Journal of Experimental Botany, Vol. 52, No. 365, pp. 2387–2388, December 2001
GENE NOTE
Expression patterns of genes encoding endomembrane proteins support a reduced function of the Golgi in wheat endosperm during the onset of storage protein deposition Galia Shy1, Linda Ehler 2, Eliot Herman2 and Gad Galili1,3 1 2
Department of Plant Sciences, the Weizmann Institute of Science, Rehovot, Israel Soybean Genomics Improvement Laboratory, USDA, Beltsville MD, USA
Received 6 June 2001; Accepted 8 August 2001
Abstract Wheat storage proteins are deposited in the vacuole of maturing endosperm cells by a novel pathway that is the result of protein body formation by the endoplasmic reticulum followed by autophagy into the central vacuole, bypassing the Golgi apparatus. This model predicts a reduced role of the Golgi in storage protein accumulation, which has been supported by electron microscopy observations. To study this issue further, wheat cDNAs encoding three distinct proteins of the endomembrane system were cloned and characterized. The proteins encoded were homologues (i) of the ER translocon component Sec61a, (ii) the vacuolar sorting receptor BP-80 which is located in the Golgi and clathrin-coated prevacuole vesicles (CCV ), and (iii) the Golgi COPI coatomer component COPa. During endosperm development, the levels of all three mRNAs were highest in young stages, before the onset of storage protein synthesis, and declined with seed maturation. However, the relative mRNA levels of BP-80uSec61a and the COPauSec61a were lower during the onset of storage protein synthesis than at earlier stages of endosperm development. These results support previous studies, suggesting a reduced function of the Golgi apparatus in wheat storage protein transport and deposition. Key words: Wheat, storage proteins, endoplasmic reticulum, storage vacuoles, endomembrane system.
Protein trafficking via the endomembrane system in plant cells is essential for many cellular processes. It occurs by vesicular transport between the Golgi and the vacuole and plasma membrane. A significant portion of data on protein trafficking through the plant cell membrane system is derived from studies on storage proteins in seeds (Galili and Herman, 1997; Galili et al., 1998; Vitale and Galili, 2001). These proteins first enter the endoplasmic reticulum (ER) and are then deposited in protein storage organelles which are located either within the ER or in storage vacuoles (Shotwell and Larkins, 1989). Electron microscopy and biochemical observations have shown in some plant species, including wheat, that storage proteins are also transported directly from the ER to the vacuoles bypassing 3
the Golgi (Galili et al., 1993; Hara-Nishimura et al., 1998; Levanony et al., 1992; Vitale and Galili, 2001). The direct ‘ER-to-vacuole’ route of wheat storage proteins was also supported by the detection of a relatively low number of Golgi organelles in electron micrographs taken from endosperm tissues during the onset of storage protein synthesis and deposition (Levanony et al., 1992). Three wheat cDNA homologues of the ER-resident membrane protein Sec61a (AtSec61a) (GenBank Accession No. AF161718), the major subunit of Golgi COPI coatomer COPa (AtCOPa) (GenBank Accession No. AF176226), and the plantspecific receptor BP-80 that is localized on Golgi and prevacuole membranes (AtBP-80) (GenBank Accession No. AF161719) have been isolated. The deduced protein encoded by TaSec61a, namely, TaSec61p, exhibits 67% identity and 76% similarity to the rat Sec61ap. The C-terminal third of the TaCOPap exhibits 43% identity and 52% similarity to its human counterpart. TaBP-80p exhibits an average of 70% identity and 75% similarity to BP-80 isoforms from Arabidopsis, pea and pumpkin plants. In this paper it is shown that the relative expression level of the three genes, as deduced from Northern blot analyses, support previous microscopic observations (Levanony et al., 1992) that the Golgi has a reduced role in the deposition of wheat storage proteins. Since the three wheat cDNAs reported here encode proteins functioning in different compartments of the endomembrane system, it was interesting to test whether they have a similar or different pattern of expression regulation. To study their expression in different tissues, total RNA was extracted from root tips (R), young leaves (L), and maturing kernels (K) at the onset of storage protein synthesis and deposition, at around 15 d after anthesis (DAA). These RNAs were then subjected to Northern blot analysis using the three cDNAs as probes. As shown in Fig. 1, Sec61a mRNA was much more abundant in maturing kernels than in root tips, and appeared only as a faint band in RNA derived from leaves. By contrast, the mRNA levels of BP-80 and COPa were most abundant in root tips as opposed to maturing kernels and leaves. The BP-80 mRNA level in leaves is slightly less abundant than in maturing kernels and COPa mRNA was more abundant in leaves than in
To whom correspondence should be addressed. Fax: q972 8 9344181. E-mail:
[email protected]
