Tahira Fatima

Extracellular Invertase Is an Essential Component of Cytokinin-Mediated Delay of Senescence

Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the delay of senescence by cytokinins and that it is a key element of the underlying molecular mechanism.

Methyl jasmonate deficiency alters cellular metabolome, including the aminome of tomato (Solanum lycopersicum L.) fruit

Exogenous treatment with jasmonates (JA) has been shown to reduce the levels of polyamines in many plants. But the role of endogenous JA on polyamine biosynthesis or other cellular metabolites has thus far remained uninvestigated. We developed transgenic tomato (Solanum lycopersicum L.) having severely reduced methyl JA levels by silencing a fruit ripening-associated lipoxygenase (LOX), SlLoxB, using a truncated LOX gene under the control of the constitutive CaMV35S promoter. The LOX suppressed and MeJA-deficient fruits had lowered polyamine levels. Thus, these transgenic fruits were used as a plant model to evaluate the effects of reduced endogenous MeJA on cellular metabolites in ripening tomato fruits using NMR spectroscopy. During on-shelf ripening, transgenic fruits were significantly reduced in the content of 19 out of 30 metabolites examined, including Ile, Val, Ala, Thr, Asn Tyr, Glu, Gln, His, Phe, Trp, GABA, citrate, succinate, myo-inositol, unidentified compound B, nucleic acid compound Nucl1, choline, and trigonelline as compared to the wild-type azygous counterparts. A significant increase in β-glucose levels in transgenic fruits was observed at the pink stage. The transgenic fruits were equivalent to the wild type in lycopene level and chlorophyll degradation rates. Taken together, these results show that intracellular MeJA significantly regulates overall primary metabolism, especially aminome (amino acids and polyamines) of ripening fruits.

Genetic Engineering to Enhance Crop-Based Phytonutrients (Nutraceuticals) to Alleviate Diet-Related Diseases

Nutrition studies have provided unambiguous evidence that a number of human health maladies including chronic coronary artery, hypertension, diabetes, osteoporosis, cancer and age- and lifestyle-related diseases are associated with the diet. Several favorable and a few deleterious natural dietary ingredients have been identified that predispose human populations to various genetic and epigenetic based disorders. Media dissemination of this information has greatly raised public awareness of the beneficial effects due to increased consumption of fruit, vegetables and whole grain cereals-foods rich in phytonutrients, protein and fiber. However, the presence of intrinsically low levels of the beneficial phytonutrients in the available genotypes of crop plants is not always at par with the recommended daily allowance (RDA) for different phytonutrients (nutraceuticals). Molecular engineering of crop plants has offered a number of tools to markedly enhance intracellular concentrations of some of the beneficial nutrients, levels that, in some cases, are closer to the RDA threshold. This review brings together literature on various strategies utilized for bioengineering both major and minor crops to increase the levels of desirable phytonutrients while also decreasing the concentrations of deleterious metabolites. Some of these include increases in: protein level in potato; lysine in corn and rice; methionine in alfalfa; carotenoids (beta-carotene, phytoene, lycopene, zeaxanthin and lutein) in rice, potato, canola, tomato; choline in tomato; folates in rice, corn, tomato and lettuce; vitamin C in corn and lettuce; polyphenolics such as flavonol, isoflavone, resveratrol, chlorogenic acid and other flavonoids in tomato; anthocyanin levels in tomato and potato; alpha-tocopherol in soybean, oil seed, lettuce and potato; iron and zinc in transgenic rice. Also, molecular engineering has succeeded in considerably reducing the levels of the offending protein glutelin in rice, offering proof of concept and a new beginning for the development of super-low glutelin cereals for celiac disease patients.

Tomato response to legume cover crop and nitrogen: differing enhancement patterns of fruit yield, photosynthesis and gene expression

Excessive use of nitrogen (N) in crop production has impacted ecosystems by contaminating soil and water. Management of N in agriculture is therefore of global concern. Sustainable agriculture systems that use leguminous cover crops such as hairy vetch (Vicia villosa Roth) to fix N and enrich soil organic matter by fixing carbon provide an alternative strategy. N signalling pathways were found associated with delayed leaf senescence and disease tolerance of hairy vetch-grown tomatoes. To test whether N in hairy vetch is the only contributing factor leading to these phenotypes, we designed a pot experiment in the field to analyse growth and gene expression in tomatoes, one set with soil overwintered without a cover crop (bare soil) and the other with soil overwintered with a hairy vetch cover crop including the vetch residue on the soil surface. Additionally, supplementary N fertiliser was also provided to aid distinguishing tomato responses to vetch from those to inorganic N. Tomato fruit yield, plant biomass and photosynthesis were higher in plants grown in vetch than bare soil. Tomato growth and photosynthesis metrics exhibited a parabolic response to inorganic N in bare soil, suggesting the potential for N toxicity in pots with the highest N rate. Vetch-grown tomato plants mitigated these effects and maintained elevated photosynthetic rates at high inorganic N levels. Vetch-grown plants also mitigated a decline in expression of several genes regulating nitrogen and carbon metabolism and upregulated the defence-related gene, osmotin, relative to plants grown in bare soil. Thus, some of the positive responses of tomatoes to a hairy vetch cover crop observed in the field seem mediated by physiological cues other than the additional N provided by the vetch cover crop.

