Globulin gene expression in embryos of viviparous mutants --Renato Paiva and Alan L. Kriz Vivipary is the phenomenon in which seed maturation fails to go to completion and, as a consequence, germination initiates while the kernel is still attached to the ear. To better understand the mechanism involved in genetically controlled vivipary, we have examined the expression of genes encoding embryo storage proteins in sib normal and viviparous (vp) kernels. In normal maize embryos, specific globulin storage proteins which accumulate to high levels during seed development and maturation are rapidly degraded during the early stages of germination. The most abundant embryo globulin components are encoded by the Glb1 and Glb2 genes, which have been characterized at the molecular level in our laboratory (see above notes). We previously used expression of these genes as markers of embryo maturation (proteins and transcripts present) and germination (proteins and transcripts absent) to determine if viviparous embryos undergoing precocious germination had switched from a "maturation program" to a "germination program" of gene expression. These experiments demonstrated that precociously germinating embryos of the Class II ABA-deficient mutants (vp2, vp5, vp7, and vp9), as well as vp8, contained significant amounts of Glb1proteins and transcripts (Kriz et al., Plant Physiol., in press; MNL 63:116, 1989), indicating that these embryos exhibit characteristics of both maturation (Glb1 expression) and germination (i.e., radicle protrusion) processes.

During this past summer, embryos were collected from ears of various ages segregating for the different vp mutants. These include normal and mutant sib embryos and endosperm from developmentally staged ears segregating for each of the viviparous mutants vp1, vp2, vp7, vp8, and vp9. With the exception of vp8, which is difficult to classify prior to the onset of precocious germination, samples were obtained at various days after pollination (DAP) prior to, and during, the period of precocious germination. Embryos were pooled from three selfed ears of the same family pollinated on the same day. Since proteins encoded by the Glb genes are rapidly degraded during the early stages of seed germination, we anticipated that these proteins might be present in vp/vp embryos prior to the onset of precocious germination, and that they would be degraded in germinating embryos older than those previously examined (as reported in last year's newsletter). This does not turn out to be the case. Proteins were extracted from sibling normal and mutant (vp) embryos and subjected to immunoblot analysis to visualize specific proteins encoded by the maize Glb1 and Glb2 genes. None of these proteins are detected in vp1/vp1 (Class I; ABA-insensitive) embryos at any of the embryo ages examined, regardless of whether precocious germination is apparent. In embryos homozygous for either vp2, vp7, or vp9 (Class II; ABA-deficient), globulins are absent in younger embryos, but are apparent in older embryos, even though these older embryos are actively germinating.

Results consistent with those obtained from the immunoblot analyses were observed when levels of Glb1 transcripts were examined in embryos by RNA blot analysis. Glb1 transcripts, usually first detectable at 15 DAP, are normally present throughout most of embryo development. These transcripts are not detectable in vp1/vp1 embryos at any age, providing additional evidence that Glb1 expression is absent in homozygous vp1 embryos; since Glb1 expression is apparently regulated by ABA (Kriz et al., 1990), this is probably due to the ABA-insensitive nature of this mutant. Although Glb1 is expressed in Class II mutant embryos after 18 DAP, the rate of accumulation of Glb1 transcripts in these mutant embryos is delayed relative to that observed in normal sib embryos.

Although the results obtained upon analysis of Glb1 expression in the Class II vp mutants were somewhat unexpected, they clearly demonstrate that the pattern of gene expression during precocious germination of the vp mutants, at least with respect to the maize Glb genes, is different from that of normal germination. The increased levels of Glb1 expression in older embryos of the Class II vp mutants may be due to accumulation of ABA as a function of embryo age. Although ABA levels in the Class II mutants are much lower than those of normal embryos (Neill et al., Planta 169:87, 1986), these low levels may be sufficient to promote expression of Glb1. We are in the process of determining ABA levels in the embryo samples collected this past summer to ascertain if there is any correlation between ABA content and level of Glb1 expression in these materials.

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