A Fusion Between Common Bean Phaseolin and the N-Terminal Domain of Maize 16 kDa Gamma Zein: Clues for Designing Nutritionally Improved Seed Storage Proteins.

Increasing the nutritional value of seed storage proteins (SSPs) has been largely hindered by limited knowledge of how the features of the various SSP domains determine high accumulation in protein storage vacuoles (PSV) or as endoplasmic reticulum (ER)-located insoluble protein bodies (PB). We had previously designed zeolin, a fusion between phaseolin (PHSL, the main bean SSP, a soluble trimeric protein located in PSV) and the N-terminal domain of 27 kDa γ-zein (27γz, a major maize PB prolamin). Zeolin has a more balanced essential amino acid composition than each parental sequence and forms insoluble homomeric PB very similar to natural maize heteropolymeric PB, indicating that the zein domain has a dominant effect over PHSL. We had also determined that the paralog 16 kDa γ-zein (16γz) remains partially soluble and forms unusual threads in the ER when ectopically expressed. We now produced zeolin2 (zeo2), by fusing PHSL to the N-terminal domain of 16γz. We show that only a minor proportion of zeo2 remains in the ER, without forming PBs or threads. Zeo2 mainly forms soluble trimers with biochemical characteristics very similar to PHSL trimers, and traffics to the vacuole via the Golgi complex. This indicates that the 16γz domain fails to significantly dominate over PHSL. In transgenic arabidopsis, zeo2 accumulates to markedly higher levels than phaseolin and only slightly lower levels than zeolin. Zeo2 is also more accessible than zeolin to proteases during the early stages of seed germination. These findings provide new information relevant for the design of modified, nutritionally enhanced storage proteins.