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Keywords:

  • lipid biosynthesis;
  • seed development;
  • seedling establishment;
  • sucrose metabolism;
  • glycolysis;
  • triacylglycerols

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

The accumulation of storage compounds during seed development ensures the survival of the young seedling, and also provides nutrition to humans and animals in the form of foods and feeds. The putative AP2/EREBP transcription factor WRINKLED1 (WRI1) is involved in the regulation of seed storage metabolism in Arabidopsis. A splicing mutant allele, wri1-1, caused the reduction of seed oil accumulation. Glycolysis was compromised in this mutant, rendering developing embryos unable to efficiently convert sucrose into precursors of triacylglycerol biosynthesis. Expression of the WRINKLED1 cDNA under the control of the cauliflower mosaic virus 35S-promoter led to increased seed oil content. Moreover, the ectopic expression of the WRINKLED1 cDNA caused the accumulation of triacylglycerols in developing seedlings. This effect depended upon the presence of glucose in the growth medium or other sugars readily metabolized to glucose. Oil-accumulating seedlings showed aberrant development consistent with a prolonged embryonic state.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

Seed development in general is governed not only by a complex network of transcription factors that integrate external cues, e.g. light, but also internal signals such as the concentration of the plant growth factor abscisic acid or sugars (Brocard-Gifford et al., 2003; Finkelstein and Gibson, 2002; Finkelstein et al., 2002; Rolland et al., 2002). Mutations in some of the best characterized transcription factors active in seeds, e.g. ABI3, LEC1, LEC2, FUS3 (Bäumlein et al., 1994; Giraudat et al., 1992; Lotan et al., 1998; Stone et al., 2001), give rise to pleiotropic phenotypes consistent with their essential role in many aspects of seed development, including seed storage compound accumulation. Storage compounds such as triacylglycerols (seed oil) serve as carbon and energy reserves, which are used during germination and growth of the young seedling. Seed (vegetable) oil is also an essential component of the human diet and a valuable commodity providing feed stocks for the chemical industry. A mutant of Arabidopsis affected in seed storage compound metabolism is wrinkled1 (wri1) (Focks and Benning, 1998), and it was named after the wrinkled appearance of the seed coat (cf. Figure 1). Detailed biochemical analysis of the wri1 mutant indicated an 80% reduction in seed oil content, an increased accumulation of soluble sugars, an inability of developing seeds to convert sucrose and glucose into precursors of fatty acid biosynthesis, and a decrease in the activity of several glycolytic enzymes (Focks and Benning, 1998). A global comparison of transcript abundance in developing seeds isolated at different stages for wild type and the wri1 mutant revealed that the expression of a limited number of structural genes involved in seed metabolism is altered (Ruuska et al., 2002). The biochemical and global expression data obtained for the mutant, therefore, are consistent with a defect in a regulatory factor controlling the glycolytic breakdown of sugars imported into the embryo and thereby the availability of precursors for fatty acid biosynthesis and ultimately oil accumulation. Here, the identification of the WRI1 locus is described and phenotypes arising from the ectopic expression of the WRI1 cDNA are investigated.

image

Figure 1. Scanning electron micrographs of wild-type and wri1 mutant seeds. Two magnifications are shown as indicated.

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The wri1 epidermis is wrinkled but otherwise normal

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

The most distinguishing morphological phenotype of the wri1-1 mutant, which also gave rise to its name, is the wrinkled appearance of the mature seeds. This phenotype is readily observed by scanning electron microscopy as shown in Figure 1. In contrast to mutants affected in seed coat development, such as apetala2 (Jofuku et al., 1994), the outer integument structure was generally intact for wri1-1. Typical features such as the central columella and the hexagonal layout for each cell were preserved. However, the outer integument cells were clearly shriveled for wri1-1 contributing to the macroscopic appearance of seed wrinkledness. Because water and solutes such as sugars replace the water-insoluble oil in the mutant seeds (Focks and Benning, 1998), the wrinkled appearance of the seeds presumably was caused by a loss of water during late seed maturation rather than impaired development of the integuments.

