4 EFFECT OF CHANGES IN CKOX, AO, AND XDH ACTIVITY ON 65 ABA, CK, AND IAA CONTENT OF DEVELOPING AVOCADO FRUIT. 5 RELATIONSHIP BETWEEN CKOX, AO AND XDH ACTIVITY AND CHANGES IN PLANT HORMONES DURING THE LINEAR PHASE OF AVOCADO FRUIT GROWTH.
1 JNTRODUCTION
THE AVOCADO
Seed coat senescence can occur at any time during development and there is no pattern regarding the distribution of the small fruit phenotype on the tree (Cowan et al. 1997). The limiting factor for the growth of the phenotypic small fruit variant therefore appears to be cell number and not cell size (Cowan et al. 1997), as found in tomato (Ho and Hewitt 1986; Bohner and Bangerth 1988a; Ho 1992), apricots (Jackson and Coombe 1966) and.
PLANT HORMONES AND FRUIT GROWTH .1 Hormones and general fruit growth
Both ABA and water stress are known to slow cell division cycle activity (Myers et al. 1990; This is highlighted by the fact that in normal fruit development the developing embryo, or seed, controls the rate and maintenance of cell division in the tissue surrounding (Gillaspy et al. 1993).
STAGE 2 STAGE 3
- HORMONE METABOLISM 1 Cytokinin metabolism
They are defined by their ability to induce cell division in tissue culture in the presence of auxin (Miller et al. A second pathway was proposed after a CK biosynthetic enzyme was found in the slime mold, Dictyostelium discoideum (Taya et al. 1978).which was able to convert adenosine monophosphate (AMP) and dimethylallyl-pyrophosphate (DMAPP) into the free CKs isopentenyladenosine-5'-monophosphate (iPMP) and the corresponding nucleoside (isopentenyladenosine; iPA) (Fig. 1.3).
APP¥EM;::MP
THE SMALL FRUIT PROBLEM IN PERSPECTIVE
The CK to ABA ratio in 'Hass' avocado fruit appears to be critical for the control of cell division and fruit growth, as evidence has been found that an imbalance in this ratio is critical for seed coat senescence and retardation of fruit growth (Moore- Gordon et al. 1998). Although the exact role of GA in fruit growth is currently poorly understood, it is suggested that it plays a role in the stimulation of cell division and maintenance of cell expansion (Gillaspy et al.
OBJECTIVES
As such, this hormone is likely to play a role in the emergence of the 'Hass' avocado small-fruit phenotype, acting in concert with CK, IAA and ABA. It is likely that GA will interact with CK, IAA and ABA through the sharing of a common biosynthetic precursor in the isoprenoid pathway or through the availability of MoCo for the MoCo-requiring enzymes.
2 MATERIALS AND METHODS
- CHEMICAL AND LABORATORY SUPPLIES 1 Radioactive and stable isotopes
- CHROMATOGRAPHIC MEDIA
- PREPARATION OF REAGENTS
- PLANT MATERIAL
- APPLICATION OF CHEMICALS .1 Excised whole fruit
- PREPARATION OF ENZYME EXTRACTS
- PROTEIN DETERMINATION
- ASSAY PROCEDURES
- EXTRACTION AND ANALYSIS OF PURINES AND PLANT HORMONES .1 Cytokinin extraction
The free IA vehicle was removed by dividing it three times against an equal volume of hexane/ethyl acetate (9:1, v/v). The hexane/ethyl acetate fraction was dried under vacuum at 35 cC using a BOchi Rotavapor\1!) R110 (BOchi Laboratoriums-Technik AG, Flawil, Switzerland), resuspended in 0.5 ml acetone and diluted to 20 ml with 0. 1 M Tris-HCl buffer (pH 8). The filtrate was evaporated to dryness in vacuo at 35°C and the residue was resuspended in 50 ml of 80% ethanol (Smith and van Staden 1978).
