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Pathways associated with lignin biosynthesis in lignomaniac jute fibres

Significance statement 

Lignin, a phenylpropanoid polymer and a major sink for carbon in tracheophytes, is deposited in plant cell wall and plays a significant role in the differentiation of bast (phloem) and wood (xylem) fibres. The xylan-type bast fibre of jute (Corchorus capsularis L.), which has one of the smallest genomes (~300 Mbp), is a dynamic model for studying lignin biosynthesis, for it not only accumulates the highest percentage of lignin (13-22 %) in its secondary wall, but also produces more syringyl (S) than guaiacyl (G) lignin, which is comparable with that found in hardwood lignins. Using de novo transcriptome sequencing based on Illumina paired-end RNA-seq, we analyzed the bast (phloem fibre cell bundles including epidermis and cortex) tissues of a deficient lignified phloem fibre (dlpf) mutant and its wild-type (C. capsularis cv. JRC-212) and resolved the pathways associated with lignin biosynthesis in jute fibres. We assembled one of the best reference transcriptomes comprising unigenes that had the longest average length (1,442 bp) and N50 (1,999 bp) and were characterized by the highest proportion (55.2 %) of ≥1 kb fragments. This allowed us to discover and validate maximum numbers of isoforms of lignin biosynthetic genes in jute fibres as compared with that obtained in other eudicot species including bast fibre crops. We found that lignin biosynthesis occurs in jute fibres via well-conserved upstream shikimate and aromatic amino acid and downstream monolignol pathways across three important metabolic branch points represented by chorismate, phenylalanine (Phe) and p-coumaryl CoA. Phe, a gatekeeper metabolite in the phenylpropanoid pathway, is essentially synthesized from prephenate (a metabolite derived from chorismate) via the arogenate route that engages PPA-AT (prephenate aminotransferase) and ADT-PDT (arogenate/prephenate dehydratase) to catalyze prephenate to arogenate and then arogenate to Phe, respectively. However, we discovered that the phenylpyruvate (PPY) route that involves PPY-AT (PPY aminotransferase) to convert PPY to Phe is also active in jute fibres. The occurrence of both the routes to Phe biosynthesis is indicative of a higher metabolic flux to Phe biosynthesis to sustain an increased production of monolignols in lignin-rich jute fibres. The p-coumaryl CoA branch point that represents an important branch point in the lignin metabolic grid bifurcates to monolignol and flavonoid biosynthetic pathways. The discovery of a large number of HCT (shikimate O-hydroxycinnamoyltransferase) isoforms- that convert p-coumaryl CoA to p-coumaryl shikimic/quinic acid- in jute fibres indicates that the Phe metabolic flux from the p-coumaryl CoA branch point is mainly routed to the monolignol instead of flavonoid biosynthetic pathway (see Figure). We demonstrated that a block upstream in the shikimate pathway is associated with PAL (phenylalanine ammonia-lyase) disruption in the mutant (dlpf) fibres, suggesting that PAL is not the critical entry step in the monolignol pathway and lignin biosynthesis entails a coordinated metabolic network. The identification of maximum numbers of homologous isoforms of CAD (cinnamyl alcohol dehydrogenase) that transforms cinnamaldehydes (p-coumaraldehyde, coniferaldehyde and sinapaldehyde) to their corresponding alcohol derivatives (coumaryl alcohol, coniferyl alcohol and sinapyl alcohol) indicates a key role of this enzyme in jute fibre lignification. Our results categorically proved that S-lignin deficiency in the dlpf fibre accrues from several folds down-regulation of CcCAD7. Since CAD down-regulation did not seriously compromise plant growth and development in the dlpf mutant, we propose CAD as a promising target for reducing lignin content in jute fibres using forward or reverse genetic approaches.

Figure legend

Lignin biosynthesis in jute fibres via monolignol biosynthetic pathway. CAD, cinnamyl alcohol dehydrogenase (EC 1.1.1.195); CCoAOMT, caffeoyl-CoA O-methyltransferase (EC 2.1.1.104); CCR, cinnamoyl-CoA reductase (EC 1.2.1.44); C3’H, 5-O-(4-coumaroyl)-D-quinate/shikimate 3′-hydroxylase (EC 1.14.13.36); C4H, cinnamate 4-hydroxylase (EC 1.14.13.11); 4CL, 4-coumarate:CoA ligase (EC 6.2.1.12); COMT, caffeic acid O-methyltransferase (EC 2.1.1.68); F5H, ferulate 5-hydroxylase (EC 1.14.-.-); HCT, shikimate O-hydroxycinnamoyltransferase (EC 2.3.1.133); PAL, phenylalanine ammonia-lyase (EC 4.3.1.24); SAD, sinapyl alcohol dehydrogenase (EC 1.1.1.195)

Pathways associated with lignin biosynthesis in lignomaniac jute fibres-	. Global Medical Discovery

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Journal Reference

Chakraborty A, Sarkar D, Satya P, Karmakar PG, Singh NK. Mol Genet Genomics. 2015 Feb 28.

Biotechnology Unit, Division of Crop Improvement, ICAR-Central Research Institute for Jute and Allied Fibres (CRIJAF), Barrackpore, Kolkata, 700 120, West Bengal, India.

Abstract

We generated the bast transcriptomes of a deficient lignified phloem fibre mutant and its wild-type jute (Corchorus capsularis) using Illumina paired-end sequencing. A total of 34,163 wild-type and 29,463 mutant unigenes, with average lengths of 1442 and 1136  bp, respectively, were assembled de novo, ~77-79 % of which were functionally annotated. These annotated unigenes were assigned to COG (~37-40 %) and GO (~22-28 %) classifications and mapped to 189 KEGG pathways (~19-21 %). We discovered 38 and 43 isoforms of 16 and 10 genes of the upstream shikimate-aromatic amino acid and downstream monolignol biosynthetic pathways, respectively, rendered their sequence similarities, confirmed the identities of 22 of these candidate gene families by phylogenetic analyses and reconstructed the pathway leading to lignin biosynthesis in jute fibres. We also identified major genes and bast-related transcription factors involved in secondary cell wall (SCW) formation. The quantitative RT-PCRs revealed that phenylalanine ammonia-lyase 1 (CcPAL1) was co-down-regulated with several genes of the upstream shikimate pathway in mutant bast tissues at an early growth stage, although its expression relapsed to the normal level at the later growth stage. However, cinnamyl alcohol dehydrogenase 7 (CcCAD7) was strongly down-regulated in mutant bast tissues irrespective of growth stages. CcCAD7 disruption at an early growth stage was accompanied by co-up-regulation of SCW-specific genes cellulose synthase A7 (CcCesA7) and fasciclin-like arabinogalactan 6 (CcFLA6), which was predicted to be involved in coordinating the S-layers’ deposition in the xylan-type jute fibres. Our results identified CAD as a promising target for developing low-lignin jute fibres using genomics-assisted molecular approaches.

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