Identification of the WRI1 locus

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

Further characterization of the WRI1 gene product required the identification of the WRI1 gene. An initial mapping population between wri1-1 (col) and WT (ler) enabled us to place WRI1 between AFC1 and BGL1. A second mapping population of 1014 randomly selected F2 plants, allowed us to narrow the region of WRI1 to between the markers CDC2BG (one recombinant) and TSA1 (two recombinants), with no recombinant line between wri1-1 and the AP3 marker as shown in Figure 2(a). One of the candidate loci (AT3AG54320) in the interval was used to probe a T-DNA cosmid library containing Col-2 wild-type-derived genomic DNA (Meyer et al., 1996). Four overlapping cosmids were identified (Figure 2b) that restored oil biosynthesis when introduced into the wri1-1 mutant (data not shown). An EST, M26E12, corresponding to locus At3G54320 was identified in the Arabidopsis seed EST database (White et al., 2000). Sequencing of M26E12 indicated that the cDNA covered locus At3G54330 as well as parts of At3G54320. The full-length cDNA obtained by 5′RACE (GenBank AY254038) corresponded to both annotated genes covering a 1239 bp open reading frame and 5′ and 3′ untranslated sequences. The gene contains four short and one fairly large intron. Analyzing the cDNA obtained by RT-PCR from the wri1-1 mutant revealed a point mutation (G3197A, GenBank AL132971) at the intron–exon border of the first intron resulting in a splicing defect (Figure 2c). This mutation caused an increased size of the mRNA visible by RNA hybridization (see below) and presumably led to a severely truncated protein due to a predicted in-frame stop codon in the first intron (Figure 2c). Introducing the open reading frame of the wild-type cDNA under the control of the cauliflower mosaic virus (CAMV) 35S promoter into the wri1-1 mutant restored oil biosynthesis in the majority of the lines analyzed (Figure 3a,b).

image

Figure 2. Map-based cloning of the WRI1 gene. (a) Markers on chromosome 3 and BAC clones. (b) Revised annotation of the region surrounding WRI1 and cosmids rescuing oil biosynthesis in transgenic wri1 plants. (c) Revised intron–exon structure of the WRI1 gene. The wri1-1 mutation (G3197A) is indicated. (d) Sequence alignment of the predicted WRI1 protein (AY254038), APETALA2 (AP2; At4g369200) and AINTEGUMENTA (ANT; At4g37750). Identical residues are shaded black, similar residues are shaded gray.

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image

Figure 3. Total fatty acids per seed for the untransformed mutant (wri1) and wild type (WT) (a), and transgenic lines in the wri1 background (b) or the wild type background (c). Transgenic lines expressing the WRI1 cDNA under the control of the CAMV 35S promoter (wri1-cDNA, WT-cDNA) are shown. In the case of the transgenic lines, 10 T2 seeds from 104 and 107 independently isolated lines were analyzed (mutant and wild-type background respectively). For wild type 10 seeds of 32 independent plants each and for the wri1 mutant 30 samples of 10 seeds each from a pooled seed batch were analyzed. The transgenic lines were grown in parallel in 16 h light, 8 h dark, at 22°C in the same chamber, while the untransformed lines were grown under identical conditions but in a different chamber to avoid cross pollination between transgenic and untransformed lines. Individual plants were grouped in 1 μg intervals according to fatty acid content per seed. For better comparison, a broken line was centered through the peak of the wild type seed distribution in (a).

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WRI1 encodes a putative transcription factor of the AP2/EREBP family

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

The WRI1-cDNA was predicted to encode a 48.4 kDa protein with two AP2/EREBP DNA binding domains (Jofuku et al., 1994; Weigel, 1995). In general, these domains represent signatures for plant-specific transcription factors involved in a wide range of developmental processes (Riechmann and Meyerowitz, 1998). For example, binding of DNA by the AP2/EREBP domains of the AINTEGUMENTA (ANT) transcription factor has been experimentally verified (Krizek, 2003; Nole-Wilson and Krizek, 2000). The second AP2/EREBP domain of WRI1 is less conserved. The WRI1 protein is also characterized by long stretches of serine and threonine as well as acidic residues toward the C-terminus. Most closely related (61% identity over 183 residues) to WRI1 is ANT, a transcription factor of Arabidopsis involved in ovule development and floral organ growth (Elliott et al., 1996; Klucher et al., 1996). A comparison of WRI1, ANT and APETALA2 (AP2) is shown in Figure 2(d). Based on this comparative sequence analysis, it is postulated that WRI1 encodes a transcription factor of the AP2/EREBP class.

