Progressive Aggregation of 16 kDa Gamma-Zein during Seed Maturation in Transgenic Arabidopsis thaliana

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.

StresSeed: The Unfolded Protein Response During Seed Development

“…The particularly strong effect of 16γz suggests that, with respect to its 27γz parent gene, this recently evolved prolamin is more dependent on partner zeins for acquiring a folded conformation that abolishes BiP binding. This is consistent with the observation that, during maturation and desiccation of transgenic Arabidopsis seeds, 16γz becomes progressively insoluble also in reducing buffers, indicating disordered aggregation ( Arcalis et al, 2021 ).…”

Generation of multi-layered protein bodies in N. benthamiana for the encapsulation of vaccine antigens

“…This is well in agreement with earlier observations in Arabidopsis leaves where 16-kDa-γ-zein formed dispersed electron-dense threads enlarging the ER lumen without assembling into PBs, and only when co-expressed with 27-kDa-γ-zein it co-assembles with the latter into insoluble polymers ( Mainieri et al., 2018 ). In transgenic Arabidopsis seeds 16-kDa-γ-zein formed more compact aggregates, but remained largely insoluble in reducing conditions, also suggesting that 16-kDa-γ-zein is unable to form well-ordered polymers on its own but relies on the ability of 27-kDa-γ-zein to drive self-assembly ( Arcalis et al., 2021 ). Although 16-kDa-γ-zein most likely evolved through gene duplication of 27-kDa-γ-zein ( Xu and Messing, 2009 ), this different behavior may be explained by the loss of the repeat region as well as some cysteine residues reducing the strength of its intermolecular interactions ( Mainieri et al., 2018 ).…”

Endomembrane‐mediated storage protein trafficking in plants: Golgi‐dependent or Golgi‐independent?

“…Although the lack of the canonical HDEL/KDEL retention or retrieval signals, prolamins destined for ER-derived PBs fail to escape from the ER in plant seeds and even vegetative tissues when ectopically expressed [34][35][36][37]. Domain truncation and mutagenesis studies have identified several conserved sequence domains and key amino acid residues essential for the ER retention of prolamins, such as the N-terminal proline-rich tandem repeat domain in maize zeins that forms an amphipathic helix, which interacts with the ER surface [38].…”