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10 páginas.- 5 figuras.- 2 tablas.- 50 referencias.- The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssuschemeng.2c01274, Innovative schemes for lignocellulosic biomass refining could optimize the chemical use and biomass conversion efficiency, providing diversified biorefinery products. A new biorefinery scheme used mild alkaline-sulfite chemithermomechanical processing to pretreat sugarcane bagasse, producing highly digestible solids and diluted lignosulfonates in the black pretreatment liquor. Most of the glucan and xylan contained in the pretreated solids were converted into monosaccharides by saccharification with commercial enzymes. The undigested biomass solids remaining from the saccharification process were recycled with the existing alkaline-sulfite black pretreatment liquor. The optimized reaction conditions (159 degrees C and 13% NaOH amendment in the liquor) enhanced the lignosulfonate concentration from 7.1 +/- 0.3 to 33.5 +/- 0.7 g/L. Mass balance for the process indicates that most of the original sugarcane bagasse lignin were split into lignosulfonates contained in the resulfonation liquor (57.3%) and in the enzymatic hydrolysate (16.2%). 2D-NMR analysis of a lignosulfonate fraction recovered by ultrafiltration confirmed that approximately 9-10% of all beta-O-4' alkyl-aryl ether substructures were alpha-sulfonated. The combined chemical characteristics of the prepared lignosulfonate suggested its suitability for use in several lignosulfonate applications., This work was supported by FAPESP (grant number 2019/25867-3), CNPq (grant number 308570/2017-0), and Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior-Brasil (CAPES)-Finance Code 001. J.R. and J.C.d.R. were funded by MCIN/AEI/10.13039/501100011033, by "ERDF A way of making Europe" (project PID2020-118968RB-I00), and by the Regional Andalusian Government, Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades/FEDER (project P20-00017).

20 páginas.- 10 figuras.- 2 tablas.- 59 referencias.- Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s10570-022-04573-y, Papyrus, produced from the white pith of Cyperus papyrus L., has been used for millennia as the major writing support by ancient cultures, but there was no continuous papyrus production until modern times. Therefore, papyrus production had to be rediscovered. Modern Egyptian papyrus producers claim that their sheets possess 'the same physical and chemical properties as ancient papyri'. To study if this is accurate, commercially available papyrus sheets were compared to ancient papyri and papyri produced according to Pliny's historic description. Material characterization was performed with a focus on the potentially color-bearing lignin. Two-dimensional nuclear magnetic resonance spectroscopy, derivatization followed by reductive cleavage, and pyrolysis-gas chromatography/mass spectrometry were complemented with microscopy and tests for surface pH and sodium content. The lignin data in the native pith and commercial sheets were compared to 10 ancient samples from the Papyrus Museum Vienna. The analytical data clearly show the involvement of a strong alkaline treatment followed by chlorine bleaching for commercial papyri, as expressed by higher pH values, altered lignin structures, and chlorinated lignin compounds. The inclusion of an alkaline step in ancient papyrus manufacture is discussed but dismissed, since the alkali-treatment causes a huge decrease in lignin content, that was not found for the analysed ancient specimen. We assume that this additional treatment was introduced to obtain yellowish papyrus sheets meeting optical expectations of modern spectators. Linguistic and art historic evidence indicates that such a step would not have made sense in antiquity, since it was desired to produce white papyri., Open access funding provided by University of Natural Resources and Life Sciences Vienna (BOKU). The authors acknowledge the financial support from the Austrian Biorefinery Centre Tulln (ABCT), the County of Lower Austria, the Spanish State Research Agency, the European Regional Development Fund (project AGL2017-83036-R) and the Junta de Andalucia (project P20-00017).

