MILAN, ITALY
University of Milan

UPTON, NEW YORK
Brookhaven National Laboratory

Characterization of new vp mutants not impaired in carotenogenesis

--Giulini, A, Bodei, S, Sirizzotti, A, Pilu, R, Burr, B, Gavazzi, G

The plant hormone abscisic acid (ABA) controls many aspects of plant growth and development under a diverse range of environmental conditions. To identify new genes functioning in ABA signalling and biosynthesis we collected a sample of 24 new mutants, derived from our collection (13) or from Dr. B. Burr (7) and Dr. D. Styles (4). Ten of these mutants are blocked in the synthesis of carotenoids, yielding albino seedlings. They represent mutational lesions on the early carotenoid pathway with down effects on ABA synthesis, whereas 14 have green seedlings and are thus not blocked in carotenogenesis. These presumably represent mutational lesions in steps of the hormone synthesis downstream of xanthophyll production, or they are response mutants impaired in the ABA receptor(s) or in the signal transduction. The data we present in this note refer to this second group of mutants.

Six of the 14 mutants isolated are allelic to vp1 (3L), three mutants (vpD*, vp374*, vp390*) represent another complementation group, do not complement vp1 or vp8 (1L) and are located on chromosome 1L, as determined by crosses with the B-A translocation stocks. Their allelism test with vp14 (1L) is underway. vp394*, whose chromosome location has not been determined, is not on chromosome 1L or 3L, thus representing a different gene from the previous two. vp105* is not allelic to vp1, vp8 and vp10 and has a unique phenotype consisting of a green shoot apex with occasional vivipary. The last of this class, rea, shows a characteristic red embryonic axis and occasional vivipary. It is uncovered by TB-3L-2S and TB-3La, but it is not allelic to vp1 on 3L. Its endogenous ABA content is normal.

Viability of the homozygous mutants is variable. Of those tested so far, rea is perfectly viable, vp390*, vp394* and vp105* are lethals, while about one-half of the vp374* mutants (13/29) can be rescued, and we succeeded in obtaining a homozygous ear (Fig. 1). Mutants deficient in ABA biosynthesis or in response to ABA signals show a difference in their sensitivity to exogenous hormone. Whereas those ABA deficients, grown in the presence of ABA are highly inhibited in their growth, those insensitive are inhibited to a much lesser extent. We tested a sample of mutants of the first class (green seedling) and they all appear less inhibited, even though to a variable extent, than their wild-type counterparts (Table 1).

An interesting result was obtained when the sensitivity of a double mutant, vp1-rea, was compared to the one of the single mutants. The double mutant, in fact, appears significantly more insensitive than either mutant taken alone (Fig. 2), a result suggesting an additive effect of vp1 and rea on the decrease in sensitivity to the hormone. This would be expected if the two genes act on separate pathways of the transduction of the ABA signal. This conclusion corroborates previous results (Sturaro et al., J. Exp. Bot. 47:755-762, 1996) indicating that vp1 and rea differ in their pattern of ABA inducible genes.

To test how the other mutants respond to ABA in terms of ABA responding genes, we started an RT-PCR analysis of vp374*. 30-day-old embryos of the mutant were collected, incubated on solidified MS medium without or with ABA (10μM) for 48h, and then assayed for the presence of globulin, an ABA inducible gene. The result (data not shown) indicates that the mutant maintains its capacity to respond to ABA by inducing globulin. This test will be extended to the other mutants.

Figure 1. Figure 2. Effect of exogenous ABA at 10 or 50 μM on growth of single (vp1 and rea) versus double (vp-rea) mutants. Growth is expressed as seedling elongation on media with ABA/ seedling elongation on media without ABA × 100. Length measurements were taken after 10 days of culture of immature embryos (30 DAP).