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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.

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Hierarchical cluster analysis of flax EST libraries. Three gene expression clusters were identified, viz., early differentiating seed tissues, maturing embryos and juvenile vegetative tissues. The tree shows hierarchical clustering of the tissue-based libraries based on similarity/distance as measured by the Pearson correlation coefficient. Values close to 1 have high degree of similarity whereas lower values indicate the degree of distance between two libraries. Globular embryo (GE), heart embryo (HE), torpedo embryo (TE), cotyledon embryo (CE), mature embryo (ME), globular stage seed coat (GC), torpedo stage seed coat (TC), pooled endosperm (EN), etiolated seedlings (ES), stem (ST), stem peel (PS), leaves (LF), and mature flower (FL).
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Figure 4: Hierarchical cluster analysis of flax EST libraries. Three gene expression clusters were identified, viz., early differentiating seed tissues, maturing embryos and juvenile vegetative tissues. The tree shows hierarchical clustering of the tissue-based libraries based on similarity/distance as measured by the Pearson correlation coefficient. Values close to 1 have high degree of similarity whereas lower values indicate the degree of distance between two libraries. Globular embryo (GE), heart embryo (HE), torpedo embryo (TE), cotyledon embryo (CE), mature embryo (ME), globular stage seed coat (GC), torpedo stage seed coat (TC), pooled endosperm (EN), etiolated seedlings (ES), stem (ST), stem peel (PS), leaves (LF), and mature flower (FL).

Mentions: In order to compare the gene expression profile in different tissues, the entire set of 261,272 EST sequences was subjected to hierarchical cluster analysis using the software HCE3.5 [22] (see Methods). Amongst the parameters required for hierarchical cluster analysis, we selected the average linkage method and the Pearson correlation coefficient for the similarity/distance measure, a technique which has been widely used in microarray analysis [23]. The results are shown in Figure 4. The analysis shows that in general gene expression is most closely related in tissues that are developmentally related and connected. For example, globular (GE) and heart (HE) embryo stages are most closely related, followed closely by the torpedo stage (TE). The maturing embryos, viz., cotyledon (CE) and mature (ME) stages clustered together but were distantly placed from the early stage embryos. The two seed coat stages (GC and TC) also shared a relatively high degree of similarity to each other. Gene expression in the pooled endosperm tissue (EN) from early developing seed stages shared some similarity with early embryonic stages but was more distant from the seed coats and maturing embryos. It is interesting to note that the CE and ME stages cluster away from the early seed tissues (GE, HE, TE, GC, TC and EN) and to a lesser extent from other non-seed tissues viz., (ES, LE, FL, ST) which is indicative of the distinct seed maturation program that is occurring in the later stages of embryo development. As the stem peel (PS) did not contain all of the tissues normally present in whole stems (ST), and was enriched for the phloem and phloem fiber cells [24], the PS gene expression profile did not cluster with ST, and as expected was distantly placed from the rest of the vegetative tissues and seed tissues. Whole stems (ST) and etiolated seedlings (ES) showed a high degree of similarity, possibly due to their polysaccharide composition. Both whole stems and etiolated seedlings are likely to be particularly enriched in xylem tissues, the secondary walls of which produce polysaccharides different from those found in the pectin-enriched phloem fibers in (PS), seed coats (GC, TC), or the primary walls of developing embryos [25]. Taken together, this analysis showed three distinct patterns of relatedness of gene expression among the 13 tissues: early seed stages, the maturing embryo stages and the juvenile vegetative tissues (ES, ST and LF).


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)

Hierarchical cluster analysis of flax EST libraries. Three gene expression clusters were identified, viz., early differentiating seed tissues, maturing embryos and juvenile vegetative tissues. The tree shows hierarchical clustering of the tissue-based libraries based on similarity/distance as measured by the Pearson correlation coefficient. Values close to 1 have high degree of similarity whereas lower values indicate the degree of distance between two libraries. Globular embryo (GE), heart embryo (HE), torpedo embryo (TE), cotyledon embryo (CE), mature embryo (ME), globular stage seed coat (GC), torpedo stage seed coat (TC), pooled endosperm (EN), etiolated seedlings (ES), stem (ST), stem peel (PS), leaves (LF), and mature flower (FL).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Hierarchical cluster analysis of flax EST libraries. Three gene expression clusters were identified, viz., early differentiating seed tissues, maturing embryos and juvenile vegetative tissues. The tree shows hierarchical clustering of the tissue-based libraries based on similarity/distance as measured by the Pearson correlation coefficient. Values close to 1 have high degree of similarity whereas lower values indicate the degree of distance between two libraries. Globular embryo (GE), heart embryo (HE), torpedo embryo (TE), cotyledon embryo (CE), mature embryo (ME), globular stage seed coat (GC), torpedo stage seed coat (TC), pooled endosperm (EN), etiolated seedlings (ES), stem (ST), stem peel (PS), leaves (LF), and mature flower (FL).
Mentions: In order to compare the gene expression profile in different tissues, the entire set of 261,272 EST sequences was subjected to hierarchical cluster analysis using the software HCE3.5 [22] (see Methods). Amongst the parameters required for hierarchical cluster analysis, we selected the average linkage method and the Pearson correlation coefficient for the similarity/distance measure, a technique which has been widely used in microarray analysis [23]. The results are shown in Figure 4. The analysis shows that in general gene expression is most closely related in tissues that are developmentally related and connected. For example, globular (GE) and heart (HE) embryo stages are most closely related, followed closely by the torpedo stage (TE). The maturing embryos, viz., cotyledon (CE) and mature (ME) stages clustered together but were distantly placed from the early stage embryos. The two seed coat stages (GC and TC) also shared a relatively high degree of similarity to each other. Gene expression in the pooled endosperm tissue (EN) from early developing seed stages shared some similarity with early embryonic stages but was more distant from the seed coats and maturing embryos. It is interesting to note that the CE and ME stages cluster away from the early seed tissues (GE, HE, TE, GC, TC and EN) and to a lesser extent from other non-seed tissues viz., (ES, LE, FL, ST) which is indicative of the distinct seed maturation program that is occurring in the later stages of embryo development. As the stem peel (PS) did not contain all of the tissues normally present in whole stems (ST), and was enriched for the phloem and phloem fiber cells [24], the PS gene expression profile did not cluster with ST, and as expected was distantly placed from the rest of the vegetative tissues and seed tissues. Whole stems (ST) and etiolated seedlings (ES) showed a high degree of similarity, possibly due to their polysaccharide composition. Both whole stems and etiolated seedlings are likely to be particularly enriched in xylem tissues, the secondary walls of which produce polysaccharides different from those found in the pectin-enriched phloem fibers in (PS), seed coats (GC, TC), or the primary walls of developing embryos [25]. Taken together, this analysis showed three distinct patterns of relatedness of gene expression among the 13 tissues: early seed stages, the maturing embryo stages and the juvenile vegetative tissues (ES, ST and LF).

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