18 páginas.- 6 figuras.- 4 tablas.- 92 referencias.-Supplementary Materials, ydroxystilbenes are a class of polyphenolic compounds that behave as lignin monomers participating in radical coupling reactions during the lignification. Here, we report the synthesis and characterization of various artificial copolymers of monolignols and hydroxystilbenes, as well as low-molecular-mass compounds, to obtain the mechanistic insights into their incorporation into the lignin polymer. Integrating the hydroxystilbenes, resveratrol and piceatannol, into monolignol polymerization in vitro, using horseradish peroxidase to generate phenolic radicals, produced synthetic lignins [dehydrogenation polymers (DHPs)]. Copolymerization of hydroxystilbenes with monolignols, especially sinapyl alcohol, by in vitro peroxidases notably improved the reactivity of monolignols and resulted in substantial yields of synthetic lignin polymers. The resulting DHPs were analyzed using two-dimensional NMR and 19 synthesized model compounds to confirm the presence of hydroxystilbene structures in the lignin polymer. The cross-coupled DHPs confirmed both resveratrol and piceatannol as authentic monomers participating in the oxidative radical coupling reactions during polymerization., This work was supported by grants from the DOE Great Lakes Bioenergy Research Center, and H.K. and J.Ra. were funded by the DOE BER Office of Science (DE- SC0018409). J.Re. and J.C.d.R. were supported by the projects AGL2017-83036-R and PID2020-118968RB-I00 (funded by MCIN/AEI/10.13039/501100011033 and, as appropriate, by “ERDF A way of making Europe”) and by the Andalusian Regional Government (project P20-00017). T.J.E. was funded by the Extreme Science and Engineering Discovery Environment (XSEDE), supported by the National Science Foundation grant number ACI-1548562. This work was made possible in part by a grant for high-performance computing resources and technical support from the Alabama Supercomputer Authority. This research was supported in part by the U.S. Department of Agriculture, U.S. Forest Service.

8 figuras.- refefencias.- Versión aceptada del artículo.- Supplementary data kiab469_Suppl. https://academic.oup.com/plphys/advance-article/doi/10.1093/plphys/kiab469/6400262#supplementary-data, Recent studies demonstrate that several polyphenolic compounds produced from beyond the canonical monolignol biosynthetic pathways can behave as lignin monomers, participating in radical coupling reactions and being incorporated into lignin polymers. Here, we show various classes of flavonoids, the chalconoid naringenin chalcone, the flavanones naringenin and dihydrotricin, and the flavone tricin, incorporate into the lignin polymer of papyrus (Cyperus papyrus L.) rind. These flavonoids were released from the rind lignin by DFRC, a chemical degradative method that cleaves the β-ether linkages, indicating that at least a fraction of each was integrated into the lignin as β-ether linked structures. Due to the particular structure of tricin and dihydrotricin, whose C-3ʹ and C-5ʹ positions at their B-rings are occupied by methoxy groups, these compounds can only be incorporated into the lignin through 4ʹ–O–β bonds. However, naringenin chalcone and naringenin have no substituents at these positions and can therefore form additional carbon-carbon linkages, including 3ʹ– or 5ʹ–β linkages that form phenylcoumaran structures not susceptible to cleavage by DFRC. Furthermore, NMR analysis indicated that naringenin chalcone can also form additional linkages through its conjugated double bond. The discovery expands the range of flavonoids incorporated into natural lignins, further broadens the traditional definition of lignin, and enhances the premise that any phenolic compound present at the cell wall during lignification could be oxidized and potentially integrated into the lignin structure, depending only on its chemical compatibility. This study indicates that papyrus lignin has a unique structure as it is the only lignin known to date that integrates such a diversity of phenolic compounds from different classes of flavonoids. This discovery will open up new ways to engineer and design lignins with specific properties and for enhanced value., This work was funded by the Spanish State Research Agency and the European Regional 51 Development Fund (project AGL2017-83036-R), the Consejería de Transformación 52 Económica, Industria, Conocimiento y Universidades, Junta de Andalucía (project P20-53 00017), the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE54 SC0018409), and the Austrian Biorefinery Center Tulln (ABCT). MJR thanks the Spanish 55 Ministry of Science, Innovation and Universities for a FPI fellowship (PRE2018-083267).

