Inhibition of zeaxanthin epoxidase activity by cadmium ions in higher plants

The effect of cadmium and zinc ions on violaxanthin cycle enzymes, violaxanthin de-epoxidase and zeaxanthin epoxidase, has been investigated on selected plant species, as well as in vitro. About 50% inhibition of zeaxanthin epoxidase by cadmium ions was found for duckweed (Lemna trisulca) and tomato (Lycopersicon esculentum) leaves but for apricot (Prunus armeniaca) leaves no cadmium inhibition of the epoxidation reaction was observed. The cadmium inhibition of zeaxanthin epoxidase in tomato was abolished by zinc ions. Zinc ions alone did not affect the activity of neither of the enzymes of the violaxanthin cycle. This suggests that mechanism of cadmium inactivation of the enzyme relies on cadmium interaction with a cysteine residue of the protein, important for the enzyme activity. The target cysteine in tomato epoxidase could be the cysteine residue present in the most conservative part of the molecule which is not present in the apricot enzyme sequence. Neither stimulation nor inhibition of violaxanthin de-epoxidase by cadmium ions both in vivo and in vitro studies was detected. It confirms the proposed mechanism of zeaxanthin epoxidation inhibition by cadmium ions because the cysteine residue in the conservative motif of violaxathin de-epoxidase is not present.     

Overexpression of zeaxanthin epoxidase gene enhances the sensitivity of tomato PSII photoinhibition to high light and chilling stress

A tomato ( Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene ( LeZE ) was isolated. The deduced amino acid sequence of LeZE showed high identities with zeaxanthin epoxidase in other plant species. Northern blot analysis showed that the mRNA accumulation of LeZE in the wild-type (WT) was not induced by light and temperature but regulated by the diurnal rhythm. The sense transgenic plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern and western blot analysis confirmed that sense LeZE was transferred into the tomato genome and overexpressed. The ratio of (A + Z)/(V + A + Z) and the values of non-photochemical quenching were lower in transgenic plants than in WT plants under high light and chilling stress with low irradiance. The O₂ evolution rate and the maximal photochemical efficiency of PSII (Fv/Fm) in transgenic plants decreased more quickly during both stresses and recovered slower than that in WT under optimal conditions. These results suggested that overexpression of LeZE impaired the function of the xanthophyll cycle and aggravated PSII photoinhibition in tomato under high light and chilling stress.     

Biochemical and phenotypical characterization of transgenic tomato plants overexpressing a basic peroxidase

Tomato plants ( Lycopersicon esculentum Mill. cv. Pera) were transformed via Agrobacterium tumefaciens with the binary vector pKYLX71 containing a tomato basic peroxidase (EC 1.11.1.7) gene, tpx1 , under the control of the cauliflower mosaic virus (CaMV35S) promoter. Transgenic plants showed a 2–5-fold increase in the activity of the peroxidase ionically bound to the cell wall, whereas soluble peroxidase activity remained similar or even lower than wild-type plants. Isoelectric focusing showed the presence of a new isoperoxidase of pI ca 9 in the ionically bound extract. Western blot also showed the presence of a new band at 41 kDa that was absent in the wild-type extract. A 40–220% increment of lignin content of the leaf was found in transgenic plants. Shoot phenotype of transgenic plants was similar to wild type, although under stress, the plants appeared wilted and the new leaves had a reduced area and were thicker than wild-type or older transgenic leaves. The root system was underdeveloped in transgenic plants, but the rooting ability of the stem was not affected by the overexpression of peroxidase. Finally, the morphogenetic response of cotyledon and hypocotyl explants from transgenic plants was evaluated. In the case of cotyledons, the percentage of explants with shoot was not different from wild-type plants. For hypocotyl, one of the transgenic lines showed a 30% reduction in the percentage of shoot organogenesis. The results are discussed in relation to the role of tpx1 in lignin synthesis.     

Structure and expression of a cDNA encoding zeaxanthin epoxidase, isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library

Zeaxanthin epoxldase (ZE) catalyses two early steps in the abscisicacid (ABA) biosynthetic pathway. The sequence of a cDNA cloneencoding ZE from Nicotiana plumbaginifolia was reported In 1996and represented the first DNA sequence data on an ABA biosyntheticenzyme. The N. plumbaginifolia cDNA has been used to providea heterologous probe to isolate a ZE cDNA from tomato (Lycopersiconesculentum Mill.). DNA and amino acid sequence differences areconsidered in relation to putative functional domans withinthe enzyme. The results of northern analysis in tomato are discussedin relation to the effects of water stress on ZE mRNA levels. Key words: ABA biosynthesis, zeaxanthin epoxidase, tomato     

Antisense-mediated suppression of tomato zeaxanthin epoxidase alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance

