By searching the Tomato Genetic Resource Center (TGRC), a set of natural dwarf tomato mutants was identified and screened to determine if any of them were due to BR deficiency. The dpy tomato mutant showed dramatic response to exogenous application of BR (Cerny, 1997), resulting in nearly complete rescue of the mutant to wild-type phenotype (Fig. 2), suggesting that the DPY gene is involved in BR biosynthesis. Lack of complete rescue of dpy to wildtype in response to exogenous application of BR was also observed in

another BR-deficient tomato mutant, dwarf (Bishop et al., 1996, Bishop et al., 1999). The authors attributed this observation to inefficient BR transport in tomato. This could also be due to the exacting requirements in terms of both temporal and spatial distribution of BR during growth and development, which would be difficult to meet by exogenous application.

In dpy, we have observed that only immature and probably

undifferentiated cells can respond to BR. Mature or differentitated cells don’t have any response to exogenous BR. BR-deficiency of dpy was also confirmed by comparing endogenous levels of BR and its intermediates in the mutant with the levels in wildtype plant of same age and grown under identical conditions, using GC-MS (Table 1). 6-deoxoCS was 25-fold less and CS was below detectable levels in dpy when compared with wildtype plants.

Two alternate pathways of BL biosynthesis, termed early and late C-6 oxidation, were identified by feeding experiments in cell cultures of Catharanthus roseus (Fig 1.; Choi et al, 1996, 1997). BR biosynthetic intermediates have also been used in feeding experiments to locate the putative site of mutation in the pathway of different BR deficient mutants. Application of both early and late C-6 oxidation pathway intermediates suggested that an enzyme activity involving the conversion 6-deoxoCT to 6-deoxoTE might be the site of mutation in dpy. This is because the deoxoTE and other downstream intermediates showed effects of rescue of the dpy mutation, with the intensity of rescue increasing as we proceed to the end of the pathway. This is consistent with earlier observations using bioassays, that the biological activity of the intermediates increases with position

along the pathway (Fujioka et al., 1995), and also that the efficiency of rescue of deficient mutants increases as the intermediates approach more closely the structure of BL (Choe et al., 1998, 1999a, 1999b; Fujioka et al., 1997; Klahre et al., 1998; Nomura et al., 1999).

In the case of Arabidopsis, both 6-oxo and 6-deoxo intermediates have been detected, indicating that both the early and late C-6 oxidation pathways are active in this species (Choi et al., 1997). Based on observations during feeding experiments it was proposed that late C-6 oxidation might be more active in the light, while early C-6 oxidation is more active in the dark (Choe et al., 1998; Fujioka et al., 1997). However, reports on tomato have shown that the early C-6 oxidation pathway might not be very active in this species (Bishop et al., 1999; Yokota et al., 1997), as intermediates such as TE and TY were not detected. Our observations in feeding experiments involving dpy also showed that responses to late C-6 oxidation pathway intermediates were more pronounced than early C-6 oxidation pathway intermediates. The rescue of dpy by early C-6 oxidation pathway intermediates was not as dramatic as late C-6 intermediates, although there was slight change in the leaf morphology. GC-MS assays for endogenous levels of intermediates also detected only late C-6 intermediates (Table 1), and early C-6 intermediates such as TE and TY were not detected. In contrast to observations in Arabidopsis (Choe et al., 1998; Fujioka et al., 1997), we found that 6-deoxo intermediates were more active than the 6-oxo intermediates in the dark also. This situation is unlikely due to differential stability or uptake, instead it

is likely due to the fact that late C-6 oxidation might be the only active pathway in tomato.

Based on sequence analysis and feeding experiments, the Arabidopsis cpd mutant has been proposed to also be due to mutation in the gene

responsible for the conversion of CT to TE (Szekeres et al., 1996). It was further proposed that this enzyme is required to hydroxylate 6-deoxoCT to 6-deoxoTE since the mutation of this gene results in a dwarf phenotype, even when both pathways are functional in Arabidopsis. Based on these observations we proposed that DPY might be the tomato homologue of CPD.

