Isolation and expression analysis of the AtSUC5 gene
A reverse genetic approach was used to unravel the molecular mechanisms involved in seed maturation in A. thaliana. The first step consisted of a non-quantitative RT-PCR study aimed at isolating the genes for carbon metabolism expressed specifically during seed development. Among the candidates thus isolated, a putative sucrose transporter gene was identified which proved to be AtSUC5 (Figure 1). AtSUC5 transcripts were detected in early developing seeds (data not shown). This was consistent with the report on expressed sequence tags (EST) from green siliques previously described [available at http://www.arabidopsis.org].
To complete these preliminary data, the expression pattern of AtSUC5 was then further investigated in various tissues of the wild-type ecotype Wassilewskija (Ws) using a quantitative RT-PCR strategy. Specific primers were designed at the 5′ end of the AtSUC5 transcript to amplify a 189 bp fragment (see Experimental procedures). The results obtained were standardized to the expression level of the EF1αA4 (EF) gene (Liboz et al., 1990; Nesi et al., 2000). In vegetative organs, in flowers and in young siliques aged 3 DAF, AtSUC5 mRNA was detected at very low levels (Figure 2a). Likewise, AtSUC5 transcripts were hardly detectable in silique walls after 4 DAF (data not shown). The relative expression profile of the gene was finally investigated in developing seeds. A six-point kinetic analysis was performed (Figure 2b) that was representative of the three stages of A. thaliana seed development (Baud et al., 2002): early embryonic morphogenesis at 4 and 6 DAF, maturation at 9, 12 and 15 DAF, and late maturation at 18 DAF. At 4 DAF, an EF expression level of 4% was measured. The AtSUC5 transcript level then increased tenfold between 4 and 6 DAF, peaking at 40%EF, before falling sharply to 8%EF at 9 DAF. The AtSUC5 transcript was not detected further during late maturation.
Figure 2. AtSUC5 expression pattern. Quantitative RT-PCR analysis of AtSUC5 expression was performed in plant organs (a) and developing seeds (b). The results obtained are standardized to the constitutive EF1αA4 gene expression level. Values are the means ± SE of three replicates carried out on cDNA dilutions obtained from three distinct mRNA extractions: Ge, germinating seeds; Ro, roots; St, stems; Le, leaves; Fl, flowers; Sil, silique aged 3 DAF. The scale is different in (a) and (b).
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Pattern of AtSUC5 promoter activity
To gain further insights in AtSUC5 expression, the spatiotemporal activity of the AtSUC5 promoter was investigated. A 1.5 kb fragment (referred as to ProSUC5) was fused translationally to both the uidA reporter gene (which encodes GUS), and the mGFP5-ER reporter gene [which encodes GFP fused to an endoplasmic reticulum (ER) retention sequence]. GUS activity was investigated using fluorometric assays. No significant activity could be found in vegetative organs (data not shown). On the contrary, a strong peak of GUS activity was measured in early developing seeds (Figure 3a), and was localized in the endosperm (data not shown). The slight shift observed when GUS activity measurements are compared with quantitative RT-PCR data might reflect both the delay due to translation and the stability of GUS protein. The ProSUC5:mGFP5-ER construct was used to monitor AtSUC5 promoter activity at various stages of seed development. A strong GFP signal was first detected 3–4 DAF in the micropylar region of the endosperm (Figure 3b,c). At 6 DAF, ProSUC5:mGFP5-ER expression progressed at the micropylar pole (Figure 3d). Observation of confocal sections at the globular stage of embryo development showed that the GFP signal was endosperm specific and confirmed that the signal was absent in the embryo and in its suspensor (Figure 3d). At the torpedo stage, expression extended to the chalazal pole of the endosperm. A strong signal was observed around the chalazal endosperm cyst, at the interface with the maternal tissues (Figure 3e). Finally, expression decayed at the upturned-U stage, 9 DAF, as the endosperm became limited to a few cell layers embedding the maturing embryo (Figure 3f).
