University of Freiburg
University of Florida

Further characterization of the double mutant opaque2 brittle2
--Winfried Hetz, Klaus Grasser, Peter Südbeck, Günter Feix and Curt Hannah

C. Y. Tsai, CY et al. previously generated a set of double mutants by combining the o2 mutant with several starch-modified or starch-deficient mutants and studied the influence of the concomitant presence of these two types of mutations on the zein protein SDS PAGE pattern of alcohol soluble proteins of mature kernels (Tsai, Larkins, BA and Glover, DV, Biochem. Genet. 16:883-896, 1978). While the majority of the analysed double mutants did not show any major effect on the zein protein PAGE pattern, a dramatic change in the composition of alcohol soluble protein was observed in the case of the o2 bt2 and o2 sh2 double mutants. The authors found no protein at the SDS PAGE running positions of the normally predominant 19 and 21kd zein proteins and observed instead a great number of proteins of larger and smaller sizes. As one of the causes for the observed changes of the protein pattern with the striking absence of 19 and 21kd size proteins, the authors proposed an effect of the elevated levels of RNAses detected in the endosperm of this material.

In line with our interest in studying assumed interconnections between the storage protein and starch syntheses in the developing endosperm, we looked again at the o2 bt2 mutant kindly given to us by Charles Tsai and were particularly interested in the question of whether a transcriptional effect is involved in the mutant behaviour. Towards this analysis we grew plants under controlled conditions in a phytochamber and analysed the zein protein SDS PAGE pattern of proteins isolated from kernels collected at two day intervals from the 9th to the 39th day post pollination, confirming basically the previous observation of Charles Tsai's group. We then isolated the predominant protein band of larger than 21kd size and identified it clearly as the 27kd zein protein by a sequence analysis of its 16 N-terminal amino acids. If analysed over development of the endosperm, the 27kd protein appeared at the 14th day post pollination and disappeared again after 28 days, indicating a breakdown of the synthesized protein normally stably accumulating in wildtype material. In Northern experiments with RNA prepared from various endosperm stages, no RNA coding for the 19 or 21kd zein proteins could be detected while RNA specific for the 27kd protein was present from the 15th to the 28th day, indicating a severe synergistic influence of the two deficient o2 and bt2 genes on zein gene transcription. It should be remembered that so far no mutants or mutant combinations with a complete deficiency in the synthesis of the major zein proteins of 19 and 21kd have been identified. 

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