Temporal and spatial expression of HSPs in developing kernels

--Carla Frova and Graziana Taramino

In a previous note (C.Frova, C, MNL 63) preliminary data on HSP synthesis in post-fertilization stages (7-20 DAP) were reported. Two genotypes were analyzed and different protocols for the analysis tested. Those first data did not reveal significant differences in the HSPs induced between genotypes and developmental stages.

Here we report the results of a second analysis performed on additional developmental stages (3-42 DAP). The reciprocal F1s between two inbred lines provided the material for this analysis. The parental lines differed for the presence/absence of a low molecular weight HSP: P1 = Mo17, 17kDa+; P2 = H95, 17kDa-. The 17kDa HSP thus served as a marker to monitor the expression of the paternally derived gene in the F1 H95 x Mo17.

The procedure was optimized as follows: for very immature stages (3-14 DAP) intact kernels were excised from the cob and incubated at 25 or 41 C for three hours in distilled water containing 150�l/ml 35S methionine. For later stages (21-42 DAP) the kernels were dissected into embryonic tissues (scutellum+embryo) and pericarp. Endosperm and aleurone were not considered in this study. The tissues were treated separately as above. After the treatment all material was extracted and analyzed by 1-D SDS PAGE.

The results are as follows:

1) In the F1 H95 x Mo17 the 17kDa HSP of paternal origin is absent up to two weeks after fertilization. In later stages (21-42 DAP) it is clearly expressed in the embryonic tissues but not in the pericarp, as expected given the entirely maternal origin of this tissue. Kernels of the reciprocal F1 express this band from the first stages analyzed.

2) Two high molecular weight HSPs (94 and 92kDa) appear in the very early stages (3 DAP), show a drastic reduction between 5 and 15 DAP, and become prominent again from 21 DAP, but only in the zygotic tissues. In the pericarp they are completely absent even in these later stages. This latter tissue however, shows a minor 35kDa HSP, not present in the embryo, from 21 DAP on.

3) The other typical maize HSPs are synthesized at all the stages considered without differences between tissues.

In conclusion, these data indicate that, as is the case in several animal systems, maize HSP synthesis in the post-zygotic phase is spatially and developmentally regulated. Three HSPs, 94, 92 and 17kDa, show temporal regulation: they begin to be synthesized and accumulated to detectable levels from two weeks after fertilization, a time corresponding to very active embryo development. The appearance of the 17kDa band indicates expression of the paternal gene. In addition, tissue specificity is observed for HSPs 94 and 92kDa, typical of embryonic tissues only, and 35kDa, apparently induced only in the pericarp.

In vivo studies of the effect of heat-shock treatments at different stages of kernel development, in particular the early ones characterized by the absence of some HSPs, are in program.

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