A tomato (Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene (LeZE) was isolated and antisense transgenic tomato plants were produced. Northern, southern, and western blot analyses demonstrated that antisense LeZE was transferred into the tomato genome and the expression of LeZE was inhibited. The ratio of (A+Z)/(V+A+Z) in antisense transgenic plants was maintained at a higher level than in the wild type (WT) plants under high light and chilling stress with low irradiance. The value of non-photochemical quenching (NPQ) in WT and transgenic plants was not affected during the stresses. The oxidizable P700 and the maximal photochemical efficiency of PSII (F_v/F_m) in transgenic plants decreased more slowly at chilling temperature under low irradiance. These results suggested that suppression of LeZE caused zeaxanthin accumulation, which was helpful in alleviating photoinhibition of PSI and PSII in tomato plants under chilling stress.     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

Engineering seed dormancy by the modification of zeaxanthin epoxidase gene expression

Abscisic acid (ABA) is a plant hormone synthesized during seed development that is involved in the induction of seed dormancy. Delayed germination due to seed dormancy allows long-term seed survival in soil but is generally undesirable in crop species. Freshly harvested seeds of wild-type Nicotiana plumbaginifolia plants exhibit a clear primary dormancy that results in delayed germination, the degree of primary dormancy being influenced by environmental culture conditions of the mother plant. In contrast, seeds, obtained either from ABA-deficient mutant aba2-s1 plants directly or aba2-s1 plants grafted onto wild-type plant stocks, exhibited rapid germination under all conditions irrespective of the mother plant culture conditions. The ABA biosynthesis gene ABA2 of N. plumbaginifolia, encoding zeaxanthin epoxidase, was placed under the control of the constitutive 35S promoter. Transgenic plants overexpressing ABA2 mRNA exhibited delayed germination and increased ABA levels in mature seeds. Expression of an antisense ABA2 mRNA, however, resulted in rapid seed germination and in a reduction of ABA abundance in transgenic seeds. It appears possible, therefore, that seed dormancy can be controlled in this Nicotiana model species by the manipulation of ABA levels.     

Ac transposition in transgenic tomato plants

Summary As an initial step towards developing a transposon mutagenesis system in tomato, the maize transposable element Ac was transformed into tomato plants via Agrobacterium tumefaciens. Southern analysis of leaf tissue indicated that in nine out of eleven transgenic plants, Ac excised from the T-DNA and reintegrated into new chromosomal locations. The comparison of Ac banding pattern in different leaves of the same primary transformant provided evidnece for transposition during later stages of transgenic plant development. There was no evidence of Ds mobilization in tomato transformants.     

Expression of recombinant human acetylcholinesterase in transgenic tomato plants

Enzyme therapy for the prevention and treatment of organophosphate poisoning depends on the availability of large amounts of cholinesterases. Transgenic plants are being evaluated for their efficiency and cost-effectiveness as a system for the bioproduction of therapeutically valuable proteins. Here we report production of a recombinant isoform of human acetylcholinesterase in transgenic tomato plants. Active and stable acetylcholinesterase, which retains the kinetic characteristics of the human enzyme, accumulated in tomato plants. High levels of specific activity were registered in leaves (up to 25 nmol min(-1) mg protein(-1)) and fruits (up to 250 nmol min(-1) mg protein(-1)).     

Functional characterization of a cotton late embryogenesis-abundant D113 gene promoter in transgenic tobacco

Previous studies have shown that mRNA and protein encoded by late embryogenesis-abundant (LEA) gene D113 from Gossypium hirsutum L. accumulate at high levels in mature seeds and also in response to abscisic acid (ABA) in young embryo. In this study, we studied the expression of four promoter 5′ deletion constructs (−1383, −974, −578 and −158) of the LEA D113 gene fused to beta-glucuronidase (GUS). GUS activity analysis revealed that the −578 promoter fragment was necessary to direct seed-specific GUS expression in transgenic tobacco plants (Nicotiana tabacum L.). To further investigate the expression pattern of LEA D113 promoter under environmental stresses, 2-week-old transgenic tobacco seedlings were exposed to ABA, dehydration, high salinity and cold treatments. GUS activity in the seedlings was quantified fluorimetrically, and expression was also observed by histochemical staining. An apparent increase in GUS activity was found in plants harboring constructs −1383, −974 and −578 after 24 h of ABA or high-salinity treatments, as well as after 10 days of dehydration. By contrast, only a slight increase was observed in all the three lines after cold treatment. Virtually no change in expression was found in construct −158 in response to dehydration, salinity and cold, but there was a moderate response to ABA, suggesting that the region between −574 and −158 was necessary for dehydration- and salinity-dependent expression, whereas ABA-responsive cis-acting elements might be located in the −158 region of the promoter.