Tissue-specific and development-dependent expression of WRI1

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

Experimental evidence for the regulation of WRI1 mRNA abundance was obtained by RNA/DNA hybridization. The expression of the WRI1 gene was tissue-specific with the highest abundance of mRNA in siliques (Figure 4a). Only very small amounts of mRNA were detectable in leaves, stems and seedlings, but the gene was moderately expressed in roots and flowers. Because intact siliques showed the highest abundance of WRI1 mRNA but leaves much less, we focused on the expression of WRI1 in developing seeds which were removed from the silique walls. Determining the abundance of WRI1 mRNA during seed development in a detailed time course revealed a distinct maximum on day 8 after flowering (Figure 4b,c). This time point approximately coincides with the onset of storage compound accumulation (Focks and Benning, 1998). In addition, abundance of WRI1 mRNA increased again toward the end of storage compound accumulation. For comparison we also analyzed the expression of the wri1-1 gene in the mutant. Noticeably, the overall abundance of the mutant mRNA was drastically increased and the mRNA abundance profile was altered in the mutant, suggesting that a functional WRI1 protein may be involved in feedback regulation of its respective gene.

image

Figure 4. Relative abundance of WRI1 and wri1-1 RNA in wild-type, mutant and transgenic lines. (a) Northern blot of total RNA from different wild type tissues/organs as indicated is shown. The ethidium bromide-stained rRNA bands indicate similar loading in each lane. (b) Northern blots of total RNA isolated from seeds dissected from the siliques at indicated days after flowering (DAF) are shown. Wild-type (W) and wri1-1 samples (M) are compared for each time point. The same membrane was hybridized with an rRNA probe for normalization purposes. Three repeats with similar results were obtained for the WRI1 Northerns. (c) Quantitation of the Northern blots in (b) using a phosphor imager. The mRNA/rRNA ratios are shown for wild type (top panel) and the wri1-1 mutant (lower panel). For better comparison, the lowest value for each graph was set to 1.

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Overexpression of the WRI1 cDNA increased seed oil content

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

In an exploratory experiment to enhance the WRI1 mRNA abundance and concomitantly increase seed oil content, and to observe other effects of ectopic WRI1 expression which might provide clues to WRI1 function, the wild-type WRI1cDNA under the control of the CAMV 35S promoter was introduced into Arabidopsis wild type. Analyzing 10 T2 seeds of 107 independent transformants each for total fatty acids as a measure of oil content, a distribution ranging from lines with very low to very high fatty acid content outside the range observed for wild-type plants was observed (Figure 3a,c). Applying Student's t-test, the distributions for the wild type, transformed and untransformed, were statistically different (P < 0.02). Although no WRI1 mRNA abundance data in the developing seeds for all lines are available, independent transgenic lines were expected to have variable amounts of the WRI1 transcript due to positional or cosuppression effects accounting for the observed wide range of the distribution. To test the reproducibility of this effect through subsequent generations, T4 seeds of a number of lines distinguished by their high seed oil content in the T2 generation were analyzed. Several lines in the wild-type and also one in the wri1-1 mutant background produced T4 seeds with 10–20% more oil per seed compared with the wild type grown in the same growth chamber. It is also important to note that the total number of seeds per plant was not affected in the transgenic lines (data not shown).