3 CHARACTERIZATION AND TISSUE DISTRIBUTION OF AO, XDH, AND CKOX IN DEVELOPING AVOCADO FRUIT
INTRODUCTION
Initial evidence for the involvement of a MoCo-AO in ABA biosynthesis came from studies with MoCo-deficient mutants of barley (Walker-Simmons et al. 1989), tobacco (Leydecker et al. 1995), tomato (Marin and Marion -PolI 1997) and Arabidopsis (Schwartz et al. 1997a). Maize AO showed a high affinity for lA-aid (Km 3-5 /JM), suggesting that even at low concentrations of lA-aid, the aldehyde can still be converted to IAA (Koshiba et al. 1996). It has been suggested that the size of the MoCo pool is not consistent but varies in response to nutritional and environmental factors (Sagi et al. 1997; Sagi and Lips 1998).
The link of these enzymes to hormone homeostasis has been alluded to before (Cowan et al.), but a direct relationship has yet to be established.
RESULTS
The results in Table 3.5 show that CKOX activity is present in 'Hass' avocado fruit tissues. Accordingly, purine levels were analyzed by HPLC in normal and small fruit tissues of 'Hass' avocado and the results are presented in Table 3.6. The results of in vivo CK treatment on CKOX activity in normal avocado mesocarp tissue are shown in Figure 3.6.
Results of in vivo treatment with auxins on COX activity in normal 'Hass' avocado mesocarp tissue are shown in Figure 3.7.
CONCLUSION AND SUMMARY
Xanthine dehydrogenase activity was detected only in seed and mesocarp tissues where it was significantly greater in these tissues than the small fruit variant. Cytokinin oxidase activity was significantly higher in seed and mesocarp tissues of young fruits, with no activity detected in old seed kidney tissue of young fruits. Although there were no differences between normal and small fruits with respect to CK glucosides, the level of CK Aase and ribosides was slightly higher in the mesocarp tissue of small fruits and the seed coat of normal fruits.
Hypoxanthine levels were significantly higher in seed tissue of small fruit and mesocarp tissue of normal fruit.
4 EFFECT OF CHANGES IN CKOX, AO, AND XDH
INTRODUCTION
This change in hormone levels was associated with higher CKOX, XDH and XAN oxidase activity, but low 1A auxiliary oxidase activity. In an attempt to establish a correlation between changes in the activity of these enzymes and changes in ABA, IAA and CK, fruit were treated with compounds known to affect CKOX and XDH activity, and the activity of AO was assessed relative to endogenous ABA and IAA. In addition, the suggestion that MoCo is limiting in plants (Sagi et al. 1997; Sagi and Lips 1998) can be partially addressed by determining the effect of exogenous molybdate on AO activity and ABA and IAA.
RESULTS
However, zeatin reduced ABA in seed tissue but had little effect on ABA in mesocarp tissue. Allopurinol increased ABA content in seed tissue but had little or no effect on ABA in mesocarp tissue (Table 4.4). In contrast, IAA in seed tissue was reduced by allopurinol treatment and significantly increased in mesocarp tissue.
Both ABA and IM were increased in mesocarp tissue from ripening fruit in response to allo+Mo treatment (Table 4.5).
CONCLUSION AND SUMMARY
Mesocarp tissue treated with molybdate showed decreased ABA and IM, but increased CK-like activity and xanthine. When applied to whole fruits, ABA increased in seed tissue, while IM increased in mesocarp tissue and decreased in seed tissue. Mesocarp tissue treated with allopurinol also showed lower CK-like activity, but increased adenine and hypoxanthine.
Cytokinin-like activity was decreased in sperm tissue treated with allo+Mo, while hypoxanthine and xanthine increased.
5 RELATIONSHIP BETWEEN ACTIVITY OF CKOX, AO AND XDH AND CHANGES IN PLANT HORMONES DURING
INTRODUCTION
The highest levels of IAA, GA and CK were found in endosperm tissue, where levels remained high provided the tissue was still present in the fruit, that is, cytokinin and IAA levels were higher in the seed and the seed coat than in the mesocarp (Blumenfeld and Gazit 1970). Gazit and Blumenfeld 1970; Gazit and Blumenfeld 1972), while levels of ABA were similar in the seed and mesocarp (Richings et al. 2000; Taylor and Cowan 2001). The previous chapters have established that differences in hormone levels exist between normal and small fruits, which likely occur due to changes in the activity of enzymes believed to play an important role in controlling hormone levels.