9 páginas.- 5 figuras.- 2 tablas.- 53 referencias.- Supplementary data associated with this article can be found in the online version at doi:10.1016/j.indcrop.2022.115658, Agro-industrial residues were used as a successful, cheap and renewable substrate, capable of increasing activity of ligninolytic enzymes for further use in melanin degradation assays to develop inputs for the treatment of skin hyperpigmentation. The analysis of melanin degradation was performed in a quantitative and qualitative way. The enzymatic crude broth extract, rich in laccases, produced by Marasmiellus palmivorus VE111 in medium formulated with lignocellulosic residues of Araucaria angustifolia subjected to hydrolysis and alcoholic fermentation, degraded almost 33% of the melanin content and enzymatic crude broth extract produced by Pycnoporus sanguineus PR_32 degraded 13%, in just 8 h, using vanillin as a mediator. Proton 2D NMR spectra show melanin changes after laccase treatment. This work has provided an insight for the development of ecofriendly, sustainable and innovative cosmetics to deal with skin hyperpigmentation. © 2022 Elsevier B.V., The Research Support Foundation of the State of Rio Grande do Sul (Process 16/2551-0000 246-0) and the National Council for Scientific and Technological Development (CNPq) (Process 407228/2018-7 and 314897/2018-6).

27 páginas.- 7 figuras.- 2 tablas.- 157 referencias.- Supplementary data The following supplementary data are available at JXB online., Phenolic metabolism is rewired during internode development and in response to cinnamyl alcohol dehydrogenase perturbation in sorghum, with different sets of genes and accumulating compounds. The molecular mechanisms associated with secondary cell wall (SCW) deposition in sorghum remain largely uncharacterized. Here, we employed untargeted metabolomics and large-scale transcriptomics to correlate changes in SCW deposition with variation in global gene expression profiles and metabolite abundance along an elongating internode of sorghum, with a major focus on lignin and phenolic metabolism. To gain deeper insight into the metabolic and transcriptional changes associated with pathway perturbations, a bmr6 mutant [with reduced cinnamyl alcohol dehydrogenase (CAD) activity] was analyzed. In the wild type, internode development was accompanied by an increase in the content of oligolignols, p-hydroxybenzaldehyde, hydroxycinnamate esters, and flavonoid glucosides, including tricin derivatives. We further identified modules of genes whose expression pattern correlated with SCW deposition and the accumulation of these target metabolites. Reduced CAD activity resulted in the accumulation of hexosylated forms of hydroxycinnamates (and their derivatives), hydroxycinnamaldehydes, and benzenoids. The expression of genes belonging to one specific module in our co-expression analysis correlated with the differential accumulation of these compounds and contributed to explaining this metabolic phenotype. Metabolomics and transcriptomics data further suggested that CAD perturbation activates distinct detoxification routes in sorghum internodes. Our systems biology approach provides a landscape of the metabolic and transcriptional changes associated with internode development and with reduced CAD activity in sorghum., This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) via the BIOEN Young Investigators Awards research grant (FAPESP no. 2015/02527-1). This study was financed in part by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior-Brasil (CAPES) -Finance Code 001, by FEDER/Agencia Estatal de Investigacion with the Spanish Projects AGL2017-83036-R and PID2020-118968RB-I00, and by Junta de Andalucia (Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades, project P20_00017). IC is indebted to Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) for the research fellowship 302927/2018-2. SSF was funded for a post-doctoral fellowship (FAPESP n0. 2016/06917-1). MSS was funded for a master's fellowship (FAPESP no. 2015/18361-5). AVAP was funded for a scientific initiation fellowship (FAPESP no. 2016/06714-3). LGAL was funded for a scientific initiation fellowship (FAPESP no. 2019/14604-1).

14 páginas.- 12 figuras.- 2 tablas.- 68 referencias.- Supplementary data related to this article can be found at https://doi.org/10.1016/j.jclepro.2022.132404, We have examined a sustainable biorefinery process for the conversion of cotton gin trash (CGT), a waste from the cotton industry to multiple high value products. Herein, we found CGT native lignin to have unique features as it not only contains the p-hydroxyphenyl (H), guaiacol (G) and syringol (S) units, but structures of flavonoids, tyrosine, and phenylalanine associated with it. We have used the lignin-first approach to exploit its features in CGT and depolymerize it using argon gas and ethanol over Pd/C. The results show that 50% of the bio-aromatics is trans-isoeugenol, a precursor to produce vanillin. The cleavage of N-compounds from CGT during the fractionation process follows similar path as the rate of formation of the bio-aromatics. CGT impurities were shown to affect the reusability of the Pd/C catalyst. The fractionated cellulose-rich portion of CGT was converted to 39 mol% (80% purity) of 5-chloromethylfurfural (CMF), and a solid fuel which has a higher heating value of 20 MJ kg−1. The liquid extract, removed prior to CGT processing, was hydrogenated to produce C13–C17 liquid hydrocarbons, and the solid extract rich in nutrients is suitable for crops and plants growth. © 2022