Transgenic seedlings grow abnormally on sucrose medium

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

During routine growth of seedlings on MS medium containing 2% sucrose, it was noticed that transgenic wri1-1 lines produced seedlings with abnormal morphology at varying frequency depending on the line. As an example, seedlings from line 107 are shown in Figure 5. This 35S-WRI1 transgenic line was distinguished by its high seed oil content and no longer segregated for hygromycin B resistance as expected for a stable homozygous 35S-WRI1 transgenic line. Plants appeared normal on medium lacking sucrose (Figure 5a,c), but approximately 15–20% of the seedlings developed atypically on medium with sucrose. These seedlings did not turn green, lacked expanded cotyledons, frequently did not open their apical hooks and often showed elongated hypocotyls. Apparently, photomorphogenesis was delayed and the seedlings accumulated triacylglycerols, a possible indication of a prolonged embryonic state. Figure 5(f) shows 11-day-old seedlings with a varying degree of severity of the morphological phenotype. Abnormal seedlings that survived to maturity usually had reduced fertility and stature. We also investigated in greater detail a second stable independent homozygous 35S-WRI1 transgenic line (106), which also was in the wri1-1 mutant background. In this line seed oil amounts were restored close to wild-type levels in the T2 generation. Nearly all seedlings of line 106 showed morphological phenotypes on agar plates containing 2% sucrose as described for line 107 (Figure 5). However, contrary to line 107 a small fraction of the plants of line 106 showed even abnormal morphological phenotypes in the absence of sucrose. To be able to distinguish between the endogenous mutant wri1-1 RNA and the transgene-derived wild-type mRNA, semi-quantitative PCR was employed using diagnostic primers flanking the intron which was not removed in the mutant (Figure 6a). The abundance of wild-type WRI1 mRNA derived from the transgene was clearly increased in 106 compared with the other lines. A clear-cut effect of sucrose on WRI1 RNA abundance was not visible in these experiments (Figure 6a).

image

Figure 5. Phenotype of representative seedlings from line 107 ectopically expressing the WRI1-cDNA in the wri1-1 background. Plants in (a) and (c) were grown on plates lacking sucrose, all others were grown on plates containing 2% sucrose. All plants were grown under a typical day/night regime. Plants in panels (a) through (e) were 4 days old (following stratification), plants shown in panel (f) were 11 days old. These plants were grown on a vertically placed agar plate, all others on horizontal plates. Size bars equal 3 mm in panels (a), (b), and (f), and 1 mm in panels (c), (d), and (e). Individual plants in (e) are numbered in the order of increasing severity of the morphological phenotype.

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image

Figure 6. Abundance of mRNA in mutant wild-type and transgenic lines. (a) Semi-quantitative RT-PCR of total RNA from wild type (WRI1), mutant (wri1-1) and the transgenic lines 106 and 107 expressing the WRI1-1 cDNA in the wri1-1 mutant background. Seedlings were grown either in the absence (0) or presence of (2) of 2% sucrose. Diagnostic fragments for the mutant (wri1-1) and wild type (WRI1) species are indicated. Actin-specific primers were used for control purposes. (b) Northern analysis of wild type (WRI1), mutant (wri1-1) and independent transgenic lines expressing the WRI1 cDNA under the control of the 35S-CAMV promoter from a CAMBIA vector-derived construct. Transgenic lines were in the wild-type (WRI1-tf) or wri1-1 mutant (wri1-1-tf) mutant backgrounds. All transgenic lines except (lane 9) were producing abnormal seedlings in the presence of 2% sucrose. Total RNA was isolated from transgenic (mixed normal and abnormal) seedlings grown for 11 days in the presence of 2% sucrose. The blot was repeatedly probed with the WRI1 cDNA (WRI1) or a cDNA encoding a predicted plastidic pyruvate kinase (Pkc, GenBank AY048198). The ethidium bromide-stained rRNA band is shown as loading control.

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Initially, we did not observe this effect in the wild-type background. However, when the experiment was repeated by expressing the WRI1 cDNA under the control of the 35S promoter in a CAMBIA binary vector, transgenic lines in the wild-type and mutant backgrounds produced abnormal seedlings in the presence of sucrose (data not shown). The abundance of WRI1 transcript was strongly increased in these transgenic lines compared with untransformed lines as shown in Figure 6(b). Moreover, the expression of one of the presumed target genes encoding a plastidic pyruvate kinase was increased (Figure 6b) in correlation with the appearance of abnormal seedlings in the presence of 2% sucrose in the medium.

Transgenic seedlings accumulate triacylglycerols in the presence of sucrose

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

The abnormal seedlings of lines 106 and 107 and other transgenic lines were reminiscent of oil-accumulating embryos that have outgrown their seed coat. Indeed, seed oil was present in seedlings of these lines when grown on sucrose (Figure 7a). Two additional independent transgenic lines, 10 and 84, both in the wri1-1 background were included to document that this phenomenon was not restricted to a few selected lines but generally occurs in lines which show abnormal seedling development on sucrose.

