Download Expression patterns of genes encoding endomembrane proteins

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
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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.
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