Journal of Experimental Botany, Vol. 54, No. 381, pp. 213-221,
January 2, 2003
© 2003 Oxford University Press
Seed germination is blocked in Arabidopsis putative vacuolar sorting receptor (atbp80) antisense transformants
Received 14 March 2002; Accepted 26 August 2002
1 UMR 5546 CNRS/Université Paul Sabatier, Pôle de Biotechnologies Végétales, 24 Chemin de Borde Rouge, BP 17 Auzeville, 31326 Castanet-Tolosan, France
2 UMR 5096 CNRS/Université Perpignan, 52 Av. de Villeneuve, 66860 Perpignan, France
3 Present address: Institute of Biomedical and Life Sciences, Glasgow University, University Avenue, Glasgow G12 8QQ, UK.
4 To whom correspondence should be addressed. Fax +33 5 62 19 35 02. E-mail: galaud{at}smcv.ups-tlse.fr
5 V. Laval and F. Masclaux contributed equally to this work.
Abbreviations: atbp80, Arabidopsis thaliana binding protein 80 kDa; EGF, epidermal growth factor; EST, expressed sequence tag; peabp80, pea binding protein 80 kDa; VSR, vacuolar sorting receptor.
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Abstract |
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The membrane receptor protein from pea, peabp80, has been shown to function by in vitro binding studies, and in vivo in yeast mutant, as a vacuolar sorting receptor (VSR). Families of proteins with homology to peabp80 have been identified in many plants including Arabidopsis. The family of membrane receptors, atbp80a–f (Arabidopsis thaliana binding protein 80 kDa) is highly homologous to peabp80 and may also function as vacuolar sorting receptors. Interactions with vacuolar sorting determinants have been shown only in vitro for atbp80b. In this paper, atbp80b was over- and under-expressed in Arabidopsis. Transgenic plants that over-expressed atbp80b showed no visible phenotype. However, antisense transformants were defective in germination. In non-germinating antisense transformants the embryo appeared to be normal, but, using several methods, it was not possible to rescue the non-germinating seeds, indicating that the mechanisms were probably independent of a seed-coat-imposed inhibition. To make a correlation between the lack of germination and gene expression, transcription analysis of all atbp80 genes was performed in the non-germinating antisense seeds indicating that all the normally transcribed genes were not detected. Then, a gene expression study of atbp80s genes was carried-out following seed imbibition and in various organs during wild-type plant development showing that all the genes from the family were transcribed and differentially expressed.
Key words: Antisense, Arabidopsis thaliana, embryogenesis, vacuolar sorting receptor, germination.
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Introduction |
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A family of type I membrane receptors identified as putative vacuolar sorting receptors (VSRs) by Paris et al. (1997), or as ELPs for EGF- Like receptor Proteins by Ahmed et al. (1997), is found in plants. The Arabidopsis genome sequencing project has identified, on the basis of sequence homology, seven putative genes named atbp80a (previously named AtELP2b), atbp80a' (AtELP2a), atbp80b (AtELP1), atbp80c (AtELP4), atbp80d (AtELP5), atbp80e (AtELP6), and atbp80f (AtELP3). The nomenclature is still confusing and that proposed by Hadlington and Denecke (2000) was chosen. EST sequencing programmes have only identified transcripts from atbp80b, atbp80a and atbp80f. Several laboratories independently identified the first member of this family from pea clathrin-coated vesicles and different preparations from Arabidopsis (Paris et al, 1997; Ahmed et al., 1997; Neuhaus and Rogers, 1998; Laval et al., 1999).
Bp80 genes encode integral membrane proteins with a large lumenal or extracellular domain containing three cys-rich regions similar to epidermal growth factor (EGF)-like repeats, and a short cytoplasmic domain. At the subcellular level, atbp80 has been found in clathrin-coated vesicles in pea cotyledons and Arabidopsis cell cultures (Ahmed et al., 1997; Hinz et al., 1999; Kirsch et al., 1994). They have also been found in the trans Golgi network, and in a prevacuolar compartment in stigmas from Nicotiana alata (Ahmed et al., 1997; Hillmer et al., 2001; Miller et al., 1999; Paris et al., 1997). Finally, atbp80 was also localized on unidentified endomembrane fractions as well as a plasma membrane rich-fraction of Arabidopsis cell cultures (Laval et al., 1999).