Figure 3. Analysis of AtSUC5 expression pattern. (a) GUS activity in transgenic ProSUC5:uidA seeds at various developmental stages. Values are the means ± SE of five replicates carried out on two independent lines. (b)–(k) Expression of ProSUC5:mGFP5-ER. GFP activity is observed with a standard fluorescence microscope in transgenic seeds (b–f) and compared with the autofluorescence of corresponding wild-type seeds (g–k). Seeds were observed 3 (b, g), 4 (c, h), 5 (d, i), 7 (e, j) and 9 DAF (f, k). Bar = 50 μm. (l–n) Analysis of AtSUC5 expression pattern by in situ hybridization. A nucleotide sequence specific to AtSUC5 was identified which allowed oligonucleotide-hybridization experiments to be carried out to characterize the AtSUC5 expression pattern in developing seeds. Wild-type seeds aged 5 (l) and 9 DAF (m) are presented. Control experiments were carried out on suc5 seeds. A suc5-1 seed aged 7 DAF is presented in (n). Bar = 100 μm. c, chalazal pole; e, endosperm; gem, globular embryo; i, integuments; m, micropylar pole; tem, torpedo-shaped embryo; uem, upturned-U shaped embryo.
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Study of AtSUC5 expression by in situ hybridization
An in situ hybridization experiment was carried out to link the activity of the AtSUC5 promoter to mRNA accumulation and to precise expression of AtSUC5 in maturing seeds, where the detection of GFP signal is severely compromised by the integuments. Due to the high level of similarity between the sequences of AtSUC1 and AtSUC5 in particular, an oligonucleotide hybridization approach was chosen. The specificity of the oligonucleotide used was demonstrated using null suc5 mutants as negative controls (see below). In the wild-type seeds, AtSUC5 transcripts were specifically detected in the endosperm. At the globular-to-heart transition stage, a strong staining was observed in the region of the endosperm surrounding the embryo, at the micropylar pole (Figure 3l). At the torpedo stage (Figure 3m) and at the bent-cotyledon stage (data not shown), the transcripts had spread to the whole endosperm, and a strong staining could be observed in the cell layer directly facing the embryo. The results obtained were consistent with the GUS and GFP expression profiles previously described (Figure 3).
Isolation and molecular characterization of three suc5 mutants
The AtSUC5 cDNA sequence corresponding to the Ws ecotype was cloned, sequenced and compared with the Ler accession characterized by Ludwig et al. (2000). Eight nucleotide substitutions were detected in the Ws background compared with Ler, resulting in an S to G substitution at position 26 of the AtSUC5 peptide and the addition of three amino acids (GFH) at the C-terminal position of the peptide. To analyse the function of the AtSUC5 protein, the cloned cDNA was used to complement a sucrose uptake-deficient yeast mutant (data not shown), thus confirming its sucrose transport capacity. To investigate the role of the AtSUC5 transporter in planta, suc5 alleles were then isolated among the 50 000 transferred (T)-DNA lines of the Versailles collection (Bechtold et al., 1993). Three flanking sequence tags (FSTs) corresponding to the left T-DNA borders were generated (Balzergue et al., 2001), which were anchored to the genome sequence of A. thaliana in the AtSUC5 gene. Plant genomic DNA flanking the right and left T-DNA borders of the three corresponding suc5 mutants were amplified by PCR and sequenced, confirming the FSTs found in the FLAGdb/FST database. In the suc5.1 mutant (line EEU50), the T-DNA insertion resulted in a 2.7 kb deletion of genomic DNA at the insertion site (Figure 4a). In the suc5.2 mutant (line DYO5), the T-DNA insertion led to a 556 bp deletion beginning in the 5′-untranslated region (UTR) of the AtSUC5 gene and ending in its first exon. In the suc5.3 mutant (line T990), the insertion occurred in the 3′-UTR of the gene and resulted in a 32 bp deletion of genomic DNA.
