Genome mapping and gene expression of NDP-sugar pathways in the giant duckweed Spirodela polyrhiza and its relevance for bioenergy

Duckweeds are fast-growing aquatic plants suitable for bioenergy due to fermentable-rich biomass with low lignin. The duckweed sub-families Lemnoideae and Wolffioideae are also distinguished by the distribution of two pectin classes 24 (apiogalacturonan and xylogalacturonan), which seem to be related to their growing capacity and the starch content. The 25 plant cell wall is built from pathways of nucleotide sugars syntheses that culminate in cell wall synthesis and deposition. 26 Therefore, understanding these pathways through mapping the genes involved and their expression would be important mannan ( CSLA ) syntheses - corroborating the chemical composition of S. polyrhiza cell wall . We further investigated the carbohydrate metabolism pathways and discussed the implications of altering the NDP pathways for bioenergy and biorefinery. We conclude that S. polyrhiza displays suitable features for future genetic transformations leading to the 35 adaptation of its cell wall for biofuels. However, such strategies will have to consider the trade-offs between fermentation and ethanol production benefits and the potential adverse effects of genetic transformation on plant growth and development. microclimate conditions, time of cultivation, and hydrolysis method were considered for comparison. The normalized EC 2.4.1.43) catalyzes the transfer of UDP-GalA into the pectic polysaccharide homogalacturonan. The mannan synthesis pathway descends directly from fructose-6P and involves the enzymes mannose-6-phosphate-isomerase (MPI, EC 5.3.1.8), phosphomannomutase (PMM, EC 5.4.2.8), and mannose-1-phosphate-guanylyltransferase (MGP, EC 2.7.7.13). Mannans, hemicelluloses of mannose chains, are synthesized by mannan synthase (MSR, EC 2.4.1-), and the backbone for glucomannan is built from 4-beta-mannosyltransferase (CSLA, EC 2.4.1.32) activity on mannan GDP-mannose. sugar found in pectins xyloglucans, derived the precursor GDP-mannose by dehydration, epimerization, and reduction of UDP-Man in UDP-Fuc by the action of GDP-mannose-4,6-dehydratase (GMD, EC 4.2.1.47), and GDP-4-keto-deoxy-d-mannose-3,5-epimerase-4-reductase EC 1.1.1.271). The salvage pathway of fucose constitutes the phosphorylation of free fucose followed by GMP attachment which involves fucokinase (FKGP, EC 2.7.1.52).


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In plants, besides the mechanical resistance and cell protection, the cell wall promotes cell adhesion, delimiting the 54 cell size and volume. Cell walls regulate the conduction of water and solutes within the plant. They also determine the 55 turgor pressure and act as a signaling molecule source [11][12][13][14][15]. This encrypted polysaccharide structure displays a glycomic 56 code [1], whose understanding holds the potential to elucidate new mechanisms associated with plant physiology and 57 creates a niche for its application.

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The cell wall is one of the most significant biomass sources, making available sugars for feedstock and industrial 59 applications such as bioenergy, cosmetology, pharmaceutical, and food inputs [16][17][18][19]. Likewise, the cell walls are also an 60 important carbon sink, fixed photosynthetically [19]. The main product of carbon assimilated through photosynthesis is

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For the cell wall synthesis, the triose-phosphate sent to the cytosol is condensed into hexose-phosphates, which will 67 generate the substrate for the nucleotide diphosphate (NDP)-sugars pathway [19]. Also, the NDP-sugars can be generated 68 from the catabolism of sugars released from storage polymers, glycoproteins, glycolipids, and polysaccharide's recycling 69 during plant growth and development (primary and secondary walls restructuring) [26]. The latter can be described as a 70 salvage pathway in which free sugars are recovered to produce sugars-1-phosphate [27]. After polymers turnover, the free 71 sugars from the cell wall must be imported to the cell again before its reactivation into NDP-sugars [27]. Here we will focus 72 on cell wall synthesis and possible applications of this pathway for biorefinery.

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The recovered genes were divided into the categories that we named as: "starch and sucrose metabolism" (

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SpCSLA, SpFKGP, and SpASD -65 orthologues), and "cellulose" (SpCESA, 23 orthologues) (Figures 1 and 3). The 257 distribution of the selected genes of this work, which focuses on the carbon pathways that lead to storage (starch and 258 sucrose) or structure (cell walls), which tends to have a higher number of genes in the chromosomes 1 and 2 (24 and 22 259 genes, respectively) that are the largest ones (14.77 and 11.37 Mb) (Figures 3 and 4).  and 20. Regarding cellulose, approximately 50% of the genes are in chromosomes 7, 10, and 11 (Figures 3 and 4).  (Table   301 2), as can be seen in the fractionation process in which this class of polysaccharides is half of the S. polyrhiza cell wall 302 ( Figure 2 and Table 1). The transcript level of SpAXS, which encodes the enzyme responsible for apiose biosynthesis, was 303 1.8 times lower than another pectin-related gene (SpGAUT1), while apiose was one of the most significant neutral 304 monosaccharides found in the S. polyrhiza cell wall (Tables 2 and 1).