To further evaluate the role of these enzymes in the control of hormone homeostasis and final fruit size of the 'Hass' avocado, enzyme activity and hormone levels were determined in the main tissues of developing fruit during the linear phase of growth.
RESULTS .1 Fruit growth
The amount of seed coat tissue was 2% and 1.4% of the total in normal and small fruit, respectively, and remained constant throughout the linear growth phase. The highest levels of CK-like activity were detected in seed coat tissue followed by seed and mesocarp tissue. In seed coat and mesocarp tissues, CK-like activity reached a maximum at 112 OAFB in both normal and small fruit (Fig. 5.4B and C).
There was little difference in XAN oxidase activity in extracts prepared from seed coat tissue of normal and small fruits, although activity did not persist in the small fruits (Fig. 5.9).
CONCLUSION AND SUMMARY
However, some of the ABA produced by the seeds may be localized in the seed coat because the decrease in seed and mesocarp ABA of normal 112 OAFB fruits (Fig. 5.10A and C) was reflected by a prolonged accumulation of ABA in the seed coat (Fig. 5.10B). Also, seed coat tissue from small fruits accumulated significant amounts of ABA (Fig. 5.10B) although this may be indicative of senescence-induced ABA synthesis. However, the early increase in seed and seed coat ABA coincided with the first peak of XAN oxidase activity (cf.
Cytokinin oxidase activity in seed and mesocarp tissue decreased over the linear phase of fruit growth, while CKOX activity in seed coat tissue increased transiently.
6 ~ENERAL DISCUSSION AND CONCLUSIONS
GENERAL DISCUSSION
This relationship is likely mediated by a hormonal interaction, as Cowan et al. 1999) demonstrated that CK stimulates the oxidative catabolism of ABA. The increase in XAN oxidation to ABA in response to CK (Cowan et al. 1999) appears to involve an increase in AO, XAN oxidase activity. Hass avocado fruit size is determined by cell number rather than cell size (Cowan et al. 1997).
Furthermore, pea LE gene expression (GA20 to GA1) in stem internodes requires IAA from the stem apex, and expression of PsGA2ox1 (inactivation of GA20 to GA29 and GA1 to GAs) is reduced by IAA (Ross et al. 2000) .
CONCLUSIONS AND FUTURE PROSPECTS
In avocado, exogenous sucrose, glucose and ABA influence changes in HMGR activity and fruit size (Cowan et al. 1997; Richings et al. 2000). Nitrate reductase is phosphorylated by SnRK1 (Sugden et al. 1999) and inactivated upon the binding of a 14-3-3 protein to the phosphorylation site (Bachman et al. 1996; Moorhead et al. 1996). Although the interaction between CK and ABA has been demonstrated in this and previous studies (Moore-Gordon et al. 1998; Cowan et al. 1999), other interactions await verification.
First, there is a need to determine whether avocado fruit is sensitive to high IAA, which results in increased ethylene and enhanced ABA biosynthesis (Grossmann et al. 1996;
7 LITERATURE CITED
Metabolism of [2-14C] abscisic acid by a cell-free system from embryonic axes of Cicer arietinumL seeds. Characterization of the 9-cis-epoxycarotenoid dioxygenase gene family and regulation of abscisic acid biosynthesis in avocado. Abscisic acid alters indole-3-acetic acid metabolism in senescent flowers of Cucumis me/oL.
The pathway of abscisic acid biosynthesis in vascular plants: a review of the current state of knowledge of ABA biosynthesis. Aldehyde oxidase and xanthine dehydrogenase in a flacca tomato mutant with abscisic acid deficiency and a wilted phenotype. Metabolism of abscisic acid and its regulation in Xanthium leaves during and after water stress.
ApPENDIX Ill: PAPERS PUBLISHED AND PRESENTED
In light of the results obtained so far from the "Hass" avocado fruiting system, the method is Minire view Fruit size: Towards an understanding of the metabolic control of fruit growth using avocado as a model system. Yet CK is apparently required for most of the fruit development program (Gillaspy et al. 1993), and mitotically active·.
A detailed biochemical study of the metabolic interaction between CK and ABA in the mesocarp of ripening avocado fruit showed that CK promotes the oxidative catabolism of ABA (Cowan et al. 1999).