Figure 4. Characterization of three suc5 mutants. (a) Structure of the AtSUC5 gene showing the position of the T-DNA insertion in suc5.1, suc5.2 and suc5.3. Closed boxes represent exons while open boxes stand for untranslated regions (UTR). Nucleotide positions are relative to the translational start site and primer positions are shown as arrows. LB, left border; RB, right border. (b) Quantitative RT-PCR analysis of AtSUC5 mRNAs in seeds aged 5–6 DAF. Values are the means ± SE of three replicates carried out on cDNA dilutions obtained from three distinct mRNA extractions. The asterisks indicate that transcripts were not detectable. (c) Characterization of chimeric AtSUC5-T-DNA transcripts in suc5.3 seeds. Three sets of primers were used for a PCR experiment on cDNA from suc5.3 and wild-type seeds aged 6 DAF, and on DNA from a hemizygous SUC5/suc5.3 plant. The position of the primers used is indicated in (a).
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Analysis of mRNAs from seeds aged 5–6 DAF was performed by quantitative RT-PCR on homozygous mutant lines (Figure 4b). AtSUC5 transcripts were not detected in suc5.1 or in suc5.2, but they were still present in the suc5.3 line. A set of different primers then allowed us to establish that the transcripts detected in the suc5.3 mutant were chimeric AtSUC5–T-DNA transcripts (Figure 4c), in agreement with the structure of the previously characterized mutation (Figure 4a). In situ hybridization experiments were carried out using the two putative null mutants, which confirmed both the lack of AtSUC5 transcripts in these mutants (Figure 3n) and the specificity of the probe used.
Study of suc5 seed development and maturation
The three mutant lines developed normally and no abnormal phenotype could be detected by visual observation of their progenies. The shape and colour of the mature and dry mutant seeds were similar to the wild type. Several cultures were conducted in the greenhouse to characterize the suc5 mutant phenotype. In each experiment the dry weight (DW) of the seed of suc5 mutants was reduced compared with the wild-type. Depending on the culture, the reduction in seed DW for the putative null alleles ranged from 5% to 29% of the wild-type. Although variations in the severity of this phenotype could be observed among the cultures conducted, the reduction in seed DW observed in the mutants was always statistically significant. Considering the very specific spatiotemporal expression pattern of AtSUC5, the whole process of seed development was then carefully investigated in the suc5 mutants.
First, microscopic observations of cleared seeds were carried out during early embryonic morphogenesis and early maturation. The structure of the three types of tissues comprising the seed, i.e. integuments, endosperm and embryo, appeared normal. However, a slight delay in embryo development was reproducibly observed in the mutant lines (Figure 5a). At 8 DAF, the wild-type embryo encountered a phase of rapid elongation, progressing from the torpedo stage (55% of embryos) to bent-cotyledon stages (walking stick and upturned-U stages; 44% of embryos). In the suc5.3 mutant line, fewer than 30% of the embryos exhibited cotyledon curvature, while in suc5.1 and suc5.2 (the putative null alleles), more than 80% of the embryos were still torpedo shaped. This observation was repeated twice on distinct embryo populations, and each time a statistically significant difference in embryo distribution between the wild-type and suc5 mutants was observed (chi-squared; α < 0.01). To confirm the link existing between this phenotype and the suc5 mutations, the mutant lines were crossed. For each of the three crosses, the progeny obtained was cultured and genotyped. No complementation of the phenotype could be observed in F2 (data not shown).