SummaryThe soil borne pathogen Ralstonia solanacearum is the causing agent of bacterial wilt, a devastating disease affecting major agricultural crops. R. solanacearum enters plants through the roots and reaches the vasculature, causing rapid wilting. We recently showed that tomato varieties resistant to bacterial wilt restrict bacterial movement in the plant. In the present work we go a step forward by identifying the physico-chemical nature of the barriers induced in resistant tomato roots in response to R. solanacearum. We describe that resistant tomato specifically responds to infection by assembling de novo a structural barrier at the vasculature formed by a ligno-suberin coating and tyramine-derived hydroxycinnamic acid amides (HCAAs). On the contrary, susceptible tomato does not form these reinforcements in response to the pathogen but instead displays lignin structural changes compatible with its degradation. Further, we show that overexpressing genes of the ligno-suberin pathway in a commercial susceptible variety of tomato restricts R. solanacearum movement inside the plant and slows disease progression, enhancing resistance to the pathogen. We thus propose that the induced barrier in resistant plants does not only restrict the movement of the pathogen, but may also prevent cell wall degradation by the pathogen and confer anti-microbial properties.

31 páginas.- 14 figuras.- 92 referencias.- Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/jof7060426/s1 .- Acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)., Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes. © 2021 by the authors. Licensee MDPI, Basel, Switzerland., This research was funded by the GENOBIOREF (BIO2017-86559-R) project of the Spanish Ministry of Science and Innovation (co-financed by FEDER funds) to F.J.R.-D. and S.C.; and by the Consejo Superior de Investigaciones CientÍficas (PIE-202120E019 to A.T.M. and PIE-201620E081 to A.G.). The work conducted by the JGI, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. DOE under contract DE-AC02-05CH11231. Part of this material is also based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (BER) under the Early Career Award Program to D.S. This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication

12 páginas.- 8 figuras.- 52 referencias.- Suplemental data in http://www.plantphysiol.org/content/suppl/2019/05/03/pp.19.00255.DC1, Recent investigations have revealed that, in addition to monolignols, some phenolic compounds derived from the flavonoid and hydroxystilbene biosynthetic pathways can also function as true lignin monomers in some plants. In this study, we found that the hydroxystilbene glucosides isorhapontin (isorhapontigenin-O-glucoside) and, at lower levels, astringin (piceatannol-O-glucoside) and piceid (resveratrol-O-glucoside), are incorporated into the lignin polymer in Norway spruce (Picea abies) bark. The corresponding aglycones isorhapontigenin, piceatannol, and resveratrol, along with glucose, were released by derivatization followed by reductive cleavage (DFRC), a chemical degradative method that cleaves β-ether bonds in lignin, indicating that the hydroxystilbene glucosides are (partially) incorporated into the lignin structure through β-ether bonds. 2D-NMR analysis confirmed the occurrence of hydroxystilbene glucosides in this lignin, and provided additional information regarding their modes of incorporation into the polymer. The hydroxystilbene glucosides, particularly isorhapontin and astringin, can therefore be considered genuine lignin monomers that participate in coupling and cross-coupling reactions during lignification in Norway spruce bark., J.C.d.R, J.Re., G.M. and A.G. were funded by the Spanish projects CTQ2014-60764-JIN and AGL2017-83036-R (financed by Ministerio de Economía y Competitividad, Agencia Estatal de Investigación, AEI, and Fondo Europeo de Desarrollo Regional, FEDER), and the CSIC project 2014-40E-097; J.Ra. and H.K. were funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-SC0018409); J.G. and H.P. were funded by Fundação para a Ciência e a Tecnologia (FCT) under UID/AGR/00239/2013; D.N. was funded by FCT through a PhD scholarship (PD/BD/52697/2014) under the SUSFOR doctoral program.

