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A deep transcriptomic analysis of pod development in the vanilla orchid (Vanilla planifolia).

Rao X, Krom N, Tang Y, Widiez T, Havkin-Frenkel D, Belanger FC, Dixon RA, Chen F - BMC Genomics (2014)

Bottom Line: The combined 454/Illumina RNA-seq platforms provide both deep sequence coverage and high quality de novo transcriptome assembly for this non-model crop species.The annotated sequence data provide a foundation for understanding multiple aspects of the biochemistry and development of the vanilla bean, as exemplified by the identification of candidate genes involved in lignin biosynthesis.This database provides a general resource for further studies on this important flavor species.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of North Texas, 1155 Union Circle #305220, Denton, TX 76203, USA. Xiaolan.Rao@unt.edu.

ABSTRACT

Background: Pods of the vanilla orchid (Vanilla planifolia) accumulate large amounts of the flavor compound vanillin (3-methoxy, 4-hydroxy-benzaldehyde) as a glucoside during the later stages of their development. At earlier stages, the developing seeds within the pod synthesize a novel lignin polymer, catechyl (C) lignin, in their coats. Genomic resources for determining the biosynthetic routes to these compounds and other flavor components in V. planifolia are currently limited.

Results: Using next-generation sequencing technologies, we have generated very large gene sequence datasets from vanilla pods at different times of development, and representing different tissue types, including the seeds, hairs, placental and mesocarp tissues. This developmental series was chosen as being the most informative for interrogation of pathways of vanillin and C-lignin biosynthesis in the pod and seed, respectively. The combined 454/Illumina RNA-seq platforms provide both deep sequence coverage and high quality de novo transcriptome assembly for this non-model crop species.

Conclusions: The annotated sequence data provide a foundation for understanding multiple aspects of the biochemistry and development of the vanilla bean, as exemplified by the identification of candidate genes involved in lignin biosynthesis. Our transcriptome data indicate that C-lignin formation in the seed coat involves coordinate expression of monolignol biosynthetic genes with the exception of those encoding the caffeoyl coenzyme A 3-O-methyltransferase for conversion of caffeoyl to feruloyl moieties. This database provides a general resource for further studies on this important flavor species.

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Validation of lignin-related gene expression in the early stage seeds and stems. qRT-PCR was performed to verify the expression patterns of PAL, 4CL, CCR, CAD, C4H and CCoAOMT in the light and dark pod tissues, seeds at 6, 8 and 10 weeks and in stems. Expression values were normalized to expression levels of actin, and the expression value in 6 W dark was set to 1. The results are the means of duplicate measurements of two biological replicates, and p-values for difference in expression between 8 week seed tissue and 6 week dark pod tissues were less than 0.01 (Student’s t-test) for all transcripts tested.
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Fig8: Validation of lignin-related gene expression in the early stage seeds and stems. qRT-PCR was performed to verify the expression patterns of PAL, 4CL, CCR, CAD, C4H and CCoAOMT in the light and dark pod tissues, seeds at 6, 8 and 10 weeks and in stems. Expression values were normalized to expression levels of actin, and the expression value in 6 W dark was set to 1. The results are the means of duplicate measurements of two biological replicates, and p-values for difference in expression between 8 week seed tissue and 6 week dark pod tissues were less than 0.01 (Student’s t-test) for all transcripts tested.

Mentions: Most of the predicted lignin-related genes shared similar expression patterns in pod tissues, with highest expression in seeds at 10 weeks post-pollination, and with lowest expression in the dark and light mesocarp tissues (Figure 7B). This pattern was observed for most of the contigs representing each gene. However, although CAD transcripts were, overall, found at highest level in developing seeds, some CAD contigs were more highly expressed in the light mesocarp tissue. More strikingly, CCoAOMT transcripts were virtually absent from developing seeds, in spite of the high expression level of the other lignin biosynthetic genes. These digital expression patterns were confirmed by quantitative real-time RT-PCR analysis of selected contigs corresponding to PAL, 4CL, CCR, CAD, C4H and CCoAOMT in early developmental stages of seeds as well as in stems. The sequences of the primers used for RT-PCR are given in Additional file 15. Consistent with the digital expression database, PAL, 4CL, CCR, CAD and C4H displayed very high transcript levels in 8 week seeds and relatively high levels in 10 week seeds, while low transcript levels in dark and light mesocarp tissues. In contrast, CCoAOMT transcripts were detected in dark and light mesocarp tissues, but barely in seed tissues (Figure 8). The low level of CCoAOMT transcripts is likely the biochemical basis for the production of the novel non-methylated C-lignin in the vanilla seed coats [5]. This result is consistent with our earlier finding of low OMT enzyme activities in the C-lignin containing seed coat of Cleome hassleriana[32]. It is currently not clear how the low expression of the O-methyltransferases is regulated in vanilla seeds. Deep mining of transcription factors in the transcriptome database should help us to answer this question.Figure 8


A deep transcriptomic analysis of pod development in the vanilla orchid (Vanilla planifolia).

