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Transcriptional profiling of sweetpotato (Ipomoea batatas) roots indicates down-regulation of lignin biosynthesis and up-regulation of starch biosynthesis at an early stage of storage root formation.

Firon N, LaBonte D, Villordon A, Kfir Y, Solis J, Lapis E, Perlman TS, Doron-Faigenboim A, Hetzroni A, Althan L, Adani Nadir L - BMC Genomics (2013)

Bottom Line: The reads, generated by the Illumina Genome Analyzer, were found to map to 31,284 contigs out of the 55,296 contigs serving as the database.A total of 8,353 contigs were found to exhibit differential expression between the two root types (at least 2.5-fold change).This resource enabled us to identify genes that are involved in the earliest stage of storage root formation, highlighting the reduction in carbon flow toward phenylpropanoid biosynthesis and its delivery into carbohydrate metabolism and starch biosynthesis, as major events involved in storage root initiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel. vcfiron@volcani.agri.gov.il

ABSTRACT

Background: The number of fibrous roots that develop into storage roots determines sweetpotato yield. The aim of the present study was to identify the molecular mechanisms involved in the initiation of storage root formation, by performing a detailed transcriptomic analysis of initiating storage roots using next-generation sequencing platforms. A two-step approach was undertaken: (1) generating a database for the sweetpotato root transcriptome using 454-Roche sequencing of a cDNA library created from pooled samples of two root types: fibrous and initiating storage roots; (2) comparing the expression profiles of initiating storage roots and fibrous roots, using the Illumina Genome Analyzer to sequence cDNA libraries of the two root types and map the data onto the root transcriptome database.

Results: Use of the 454-Roche platform generated a total of 524,607 reads, 85.6% of which were clustered into 55,296 contigs that matched 40,278 known genes. The reads, generated by the Illumina Genome Analyzer, were found to map to 31,284 contigs out of the 55,296 contigs serving as the database. A total of 8,353 contigs were found to exhibit differential expression between the two root types (at least 2.5-fold change). The Illumina-based differential expression results were validated for nine putative genes using quantitative real-time PCR. The differential expression profiles indicated down-regulation of classical root functions, such as transport, as well as down-regulation of lignin biosynthesis in initiating storage roots, and up-regulation of carbohydrate metabolism and starch biosynthesis. In addition, data indicated delicate control of regulators of meristematic tissue identity and maintenance, associated with the initiation of storage root formation.

Conclusions: This study adds a valuable resource of sweetpotato root transcript sequences to available data, facilitating the identification of genes of interest. This resource enabled us to identify genes that are involved in the earliest stage of storage root formation, highlighting the reduction in carbon flow toward phenylpropanoid biosynthesis and its delivery into carbohydrate metabolism and starch biosynthesis, as major events involved in storage root initiation. The novel transcripts related to storage root initiation identified in this study provide a starting point for further investigation into the molecular mechanisms underlying this process.

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Changes in the phenylpropanoid biosynthesis pathway map between fibrous roots (FRs) and initiating storage roots (ISRs). Enzymes exhibiting up-regulated expression in ISRs and FRs are marked in green and in red, respectively. Marked in light green are enzymes representing gene sequences that exhibit up-regulated expression in both ISRs and FRs (in most cases, a larger number of contigs exhibited higher expression in FRs compared to ISRs (Table 5)). Marked in white are enzymes representing gene sequences that were not detected in the Illumina-generated transcription profiles (exhibited less than 10 reads) or general enzyme categories representing an enzyme class (such as 4.1.1.-, 2.1.1- and 5.2.1-, representing lyases, methyltransferases and isomerases, respectively). Enzyme annotation was obtained from the sequence annotation and GO classification data.
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Figure 11: Changes in the phenylpropanoid biosynthesis pathway map between fibrous roots (FRs) and initiating storage roots (ISRs). Enzymes exhibiting up-regulated expression in ISRs and FRs are marked in green and in red, respectively. Marked in light green are enzymes representing gene sequences that exhibit up-regulated expression in both ISRs and FRs (in most cases, a larger number of contigs exhibited higher expression in FRs compared to ISRs (Table 5)). Marked in white are enzymes representing gene sequences that were not detected in the Illumina-generated transcription profiles (exhibited less than 10 reads) or general enzyme categories representing an enzyme class (such as 4.1.1.-, 2.1.1- and 5.2.1-, representing lyases, methyltransferases and isomerases, respectively). Enzyme annotation was obtained from the sequence annotation and GO classification data.