Several publications point to a role for atbp80 as VSRs in plants, equivalent to animal mannose 6-phosphate receptors (Kornfeld, 1992), or the VPS10p receptor from yeast (Marcusson et al., 1994). Peabp80 (also named BP80 or VSR-PS-1) binds as a monomer to polypeptide ligands containing a central NPIR motif at pH 6.0 to 6.5 and this binding was abolished at acidic pH leading to ligand release into the prevacuolar compartment (Kirsch et al., 1994; Cao et al., 2000). In vitro binding to this motif has been also demonstrated for atbp80b (Ahmed et al., 2000). Humair et al. (2001) have recently demonstrated by using an in vivo system, that peabp80 was able to function as a VSR in yeast mutant.
Unlike other eukaryotes, many plants cells contain two distinct vacuolar compartments. One functions as a digestive organelle, similar to the lysosome in animal cells. It contains hydrolytic enzymes and accumulates secondary metabolites (Wink, 1993). The second type functions as a protein storage compartment, especially in seeds but also in vegetative tissues such as roots and tubers (Muntz, 1998; Staswick, 1994; Neuhaus and Rogers, 1998). It is now clear that these two types of vacuoles are separate organelles (Hoh et al., 1995; Paris et al., 1996). The bp80 proteins have been found in vesicles containing proteins targeted to the lytic vacuole, but not in the dense vesicles containing storage proteins (Hinz et al., 1999; Jiang and Rogers, 1999; Miller et al., 1999). However, there is one report that suggests that these proteins may function as sorting receptors for pumpkin storage proteins (Shimada et al., 1997).
With the exception of peabp80, none of the other proteins have been shown to function as VSRs in vivo. In contrast to the vacuolar sorting receptors in animal cells or in yeast, atbp80s, the putative plant vacuolar sorting receptors in Arabidopsis, are members of a large gene family. Are these proteins expressed differentially during plant development, and do all members of this family share the same function? In this paper, a gain or loss of function strategy was used on whole plants to get new data on the biological role of this protein family in planta. Transgenic Arabidopsis plants were constructed over- or under-expressing atbp80 genes, using sense or antisense atbp80b constructs. Antisense transformants produced apparently normal seeds, but in some lines fewer than 10% of the seeds germinated. A gene expression analysis of all the atbp80 was performed in non-germinating antisense seeds indicating that none of the atbp80 is detected. At present, only three atbp80 genes (atbp80b, a and f) are known to be transcribed referring to the EST sequencing programs. Atbp80 gene expression was analysed during the first days following imbibition in various organs of wild-type plants. All the genes were expressed and showed different patterns of expression.
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Materials and methods |
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Plant material
Arabidopsis thaliana, ecotype Columbia (Col-0), was cultured in a grown chamber with 16/8 h light/dark photoperiod, given by fluorescent tubes 36W (12 W m–2) at 20 °C temperature. Seeds were sterilized by 5 min incubation in 2.6% sodium hypochlorite and washed three times with 70% ethanol. Plants were grown in pots filled with TKS2 peat Floratorf supplemented with 1
(w/w) nitrate.
Sense and antisense constructs
For the antisense construct, the coding region of atbp80b (accession No. U 86700; Laval et al., 1999), from the ATG to the stop codon was introduced in antisense orientation in the expression vector pBin19pJR1, which contains the 35S CaMV promoter (Smith et al., 1988). Ten pmoles of primer 5'-ATATGTCGACATGAAGCTTG GGCTTTTCAC-3' and 5'-TAATGGTACCCCACTTATTTCTG TTTGTGGC-3', allowing the introduction of SalI and KpnI restriction sites, were used for PCR amplification. The amplified DNA was cloned in pGEM-T vector (Promega) before sequencing (Sanger et al., 1977). The vector was digested and the fragment of interest was cloned into the predigested KpnI-SalI pBin19pJR1 vector.
A similar procedure was followed for the sense construct, using primers 5'-ATATGTCGACGCACAGTTGAAGTGAACT TGC-3' and 5'-ATTAGGTACCATGAAGCTTGGGCTTTTCAC-3' to introduce the coding region of atbp80b from the ATG to poly A tail in the forward orientation under the 35S-promoter control in pBin19pJR1.
Plant transformation
The constructs were mobilized in Agrobacterium tumefaciens strain C58Ci (pMP90) (Koncz and Schell, 1986) by the heat shock method derived from (Holsters et al., 1978). Agrobacterium-mediated transformation of Arabidopsis was performed by incubating standard floral tip. Sterilized seeds obtained from self-fertilized primary transformant (T1) were germinated on MS medium supplemented with 50 µg ml–1 kanamycin, 10% sucrose and 7% agar. After 2 weeks, kanamycin T1-resistant seedlings were individually transplanted into pot filled with TKS2, grown to maturity self-fertilized and T2 seeds were harvested.