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polyrhiza, represent only 13% of the cell wall (Figure 2 and    here it is reported that pectins represent 49% of the S. polyrhiza cell wall (Figure 2 and Table 1). Some of these pectins 386 are likely to be linked to the cellulose microfibrils since pectin monosaccharides (apiose and rhamnose) were found on the 387 cellulose-containing residue fractions (Table 1) [129], blood glucose levels [130], and stimulate the immune response [131]. Thus, apiogalacturonan properties should also be investigated for these alternative purposes, and as its biosynthesis is dependent on a unique copy 425 of the S. polyrhiza genome, molecular approaches should be interesting.

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Fucose is a monosaccharide present in rhamnogalacturonan type-II (RG-II) and xyloglucan. It is derived from    (Figure 1). SpRHM relative expression level was Medium (0.91) (Table   436 2), and rhamnose represented 8.8% of the cell wall monosaccharides (28.36 µg.mg -1 AIR -   Table 2). The pathway to mannan synthesis has a low number of gene copies, except for the glucomannan 485 synthase (MSR) and glucomannan-4β-mannosyltransferase (CSLA) gene with 21 and 16 paralogs, respectively 486 (Supplemental Table 1 from previous studies has been compared with the ones obtained in this work and unpublished lab data from our lab. They 501 are presented in Supplemental Table 13. The quantitative proportions of the sugars vary according to growth conditions 502 (light intensity, photoperiod, temperature). However, when the data is normalized for comparative purposes of the 503 percentages, a pattern emerges with 20.6% galactose, 17.4% arabinose, 15.5% xylose, 21.9% apiose, 5.4% mannose, 7.5% 504 rhamnose, 9.3% glucose, and 2.4% fucose (Supplemental Table 13 heatmap average). Therefore, quantitative data must 505 be evaluated individually in each study, considering the plant growth conditions and the cell wall composition. The 506 fermented sugars make up 60.7%, which is suitable for bioenergy applications.

Conclusion 508
The present work contributes to the elucidation of polysaccharides' metabolism in the cell wall and their relevance 509 for industrial applications. Additionally, the determination of the reference genes and family structures in S. polyrhiza can 510 contribute to further studies of the duckweed plant family. We found that the distribution of genes throughout the 511 chromosomes may not be random, with clusters of genes that differ according to their functional category.

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Furthermore, 190 genes were found in the genome of S. polyrhiza associated with pathways related to the 513 formation of the cell walls and starch. We selected 38 of these genes to study expression in developing plants of S. polyrhiza.

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Their expression corroborated the composition of the sugars found in the plant. We conclude that S. polyrhiza carbohydrates 515 display potential applications as adjuvants, cosmetics, food supplemental, stabilizing and gelling agents, and biofuels. The 516 non-structural carbohydrates can be quickly accumulated by growth conditions, not needing a biomolecular approach. On 517 the other hand, the cell wall could be modified to produce more galactose, mannose, and glucose, hexoses that are more 518 readily fermented. Simultaneously, some pectin-related genes could be depleted to facilitate their conversion into 519 bioproducts.

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According to the mapping performed in this work, S. polyrhiza could be suitable for future genetic modifications 521 capable of turning biomass more ideal for bioenergy production. However, any change in cell wall polymer proportions 522 should be carefully planned to assess the trade-offs between the benefits for industry and the plant's growth capacity, 523 directly related to the composition of the cell walls in duckweeds.       Chromosome's ideogram of the nucleotide sugar pathway genes evaluated in the present study. S. polyrhiza has 20 chromosomes which are represented in the gure. The colors on the chromosomes represent each class of polysaccharide or its involvement on the pathway, being blue for cellulose, green hemicelluloses, red for pectin, and black for starch and sucrose metabolism. For gene names, see Figure  Figure 4 Distribution of genes throughout the chromosomes in the genome of Spirodela polyrhiza. Heatmap of the number of genes in each chromosome of S. polyrhiza divided into the categories "sucrose and starch metabolism," "pectin," "hemicellulose," and "cellulose."

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