Figure 5. Effect of the suc5 mutation on seed development. (a) Delayed elongation of the embryo in suc5 seeds. Microscopic observations of developing seeds aged 8 DAF produced by mutant and wild-type plants were made. The quick-clearing solution of chloral hydrate in which the seeds were mounted allowed us to distinguish the developmental stage of the embryo. The relative distribution of the embryos amongst four developmental categories, e.g. small torpedo, torpedo, walking stick and upturned-U stages, is presented. For each genotype, the number of embryos observed is indicated. (b) Altered accumulation of fatty acid in suc5 seeds. A time course analysis of total fatty acid concentration was made for suc5.1, suc5.2, suc5.3 and wild-type seeds. For each point of the kinetic and for each genotype, three independent measurements on batches of 20 seeds were performed. The three values obtained were averaged and the difference in fatty acid concentration compared with the wild-type, in %, was calculated for each mutant. The vertical arrow represents the peak in AtSUC5 expression. (c) Decreased content of fatty acids in mature suc5 embryos and endosperms. Measurements were first carried out on batches of 15 mature dry seeds. To investigate the fatty acid content of separate embryos and endosperms, dry seeds were imbibed for 2 h at 4°C and then dissected under a binocular microscope. Batches of 15 embryos or endosperms were prepared and analysed. Each value is the mean ± SE of five replicates. Statistical analysis of the results was performed using the Dunnet multiple comparisons test, the control corresponding to the wild-type values. One asterisk indicates a significant difference at the 95% level and two asterisks a very significant difference at the 99% level.
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Determinations of glucose, fructose, sucrose and starch were carried out on developing seeds aged 4–22 DAF. Sugar content exhibited a similar evolution in mutant and wild-type seeds (data not shown). To investigate the putative effect of the suc5 mutations on the accumulation of storage compounds, time-course analyses of fatty acid content were then made. A classical sigmoid pattern of oil accumulation which closely paralleled the increase in seed DW was observed in the wild-type (see Figure 5 in Baud et al., 2003). Until 6 DAF, the fatty acid concentration in mutant seeds was fairly similar to the wild-type (Figure 5b; Table 1). Then at 8 DAF, the suc5.1 and suc5.2 seeds exhibited an important reduction (−45%) in fatty acid concentration compared with the wild-type. A smaller reduction of 20% was measured in the seed of the leaky suc5.3 allele. This marked delay in lipid accumulation in the mutant backgrounds was transient. After 8 DAF, the differences observed between the wild-type and the suc5 lines steadily decreased, so that the concentration of fatty acid in dry mutant seeds was only 2–13% lower than in the wild-type, depending on the culture. The composition of the fatty acid was then considered (Table 1). At 4 DAF, both wild-type and mutant seeds contained high amounts of 16:0 (palmitic acid), 18:0 (stearic acid), 18:2 (linoleic acid) and 18:3 (alpha-linolenic acid), plus minor amounts of 18:1 (oleic acid) and 20:0 (behenic acid). With the onset of active synthesis of fatty acids at 8 DAF, this composition evolved; the proportion of 16:0 and 18:0 dropped markedly, while relative levels of 18:1 and 20:1 (eicosenoic acid) increased. Yet the fatty acid composition of the mutants shifted from the wild-type one at this stage. Compared with the wild-type, suc5 seeds aged 8 DAF exhibited a significant enrichment in 16:0, 18:2, 18:3 and a decrease in 18:1 and 20:1 (Table 1). These differences then gradually disappeared during seed maturation, so that the fatty acid compositions of wild-type and mutant dry seeds aged 22 DAF were very similar, with very long chains accounting for more than 25 mol.%.