10 páginas.-- 5 figuras.-- 3 tablas.-- 41 referencias.-- El archivo adjunto no es la última versión postprint, It was demonstrated for the first time that a laccase-based enzymatic pretreatment is able to delignify fast-growing paulownia species. The treatment was performed with a commercial low-redox potential laccase isolated from Myceliophthora thermophila (Apinis) Oorschot and methyl syringate (MeS) as a natural phenolic mediator. Up to 24% lignin removal was attained by the laccase-MeS treatment (L/MeS), followed by alkaline peroxide extraction in a multistage sequence. The reduction in lignin content was accompanied by a significant improvement in the subsequent enzymatic saccharification, with increases of up to 38% glucose and 34% xylose yields. The structural modifications of the lignin were analyzed in situ by two dimensional-nuclear magnetic resonance (2D-NMR) spectroscopy. A considerable removal of guaiacyl and syringyl lignin units with respect to the carbohydrate signals was visible as well as the cleavage of β-O-4′, β-5′ and β-β′ linkages leading to elevated amounts of Cα-oxidized guaiacyl and syringyl units. The presence of oxidized lignin compounds in the filtrates of the enzymatic treatments - such as vanillin, vanillic acid, syringaldehyde and syringic acid - conclusively demonstrates the ability of L/MeS treatment to oxidize and depolymerize the lignin in paulownia wood, This study has been funded by the Spanish projects CTQ2014-60764-JIN, AGL2014-53730-R and AGL2017-83036-R (financed by Agencia Estatal de Investigación, AEI, and Fondo Europeo de Desarrollo Regional, FEDER) and the CSIC project 2014-40E-097.

17 páginas.- 6 figuras.- 5 tablas.- referencias.- We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)., Rice (Oryza sativa L.) is a major cereal crop used for human nutrition worldwide. Harvesting and processing of rice generates huge amounts of lignocellulosic by-products such as rice husks and straw, which present important lignin contents that can be used to produce chemicals and materials. In this work, the structural characteristics of the lignins from rice husks and straw have been studied in detail. For this, whole cell walls of rice husks and straw and their isolated lignin preparations were thoroughly analyzed by an array of analytical techniques, including pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS), nuclear magnetic resonance (NMR), and derivatization followed by reductive cleavage (DFRC). The analyses revealed that both lignins, particularly the lignin from rice husks, were highly enriched in guaiacyl (G) units, and depleted in p-hydroxyphenyl (H) and syringyl (S) units, with H:G:S compositions of 7:81:12 (for rice husks) and 5:71:24 (for rice straw). These compositions were reflected in the relative abundances of the different interunit linkages. Hence, the lignin from rice husks were depleted in β–O–4′ alkyl-aryl ether units (representing 65% of all inter-unit linkages), but presented important amounts of β–5′ (phenylcoumarans, 23%) and other condensed units. On the other hand, the lignin from rice straw presented higher levels of β–O–4′ alkyl-aryl ethers (78%) but lower levels of phenylcoumarans (β–5′, 12%) and other condensed linkages, consistent with a lignin with a slightly higher S/G ratio. In addition, both lignins were partially acylated at the γ-OH of the side-chain (ca. 10–12% acylation degree) with p-coumarates, which overwhelmingly occurred over S-units. Finally, important amounts of the flavone tricin were also found incorporated into these lignins, being particularly abundant in the lignin of rice straw., his study has been funded by the Spanish Project AGL2017-83036-R (financed by Agencia Estatal de Investigación, AEI, and Fondo Europeo de Desarrollo Regional, FEDER). MJR thanks the Spanish Ministry of Science, Innovation and Universities for a FPI fellowship (PRE2018-083267). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

