Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility.

BACKGROUND: Lignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing. RESULTS: Arabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes. CONCLUSIONS: Simultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.

Xylobiose treatment triggers a defense-related response and alters cell wall composition.

Plant cell wall-derived oligosaccharides, i.e., damage-associated molecular patterns (DAMPs), could be generated after pathogen attack or during normal plant development, perceived by cell wall receptors, and can alter immunity and cell wall composition. Therefore, we hypothesised that xylo-oligosaccharides (XOS) could act as an elicitor and trigger immune responses. To test this, we treated Arabidopsis with xylobiose (XB) and investigated different parameters. XB-treatment significantly triggered the generation of reactive oxygen species (ROS), activated MAPK protein phosphorylation, and induced callose deposition. The combination of XB (DAMP) and flg22 a microbe-associated molecular pattern (MAMP) further enhanced ROS response and gene expression of PTI marker genes. RNA sequencing analysis revealed that more genes were differentially regulated after 30 min compared to 24 h XB-treated leaves, which correlated with ROS response. Increased xylosidase activity and soluble xylose level after 30 min and 3 h of XB-treatment were observed which might have weakened the DAMP response. However, an increase in total cell wall sugar and a decrease in uronic acid level was observed at both 30 min and 24 h. Additionally, arabinose, rhamnose, and xylose levels were increased in 30 min, and glucose was increased in 24 h compared to mock-treated leaves. The level of jasmonic acid, abscisic acid, auxin, and cytokinin were also affected after XB treatment. Overall, our data revealed that the shortest XOS can act as a DAMP, which triggers the PTI response and alters cell wall composition and hormone level.

Morpho-biochemical characterization of a RIL population for seed parameters and identification of candidate genes regulating seed size trait in lentil (Lens culinaris Medik.).

The seed size and shape in lentil (Lens culinaris Medik.) are important quality traits as these influences the milled grain yield, cooking time, and market class of the grains. Linkage analysis was done for seed size in a RIL (F5:6) population derived by crossing L830 (20.9 g/1000 seeds) with L4602 (42.13 g/1000 seeds) which consisted of 188 lines (15.0 to 40.5 g/1000 seeds). Parental polymorphism survey using 394 SSRs identified 31 polymorphic primers, which were used for the bulked segregant analysis (BSA). Marker PBALC449 differentiated the parents and small seed size bulk only, whereas large seeded bulk or the individual plants constituting the large-seeded bulk could not be differentiated. Single plant analysis identified only six recombinant and 13 heterozygotes, of 93 small-seeded RILs (<24.0 g/1000 seed). This clearly showed that the small seed size trait is very strongly regulated by the locus near PBLAC449; whereas, large seed size trait seems governed by more than one locus. The PCR amplified products from the PBLAC449 marker (149bp from L4602 and 131bp from L830) were cloned, sequenced and BLAST searched using the lentil reference genome and was found amplified from chromosome 03. Afterward, the nearby region on chromosome 3 was searched, and a few candidate genes like ubiquitin carboxyl-terminal hydrolase, E3 ubiquitin ligase, TIFY-like protein, and hexosyltransferase having a role in seed size determination were identified. Validation study in another RIL mapping population which is differing for seed size, showed a number of SNPs and InDels among these genes when studied using whole genome resequencing (WGRS) approach. Biochemical parameters like cellulose, lignin, and xylose content showed no significant differences between parents and the extreme RILs, at maturity. Various seed morphological traits like area, length, width, compactness, volume, perimeter, etc., when measured using VideometerLab 4.0 showed significant differences for the parents and RILs. The results have ultimately helped in better understanding the region regulating the seed size trait in genomically less explored crops like lentils.

Comparative transcriptome analysis, unfolding the pathways regulating the seed-size trait in cultivated lentil (Lens culinaris Medik.).

Market class, cooking time, quality, and milled grain yield are largely influenced by the seed size and shape of the lentil (Lens culinaris Medik.); thus, they are considered to be important quality traits. To unfold the pathways regulating seed size in lentils, a transcriptomic approach was performed using large-seeded (L4602) and small-seeded (L830) genotypes. The study has generated nearly 375 million high-quality reads, of which 98.70% were properly aligned to the reference genome. Among biological replicates, very high similarity in fragments per kilobase of exon per million mapped fragments values (R > 0.9) showed the consistency of RNA-seq results. Various differentially expressed genes associated mainly with the hormone signaling and cell division pathways, transcription factors, kinases, etc. were identified as having a role in cell expansion and seed growth. A total of 106,996 unigenes were used for differential expression (DE) analysis. String analysis identified various modules having certain key proteins like Ser/Thr protein kinase, seed storage protein, DNA-binding protein, microtubule-associated protein, etc. In addition, some growth and cell division-related micro-RNAs like miR3457 (cell wall formation), miR1440 (cell proliferation and cell cycles), and miR1533 (biosynthesis of plant hormones) were identified as having a role in seed size determination. Using RNA-seq data, 5254 EST-SSR primers were generated as a source for future studies aiming for the identification of linked markers. In silico validation using Genevestigator® was done for the Ser/Thr protein kinase, ethylene response factor, and Myb transcription factor genes. It is of interest that the xyloglucan endotransglucosylase gene was found differentially regulated, suggesting their role during seed development; however, at maturity, no significant differences were recorded for various cell wall parameters including cellulose, lignin, and xylose content. This is the first report on lentils that has unfolded the key seed size regulating pathways and unveiled a theoretical way for the development of lentil genotypes having customized seed sizes.

Arabidopsis GELP7 functions as a plasma membrane-localized acetyl xylan esterase, and its overexpression improves saccharification efficiency.

Acetyl substitution on the xylan chain is critical for stable interaction with cellulose and other cell wall polymers in the secondary cell wall. Xylan acetylation pattern is governed by Golgi and extracellular localized acetyl xylan esterase (AXE). We investigated the role of Arabidopsis clade Id from the GDSL esterase/lipase or GELP family in polysaccharide deacetylation. The investigation of the AtGELP7 T-DNA mutant line showed a decrease in stem esterase activity and an increase in stem acetyl content. We further generated overexpressor AtGELP7 transgenic lines, and these lines showed an increase in AXE activity and a decrease in xylan acetylation compared to wild-type plants. Therefore, we have named this enzyme as AtAXE1. The subcellular localization and immunoblot studies showed that the AtAXE1 enzyme is secreted out, associated with the plasma membrane and involved in xylan de-esterification post-synthesis. The cellulose digestibility was improved in AtAXE1 overexpressor lines without pre-treatment, after alkali and xylanases pre-treatment. Furthermore, we have also established that the AtGELP7 gene is upregulated in the overexpressor line of AtMYB46, a secondary cell wall specific transcription factor. This transcriptional regulation can drive AtGELP7 or AtAXE1 to perform de-esterification of xylan in a tissue-specific manner. Overall, these data suggest that AtGELP7 overexpression in Arabidopsis reduces xylan acetylation and improves digestibility properties of polysaccharides of stem lignocellulosic biomass.