image

Figure 7. Accumulation of triacylglycerols in transgenic seedlings. (a) Thin layer chromatogram of lipid extracts from 10 seeds or ten 10-day-old seedlings as indicated. Visibly morphologically altered seedlings were selected for the transgenic lines. In addition to transgenic lines 106 and 107, two additional independent transgenic lines, 10 and 84, were included to demonstrate broad occurrence of the phenomenon in the transgenic lines, which all expressed the WRI1 cDNA in the wri1-1 mutant background. Pig, pigments; TAG, triacylglycerol. (b–d) Long-chain fatty acids as makers for seed oil in developing seedlings of wild type, wri1-1, and line 106 ectopically expressing the WRI1-cDNA in the wri1-1 mutant background. (b) Time courses of plants grown on MS agar plates without sucrose, and (c) plants grown on plates with 2% sucrose. Squares, wild type; open triangles, line 106; circles, wri1-1 mutant. In general, six groups of 15 plants each were analyzed and averaged (±SE). In (c) from 7 days on, individual seedlings of line 106 (15–25 individuals) grown on sucrose were analyzed and averaged (±SD). These seedlings (closed bar, 7 day; hatched, 9 day; open, 11-day-old seedlings) were also grouped into classes according to their long-chain fatty acid content as shown in (d) to provide an impression of the large spread of phenotypes.

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To obtain more quantitative results, long-chain fatty acids (LFA) as markers for seed oil (Lu and Hills, 2002) were compared for transgenic line 106, the wild type, and the wri1-1 mutant during a time course of 11 days following the incubation of stratified seeds on agar plates containing or lacking sucrose (Figure 7b–d). In the absence of sucrose, line 106 and wild type were indistinguishable (Figure 7b), while the wri1-1 mutant showed the characteristic low LFA content at all stages. After 3 days, LFAs were no longer detectable consistent with complete catabolism of seed oil. In the presence of sucrose (Figure 7c), degradation of seed oil was delayed for the wild type and line 106 for approximately 1 day. Beginning with day 4, visibly aberrant seedlings were present for line 106 with yellow plants showing increasing amounts of LFAs. Following day 4, plants were no longer pooled but individually analyzed. The increasing standard deviation was due to the large variability among individual plants of line 106. Because line 106 is genetically stable showing no segregation of antibiotic-resistant and -sensitive plants, this variability reflects differences between seedlings in their ability to metabolize carbohydrates from the medium to triacylglycerols. To show this variability more explicitly, the class distribution for the amount of LFAs per individual plant of line 106 grown for 7, 9, and 11 days on sucrose-containing agar plates was determined (Figure 7d). Remarkably, among the plants were individuals with amounts of LFAs in excess of 10-fold compared with the amount present in individual wild type seeds. Apparently, these seedlings actively synthesized triacylglycerols containing LFAs consistent with the TLC analysis (Figure 7a). Similar, but less pronounced trends were observed for line 107 (data not shown).

To explore whether the effect was specific for sucrose, LFAs in 11-day-old 35S-WRI1 transgenic seedlings were analyzed, which were grown on different sugars as indicated in Table 1. Pools of 10 randomly picked seedlings included morphological normal and abnormal individuals of lines 106 and 107. Glucose or disaccharides giving rise to glucose seemed to be most effective (Table 1). Neither galactose, the epimer of glucose, nor the sugar alcohol sorbitol had an effect.

Table 1.  Sugar specificity for ectopic oil accumulation in 35S-WRI1 cDNA transgenic lines
 Long-chain fatty acids (μg/seedling)
Line 106Line 107
  1. Long-chain fatty acids (20:0, 20:1, 22:0, 22:1) are shown. Three samples of 10 seedlings each were averaged (±SE). All sugars were present at 1% (w/v) except sucrose and mannose (2%, w/v). Frc, fructose; Gal, galactose; Glc/Frc, glucose and fructose (1% each); Mal, maltose; Srb, sorbitol; Suc, sucrose; nd, not detected (detection limit 0.1 μg).

No sugar0.1 ± 0nd
Glc5.1 ± 0.40.6 ± 0.2
Frc1.8 ± 0.7nd
Suc5.3 ± 0.30.9 ± 0.1
Glc/Frc3.7 ± 0.30.9 ± 0.2
Mal2.9 ± 0.1nd
Srb0.4 ± 0nd
Gal0.5 ± 0.1nd

WRI1, a node in the regulatory network controlling metabolism during seed development?