Germination assays
Sterilized T2 antisense (AST2) seeds were sown on MS culture medium, complemented or not with 10–4 M GA (Sigma), or with a mix of amino acids (Sigma). Seeds were incubated 2 d at 4 °C prior to the transfer to culture chamber at 20 °C with 16/8 h light/dark photoperiod. Germination was scored after 10 d of incubation.
To determine if seed integuments inhibit germination, wild-type and T2 antisense integuments were mechanically removed. Embryos were placed on MS culture medium recovered by 50 µl of MS liquid and observed every day for 3 weeks.
Analysis of embryo cell viability
To estimate embryo viability, integuments from wild-type and T2 antisense seeds were removed and embryos were incubated in solution containing 1% FDA (fluorescein diacetate) for 30 min, washed in distilled water and observed under fluorescent microscopy at 470–490 nm excitation.
Histological analysis
Seeds or plantlets were fixed with 3% glutaraldehyde in sodium cacodylate 0.1 M pH 7.2 for 2 h, post-fixed with osmium tetroxyde (1%) for 1 h, then dehydrated by serial incubation in ethanol for 15 min increasing the alcohol concentration and embedded in spurr resins for 1 week (Spurr, 1969). Resin polymerization was carried out for 48 h at 60 °C. Semi-thin sections were cut with an ultracut E microtome and stained with toluidine blue 0.5% in water before microscopic observation.
DNA analysis
PCR assays on genomic DNA were performed to verify the presence of the transgene in the different plants or seeds. The presence of the NptII gene was confirmed by PCR by using primers 5'-GA GGCTATTCGGCTATGACTG-3' and 5'-ATCGGGAGCGGCGA TACCGTA-3'. Genomic DNA was prepared according to Dellaporta et al. (1983). Fifty ng of genomic DNA were used for PCR amplification with 50 pmol of the corresponding primers. PCR amplification was carried for 40 cycles of 1 min denaturation at 95 °C, 1 min annealing at 50 °C and 1 min extension at 72 °C. The amplification products were loaded on 2% agarose gel, separated by electrophoresis, and stained with ethidium bromide.
RT-PCR analysis
Total RNA was extracted from various organs at different stages. First strand cDNA synthesis was performed using oligo-dT and specific oligonucleotides used for the PCR reactions were complementary to two separated exons, allowing the products from genomic DNA and cDNA to be differentiated. The primers used for atbp80b (at3g52850) were 5'-TTGGTGACGGTTAC ACTCAC-3' and 5'-TCCATATGGTGACCACTTGTGTTGG-3'; primers 5'-AAGTGAGATCAGCATGGGC-3' and 5'-AGGCGA GAAAACATCGTCGTT-3' for atbp80a’ (at2g14740); primers 5'-AAGTGAAATCAGCGTGGGC-3' and 5'-CAGTTCTCCGTAG CTACATCG-3' for atbp80a (at2g14720); primers 5'-ACGGCTTA ACTTTCTCTGCTTGC-3' and 5'-GATCTCAAACATTTCTTT GTGTATG-3' for atbp80f (at4g20110); primers 5'-GAGTGA AGCGAGTGTAGCCTCC-3' and 5'-CGGTGGGGTGGAGTTC ATGCACAGGGAGG-3' for atbp80c (at2g30290); primers 5'-GGT GATCCTGATGCTGATGTAGAGAATG-3' and 5'-GAGGGCA ACGACATCCTGATGTCTCTGAG-3' for atbp80d (at2g34940); primers 5'-GTGCAGAGAGAGTTGTTGAATCTCTAG-3' and 5'-GAGATCACAGTCATTGGATCGGTTGTG-3' for atbp80e (at1g30900). Primers 5'-GTCCAGTGTCTGTGATATTGCACC-3' and 5'-GCTTACGAATCCGAGGGTGCC-3' for ß-tubulin (M21415) were used as a control. PCR amplification was carried for 35 cycles of 1 min denaturation at 95 °C, 1 min annealing at 50 °C and 1 min extension at 72 °C.