Table 1. Fatty acid composition of WS and suc5 seeds. Total lipid extraction was carried out on batches of 20 seeds. The total fatty acid composition (in mol.%) of the seeds was determined by gas chromatographic analysis of an aliquot. Values are the means ± SE of three independent replicates. TFA, total fatty acids. Values in bold are significantly different from the WT (unpaired t-test)
|Age (DAF)|| ||Fatty acid composition (mol.%)||TFA (μg mg−1 DW)|
| 4||WS||28.8 ± 0.2||15.2 ± 0.5||6.2 ± 0.2||34.8 ± 0.3||13.2 ± 0.4||1.9 ± 0.2||0.0 ± 0.0||0.0 ± 0.0||57.4 ± 5.0|
|suc5.3||30.5 ± 0.9||16.9 ± 3.4||4.7 ± 0.6||33.0 ± 3.1||13.2 ± 1.7||1.7 ± 0.3||0.0 ± 0.0||0.0 ± 0.0||53.9 ± 6.5|
|suc5.2||29.4 ± 0.5||15.5 ± 0.3||5.6 ± 0.3||34.9 ± 0.4||12.9 ± 0.3||1.7 ± 0.1||0.0 ± 0.0||0.0 ± 0.0||56.6 ± 1.3|
|suc5.1||28.9 ± 0.9||12.9 ± 1.0||4.2 ± 0.2||36.8 ± 1.1||15.7 ± 0.7||1.6 ± 0.2||0.0 ± 0.0||0.0 ± 0.0||52.9 ± 2.5|
| 8||WS||17.2 ± 0.8||6.3 ± 0.1||23.7 ± 1.7||35.0 ± 0.8||12.4 ± 0.7||1.2 ± 0.0||4.3 ± 0.4||0.0 ± 0.0||78.2 ± 5.8|
|suc5.3||21.0 ± 1.2||6.1 ± 0.1||19.4 ± 0.2||35.2 ± 0.7||13.6 ± 0.2||1.1 ± 0.1||3.6 ± 0.3||0.0 ± 0.0||62.8 ± 0.5|
|suc5.2||24.0 ± 0.4||5.9 ± 0.1||10.0 ± 0.8||40.6 ± 0.7||15.9 ± 0.1||1.1 ± 0.1||2.5 ± 0.4||0.0 ± 0.0||43.5 ± 1.3|
|suc5.1||23.1 ± 0.4||5.5 ± 0.3||8.3 ± 0.2||42.8 ± 0.4||16.9 ± 0.2||1.0 ± 0.1||2.5 ± 0.3||0.0 ± 0.0||43.0 ± 1.4|
|22||WS||7.4 ± 0.0||3.3 ± 0.0||12.7 ± 0.1||28.5 ± 0.1||20.3 ± 0.1||2.4 ± 0.0||23.3 ± 0.1||2.3 ± 0.0||331.9 ± 1.4|
|suc5.3||7.2 ± 0.0||3.1 ± 0.0||12.6 ± 0.0||28.3 ± 0.0||20.5 ± 0.0||2.3 ± 0.0||23.6 ± 0.0||2.4 ± 0.0||324.9 ± 3.8|
|suc5.2||7.2 ± 0.0||3.0 ± 0.0||12.6 ± 0.1||28.4 ± 0.1||19.6 ± 0.1||2.4 ± 0.0||24.2 ± 0.0||2.7 ± 0.0||322.0 ± 1.6|
|suc5.1||7.3 ± 0.0||3.1 ± 0.0||12.9 ± 0.2||28.3 ± 0.1||19.9 ± 0.1||2.4 ± 0.0||23.8 ± 0.1||2.5 ± 0.0||324.9 ± 2.6|
If the small size of A. thaliana seed impaired the dissection and analysis of its constitutive tissues during their development (Hill et al., 2003), fatty acid measurements could be carried out separately on the embryo and the surrounding cellular endosperm of mature imbibed seeds (Penfield et al., 2004). In the suc5 mutant background, the two tissues exhibited a reduction in fatty acid content compared with the wild-type (Figure 5c). A 15% loss was measured in embryos of the suc5.1 and suc5.2 lines, whereas the corresponding mature mutant endosperm exhibited a 35% loss of fatty acids. Due to the small size of the material considered, it was not possible to weigh the samples and thus to determine whether these variations in total fatty acid content corresponded to significant differences in terms of fatty acid concentrations or merely reflected a reduction in the size of the tissues analysed, as suggested by whole seed analyses (Table 1). Penfield et al. (2004) demonstrated that wild-type endosperm contained proportionally higher levels of 18:1n7 and 20:1n7 long-chain fatty acids compared with embryos. In the suc5 lines, the relative proportion of n7 monounsaturated fatty acids exhibited a 20% increase compared with the wild-type (data not shown). Correspondingly, levels of n9 monounsaturated fatty acids were proportionally decreased. In spite of this altered lipid content, we were unable to detect any germination phenotype, either in the light or in the dark.