To verify this assumption we transformed dpy with CPD cDNA to

determine if the mutation could be complemented. We used the same construct and Agrobacterium strain that was succesfully used to complement the

Arabidopsis cpd mutation (Szekeres et al., 1996). This experiment resulted in the recovery of transgenic plants that had no phenotypic differences when compared to the untransformed dpy. Although it might be premature to draw absolute conclusions since we had only three transgenic lines that were recovered from each transformation, and expression levels of the transformed gene were not experimentally determined. Hence, we cannot rule out the possibility of the tomato protein that cross-reacts with the Arabidopsis CPD antibodies actually being the CPD homologue in tomato and that it might be functionally inactive due to a mutation in the protein. Due to difficulty in evaluating the activity of the CPD protein the best approach to resolve these questions would be to clone the DPY gene.

Using antibodies to CPD protein, we observed that dpy actually had more protein cross-reacting to the CPD antibody than the wildtype (Fig 6). The

Arabidopsis CPD gene has been demonstrated to be negatively regulated by BR (Mathur et al., 1998). This suggests that due to BR deficiency, BR feed back regulation of the CPD gene might not be functional and, may result in over accumulation of CPD protein in dpy when compared with protein levels in the wildtype. Based on these two observations, we propose that dpy and CPD are unlikely to be homologues; of course the only way to be certain about this is to do protein activity assays. CPD is a member of the cytochrome P450 gene family, and activity assays may not be feasible, since this family is large and specificity of the assay is difficult to control.

Instead, DPY enzyme might be acting on intermediates one or two steps before the CPD enzyme. It is also possible that there are intermediate steps in the conversion of 6-deoxoCT to 6-deoxoTE that have not been demonstrated yet. This idea is encouraged by a recent report showing that the conversion of CR to CN, in the early steps of the pathway, actually has three more intermediate stages than originally thought (Noguchi et al., 1999), and that DET2, which was thought to be solely responsible for converting CR to CN, is only involved in the conversion of 4-en-3-one to 3-one (Noguchi et al., 1999). It is also possible that DPY might encode a regulatory protein that is required for the transcription or activity of the true CPD homologue.

Since there is no direct evidence for the existence of other intermediates between CT and TE intermediates, cloning and sequence of the DPY gene will

go a long way in answering these questions. We have tried to transposon-tag the gene (Fig. 7), but have not been successful in isolating a tagged-allele of DPY yet. We have only screened 12,000 seeds to date, and have not yet reached the numbers that increase the odds of find the tagged allele. Other mutants similar to dpy, including dwarf, have been cloned using this approach (Bishop et al., 1996). The DWARF gene encodes a cytochrome P-450 and feeding experiment have shown that this enzyme is responsible for the conversion of 6-deoxoCS to CS (Bishop et al., 1999). Although both dwarf and dpy have been mapped to similar regions of the long arm of chromosome 2 (Miller and Tanksley, 1990), allelism tests (Yu, 1998) have shown that they are not alleles. Fine mapping of dpy might give us better information to use the correct Ac tagged line that is most proximal to the DPY gene.

However, before continuing the transposon-tagged approach, I would redo the mapping of the dpy mutation to confirm the report that it is localized to

chromosome 2 specifically. Since working with the dpy population, I have

observed that there is some segregation with respect to height in the population. This was also observed by Cerny (1997), who identified dpy to be BR-deficient. However, he observed that ratio of segregation in the population to be 1: 49 (short: tall). On the contrary, my observation is that the segregation ratio is more like 1:3. To better characterize the genetics of the segregation I did progeny testing to identify homozygous lines within the population that are not

genetics and the only interpretation I could draw was that the segregation pattern does not fit Mendelian ratios.

In summary, we have documented physiological and biochemical evidence to prove that dpy is a BR-deficient mutant. This contributes to the growing evidence that BRs are critical for the growth and development of normal tomato similar to the roles played by BRs in Arabidopsis growth and

development. Cloning of the DPY gene will be very helpful in further

characterizing this mutation, contributing towards better understanding of BR biosynthesis.

In document Physiological and Molecular Characterization of a Brassionosteroid-Deficient Tomato Mutant (Page 62-68)

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