Mentions: Among the significantly enriched pathways in FRs, high enrichment was observed for the phenylpropanoid biosynthesis pathway (Table 4). The first three biosynthetic reactions in this pathway, referred to as the general phenylpropanoid pathway, produce p-coumaroyl CoA, which is a major branch-point metabolite between the production of the flavonoids and the pathway that produces monolignols, lignans and hydroxy-cinnamate conjugates [[52] and references therein]. The first of these reactions is the deamination of phenylalanine by phenylalanine ammonia-lyase (PAL) to generate trans-cinnamic acid. Cinnamic acid is then para-hydroxylated by cinnamate 4-hydroxylase (C4H) to produce p-coumaric acid [[52] and references therein], which is then activated to its corresponding CoA thioester by 4-coumarate CoA ligase (4 coumaroyl-CoA synthase; 4CL). All phenylalanine-derived units destined to be incorporated into the lignin polymer must be hydroxylated by C4H, because the p-hydroxy group is required for the activation of monolignols to their corresponding free radicals, and for polymerization into lignin. The phenylpropanoid biosynthesis pathway map is presented in Figure 11, with the enzymes exhibiting up-regulated expression in ISRs and FRs marked in green and red, respectively. Over twofold up-regulation in FRs of contigs representing C4H and 4CL, as well as of contigs of coniferyl-alcohol glucosyltransferase was apparent (Figure 11 and Table 5). In addition, high expression of PAL was detected in the FR sample, whereas an over fourfold reduction in read number was observed in the ISR sample (Additional file 5). The presence of several contigs representing these enzymes may indicate the presence of isoenzymes. 4CL catalyzes the formation of CoA esters of caffeic acid, ferulic acid, 5-hydroxyferulic acid, and sinapic acid, in addition to p-coumaric acid [[53] and references therein]. The plethora of additional potential substrates may explain why there are many 4CL isoenzymes in most plants. In addition to the different substrate specificities, the genes may have a distinct spatiotemporal expression pattern [[53] and references therein]. Looking into the read number of contigs representing genes of the lignin pathway, such as cinnamyl alcohol dehydrogenase (CAD), more than fivefold lower expression was detected in the ISR vs. FR sample (Table 5 and Additional file 5). Taken together, the results indicate down-regulation in the expression of key genes of the phenylpropanoid biosynthesis pathway upon the change in root fate from FR to a storage organ, which may be responsible for the significant reduction in lignin levels (Figure 1), representing novel data not previously described in sweetpotato. Indeed, it has been demonstrated in Arabidopsis and tobacco that down-regulating 4CL results in reduced lignin content [54-56]. Hu et al. [57] showed that down-regulating the expression of 4CL in transgenic aspen (Populus tremuloides Michx.) by antisense inhibition causes up to 45% reduction in lignin. Reductions in lignin content in Arabidopsis plants carrying a mutation in the second enzyme of this pathway, C4H, were shown to accumulate decreased levels of several different classes of phenylpropanoid end products and to exhibit reduced lignin deposition, altered lignin monomer content and a collapsed xylem phenotype [52].


Transcriptional profiling of sweetpotato (Ipomoea batatas) roots indicates down-regulation of lignin biosynthesis and up-regulation of starch biosynthesis at an early stage of storage root formation.

Firon N, LaBonte D, Villordon A, Kfir Y, Solis J, Lapis E, Perlman TS, Doron-Faigenboim A, Hetzroni A, Althan L, Adani Nadir L - BMC Genomics (2013)

Changes in the phenylpropanoid biosynthesis pathway map between fibrous roots (FRs) and initiating storage roots (ISRs). Enzymes exhibiting up-regulated expression in ISRs and FRs are marked in green and in red, respectively. Marked in light green are enzymes representing gene sequences that exhibit up-regulated expression in both ISRs and FRs (in most cases, a larger number of contigs exhibited higher expression in FRs compared to ISRs (Table 5)). Marked in white are enzymes representing gene sequences that were not detected in the Illumina-generated transcription profiles (exhibited less than 10 reads) or general enzyme categories representing an enzyme class (such as 4.1.1.-, 2.1.1- and 5.2.1-, representing lyases, methyltransferases and isomerases, respectively). Enzyme annotation was obtained from the sequence annotation and GO classification data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716973&req=5

