaction (PCR) and Southern analyses. Except for two lines, all results were consistent with a stable integration of T-DNA into the persimmon genome. The production of CryIA(c) protein in transformed shoot lines was confirmed with Western analysis using anti-CryIA(c) serum. Insect bioassays were conducted with 10 lines showing GUS activity. Many of these lines showed high significant mortality of the test insects, Plodia interpunctella Hüber and Monema flavescens Walker, when compared to nontransformed controls.
The California almond industry is the largest supplier of almonds [Prunus dulcis (Miller) D.A. Webb] in the United States and throughout the world. Self-incompatibility is a major issue in almond production as it greatly affects nut set. In this study, we determined full-length sequences for alleles Sa - Si, determined the genotypes of 44 California cultivars, and assigned the cultivars to cross-incompatibility groups (CIGs). Newly identified S-alleles led to an increase in the number of CIGs. A pairwise distance tree was constructed using the aligned amino acid sequences showing their similarity. Four pairs of alleles (Sc and Se, Sg and Sh, Sd and Sj, and Sb and Sf) showed high sequence similarity. Because of its simplicity, reproducibility, and ease of analysis, PCR is the preferred method for genotyping S-alleles.
The enzyme polyphenol oxidase (PPO) is nearly ubiquitous in Kingdom Plantae and catalyzes the oxidation of phenolic compounds into highly reactive quinones. Although the functional importance of PPO in plants remains uncertain, a putative antipathogen role for walnut (Juglans regia) PPO was posited as early as 1911. However, despite the rich diversity of phenolics present in walnut leaves and hulls, walnut PPO has been little studied since the early 1900s. We cloned a PPO-encoding gene from a walnut pistillate flower cDNA library and designated the gene jrPPO1. Genomic Southern analysis demonstrated that jrPPO1 is the sole PPO gene in walnut. Transgenic tobacco (Nicotiana tabacum) plants expressing jrPPO1 display greater than 10-fold increases in leaf PPO activity compared with wild-type tobacco, demonstrating that jrPPO1 encodes a functional enzyme. The jrPPO1 protein is expressed primarily in the leaves, hulls, and flowers of walnut trees and is not regulated by wounding or methyl jasmonate. To examine whether walnut PPO could affect pathogen resistance, tobacco plants expressing jrPPO1 were challenged with Pseudomonas syringae pv. tabaci. Based on both symptom development and quantitative analyses of bacterial growth in planta, the PPO-expressing plants did not display increased resistance to this pathogen. Leaf extract browning assays indicated that tobacco leaves lack the endogenous phenolic substrates required for significant jrPPO1 activity and quinone production in planta.