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

The WRI1 gene was identified by map-based cloning of the wri1-1 locus. It encodes a putative transcription factor of the AP2/EREBP class (Jofuku et al., 1994; Riechmann et al., 2000; Weigel, 1995). Reduced function of WRI1 or complete absence in the wri1-1 mutant causes a deficiency in the glycolytic pathway during a phase of seed development, which is critical for storage oil accumulation (Focks and Benning, 1998). Considering these two facts it is hypothesized that WRI1 represents a specific node of the seed metabolic network that controls the pathway from sucrose import to oil accumulation. In support of this hypothesis, global comparative expression analysis in developing seeds of wild type and the wri1-1 mutant (Ruuska et al., 2002) identified a limited number of potential target genes for WRI1, primarily encoding enzymes of glycolysis and triacylglycerol biosynthesis. Moreover, the ectopic expression of WRI1 under the control of a CAMV 35S promoter led to an increase in WRI1 mRNA abundance in developing seedlings and concomitantly to an increase in the abundance of mRNA predicted to encode a plastidic isoform of the glycolytic enzyme pyruvate kinase (Figure 6b). The gene for this isoform was previously found to be highly expressed in developing seeds as expected for a potential WRI1 target (White et al., 2000). In addition, the WRI1 gene itself might be a target of WRI1 action. Strongly increased expression of the mutant gene in developing seeds was observed in the wri1-1 mutant background (Figure 4c), suggesting that the expression of WRI1 is negatively feedback-regulated in wild-type developing seeds.

Does WRI1 have functions beyond the control of seed metabolism?

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

The expression of the WRI1 gene was not only observed in developing siliques and seeds isolated from the siliques, but also in roots and to a lower extent in young seedlings and flowers (Figure 4a). Interestingly, expression in strictly photosynthetic tissues was not detected by RNA/DNA hybridization. Non-photosynthetic tissues such as roots require ATP and reducing equivalents for metabolic processes or for the active uptake of nutrients. In young developing seedlings, oil is metabolized into sugars by the process of gluconeogenesis. Some of the enzymes of the glycolytic pathway participate in gluconeogenesis (Plaxton, 1996). Thus, of the tissues tested in this study, those with elevated needs for glycolytic enzyme activity also show the highest abundance of WRI1 mRNA.

The appearance of oil-accumulating seedlings in lines, which ectopically express WRI1, might imply a function for WRI1 beyond the regulation of metabolism. Essentially, the phenotype of these abnormal seedlings is consistent with an extension or reinitiation of the embryonic state of developing seeds during oil accumulation. It should be pointed out, though, that ectopic expression of WRI1 did not lead to ectopic embryo formation as observed for LEC1 (Lotan et al., 1998) in Arabidopsis or BABY BOOM in Brassica (Boutilier et al., 2002). In the absence of sugar in the medium, seedlings of most of the transgenic lines were generally indistinguishable from wild type and developed into Arabidopsis plants which did not form ectopic embryos or show other obvious abnormalities. The requirement for sugar to bring about the aberrant phenotype may be simply explained by the fact that carbon skeletons and reducing power are needed to synthesize the large amounts of oil in these compromised seedlings. Plants grown from abnormal seedlings were often stunted in growth and yellowish suggesting reduced photosynthetic capability. They were often sterile as well.

WRI1 as a second-generation target to engineer seed oil yield?

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

In the past, several schemes have been developed to modify the fatty acid composition of seed oils by genetic engineering resulting in increased nutritional and industrial value (Voelker et al., 1996). However, strategies targeting single rate-limiting enzymes to increase seed oil content have been, to date, only moderately successful (Thelen and Ohlrogge, 2002). The putative AP2/EREB WRI1 protein described here seems to hold the promise expected for a second-generation target to modify oil content in developing oil seeds and possibly other tissues, because all current data suggest that it controls metabolic processes critical to seed oil accumulation. The ectopic expression of WRI1 under the control of the CAMV 35S promoter described here was directed at the investigation of WRI1 function and not at engineering of oil content. However, even this crude expression system led to increases in seed oil content as well as the ectopic production of triacylglycerols in developing seedlings. More refined approaches toward the tissue-specific engineering of WRI1 RNA abundance may well provide the means to control triacylglycerol biosynthesis in desirable tissues or at extended times of the life cycle of the plant.

