The expression of 16KDa γ-zein in Arabidopsis induces proliferation of ER membranes and the formation of unconventional structures

Prolamins represent the major seed storage proteins in cereals and are peculiar of these plants, unlike the 7/11S globulins and 2S albumins present in all plants (1). Many prolamins form large insoluble polymers, termed protein bodies (PB), within the ER lumen, where they accumulate (2). In particular, maize prolamins (zeins) are grouped into four classes (α, β, γ, δ) being γ and β evolutively the most ancient and synthesized before α and δ during seed development (3). Three genes encode γ-zein polypeptides, of 16, 27 and 50 kD. The 27 kD γ-zein forms ER-located PB also when expressed in vegetative tissues of transgenic plants, indicating that it contains all the required information to form a PB (4). Its C-terminal region has high homology to 2S albumins, with four conserved intra-chain disulfide bonds, while the N-terminal region contains an amphypathic repeated domain and seven Cys residues (4, 5, 6). The insolubility of 27 kD γ-zein is related to the number of Cys residues in the first domain, that form inter-chain disulfide bonds; substitution of all these Cys with Ser residues leads to full solubility and secretion (7). The role of other structural determinants, such as the repeated domain is however less clear. 16 kD γ-zein almost completely lacks the amphypathic repeats has only four Cys in the N-terminal domain. It may lack also one of the intra-chain disulfide bonds in the C-terminal domain. Arabidopsis plants constitutively expressing the 27 or the 16 kD polypeptides were produced. Biochemical analysis of leaves showed that 16 kD γ-zein forms polymers that remain in the ER but, unlike the 27 kD polypeptide, is does not form the insoluble PBs with high efficiency. Comparative ultrastructural observations by TEM showed substantial differences in the ability of the two prolamins to form PBs. As expected, the 27 KDa γ-zein was able to form canonical, round shaped, compact PBs in the ER. Similar structures were rarely seen in leaves expressing 16 kD γ-zein, which instead formed layers of different thickness leaning ER membranes, as also confirmed by immunolabeling experiments. Intriguingly, these membranes abnormally proliferated to form large membranous bodies of different size up to 10-20 µm long in mesophyll cells. Occasionally, in the center of these membranous systems compact structures were present, however very irregular in shape. No other apparent ultrastructural changes were observed in the transformed plants in comparison with wt Arabidopsis. The above results indicate that 16 KDa γ-zein is competent to polymerize, hence be retained in the ER. However, its polymerization mostly occurs in layers leaning ER membranes, which are somehow induced to proliferate to sustain this unusual process. 1. Shewry et al (1995) Plant Cell 7: 945-956; 2. Herman (2008) Curr Opin Plant Biol 11:672–679 3. Xu and Messing (2009) Theor Appl Genet 119:1397–1412; 4. Geli et al (1994) Plant Cell 6: 1911-1922; 5. Vitale et al (1982) Jof Exp Bot 33: 439-448; 6. Mainieri et al (2004) Plant Physiol 136:3447-3456: 7. Mainieri et al (2014) Front Plant Sci 5:331