Abstract
Prolamins constitute a unique class of seed storage proteins, present only in grasses. In the lumen of the endoplasmic reticulum (ER), prolamins form large, insoluble heteropolymers termed protein bodies (PB). In transgenic Arabidopsis (Arabidopsis thaliana) leaves, the major maize (Zea mays) prolamin, 27 kDa γ-zein (27γz), assembles into insoluble disulfide-linked polymers, as in maize endosperm, forming homotypic PB. The 16 kDa γ-zein (16γz), evolved from 27γz, instead forms disulfide-bonded dispersed electron-dense threads that enlarge the ER lumen without assembling into PB. We have investigated whether the peculiar features of 16γz are also maintained during transgenic seed development. We show that 16γz progressively changes its electron microscopy appearance during transgenic Arabidopsis embryo maturation, from dispersed threads to PB-like, compact structures. In mature seeds, 16γz and 27γz PBs appear very similar. However, when mature embryos are treated with a reducing agent, 27γz is fully solubilized, as expected, whereas 16γz remains largely insoluble also in reducing conditions and drives insolubilization of the ER chaperone BiP. These results indicate that 16γz expressed in the absence of the other zein partners forms aggregates in a storage tissue, strongly supporting the view that 16γz behaves as the unassembled subunit of a large heteropolymer, the PB, and could have evolved successfully only following the emergence of the much more structurally self-sufficient 27γz.
Highlights
The polypeptides of seed storage proteins undergo different interchain interactions that are important for their synthesis, intracellular traffic, and accumulation
The results reported here show that electron-dense 16γzf threads, similar to those accumulated in the endoplasmic reticulum (ER) of Arabidopsis leaf cells, are present in developing embryos and that these coalesce into smaller, nearly homogeneously electron-dense structures that accumulate in mature seeds
The results show that most 16γzf present in mature, dry seeds is insoluble in the presence of reducing agents that fully solubilize seed-accumulated 27γzf, as well as both recombinant zeins accumulated in leaf cells [14]
Summary
The polypeptides of seed storage proteins undergo different interchain interactions that are important for their synthesis, intracellular traffic, and accumulation. In seeds of certain plants, such as pumpkin (Cucurbita sp cv Kurokawa Amakuri Nankin), transient polymerization occurs already in the ER lumen, leading to the formation of electron-dense precursoraccumulating (PAC) vesicles that reach storage vacuoles bypassing the Golgi apparatus [7]. All these transient, large structures that may involve the two major classes of vacuolar storage proteins—7S/11S globulins and the monomeric 2S albumins—are solubilized in aqueous buffers, whereas the major storage proteins of cereals—prolamins—form very large insoluble PBs in the ER [8]. We have investigated whether the peculiar features of 16γz are maintained when the protein is synthesized in a storage seed tissue, where other types of storage proteins are synthesized as well, and where a development program is active to favor their optimal accumulation
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