Immunodetection
Antibodies were raised, as previously described by Laval et al. (1999), against selected peptides derived from atbp80b, peptide 63 (AEQESQIGKGSRGDC) and peptide 64 (NNRQYRGKLEC) which are conserved in most of the members of the family. A mixture of those two antibodies was used to detect atbp80 in wild-type, antisense T2 lines, and sense T2 lines. To extract proteins, tissues from 4-week-old plants were ground in liquid nitrogen and extracted with 62.5 mM Tris-HCl, pH 6.8, and 2% SDS at 65 °C for 30 min. Extracts were centrifuged for 30 min at 10 000 g and the protein concentration was determined (Smith et al., 1985). Gel electrophoresis and antibody binding competition assays were carried-out as previously reported (Laval et al., 1999).
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Analysis of atbp80b T2 sense and antisense transformants
Transgenic plants with constructs designed to over- or under-express atbp80b, were obtained. The nucleotide sequence of atbp80b is 61–84% identical to the other members of the family in Arabidopsis. The complete coding region of atbp80b (1872 bp) was used for the antisense construct to inhibit the expression of multiple members of the gene family (Fig. 1A). For the sense transformants, the coding region of atbp80b including the 3'-UTR (which is different for each gene in the family) was used (Fig. 1B).
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Around 500 seeds for each transformant were tested for germination. Wild type seeds show between 80% and 100% germination as did five sense transformants (ST2 lines C, D, E, F, and G) tested (Fig. 1C). All of the ST2 transformants were similar to wild type in appearance. Most of the eleven AST2 seed lines showed significantly reduced germination (Fig. 1C, AST2, lines M, D, O, G, N, F, L, K, I, E, J), ranging from 4% for line J to 100% for line M. It is assumed that the defect of germination is not the consequence of a T-DNA disruption of a functional gene because most of the antisense transformants have the same phenotype. Line to line variability in germination is probably due to different levels of expression, as expected for antisense transformants.
Transformants AST2E, J, I and K showed the strongest inhibition of germination. In these four lines, less than 25% of the seeds germinated and developed as in the wild type. PCR on antisense lines using primers to the NPTII and atbp80b constructs confirmed that all contained a complete construct (data not shown). Also, southern blots (data not shown) revealed that AST2E, I and K developing plants contain at least two insertions and AST2J, four insertions of the T-DNA.
Analysis of AST2 non-germinating seeds.
Four antisense lines showing the lowest level of germination: line K (25% germination), lines I and E (10% germination), and J (4% germination) were analysed further. In these lines, germinating seeds were distinguished from non-germinating, sowing the T2 on MS medium for 10 d. The germinated ones were isolated and T3 seeds were collected from these individuals. Eighty to 100% of these T3 seeds germinated. Possibly, in those seeds that germinated, the antisense construct has been heritably silenced at meiosis by epigenetic mechanisms (Kooter et al., 1999).
To establish a correlation between the absence of germination and atbp80 gene expression level, a transcriptional analysis by RT-PCR was carried-out on non-germinating AST2K and wild type dry seeds, imbibed wild-type seeds, and on wild-type plants at the cotyledon stage (Fig. 2). Results show that only the atbp80b, atbp80a' and atbp80a genes were transcribed in mature dry seed and following imbibition. In AST2K non-germinating seeds, none of these genes were expressed, indicating that the antisense strategy was efficient to inhibit the expression of several members of the atbp80 family. From this analysis, it was showed that atbp80d and atbp80e mRNAs were detected only when the plants reached the cotyledon stage. At present, these genes were not found in EST databases and it can be concluded that these genes are not pseudogenes. However, for atbp80c, no mRNA was detected in the conditions tested.
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Failure to germinate may be caused by various factors, seed viability, dormancy, or seed-coat-imposed inhibition (Dubreucq et al., 1996). To check if the sterilization procedure can influence the germination rate of non-germinating antisense seeds, these were sown directly in soil, but the germination rate was unmodified. To evaluate seed viability, embryos were isolated from seeds that failed to germinate after 8 d, as well as from non-transformed seeds, and incubated with 1% FDA. Wild-type embryos boiled for 15 min did not fluoresce, but untreated wild-type, AST2E, and AST2I embryos fluoresced strongly (Fig. 3) indicating that the embryo cells are living. Histological analysis of wild-type, AST2E, and AST2I embryos from non-germinating seeds confirmed that the shape of the embryos, and the cellular organization was similar between types (data not shown). To break putative dormancy, chilling or gibberellin treatment (10–4 M) was carried out; however, neither treatment improved germination. To distinguish between seed-coat-imposed inhibition and embryo-blocked germination, naked embryos from wild-type, AST2E, I, and J seeds were sown in MS medium. Under such conditions, less than 10% of the AST2 transformants germinated compared to 80% of wild-type embryos. Also, both control and antisense seeds show a normal uptake of water in the first 24 h of imbibition confirming that the seed coat was not impermeable.
