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The first enzyme committed directly to ABA biosynthesis is 9-cis-epoxycarotenoid dioxygenase (NCED) (Fig. 1.4), NCED catalyses the formation of xanthoxin by oxidatively cleaving an epoxycarotenoid precursor, i.e. 9'-cis-neoxanthin and/or possibly 9-cis-violaxanthin (Cutler and Krochko, 1999). Xanthoxin is the first C15 intermediate of ABA biosynthesis (Tung et al., 2008). Xanthoxin is subsequently converted to ABA following further steps in the ABA biosynthesis pathway (Cutler and Krochko, 1999).
It has been reported on many occasions that an increase in ABA concentration can increase water use efficiency (WUE) by restricting stomatal opening. This is a beneficial phenotypic trait and is potentially very important for agriculture. Understanding the ABA biosynthetic pathway and genetically manipulating key regulatory enzymes could facilitate increased ABA accumulation. Advances in transgene technology have resulted in the production of transgenic plants with elevated activity of one or more biosynthetic enzymes, i.e. NCED and ZEP (Smeeton, 2010). A plant's response to ABA is dependent on the sensitivity of the plant tissue to ABA and the concentration of the ABA phytohormone (Taiz and Zeiger, 2006). Expression of one or more members of the NCED gene family is thought to regulate the accumulation of ABA in seed (Nambara and Marion-Poll, 2005). Genes that encode enzymes in the ABA pathway have been cloned recently. Two of these cloned genes encode the enzymes zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED) (Thompson et al., 2000b). The first gene to be isolated from the tomato plant Lycopersicon esculentum (now usually called Solanum lycopersicum) was named LeNCED1. The tomato NCED encoding gene LeNCED1 is particularly important in ABA biosynthesis in response to water stress. Transgenic tomato plants were produced with the gene encoding regions open reading frame for LeNCED1, under the control of the Gelvin chimeric super-promoter (sp) (Thompson et al., 2000a). One of the resulting transgenic genotypes was named sp5. This name derived from the Gelvin chimeric super-promoter that its expression was driven by (Thompson et al., 2000a). Transgenic tomato plants, which over-express LeNCED1, including the genotype sp5 were found, to various extents, to have increased seed dormancy.
Thompson et al. (2000a) investigated seed dormancy and germination in sp5 and wild type (WT) genotypes. The final percentage germination was 15% and 100% in sp5 and WT genotypes respectively. These results indicate that over-expression of LeNCED1 in the sp5 genotype increases ABA synthesis. The induced over-expression of LeNCED1 led to whole plant increases in ABA concentration. The increased dormancy is due to the increased ABA synthesis in seed. The herbicide norflurazon was used to restore the percentage germination of sp5 genotypes to that found in WT seeds (Fig. 1.5). It is important to note that the sp5 genotypes had no dramatic impact on growth rate beyond the germination and early seedling establishment phases. Following this work, it was concluded that over-accumulation of ABA should ideally be confined to plant tissues only where it is beneficial, i.e. shoots and not seeds (Smeeton, 2010).
The importance of the key regulatory step catalysed by the enzyme NCED in 'latter ABA biosynthesis' is well documented. Earlier stages of ABA biosynthesis have not been as extensively researched. One early step in ABA biosynthesis involves the enzyme ï¢-carotene hydroxylase (BCH). ï¢-carotene is hydroxylated on both rings to produce all-trans-zeaxanthin and the reaction is catalyzed by BCH (Taylor et al., 2005). In tomato plants, two genes (CrtR-b1 and CrtR-b2), encode BCH. CrtR-b1 is constitutively expressed in green tissue for the production of all-trans-zeaxanthin pools that serve as a method of photoprotection. CrtR-b2 is expressed to varying degrees in mature flowers, leaves and roots (Hirschberg, 2001; Galpaz et al., 2006). A construct encoding BCH was over-expressed in transgenic Arabidopsis plants, which elevated violaxanthin levels in the leaves (Davison et al., 2002). Similar transformation experiments in tomato resulted in BCH12 lines, which over express the gene encoding LeBCH2. A DNA construct was produced by combining the LeBCH2 open reading frame with the constitutive 35s promoter. This DNA construct was used in the creation of transgenic plants, which led to the selection of the BCH12 over-expresser line. The gene CrtR-b2, also known as LeBCH2, is active in cells constantly because the 35s promoter is constitutive. Its omnipresence is likely to have resulted in raised levels of the enzyme BCH in all tissues. The greater concentration of BCH could increase the conversion rate of ï¢-carotene to all-trans-zeaxanthin, which could ultimately increase the level of ABA in cells. Production of the BCH12 tomato genotype is a comparatively recent development. BCH12 lines produce elevated levels of ABA (Balasubramanian, 2007). It is accurate to call BCH12 a 'mildly high-ABA' genotype. Because, in comparison to other 'high-ABA' genotypes such as sp5, the level of ABA in plant tissue is not as concentrated.
