Zein protein interactions and stabilization: Utilizing high-methionine zein proteins to improve the nutritional quality of vegetative plant tissues

Expression of the 18-kD zein gene under the control of the CaMV 35S promoter results in stable transcript and protein accumulation in leaf tissues of transgenic tobacco. Co-expression of the 15-kD and 18-kD zein genes followed by immunolocalization revealed that both proteins accumulate in the same protein bodies. Furthermore, there is a 16-fold increase in accumulation of the 18-kD zein protein in co-expressing tobacco plants. Transgenic tobacco leaves co-expressing the 15-kD and 18-kD zein genes, or expressing only the 18-kD zein gene, accumulate the 18-kD zein transcript to equal levels. This suggests that the 15-kD zein protein stabilizes the 18-kD zein protein when sequestered in protein bodies. The possibility of 18-kD zein protein stabilization by the 15-kD zein protein is further evidenced by L-[35S] methionine pulse-chase experiments. A dramatic decrease in the rate of 18-kD protein degradation in transgenic tobacco leaves co-expressing the 15-kD and 18-kD zein genes was observed when compared to 18-kD protein degradation in transgenic tobacco leaves expressing only the 18-kD zein gene.; Promoter analysis studies were peformed on a chimeric, bi-directional mannopine synthase (mas) promoter with an enhancer element from the octopine synthase (ocs) gene, called ΔMAS-IOCS. The ΔMAS-1OCS promoter was used to control expression of the 15-kD β-zein gene in transgenic tobacco leaf tissues. Regulation of β-zein expression was observed in response to endogenous and environmental factors in the transgenic tobacco leaf tissues, with highest expression occurring in leaf tissues nearest the plant base and in roots. Analysis of the promoter sequence revealed an AS-1 motif and a root-specific expression motif of the rolD promoter of A. rhizogenes.; Amino acid analysis of T2 transgenic tobacco leaf tissues co-expressing the 15-kD and 18-kD zein genes showed an average increase in bound methionine of ∼11% when compared with non-transformed tobacco leaf tissues, and accumulated zein proteins to ∼4.1% of total leaf protein. Accumulating the 15-kD and 18-kD zein proteins to 4.1% of total alfalfa leaf protein should increase the methionine content such that alfalfa forage alone would supply adequate levels of all 10 essential amino acids required for cattle growth and milk production.