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Atbp80 has been implicated in the transport of vacuolar peptidases (Ahmed et al., 2000; Paris et al., 1997), and antisense transformants defective in the VSR might be unable to mobilize reserve proteins for germination. The medium was supplemented with a mixture of the 20 amino acids to support protein synthesis during germination, but no change in the germination pattern was observed.
AST2 seedlings show an arrest in development.
As stated before, about 1% of AST2 lines E, and J germinated, but an abnormal development of the seedling was observed. Seedlings showed a long root, a thick but short hypocotyl, and no photosynthetic tissues. Cotyledons never developed and became green. They were frequently enclosed in the integument (Fig. 4A). Histological sections (Fig. 4B) of 2-month-old AST2E transformants were compared to 5-d-old wild-type plants. At the root level, vascular tissue contained bigger cells than the wild type (Fig. 4B1, 2), and the cortex and epidermal cells were collapsed and without structure. Hypocotyl sections (Fig. 4B3, 4) showed a disorganized structure in the AST2 seedlings. The epidermal layer was detached from the cortex cells. Vascular tissues were not clearly organized and many cells looked collapsed (cf. Fig. 4B5 and 4B6).
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Atbp80 protein content in the various different transgenic lines was detected using antibodies raised against two different synthetic peptides derived from atbp80b (Laval et al., 1999). Extracts from 1-month-old plants transformed with the sense construct showed a strong band of the expected size of 80 kDa (Fig. 5). However, the antibodies can potentially recognize any of seven atbp80 gene products, since the peptide sequence used for raising the antibodies is highly conserved in all atbp80 proteins. Some weaker upper and lower bands were detected. These could correspond to modified forms of atbp80 glycosylated product, degradation products and/or cross-reacting unrelated proteins. As in the wild-type plants, atbp80 protein was also detectable serologically in 5–10% T2 plants of antisense lines AST E, I, and J that germinate. These data suggest that the level of atbp80s is sufficient for germination and agreed with a probable silencing of antisense constructs.
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Atbp80 gene expression in various organs during plant development
At present, only atbp80b, atbp80a, and atbp80f are found in EST databases. Are all atbp80 transcribed, or some of them are pseudogenes? An expression study of the all atbp80 genes was performed on roots, young and old leaves, floral stalk, flowers, immature siliques (days 4–9 after pollination) or pre-mature siliques (days 10–17 after pollination). The beta tubulin gene was used as a control (Fig. 6). Results indicate that all the atbp80 are transcribed either in all the organs analysed (atbp80a', atbp80a and atbp80e) or in a particular organ such as atbp80c, which is only expressed in flowers. Except for atbp80a' and atbp80a genes, which are expressed in all the conditions and in all the organs analysed (Figs 2, 6), the other genes present a specific expression profile. Atbp80b gene was transcribed in all the conditions except in pre-mature siliques. Atbp80f mRNAs were detected in roots, floral stalk, and young organs (leaves and siliques), but not in old leaves or in old-siliques or in the flowers. A relationship can be established between atbp80f gene expression and the age of the tissue. Atbp80d mRNAs were detected in every tissue except in pre-mature siliques. Finally, the atbp80e gene was expressed in each tissue except in dry seeds and after imbibition as reported in Fig. 2.
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Discussion |
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Arabidopsis was transformed with sense and antisense atbp80b. All the sense lines germinated, grew, and reproduced normally. The sense transformants indicated an increase in atbp80 proteins as revealed by atbp80 antibodies, suggesting that over-expression of the gene and accumulation of atbp80b protein does not change the phenotype in the growing conditions described here. Eleven independent antisense transformants showed variable degrees of germination from 4–100%. From each line seeds could be divided into three categories; (1) those that germinated, grew, and reproduced normally; (2) non-germinating seeds; and (3) seeds that germinated but then failed to develop further. In antisense plants derived from seeds that germinated normally (group 1), atbp80 proteins were detectable serologically. Thus, it can be assumed that the non-germinating phenotype (group 2) may be linked to the lack of atbp80s in those antisense transformants as indicated by the atbp80 gene expression analysis. Among the seven members of atbp80 genes, only the atbp80b, atbp80a', and atbp80a transcripts are present in dry seed and following imbibition. Even if atbp80 proteins are well conserved at the amino acid level, the expression data reported here clearly suggest that atbp80 proteins may participate in specific developmental steps. In non-germinating antisense seeds, none of these genes were detected. This indicates that the complete coding region of atbp80b, used for the antisense construct, was able to inhibit the expression of other members of the family. The embryo appeared to be normal in group 2 antisense transformants, and it was not possible to rescue the non-germinating seeds, indicating that the defect in germination was independent of a seed-coat-imposed inhibition.