Plant growth conditions

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

Wild type and wri1-1 Arabidopsis plants were of the Col-2 or Ler ecotypes. Except when otherwise mentioned, all plants were started on half-strength MS medium (Murashige and Skoog, 1962), pH 6.2, 2% sucrose and 1.2% agar. Seeds were sterilized for 20 min in 20% bleach 0.5% triton X100 and rinsed six times with excess sterile water. Hygromycin B and kanamycin were added to final concentrations of 25 μg ml−1 for hygromycin B and 50 μg ml−1 kanamycin. Seeds were pre-incubated for 3 days in the dark at 4°C before placing them into an incubator (AR-75; Percival Scientific, Boone, IA, USA) at a photon flux density of 60–80 μmol m−2 sec−1 and a light period of 16 h (22°C), and a dark period of 8 h (18°C). Seedlings were transferred to 3.5 inch square pots containing a soil mixture as previously described (Xu et al., 2002) and were grown under a 16-h photoperiod with a day temperature of 24°C and a night temperature of 22°C at a photon flux density of 100–120 μmol m−2 sec−1. The plants were fertilized with over the counter Miracle-Gro (Scotts, Marysville, OH, USA) water-soluble plant food at 3-week intervals.

Genetic mapping

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

Genetic mapping of the WRI1 locus was carried out using CAPS markers (Konieczny and Ausubel, 1993). Initial mapping relative to the chromosome 3 markers BGL1 (no gene number available at http://www.arabidopsis.org) and AFC1 (At3g53570) was carried out with the original mapping population (Focks and Benning, 1998). The mapping population was then expanded to include 1014 F2 plants from a second cross between wri1-1 (col-2) and wild type ecotype Ler. The plants were scored with the CAP markers TT5 (At3g55120), AFC1, BGL1, TSA1 (At3g54640), CDC2B (At3g54180), AP3-L (At3g54340) using the primers and conditions listed by The Arabidopsis information resource (http://www.arabidopsis.org). A contig of overlapping cosmids was constructed to cover the 160 kb region defined by the last break points between these CAPs markers and wri1-1 using a transformable pBIC20 cosmid library (Meyer et al., 1996).

Isolation of the WRI1 cDNA

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

A partial cDNA of the wri1 locus was represented by clone M26E12 from the seed-specific expressed sequence tag (EST) set (White et al., 2000). The 5′ end was obtained by 5′-RACE using a kit (Takara, Madison, WI, USA). The complete cDNA was obtained by RT-PCR employing a kit from Qiagen (Valencia, CA, USA) using wild type (Col-2) silique RNA and the PCR primers 5′-GGTACCAAATCTAAACTTTCTCAGAG-3′ and 5′-GGTACCGGCAAAGACATTGATTATTC-3′. The RT-PCR product was cloned using TA-overhangs into the pCR2.1TOPO vector from Invitrogen (Carlsbad, CA, USA). Sequencing was performed at the MSU Genomics Technology Support Facility. Single sequence alignments were performed with blast (Altschul et al., 1997) and multiple alignments with clustal W (Thompson et al., 1994). The cDNA sequence was deposited at GenBank (AY254038). The cDNA was excised with KpnI and inserted into the binary vector pBinAR-Hyg (Dörmann and Benning, 1998) or into a pCAMBIA1300 derivative (CAMBIA, Canberra, Australia), which contained the EcoRI/HindIII expression cassette from pBIN121 (Clontech, Palo Alto, CA, USA). Proper orientation and integrity were confirmed by restriction analysis and sequencing.

Generation of transgenic plants

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

Seedlings for wild type and wri1-1 were grown as cited above and transferred to 3.5 inch pots that had been set up for dipping by mounding with potting soil and were covered with a plastic window screen with 20 evenly spaced holes of 0.5 cm diameter. Growth conditions were as described above. When the primary inflorescence had bolted and the first three or four flowers had opened (approximately 4 weeks), the plants were transformed using the floral dip method (Clough and Bent, 1998). Three to four pots were used per construct. The plants were dipped again in the same manner 2 or 3 weeks later. Competent cells of Agrobacterium tumefaciens strain GV3101 C58C1 pMP90 (Koncz and Schell, 1986) were prepared and transformed according to Shen and Forde (1989). Transformation efficiencies ranged from 1 to 2% for pBinAR-Hyg or pCAMBIA constructs and 0.5–0.8% for pBIC20. No difference was observed in transformation efficiencies between wild type and wri1-1. Wild type and wri1-1 transformed with the WRI1 cDNA were grown in separate flats in the same walk-in chamber and plants were individually tied to stakes.

