3 CHARACTERIZATION AND TISSUE DISTRIBUTION OF AO, XDH, AND CKOX IN DEVELOPING AVOCADO FRUIT
3.1 INTRODUCTION
3 CHARACTERIZATION AND TISSUE DISTRIBUTION OF
Table 3.1 Previous findings of hormone content and composition in the tissues of developing avocado fruit. (RIA, Radioimmunoassay).
Hormone Method of
Seed Seed coat Mesocarp Reference
determination
high initially initially very high moderate "bound" CK-like
Blumenfeld and Gazit 1970;
Bioassay levels
declines as fruit matures declines as fruit matures
declines as fruit matures Gazit and Blumenfeld 1970
CK
RIA iP: >30 to 14n~g-1 FW iP: 30 to 0 ng g-1 FW iP: 20 to 5 ng
p-'
FW Cutting et al. 1986 iPA: 5 ng g- FW . iPA: 20 to 0 ng g-1 FW iPA: 5 ng g- FWmoderate Bioassay high initially high initially declines during fruit
Gazit and Blumenfeld 1972 declines as fruit matures declines as fruit matures growth, small peak close
~ IAA to maturity
....,
RIA 70 to 15 ng g-1 FW at 70 to 0 ng g01 FW at steady decline from 30 to
Cuttinget al. 1986
maturity maturity 10 ng g-1 FW
varied between 35 and 65 varied between 35 and
40 to 100 ng g-1 FW from
RIA ng g-1 FW 65 ng g01 FW declined to Cuttinget al. 1986
o
at maturity mid growth to maturity ABAReversed-phase 14~gg-1 DW during linear 29.3 to 10.9 ~gg-1 DW
Cowan et al. 1997
HPLC phase of growth
-
over course linear phaseRichings etsI. 2000 of growth
GA Bioass'ay no measurable activity initially very high
no measurable activity Blumenfeld and Gazit 1972 declines as fruit matures
Initial evidence for the involvement of a MoCo-AO in ABA biosynthesis came from studies using MoCo-deficient mutants of barley (Walker-Simmons etal. 1989), tobacco (Leydecker et al. 1995), tomato (Marin and Marion-PolI 1997) and Arabidopsis (Schwartz et al. 1997a).
These mutants lacked MoCo-containing AO and XDH activities and had impaired ABA production. Lee and Milborrow (1997) presented further evidence of the involvement of a MoCo-AO in ABA biosynthesis by demonstrating XAN accumulation in avocado fruit treated with tungstate. Through the addition of cinchonine, which forms an insoluble complex with tungstate, they were also able to show restoration of ABA production. These findings, taken together, lead to the distinct possibility that XAN oxidase is a MoCo-containing AO. Recently two AO genes have been identified in Arabidopsis leaves that are rapidly induced after desiccation (Seo et al. 1999) and an AO isoform, designated AOo, has been found that has high specificity for AB-aid and is expressed mainly in rosette leaves ofArabidopsis(Seo etal.
2000a;b).
Mutants impaired in MoCo biosynthesis would also be expected to exhibit impaired IAA biosynthesis, if a MoCo-AO is indeed involved in the final step of its biosynthesis. However, MoCo mutants exhibit no obvious IAA deficiency or auxotrophy phenotype (Seo et al. 1998).
One possible explanation is that the mutants are leaky and small quantities of IAA are sufficient to promote normal growth. Another possibility is that several parallel pathways for IAA biosynthesis exist in plants, operating at different stages of development and/or in different organs or tissues (Normanly et al. 1995; Kawaguchi and Syono 1996; Normanly 1997). Some investigators have shown that an AO, tentatively designated lA-aid oxidase, may be involved in IAA synthesis (RajagopaI1971; Bower etal. 1978; Miyata etal. 1981), but the actual function of the enzyme in IAA biosynthesis has not been definitively confirmed. Maize AO has exhibited a high affinity for lA-aid (Km 3-5 /JM), which indicates that even at low concentrations of lA-aid, the aldehyde could still be converted to IAA (Koshiba et al. 1996).
Subsequently, Seo et al. (1998) demonstrated that an AO isoform, designated AOa, in Arabidopsis plants had high affinity for I-aid and lA-aid and was overexpressed in the IAA overproducing sur1 mutant, as compared to the wild type, suggesting the involvement of this enzyme in the final step of IAA biosynthesis.
Xanthine dehydrogenase is another MoCo-requiring enzyme and catalyses the first oxidative step in purine catabolism (Nguyen 1986). It is ubiquitous and ensures that purine compounds, originating from CK and nucleic acid degradation, are irreversibly committed into pUrine catabolism (Wastemack 1982; Nguyen 1986). Xanthine dehydrogenase catalyses the formation of uric acid from xanthine and hypoxanthine and is thus necessary for the synthesis of ureides in higher plants (Nguyen 1986). This enzyme is inhibited by excess substrate (Bray
1963) and product (Nguyen 1979; Boland 1981), as well as substrate and product analogues (Nguyen 1986), such as adenine and guanine(yVoo et al. 1981).
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). It is therefore hypothesized that under conditions where nitrate assimilation is reduced and/or XOH inhibited, more MoCo might be expected to be available for AO required for ABA and IAA biosynthesis.
One possible means by which XOH might be inhibited is through elevated adenine levels, one source of which is the irreversible degradation of isoprenylated CK. The degradation of CK is catalysed by the enzyme CKOX, which raises the possibility that changes in activity of this enzyme might impact on the activity of XOH, through the production of adenine. The result of CKOX activity is the irreversible loss of CK structure and thus biological activity and in this way CKOX is thought to play an important role in controlling the internal pool of CK in plants.
Available evidence suggests that the activity of CKOX and the degradative metabolism of CKs can be mediated by four principle mechanisms: (1) CK supply; (2) phenylurea compounds; (3) auxin levels; and (4) glycosylation and/or isozyme variation (Jones and Schreiber 1997).
There have been very few investigations of the activity of AO, XOH and CKOX in fruit tissues and certainly none considering all three simultaneously. The linkage of these enzymes to hormone homeostasis has been alluded to before (Cowan et al. 1999; 2001), but a direct relationship has yet to be established. The plants of choice used to investigate these enzymes remain those with a rapid growth cycle, e.g.Arabidopsis thaliana, Nicotiana sp., Zea mays and other cereal crops. The tissues extracted to evaluate activity of these enzymes are mainly leaves or roots and in the case of CKOX, maize kernels. The use of the avocado fruit is thus a first, with the 'Hass' small fruit phenotype providing an ideal system in which to probe more detailed aspects of the control of final fruit size. To further characterize the 'Hass' avocado small fruit phenotype, it was therefore necessary to compare and contrast the endogenous hormone profile of seed, seed coat, and mesocarp tissue in normal and small fruit. In addition, certain key enzymes involved either directly or indirectly in hormone metabolism were assayed to determine activity and tissue distribution in normal and small fruit.