One per cent of the AST2 germinating-seeds from lines E and J did not develop and the seedlings remained at the cotyledon stage (group 3). The level of gene expression or atbp80 protein content in those plants cannot be evaluated. Histological studies revealed alterations in the structure of cell walls, mainly in the root and hypocotyl cortex. In the hypocotyl, the epidermis detached from cortical cells suggesting weak cell adhesion in these lines. The mechanism by which the absence or low levels of atbp80s may alter cell wall structure is unclear, but two hypotheses can be stated. First, if atbp80 is missing, some vacuolar hydrolases may take the default pathway to the extracellular space and degrade cell wall components. Second, some members of the atbp80 family may be located either in the plasma membrane or associated vesicles (Laval et al., 1999) and therefore could participate in the deposition of cell wall material or trafficking between the ER and the plasma membrane. Recent results published by Brandizzi et al. (2002) show the importance of the length of the hydrophobic domain on subcellular location of peabp80. By using the same predictive method (TMHMM version 2.0 program), all the transmembrane domains of atbp80 proteins are 23 amino acids long and with reference to Brandizzi et al. (2002), these proteins may potentially be located on the plasma membrane. It can reasonably be proposed that suppression of atbp80 expression is responsible for the germination defect and abnormal development. These results are the first demonstration of phenotypes with a reduced level of atbp80 in planta.
Bp80 proteins are believed to function as VSRs. They target to the lytic vacuole soluble proteins containing the sorting signal (NPIR) within the NH2-terminal propeptide (Ahmed et al., 2000; Cao et al., 2000). The cargo proteins transported by the receptors can be proteases such as aleurain in barley and Arabidopsis. In aleurone cells of barley, the degradation of the storage proteins in the protein storage vacuoles depends on the synthesis of cysteine proteases. It was not possible to verify the presence of the protease AtALEU (a protein highly homologous to barley aleurain) in either wild-type or antisense seeds, using an antibody raised against the barley protein (kindly supplied by J Rogers, Washington State University). Humair et al. (2001) showed that the peabp80 protein functions as a VSR in vivo using the vps10p yeast mutant. Peabp80 was able to target proteins sharing the motif NPIR and not the typical C-terminal vacuolar determinant signal. The non-germinating antisense phenotype described in this paper seems to confirm that function a priori, since the absence of specific proteases in the seed will produce an embryo unable to mobilize reserve proteins. A low level of atbp80 may be responsible for a failure to target cysteine proteases to the lytic vacuole. However, the cotyledons developmental arrest observed in the group 3 antisense transformants suggests that germination itself is independent of mobilization of cotyledon reserves.
It has been suggested that all the members of the atbp80 family may share the same function. This is based on the presence of a much-conserved region on the C-terminal cytoplasmic tail, conserved not only within the Arabidopsis proteins, but also in orthologous proteins from monocots and dicots (Cao et al., 2000; Hadlington and Denecke, 2000). However, if the conserved cytoplasmic sequence is certainly involved in the recruitment of coat proteins for clathrin coated vesicle formation and, such as in yeast, for the recycling of the receptor, only the transmembrane domain seems to be necessary for aleurain targeting to the vacuole (Jiang and Rogers, 1998). It will be important to determine if other bp80s are also capable of functioning in the vps10p yeast mutant targeting to the vacuole NPIR-containing or other vacuolar proteins. This will support the putative VSR function. Atbp80 proteins are members of a multigenic family and these results on expression of atbp80s during plant development in Arabidopsis indicate that all the genes are expressed differently and this implies different functions during development. Atbp80s may have a role in early germination, in the deposition of cell wall material or trafficking between the ER and the plasma membrane and in mobilization of storage proteins.
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Acknowledgements |
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The University Paul Sabatier and the CNRS supported this work. VL and FM are fellows from the Ministère de l’Education Nationale, de la Recherche et de la Technologie, France. We are grateful to A Jauneau (IFR40, Toulouse, France) for the microscopic analysis and to Dr JJ Milner for valuable advice and for reading this manuscript.
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