Figure 11: Changes in the phenylpropanoid biosynthesis pathway map between fibrous roots (FRs) and initiating storage roots (ISRs). Enzymes exhibiting up-regulated expression in ISRs and FRs are marked in green and in red, respectively. Marked in light green are enzymes representing gene sequences that exhibit up-regulated expression in both ISRs and FRs (in most cases, a larger number of contigs exhibited higher expression in FRs compared to ISRs (Table 5)). Marked in white are enzymes representing gene sequences that were not detected in the Illumina-generated transcription profiles (exhibited less than 10 reads) or general enzyme categories representing an enzyme class (such as 4.1.1.-, 2.1.1- and 5.2.1-, representing lyases, methyltransferases and isomerases, respectively). Enzyme annotation was obtained from the sequence annotation and GO classification data.
Mentions: Among the significantly enriched pathways in FRs, high enrichment was observed for the phenylpropanoid biosynthesis pathway (Table 4). The first three biosynthetic reactions in this pathway, referred to as the general phenylpropanoid pathway, produce p-coumaroyl CoA, which is a major branch-point metabolite between the production of the flavonoids and the pathway that produces monolignols, lignans and hydroxy-cinnamate conjugates [[52] and references therein]. The first of these reactions is the deamination of phenylalanine by phenylalanine ammonia-lyase (PAL) to generate trans-cinnamic acid. Cinnamic acid is then para-hydroxylated by cinnamate 4-hydroxylase (C4H) to produce p-coumaric acid [[52] and references therein], which is then activated to its corresponding CoA thioester by 4-coumarate CoA ligase (4 coumaroyl-CoA synthase; 4CL). All phenylalanine-derived units destined to be incorporated into the lignin polymer must be hydroxylated by C4H, because the p-hydroxy group is required for the activation of monolignols to their corresponding free radicals, and for polymerization into lignin. The phenylpropanoid biosynthesis pathway map is presented in Figure 11, with the enzymes exhibiting up-regulated expression in ISRs and FRs marked in green and red, respectively. Over twofold up-regulation in FRs of contigs representing C4H and 4CL, as well as of contigs of coniferyl-alcohol glucosyltransferase was apparent (Figure 11 and Table 5). In addition, high expression of PAL was detected in the FR sample, whereas an over fourfold reduction in read number was observed in the ISR sample (Additional file 5). The presence of several contigs representing these enzymes may indicate the presence of isoenzymes. 4CL catalyzes the formation of CoA esters of caffeic acid, ferulic acid, 5-hydroxyferulic acid, and sinapic acid, in addition to p-coumaric acid [[53] and references therein]. The plethora of additional potential substrates may explain why there are many 4CL isoenzymes in most plants. In addition to the different substrate specificities, the genes may have a distinct spatiotemporal expression pattern [[53] and references therein]. Looking into the read number of contigs representing genes of the lignin pathway, such as cinnamyl alcohol dehydrogenase (CAD), more than fivefold lower expression was detected in the ISR vs. FR sample (Table 5 and Additional file 5). Taken together, the results indicate down-regulation in the expression of key genes of the phenylpropanoid biosynthesis pathway upon the change in root fate from FR to a storage organ, which may be responsible for the significant reduction in lignin levels (Figure 1), representing novel data not previously described in sweetpotato. Indeed, it has been demonstrated in Arabidopsis and tobacco that down-regulating 4CL results in reduced lignin content [54-56]. Hu et al. [57] showed that down-regulating the expression of 4CL in transgenic aspen (Populus tremuloides Michx.) by antisense inhibition causes up to 45% reduction in lignin. Reductions in lignin content in Arabidopsis plants carrying a mutation in the second enzyme of this pathway, C4H, were shown to accumulate decreased levels of several different classes of phenylpropanoid end products and to exhibit reduced lignin deposition, altered lignin monomer content and a collapsed xylem phenotype [52].

Bottom Line: The reads, generated by the Illumina Genome Analyzer, were found to map to 31,284 contigs out of the 55,296 contigs serving as the database.A total of 8,353 contigs were found to exhibit differential expression between the two root types (at least 2.5-fold change).This resource enabled us to identify genes that are involved in the earliest stage of storage root formation, highlighting the reduction in carbon flow toward phenylpropanoid biosynthesis and its delivery into carbohydrate metabolism and starch biosynthesis, as major events involved in storage root initiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel. vcfiron@volcani.agri.gov.il

ABSTRACT

Background: The number of fibrous roots that develop into storage roots determines sweetpotato yield. The aim of the present study was to identify the molecular mechanisms involved in the initiation of storage root formation, by performing a detailed transcriptomic analysis of initiating storage roots using next-generation sequencing platforms. A two-step approach was undertaken: (1) generating a database for the sweetpotato root transcriptome using 454-Roche sequencing of a cDNA library created from pooled samples of two root types: fibrous and initiating storage roots; (2) comparing the expression profiles of initiating storage roots and fibrous roots, using the Illumina Genome Analyzer to sequence cDNA libraries of the two root types and map the data onto the root transcriptome database.

Results: Use of the 454-Roche platform generated a total of 524,607 reads, 85.6% of which were clustered into 55,296 contigs that matched 40,278 known genes. The reads, generated by the Illumina Genome Analyzer, were found to map to 31,284 contigs out of the 55,296 contigs serving as the database. A total of 8,353 contigs were found to exhibit differential expression between the two root types (at least 2.5-fold change). The Illumina-based differential expression results were validated for nine putative genes using quantitative real-time PCR. The differential expression profiles indicated down-regulation of classical root functions, such as transport, as well as down-regulation of lignin biosynthesis in initiating storage roots, and up-regulation of carbohydrate metabolism and starch biosynthesis. In addition, data indicated delicate control of regulators of meristematic tissue identity and maintenance, associated with the initiation of storage root formation.

Conclusions: This study adds a valuable resource of sweetpotato root transcript sequences to available data, facilitating the identification of genes of interest. This resource enabled us to identify genes that are involved in the earliest stage of storage root formation, highlighting the reduction in carbon flow toward phenylpropanoid biosynthesis and its delivery into carbohydrate metabolism and starch biosynthesis, as major events involved in storage root initiation. The novel transcripts related to storage root initiation identified in this study provide a starting point for further investigation into the molecular mechanisms underlying this process.

Show MeSH
Related in: MedlinePlus