Rao X, Krom N, Tang Y, Widiez T, Havkin-Frenkel D, Belanger FC, Dixon RA, Chen F - BMC Genomics (2014)

Validation of lignin-related gene expression in the early stage seeds and stems. qRT-PCR was performed to verify the expression patterns of PAL, 4CL, CCR, CAD, C4H and CCoAOMT in the light and dark pod tissues, seeds at 6, 8 and 10 weeks and in stems. Expression values were normalized to expression levels of actin, and the expression value in 6 W dark was set to 1. The results are the means of duplicate measurements of two biological replicates, and p-values for difference in expression between 8 week seed tissue and 6 week dark pod tissues were less than 0.01 (Student’s t-test) for all transcripts tested.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4233054&req=5

Fig8: Validation of lignin-related gene expression in the early stage seeds and stems. qRT-PCR was performed to verify the expression patterns of PAL, 4CL, CCR, CAD, C4H and CCoAOMT in the light and dark pod tissues, seeds at 6, 8 and 10 weeks and in stems. Expression values were normalized to expression levels of actin, and the expression value in 6 W dark was set to 1. The results are the means of duplicate measurements of two biological replicates, and p-values for difference in expression between 8 week seed tissue and 6 week dark pod tissues were less than 0.01 (Student’s t-test) for all transcripts tested.
Mentions: Most of the predicted lignin-related genes shared similar expression patterns in pod tissues, with highest expression in seeds at 10 weeks post-pollination, and with lowest expression in the dark and light mesocarp tissues (Figure 7B). This pattern was observed for most of the contigs representing each gene. However, although CAD transcripts were, overall, found at highest level in developing seeds, some CAD contigs were more highly expressed in the light mesocarp tissue. More strikingly, CCoAOMT transcripts were virtually absent from developing seeds, in spite of the high expression level of the other lignin biosynthetic genes. These digital expression patterns were confirmed by quantitative real-time RT-PCR analysis of selected contigs corresponding to PAL, 4CL, CCR, CAD, C4H and CCoAOMT in early developmental stages of seeds as well as in stems. The sequences of the primers used for RT-PCR are given in Additional file 15. Consistent with the digital expression database, PAL, 4CL, CCR, CAD and C4H displayed very high transcript levels in 8 week seeds and relatively high levels in 10 week seeds, while low transcript levels in dark and light mesocarp tissues. In contrast, CCoAOMT transcripts were detected in dark and light mesocarp tissues, but barely in seed tissues (Figure 8). The low level of CCoAOMT transcripts is likely the biochemical basis for the production of the novel non-methylated C-lignin in the vanilla seed coats [5]. This result is consistent with our earlier finding of low OMT enzyme activities in the C-lignin containing seed coat of Cleome hassleriana[32]. It is currently not clear how the low expression of the O-methyltransferases is regulated in vanilla seeds. Deep mining of transcription factors in the transcriptome database should help us to answer this question.Figure 8

Bottom Line: The combined 454/Illumina RNA-seq platforms provide both deep sequence coverage and high quality de novo transcriptome assembly for this non-model crop species.The annotated sequence data provide a foundation for understanding multiple aspects of the biochemistry and development of the vanilla bean, as exemplified by the identification of candidate genes involved in lignin biosynthesis.This database provides a general resource for further studies on this important flavor species.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of North Texas, 1155 Union Circle #305220, Denton, TX 76203, USA. Xiaolan.Rao@unt.edu.

ABSTRACT

Background: Pods of the vanilla orchid (Vanilla planifolia) accumulate large amounts of the flavor compound vanillin (3-methoxy, 4-hydroxy-benzaldehyde) as a glucoside during the later stages of their development. At earlier stages, the developing seeds within the pod synthesize a novel lignin polymer, catechyl (C) lignin, in their coats. Genomic resources for determining the biosynthetic routes to these compounds and other flavor components in V. planifolia are currently limited.

Results: Using next-generation sequencing technologies, we have generated very large gene sequence datasets from vanilla pods at different times of development, and representing different tissue types, including the seeds, hairs, placental and mesocarp tissues. This developmental series was chosen as being the most informative for interrogation of pathways of vanillin and C-lignin biosynthesis in the pod and seed, respectively. The combined 454/Illumina RNA-seq platforms provide both deep sequence coverage and high quality de novo transcriptome assembly for this non-model crop species.

Conclusions: The annotated sequence data provide a foundation for understanding multiple aspects of the biochemistry and development of the vanilla bean, as exemplified by the identification of candidate genes involved in lignin biosynthesis. Our transcriptome data indicate that C-lignin formation in the seed coat involves coordinate expression of monolignol biosynthetic genes with the exception of those encoding the caffeoyl coenzyme A 3-O-methyltransferase for conversion of caffeoyl to feruloyl moieties. This database provides a general resource for further studies on this important flavor species.

Show MeSH