Limits...
Gene expression analysis of flax seed development.

Venglat P, Xiang D, Qiu S, Stone SL, Tibiche C, Cram D, Alting-Mees M, Nowak J, Cloutier S, Deyholos M, Bekkaoui F, Sharpe A, Wang E, Rowland G, Selvaraj G, Datla R - BMC Plant Biol. (2011)

Bottom Line: When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis.We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors.Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Biotechnology Institute, NRC, 110 Gymnasium Place, Saskatoon, Saskatchewan, S7N 0W9, Canada.

ABSTRACT

Background: Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed.

Results: We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development.

Conclusions: We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors. This has allowed us to delineate the spatio-temporal aspects of gene expression underlying the biosynthesis of a number of important seed constituents in flax. Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well.

Show MeSH

Related in: MedlinePlus

EST distribution of fatty acid biosynthetic genes during seed development and maturation across tissue libraries. (A) acyl chain elongation (Keto Acyl Synthases); (B) acyl chain termination (Fatty Acyl Thioesterases); (C) desaturation (Desaturases); (D) triacylglycerol (TAG) biosynthesis. EST distribution of flax unigenes used to compile these graphs is listed in Additional File 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3107784&req=5

Figure 9: EST distribution of fatty acid biosynthetic genes during seed development and maturation across tissue libraries. (A) acyl chain elongation (Keto Acyl Synthases); (B) acyl chain termination (Fatty Acyl Thioesterases); (C) desaturation (Desaturases); (D) triacylglycerol (TAG) biosynthesis. EST distribution of flax unigenes used to compile these graphs is listed in Additional File 3.

Mentions: Mature flax seeds consist of approximately 43% oil, mostly in the form of triacylglycerols (TAGs) within oil bodies located in the embryo [11]. In order to study the timing and source of lipid synthesis within the developing seeds, enzymes representing the four key steps of fatty acid synthesis were studied: acyl-chain elongation, termination, desaturation and TAG synthesis [43,44] (Figure 8A, Figure 9; Additional File 3). Based on the preponderance of ESTs representing the 3-ketoacyl-acyl carrier protein synthases (KAS1, KAS2 and KAS3) in the various tissues, it appears that acyl chain elongation activity increases during the torpedo stage and that the embryo, endosperm and seed coat all contribute to this activity in the seed (Figure 9A). Although the number of ESTs representing termination of elongation by fatty acyl-ACP thioesterases (FATA and FATB) was lower than KAS ESTs, this activity also appears to peak during the torpedo stage (Figure 9B). Within the developing embryos, fatty acids are transferred onto a glycerol backbone to form triacylglycerols by the activity of diacylglycerol acyltransferase (DGAT). TAGs are stored in oil bodies, the outer membrane of which is a spherical phospholipid monolayer interspersed with the protein oleosin [44]. ESTs representing DGAT were found in quantities similar to the FATA and FATB ESTs, i.e. in very low quantities. The key difference is that this activity seems to peak later, during the cotyledon embryonic stage rather than the torpedo stage (Figure 9D). Also, while termination of elongation and release of free FAs appears to occur in both seed tissues as well as in some of the vegetative tissues, DGAT expression in vegetative tissues is too low to detect with the EST counts. Desaturation is the key step that results in the desirable omega-3 and omega-6 fatty acids [44]. This seems to occur later during seed development as the spike in the number of ESTs representing the Fatty Acid Desaturases (FAD) 2, 3, 5 and 8 occurs within the mature embryo (Figure 9C). One of the omega-3 fatty acids found in flax, alpha-linolenic acid (ALA, 18:3n-3), constitutes up to 55% of the total seed oil [41]. ALA is an essential fatty acid in human diet and it is converted to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) which are then incorporated into membrane phospholipids. Some fatty acids are used in plant membrane synthesis, wax formation and pigmentation. The repertoire of lipid synthesis ESTs found in stem, stem peel and flowers provide a basis to probe these processes in these tissues (Figures 8A and 9).