G29 is a relatively new genotype. To create transgenic lines, which simultaneously over-express LeBCH2 and LeNCED1, a homozygous LeBCH2 over-expressing line was crossed with the homozygous sp5 line, eventually resulting in the double transgenic line G29 (sp5/BCH12) being selected. This simultaneously over-expresses both the LeBCH2 and LeNCED1 transgenes (Smeeton, 2010) and has higher levels of ABA over-accumulation than either the BCH12 or sp5 genotypes, which gave rise to it.
The TRIPLE genotype simultaneously over-expresses LeBCH2, LeNCED1 and LePsy1, it also over-accumulates ABA to a greater extent than even G29 genotypes. White, (2010) reported that roots of a selected TRIPLE genotype accumulated increased concentrations of ABA. Rootstock grafts often showed increased ABA concentration. The TRIPLE genotype appeared to conserve water modestly but consistently when used as a rootstock for WT scions. The author concluded that TRIPLE genotypes display the positive phenotype of WUE, and presented preliminary evidence that this was without reduced biomass production. There are numerous enzymes involved in catalysing carotenoid biosynthesis one of the first genes involved in carotenoid biosynthesis to be isolated from plants was the gene for phytoene synthase (Psy1). In the first committed step of the carotenoid biosynthesis pathway, the enzyme Psy1 catalyses the conversion of GGDP to phytoene (Bartley et al., 1992). Ectopic expression of the gene Psy1 in tomato plants resulted in accumulation of carotenoids in many different plant organs (Fray and Grierson, 1993). These plants, which showed accumulation of carotenoids also showed a reduction in height. A possible explanation may be competition between GA, phytol and carotenoid pathways for GGDP. The over-expression of Psy1 led to a reduction in GA concentrations and ultimately dwarfism in the plants (Fray and Grierson, 1993). The constitutive expression of Psy1 in transgenic tomatoes causes dwarfism, possibly by redirecting metabolites from the gibberellin pathway. Fray et al., (1995) reported that seedlings associated with the most extreme dwarf phenotype showed the highest expression of the Psy1 transgene.