Expression analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.

Leaf tissue taken for RNA analysis was harvested from 28-day-old rosettes. The stems used were taken from 35-day-old plants. Seedlings were 8 days old from the time the plates were placed into the incubator. Roots and shoots were excised from 10-day-old plantlets. All tissues were snap frozen in liquid nitrogen. Flowers consisted of the inflorescence meristem through the first three opened flowers. Whole silique tissue was harvested from approximately 40-day-old plants. Green siliques of all stages were taken. The age of the seed for the time course was determined by staging flowers as previously described (Focks and Benning, 1998). Seeds were dissected out of the silique using fine tweezers. Seeds from 10 to 20 siliques were harvested onto a glass slide on ice and then gathered and placed into a 1.5 ml polypropylene tube in dry ice. This was repeated until all of the tagged siliques for a particular time point were harvested (10–14 h for one person). Tissue was stored at −80°C until extraction. RNA extraction was performed according to the methods of Hosein (2001). Northern analysis (5 μg total RNA) was performed as previously described (Dörmann and Benning, 1998). The blots were analyzed using a phosphor imager (Molecular Dynamics, Amersham, Piscataway, NJ, USA). Background integrals of areas equal in size to those of the bands located between the wells and the bands on the blots were subtracted. Using the image quantification software provided by the manufacturer (image quant 5.2 and fragment analysis 1.1), the ratio of each background-corrected band intensity to the 16S rRNA band intensity for the respective time point was calculated and the lowest ratio was arbitrarily set to 1, to which all other signals in each series were normalized.

In experiments using RT-PCR, RNA was extracted from 11-day-old seedlings of each genotype using a Qiagen RNeasy kit, as per the manufacturer's instructions. Reverse transcription was performed on 1 μg of total RNA using a Qiagen Omniscript kit. The primers used to distinguish between WRI1 and wri1-1 were designed to span the region of the first intron (5′-CCGACGCAGC-TCTATCTACA-3′ and 5′-AGCCTCCCATCTTCCGTTGT-3′). Actin (At2g3762) was used for control purposes (5′-TGCGACAATGGAACTGGAATGG-3′ and 5′-AACAATCGATGGACCTGACTCG-3′). The cycle conditions were 3.5 min at 94°C, 0.5 min at 94°C, 0.75 min at 54°C, 1 min at 72°C, 30 cycles followed by 5 min at 72°C. In the wild type, the predicted PCR product is 358 bp and in wri1-1 it is 464 bp, in the event of genomic DNA contamination, a product of 1080 bp is expected. The same reaction conditions were used for the actin mRNA quantification, except that 25 cycles were used. For both, 1 unit of Invitrogen Taq polymerase was used with a final MgCl2 concentration of 1.5 mm, dNTP concentration of 0.125 mm, and 10 pmol of each primer.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. The wri1 epidermis is wrinkled but otherwise normal
  6. Identification of the WRI1 locus
  7. WRI1 encodes a putative transcription factor of the AP2/EREBP family
  8. Tissue-specific and development-dependent expression of WRI1
  9. Overexpression of the WRI1 cDNA increased seed oil content
  10. Transgenic seedlings grow abnormally on sucrose medium
  11. Transgenic seedlings accumulate triacylglycerols in the presence of sucrose
  12. Discussion
  13. WRI1, a node in the regulatory network controlling metabolism during seed development?
  14. Does WRI1 have functions beyond the control of seed metabolism?
  15. WRI1 as a second-generation target to engineer seed oil yield?
  16. Experimental procedures
  17. Plant growth conditions
  18. Genetic mapping
  19. Isolation of the WRI1 cDNA
  20. Generation of transgenic plants
  21. Expression analysis
  22. Lipid analyses
  23. Scanning electron microscopy
  24. Acknowledgements
  25. References
  26. GenBank Accession for the WRI1 cDNA: AY254038.
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