ß Published by Oxford University Press
2388 Shy et al.
Fig. 1. Northern blot analysis of TaSec61a, TaBP-80 and TaCOPa in different tissues. Total RNA extraction was as described ( Tang et al., 1997) with a few modifications for kernels. Ground kernels were extracted twice in phenol, dissolved in a 1 : 1 mixture of phenol and 1 M TRIS–HCl pH 9, centrifuged at 13 000 g for 5 min, and the supernatant was further extracted with phenol : chloroform : isoamilalcohol (25 : 24 : 1 by vol.) and ethanol precipitated. The RNA pellet was dissolved in 2.5 M LiCl2 for 2 h and ethanol precipitated. Northern blot analysis was performed as previously described (Tang et al., 1997). Equal amounts of total RNA from young leaves (L), root tips (R) and developing seeds at 15 d after anthesis (S) were fractionated on a gel, transferred to membrane and hybridized TaSec61a, TaBP-80 and TaCOPa as probes.
maturing kernels. These results indicate that Sec61a mRNA is significantly enriched in maturing kernels than in root tips and young leaves relative to the mRNA levels of BP-80 and COPa. The relative mRNA levels of these three endomembrane genes were also compared during wheat kernel development at 8, 15 and 20 DAA. As shown in the Northern blot of Fig. 2, mRNA levels of all three were highest at the early stages of kernel maturation (8 DAA), and were reduced with the increasing age of the kernels. This confirmed previous observations from maturing barley kernels (Mogelsvang and Simpson, 1998), showing that the levels of different endomembraneassociated proteins (BiP, PDI, Sar1, Sec12, calreticulin, and calnexin) were high in young kernels and decreased as the kernel matures. These observations suggest that young kernels, at the stage of embryo and endosperm differentiation and development, possess an elaborate endomembrane system while the maturing kernels, at the stage of synthesis and accumulation of reserves have a simpler one. Despite the general reduction of the Sec61a, COPa and BP-80 mRNAs levels with kernel maturation, the level of Sec61a mRNA, compared to the other two, is higher in kernels during storage proteins synthesis (15–20 DAA)
Fig. 2. Northern blot analysis of TaSec61a, TaBP-80 and TaCOPa in developing seeds. RNA extraction and Northern blot analysis were performed as described in Fig. 1. Equal amounts of total RNA from seeds at different developmental stages (8, 15 and 30 DAA) were fractionated on a gel, transferred to membrane and hybridized TaSec61a, TaBP-80 and TaCOPa as probes.
relative to young kernels before that stage. This result is consistent with prior electron microscopy observations that indicate a paucity of Golgi in wheat endosperm cells in which the primary assembly of proteins is into ER-derived protein bodies.
References Galili G, Altschuler Y, Levanony H. 1993. Assembly and transport of seed storage proteins. Trends in Cell Biology 3, 437–443. Galili G, Herman EM. 1997. Protein bodies: storage vacuoles in seeds. Advances in Botanical Research 25, 113–140. Galili G, Sengupta-Gopalan C, Ceriotti A. 1998. The endoplasmic reticulum of plant cells and its role in protein maturation and biogenesis of oil bodies. Plant Molecular Biology 38, 1–29. Hara-Nishimura I, Shimada T, Hatano K, Takeuchi Y, Nishimura M. 1998. Transport of storage proteins to protein storage vacuoles is mediated by large precursor-accumulating vesicles. The Plant Cell 10, 825–836. Levanony H, Rubin R, Altschuler Y, Galili G. 1992. Evidence for a novel route of wheat storage proteins to vacuoles. Journal of Cell Biology 119, 1117–1128. Mogelsvang S, Simpson DJ. 1998. Changes in the levels of seven proteins involved in polypeptide folding and transport during endosperm development of two barley genotypes differing in storage protein localisation. Plant Molecular Biology 36, 541–552. Shotwell MA, Larkins BA. 1989. The biochemistry and molecular biology of seed storage proteins. In: Marcus A, ed. The biochemistry of plants, Vol. 15. San Diego, CA: Academic Press, 297–345. Tang G, Miron D, Zhu-Shimoni JX, Galili G. 1997. Regulation of lysine catabolism through lysine-ketoglutarate reductase and saccharopine dehydrogenase in Arabidopsis. The Plant Cell 9, 1305–1316. Vitale A, Galili G. 2001. The endomembrane system and the problem of protein sorting. Plant Physiology 125, 115–118.