Metabolic control of seedling development by invertases

Invertases are important enzymes in higher plants, which are involved in regulating developmental processes and responses to external factors. In a functional approach the role of invertases was investigated using transgenic plants ectopically expressing inhibitor proteins to decrease invertase activity. For generating specific effects, these inhibitor proteins were expressed in Arabidopsis under the control of synthetic promoters consisting of tetramers of pathogen-inducible elements, which were reported to yield low constitutive expression. Unexpectedly, seedling growth of putative transgenic plants was arrested at the four-leaf stage. Analysis of β-glucuronidase activity of corresponding reporter gene lines showed a correlation of the growth arrest with high activity of these promoters in seedlings grown under tissue culture conditions. The negative effect of invertase inhibition on seedling growth was substantiated by transgenic tobacco plants expressing an invertase inhibitor under control of a tetracycline inducible promoter. Ectopic induction of the invertase inhibitor during early seedling development resulted in a reduced fresh weight of seedlings. The importance of invertase in seedling development is further supported by results of expression profiling of invertases in Arabidopsis, which was confirmed by expression analyses. The mRNA for the vacuolar invertases Atßfruct3 and Atßfruct4 and cell wall invertase AtcwINV1 are specifically and strongly expressed during seedling development. These complementing results show that invertase activity is required for normal seedling development.

Pathogenesis-Related Protein 1b1 (PR1b1) Is a Major Tomato Fruit Protein Responsive to Chilling Temperature and Upregulated in High Polyamine Transgenic Genotypes

Plants execute an array of mechanisms in response to stress which include upregulation of defense-related proteins and changes in specific metabolites. Polyamines – putrescine (Put), spermidine (Spd), and spermine (Spm) – are metabolites commonly found associated with abiotic stresses such as chilling stress. We have generated two transgenic tomato lines (556HO and 579HO) that express yeast S-adenosylmethionine decarboxylase and specifically accumulate Spd and Spm in fruits in comparison to fruits from control (556AZ) plants (Mehta et al., 2002). Tomato fruits undergo chilling injury at temperatures below 13°C. The high Spd and Spm tomato together with the control azygous line were utilized to address role(s) of polyamines in chilling-injury signaling. Exposure to chilling temperature (2°C) led to several-fold increase in the Put content in all the lines. Upon re-warming of the fruits at 20°C, the levels of Spd and Spm increased further in the fruit of the two transgenic lines, the higher levels remaining stable for 15 days after re-warming as compared to the fruit from the control line. Profiling their steady state proteins before and after re-warming highlighted a protein of ∼14 kD. Using proteomics approach, protein sequencing and immunoblotting, the ∼14-kD protein was identified as the pathogenesis related protein 1b1 (PR1b1). The PR1b1 protein accumulated transiently in the control fruit whose level was barely detectable at d 15 post-warming while in the fruit from both the 556HO and 579HO transgenic lines PR1b1 abundance increased and remained stable till d 15 post warming. PR1b1 gene transcripts were found low in the control fruit with a visible accumulation only on d 15 post warming; however, in both the transgenic lines it accumulated and increased soon after rewarming being several-fold higher on day 2 while in 556HO line this increase continued until d 6 than the control fruit. The chilling-induced increase in PR1b1 protein seems independent of ethylene and methyl jasmonate signaling but may be linked to salicylic acid. We propose that polyamine-mediated sustained accumulation of PR1b1 protein in post-warmed chilled tomato fruit is a pre-emptive cold stress response and possibly a defense response mechanism related to Cold Stress-Induced Disease Resistance (SIDR) phenomenon.

Genetic introgression of ethylene-suppressed transgenic tomatoes with higher-polyamines trait overcomes many unintended effects due to reduced ethylene on the primary metabolome

Ethylene regulates a myriad physiological and biochemical processes in ripening fruits and is accepted as the ripening hormone for the climacteric fruits. However, its effects on metabolome and resulting fruit quality are not yet fully understood, particularly when some of the ripening-associated biochemical changes are independent of ethylene action. We have generated a homozygous transgenic tomato genotype (2AS-AS) that exhibits reduced ethylene production as a result of impaired expression of 1-aminocyclopropane-1-carboxylate synthase 2 gene by its antisense RNA and had a longer shelf life. Double transgenic hybrid (2AS-AS × 579HO) developed through a genetic cross between 2AS-AS and 579HO (Mehta et al., 2002) lines resulted in significantly higher ethylene production than either the WT or 2AS-AS fruit. To determine the effects of reduced ethylene and introgression of higher polyamines’ trait, the metabolic profiles of ripening fruits from WT (556AZ), 2AS-AS, and 2AS-AS × 579HO lines were determined using 1H-NMR spectroscopy. The levels of Glu, Asp, AMP, Adenosine, Nucl1, and Nucl2 increased during ripening of the WT fruit. The increases in Glu, Asp, and AMP levels were attenuated in 2AS-AS fruit but recovered in the double hybrid with higher ethylene and polyamine levels. The ripening-associated decreases in Ala, Tyr, Val, Ile, Phe, malate, and myo-inositol levels in the 2AS-AS line were not reversed in the double hybrid line suggesting a developmental/ripening regulated accumulation of these metabolites independent of ethylene. Significant increases in the levels of fumarate, formate, choline, Nucl1, and Nucl2 at most stages of ripening fruit were found in the double transgenic line due to introgression with higher-polyamines trait. Taken together these results show that the ripening-associated metabolic changes are both ethylene dependent and independent, and that the fruit metabolome is under the control of multiple regulators, including ethylene and polyamines.