Gene expression analysis of flax seed development.

Venglat P, Xiang D, Qiu S, Stone SL, Tibiche C, Cram D, Alting-Mees M, Nowak J, Cloutier S, Deyholos M, Bekkaoui F, Sharpe A, Wang E, Rowland G, Selvaraj G, Datla R - BMC Plant Biol. (2011)

EST distribution of fatty acid biosynthetic genes during seed development and maturation across tissue libraries. (A) acyl chain elongation (Keto Acyl Synthases); (B) acyl chain termination (Fatty Acyl Thioesterases); (C) desaturation (Desaturases); (D) triacylglycerol (TAG) biosynthesis. EST distribution of flax unigenes used to compile these graphs is listed in Additional File 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3107784&req=5

Figure 9: EST distribution of fatty acid biosynthetic genes during seed development and maturation across tissue libraries. (A) acyl chain elongation (Keto Acyl Synthases); (B) acyl chain termination (Fatty Acyl Thioesterases); (C) desaturation (Desaturases); (D) triacylglycerol (TAG) biosynthesis. EST distribution of flax unigenes used to compile these graphs is listed in Additional File 3.
Mentions: Mature flax seeds consist of approximately 43% oil, mostly in the form of triacylglycerols (TAGs) within oil bodies located in the embryo [11]. In order to study the timing and source of lipid synthesis within the developing seeds, enzymes representing the four key steps of fatty acid synthesis were studied: acyl-chain elongation, termination, desaturation and TAG synthesis [43,44] (Figure 8A, Figure 9; Additional File 3). Based on the preponderance of ESTs representing the 3-ketoacyl-acyl carrier protein synthases (KAS1, KAS2 and KAS3) in the various tissues, it appears that acyl chain elongation activity increases during the torpedo stage and that the embryo, endosperm and seed coat all contribute to this activity in the seed (Figure 9A). Although the number of ESTs representing termination of elongation by fatty acyl-ACP thioesterases (FATA and FATB) was lower than KAS ESTs, this activity also appears to peak during the torpedo stage (Figure 9B). Within the developing embryos, fatty acids are transferred onto a glycerol backbone to form triacylglycerols by the activity of diacylglycerol acyltransferase (DGAT). TAGs are stored in oil bodies, the outer membrane of which is a spherical phospholipid monolayer interspersed with the protein oleosin [44]. ESTs representing DGAT were found in quantities similar to the FATA and FATB ESTs, i.e. in very low quantities. The key difference is that this activity seems to peak later, during the cotyledon embryonic stage rather than the torpedo stage (Figure 9D). Also, while termination of elongation and release of free FAs appears to occur in both seed tissues as well as in some of the vegetative tissues, DGAT expression in vegetative tissues is too low to detect with the EST counts. Desaturation is the key step that results in the desirable omega-3 and omega-6 fatty acids [44]. This seems to occur later during seed development as the spike in the number of ESTs representing the Fatty Acid Desaturases (FAD) 2, 3, 5 and 8 occurs within the mature embryo (Figure 9C). One of the omega-3 fatty acids found in flax, alpha-linolenic acid (ALA, 18:3n-3), constitutes up to 55% of the total seed oil [41]. ALA is an essential fatty acid in human diet and it is converted to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) which are then incorporated into membrane phospholipids. Some fatty acids are used in plant membrane synthesis, wax formation and pigmentation. The repertoire of lipid synthesis ESTs found in stem, stem peel and flowers provide a basis to probe these processes in these tissues (Figures 8A and 9).

Bottom Line: When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis.We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors.Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Biotechnology Institute, NRC, 110 Gymnasium Place, Saskatoon, Saskatchewan, S7N 0W9, Canada.

ABSTRACT

Background: Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed.

Results: We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development.

Conclusions: We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors. This has allowed us to delineate the spatio-temporal aspects of gene expression underlying the biosynthesis of a number of important seed constituents in flax. Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well.

Show MeSH
Related in: MedlinePlus