FOLDING HELPERS INVOLVED IN CEREAL PROTEIN BODY BIOGENESIS

Cereals and legumes are the primary source of proteins for human and animal consumption. Cereal storage proteins belong to the large prolamin family. In maize, prolamins ?alpha-,??beta-, ?gamma- and ?delta-zeins polymerize in the endoplasmic reticulum (ER) of developing endosperm to form ordered structures termed protein bodies (PBs). 27kD gamma-zein (27gamma-zein), possibly the most ancient zein, is among the first zeins synthesized during seed filling and forms PBs when expressed alone in vegetative tissues of transgenic plants. Its N-terminal domain, which contains eight repeats of the hexapeptide VHLPPP and seven cysteine residues, is necessary for retention and deposition into the ER and promotes PB formation when fused to other proteins inserted into the secretory pathway. While extensive data are available on the structural requirements of 27gamma-zein for PB biogenesis, few data exist on the molecular machinery engaged in this process. With the aim to identify molecular helpers involved in PB formation, we have analyzed the transcriptome of A.thaliana expressing zeolin, a fusion between phaseolin (PHSL, a 7S globulin of P. vulgaris that accumulates in the storage vacuoles) and the N-terminal domain of 27gamma-zein. We previously showed that zeolin forms PBs in N. tabacum. The synthesis of zeolin determines an increase of a number of Arabidopsis transcripts encoding ER proteins, including CYP21-2 (At3g55920), a protein belonging to the peptidyl-prolyl cis-trans isomerase/cyclophilin family. After selecting and analysing cyp21-2 knockout homozygous A.thaliana plants, we produced plants expressing the native 27kD gamma-zein in the cyp21-2 (cyp21-2/27gamma-zein) or wild type (27gamma-zein) background. mRNA and protein levels of ER-resident chaperones in wt, PHSL, 27gamma-zein, cyp21-2 and cyp21-2/27gamma-zein plants will be assayed by real-time PCR and protein analysis. Supported by the FILAGRO Project of CNR-Regione Lombardia