Dormant, colorless alleles of vp1

--Christian Carson and Donald McCarty

Null mutant alleles of vp1 produce viviparous, anthocyanin deficient kernels. The structure and expression of dormant vp1 alleles was determined and a summary is presented in the table. Leaky expression is exhibited by the vp1-mum3 allele; the level of anthocyanin expression is comparable to the apparent level of vivipary. Alternatively, full expression of the dormant, colorless alleles effectively promotes maturation, but not anthocyanins.

The maturation-related genes, Glb1 and Em, and the anthocyanin regulatory gene, C1, are activated by Vp1. Expression of Glb1 and Em mRNA is relatively normal in vp1-McWhirter and vp1-c821708 embryos; and consistent with the dormant, colorless phenotype, C1 expression is undetectable.

vp1-McWhirter mRNA is 3' truncated and the cDNA sequence encodes a C-terminal truncated protein. vp1-Mc protein, immunoprecipitated from in vitro translation and resolved on SDS-PAGE, is proportionately smaller than wildtype. In fact, all of the dormant, colorless alleles encode similarly sized, apparently truncated protein. In contrast, vp1-mum3 expresses normal sized message and protein.
 
 
  Phenotype vp1 expression
  kernels Glb1* Em* C1* mRNA (bases) Protein (~M in kDa)
WT (W22) d/p + + + 2500 90/92
1 vp1-mum3 leaky + + + 2500 90/92
2 vp1-McWhirter d/c + + - 2300 69/74
3 vp1-c821708 d/c + + - 2600 66/68
4 vp1-1695 d/c       2600/4000 61/62/65/67
5 vp1-a1 d/c       3000/6000 65/68/75
6 vp1-Ref. (null) v/c - - -   none
d=dormant, v=vivipary, p=anthocyanin pigmented, c=colorless; leaky indicates variable none to weak vivipary and anthocyanins; + and - indicate qualitative expression detected on Northern blot; * probes for Northern blots: Glb1-A. Kriz, A, Em-R. Quatrano, R (from wheat), C1-K. Cone, K; allele donations: 1-P. Stinard, P, 2-H. Dooner, H, 3-P. Peterson, PA, 4-M. Alleman, M, 5-E. Patterson, EB; blanks are undetermined
 

We have compared vp1-Mc with wildtype for activation of both the Em and C1 promoters during transient expression in electroporated maize protoplasts. The result is again consistent with the phenotype. In summary, vp1-Mc activates Em-GUS expression 10-20 fold, about 5% of wildtype. C1-GUS is activated 6 fold by wildtype, but it is not activated by vp1-Mc.

We conclude that the Vp1 gene is composed of at least two distinct functional domains. The N-terminal portion of the protein, which is encoded mostly by the first exon, is sufficient in activating maturation and the Em promoter, while the C-terminal structure is required additionally for the activation of C1.


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