Journal of Experimental Botany, Vol. 52, No. 365, pp. 2387-2388,
December 1, 2001
© 2001 Oxford University Press
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
1 Department of Plant Sciences, the Weizmann Institute of Science, Rehovot, Israel
2 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 Sec61
, (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 COP. 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-80/Sec61 and the COP/Sec61 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 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 Sec61 (AtSec61) (GenBank Accession No. AF161718), the major subunit of Golgi COPI coatomer COP (AtCOP) (GenBank Accession No. AF176226), and the plant-specific 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 TaSec61, namely, TaSec61p, exhibits 67% identity and 76% similarity to the rat Sec61p. The C-terminal third of the TaCOPp 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, Sec61 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 COP 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 COP mRNA was more abundant in leaves than in maturing kernels. These results indicate that Sec61 mRNA is significantly enriched in maturing kernels than in root tips and young leaves relative to the mRNA levels of BP-80 and COP.
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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 endomembrane-associated 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 Sec61, COP and BP-80 mRNAs levels with kernel maturation, the level of Sec61 mRNA, compared to the other two, is higher in kernels during storage proteins synthesis (15–20 DAA) 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.
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Notes
3 To whom correspondence should be addressed. Fax: +972 8 9344181. E-mail: lpgad{at}wiccmail.weizmann.ac.il 
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