NCED catalyses a key rate limiting step in ABA biosynthesis. The over-expression of LeNCED1 in sp5 results in elevated levels of ABA in leaves, roots and seeds. The increased ABA levels could have many benefits including increased WUE. However, the additional ABA that accumulated in the imbibed sp5 seeds caused a substantial delay in germination (Thompson et al., 2000a). A new genotype that minimizes the negative phenotypic effects of increased dormancy should ideally be produced. It is anticipated that this genotype would hopefully still express the positive phenotypic effects, such as high levels of ABA in roots and leaves leading to increased WUE in plants. Tung et al., (2008) investigated an alternative strong promoter to drive LeNCED1 expression instead of the Gelvin superpromoter. The objective was to derive a promoter able to drive LeNCED1 over-expression in shoots and eliminate the undesirable increased seed dormancy phenotype observed in the sp lines (Thompson et al., 2000a). The promoter was derived from rbcS3C, a tomato gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The rbcS3C promoter is regulated by light and highly active in photosynthetic tissues (Gittins et al, 2000). The rbcS3C gene provides a high level of rbsS mRNA in leaves and cotyledons (Sugita and Gruissem, 1987; Wanner and Gruissem, 1991). Tung et al., (2008) proposed that rbcS3C promoter activity might be regulated in a circadian way. The authors measured LeNCED1 mRNA in the rbcs-10 genotype during a 12-hour photoperiod, a period of total darkness and another 12-hour photoperiod. LeNCED1 mRNA peaked during the first light period and was generally low during the dark period. During the second light period LeNCED1 mRNA peaked again. This light-inducible promoter allowed the production of new transgenic lines, which over-produce ABA to a greater extent than previous sp lines. The rbcS3C promoter facilitated the production of transgenic plants with high levels of ABA in leaves. The negative phenotypic effect of extended seed dormancy seen in earlier studies was greatly diminished. However, these plants displayed another negative set of symptoms including: greatly reduced growth, interveinal leaf flooding, photobleaching in seedlings and perturbed cotyledon release from the testa (a 'lollipop' phenotype was reported in germinating seed of the rbcS lines, which resulted in cotyledons being unable to emerge from the testa). These negative phenotypic effects revealed the severe consequences of acute high ABA accumulation in plant tissue (Tung et al., 2008). This highlights the need for new genotypes that display more measured/less dramatic increases in ABA biosynthesis. In the present study, the 'ultra-high ABA' over-producer genotype rbcS-17 was re-investigated. This provided the opportunity to confirm the findings of Tung et al., (2008) that its positive phenotypic effect in diminishing seed dormancy was accompanied by undesirable phenotypic effects on shoot growth.
ZEP was initially cloned in Nicotiana and is a gene encoding an enzyme located earlier in the ABA biosynthetic pathway than NCED (Audran et al., 1998). Thompson et al., (2000b) studied the mRNA patterns of LeNCED1 and LeZEP1 to determine the regulatory mechanisms for ABA biosynthesis. The authors reported strong diurnal expression patterns for both NCED and ZEP, with the NCED and ZEP genes displaying clearly different patterns. The diurnal expression pattern of NCED mRNA was unforeseen by the authors. As is true for light-harvesting complex II (LHCII) mRNA, during a 48-hour period of continuous darkness, two oscillations in ZEP mRNA were observed, proposing that ZEP mRNA may be regulated by a circadian oscillator (Beator and Kloppstech, 1996). NCED mRNA also oscillated diurnally, the peak of this oscillation was always at the end of the light period as opposed to the middle. In total darkness (a 48-hour dark period), no oscillations were observed, as NCED mRNA was low (Thompson et al., 2000b). The authors concluded that NCED mRNA is positively regulated by light. The authors demonstrated that down regulation of the tomato version of the ZEP gene (LeZEP1) increased zeaxanthin levels in transgenic tomato leaves. This result was consistent with the suggestion that this gene encodes the enzyme ZEP in tomato. ZEP expression is important in the mediation of ABA biosynthesis in seed. Operation of the xanthophyll cycle, necessitates ZEP accumulation, required for the protection of photo-system II, an important light-harvesting complex (LHC) required for photosynthesis (Thompson et al., 2000b). ZEP mRNA and ABA accumulation appears to climax at the same stage of seed development, hinting at a possible role for ZEP in mediating ABA concentration in seeds (Audran et al., 1998). Previous work on seed over-expressing ZEP has shown the seeds display slightly more delayed germination than WT seed of Nicotiana (Frey et al., 1999). The present investigation included constructs based on the tomato NCED1 coding sequence driven by the tomato ZEP promoter (built by E. Harrison and A.J. Thompson, Warwick-HRI, University of Warwick). Transformation of tomato variety Ailsa Craig (Tm2a) was carried out by E. Harrison resulting in seed derived from the ZEP primary transformants ZEP-6, ZEP-10A and ZEP-11. This is very recent work and prior to the present investigation there was no information on the phenotype of transgenic plants containing this ZEP::NCED1 construct (Taylor, I., personal correspondence).