Accumulation of 15-Kilodalton Zein in Novel Protein Bodies in Transgenic Tobacco

Abstract

Zeins, the seed storage proteins of maize, are a group of alcoholsoluble polypeptides of different molecular masses that share a similar amino acid composition but vary i n their sulfur amino acid composition. They are synthesized on the rough endoplasmic reticulum (ER) in the endosperm and are stored in ER-derived protein bodies. Our goal i s to balance the amino acid composition of the methionine-deficient forage legumes by expressing the sulfur amino acid-rich 15-kD zeins in their leaves. However, it is crucial to know whelher this protein would be stable i n nonseed tissues of transgenic plants. The major focus of this paper is to compare the accumulation pattern of the 15-kD zein protein with a vacuolar targeted seed protein, p-phaseolin, in nonseed tissues and to determine the basis for i t s stability/instability. We have introduced the 15-kD zein and bean p-phaseolin-coding sequences behind the 35s cauliflower mosaic virus promoter into tobacco (Nicofiana fabacum) and analyzed the protein's accumulation pattern in different tissues. Our results demonstrate that the 15-kD seed protein i s stable not only i n seeds but in all nonseed tissues tested, whereas the P-phaseolin protein accumulated only i n mid-and postmaturation seeds. Interestingly, zein accumulates in novel protein bodies both in the seeds and in nonseed tissues. We attribute the instability of the p-phaseolin protein i n nonseed tissues to the fact that it is targeted to protease-rich vacuoles. The stability of the 15-kD zein could be attributed to its retention i n the ER or to the proteaseresistant nature of the protein.Seed storage proteins constitute a potentially useful class of proteins for the improvement of forage crops if they can be made to accumulate in leaves. It is a fairly simple genetic engineering feat to introduce a seed protein-coding sequence behind a strong constitutive promoter into transgenic plants and ensure high rates of synthesis of the corresponding protein. However, stability of the protein in an alien environment is still not clearly defined and has to be treated on a case by case basis.Seed proteins are synthesized during seed development and accumulate in protein bodies. These proteins are then utilized by the emerging seedling during germination. The major seed protein in dicotyledonous plants are the saltsoluble globulins, which are stored in vacuole-derived protein bodies. Most monocot seed storage proteins are '

Coexpression of the maize delta-zein and beta-zein genes results in stable accumulation of delta-zein in endoplasmic reticulum-derived protein bodies formed by beta-zein.

“…The leaves showed the highest level of accumulation of 8-zein, with stems having the next highest level of accumulation. The seeds had añ 10-fold lower level of 8-zein compared with leaves, as was the case with p-zein in transgenic tobacco (Bagga et al, 1995). Taken together, our results suggest that 8-zein accumulates to significant levels in all of the organs of tobacco plant, as we had reported earlier for p-zein (Bagga et al, 1995).…”

“…In an earlier study, we showed that p-zein accumulates in ER-derived protein bodies in both tobacco leaves and seeds (Bagga et al, 1995); however, Hoffman et al (1987) reported that p-zein, when expressed in a seed-specific manner in transgenic tobacco, accumulated in vacuolar protein bodies.…”

“…Electron microscopy and immunocytochemical studies of the transgenic tobacco plants expressing the p-zein gene showed that this protein accumulated in special ER-derived protein bodies in the leaves as well as in the seeds (Bagga et al, 1995). Electron microscopy was also performed using leaf tissue from transgenic tobacco plants expressing 8-zein to determine whether any protein bodies were present.…”

“…The -y-zein and p-zein coding sequences have been expressed individually in transgenic Arabidopsis (-y-zein) and tobacco 0-zein) plants by using the cauliflower mosaic virus (CaMV) 35S promoter, and in both cases, the proteins were retained in ER-derived protein bodies (Geli et al, 1994;Bagga et al, 1995). In the case of -y-zein, Geli et al (1994) determined that a proline-rich region composed of repeats of a hexapeptide (PPPVHL) in the N-terminal domain is responsible for ER retention, whereas both the proline-rich repeat and the C-terminal cysteine-rich domains are needed for protein body biogenesis.…”

Nonredundant Function of Zeins and Their Correct Stoichiometric Ratio Drive Protein Body Formation in Maize Endosperm

“…It is thought that direct interactions between zeins are essential for their retention in the ER as properly formed protein bodies (Kim et al, 2002). Experiments using transgenic expression of both a-and g-zeins in heterologous species shed some light on their interaction in protein body formation (Williamson et al, 1988;Bagga et al, 1995Bagga et al, , 1997Coleman et al, 1996). However, interpretation of such studies was limited because they did not take into account endogenous nonzein accessory factors such as Floury1 (Fig.…”