14 páginas.- 6 figuras.- 3 tablas.- referencias.- The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2021.740923/full#supplementary-material, In the present work, lignin-like fractions were isolated from several ancestral plants –including moss (Hypnum cupressiforme and Polytrichum commune), lycophyte (Selaginella kraussiana), horsetail (Equisetum palustre), fern (Nephrolepis cordifolia and Pteridium aquilinum), cycad (Cycas revoluta), and gnetophyte (Ephedra fragilis) species– and structurally characterized by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. Py-GC/MS yielded marker compounds characteristic of lignin units, except in the H. cupressiforme, P. commune and E. palustre “lignins,” where they were practically absent. Additional structural information on the other five samples was obtained from 2D-NMR experiments displaying intense correlations signals of guaiacyl (G) units in the fern and cycad lignins, along with smaller amounts of p-hydroxyphenyl (H) units. Interestingly, the lignins from the lycophyte S. kraussiana and the gnetophyte E. fragilis were not only composed of G- and H-lignin units but they also incorporated significant amounts of the syringyl (S) units characteristic of angiosperms, which appeared much later in plant evolution, most probably due to convergent evolution. The latter finding is also supported by the abundance of syringol derivatives after the Py-GC/MS analyses of these two samples. Regarding lignin structure, β−O−4′ alkyl-aryl ethers were the most abundant substructures, followed by condensed β−5′ phenylcoumarans and β−β′ resinols (and dibenzodioxocins in the fern and cycad lignins). The highest percentages of alkyl-aryl ether structures correlated with the higher S/G ratio in the S. Kraussiana and E. fragilis lignin-like fractions. More interestingly, apart from the typical monolignol-derived lignin units (H, G and S), other structures, assigned to flavonoid compounds never reported before in natural lignins (such as amentoflavone, apigenin, hypnogenol B, kaempferol, and naringenin), could also be identified in the HSQC spectra of all the lignin-like fractions analyzed. With this purpose, in vitro synthesized coniferyl-naringenin and coniferyl-apigenin dehydrogenation polymers were used as standards. These flavonoids were abundant in H. cupressiforme appearing as the only constituents of the moss lignin-like fraction (including 84% of dimeric hypnogenol B) and their abundance decreased in those of S. Kraussiana (with amentoflavone and naringenin representing 14% of the total aromatic units), and the two ancient gymnosperms (0.4–1.2%) and ferns (0–0.7%)., This work was funded by the Spanish Ministry of Industry, Economy and Competitiveness projects BIO2017-86559-R, AGL2017-83036-R, and PID2020-118968RB-I00, cofinanced by FEDER funds, by the Junta de Andalucía project P20-00017, and by the CSIC projects PIE-202120E019 and PIE-202040E185. We would like to acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

13 pág.- 9 figuras.- 1 tabla.- 38 referencias.- The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2021.642848/full#supplementary-material, he monolignols, p-coumaryl, coniferyl, and sinapyl alcohol, arise from the general phenylpropanoid biosynthetic pathway. Increasingly, however, authentic lignin monomers derived from outside this process are being identified and found to be fully incorporated into the lignin polymer. Among them, hydroxystilbene glucosides, which are produced through a hybrid process that combines the phenylpropanoid and acetate/malonate pathways, have been experimentally detected in the bark lignin of Norway spruce (Picea abies). Several interunit linkages have been identified and proposed to occur through homo-coupling of the hydroxystilbene glucosides and their cross-coupling with coniferyl alcohol. In the current work, the thermodynamics of these coupling modes and subsequent rearomatization reactions have been evaluated by the application of density functional theory (DFT) calculations. The objective of this paper is to determine favorable coupling and cross-coupling modes to help explain the experimental observations and attempt to predict other favorable pathways that might be further elucidated via in vitro polymerization aided by synthetic models and detailed structural studies., JRa and HK were funded by the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-SC0018409). JRe and JCR were funded by the Spanish Projects CTQ2014-60764-JIN and AGL2017-83036-R (financed by Agencia Estatal de Investigación, AEI and Fondo Europeo de Desarrollo Regional, FEDER).

23 páginas.- 7 figuras.- 3 tablas.- 81 referencias.- : The following are available online at https://www.mdpi.com/article/10.3390/antiox10091446/s1 .- Support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)., Lignin biodegradation has been extensively studied in white-rot fungi, which largely belong to order Polyporales. Among the enzymes that wood-rotting polypores secrete, lignin peroxidases (LiPs) have been labeled as the most efficient. Here, we characterize a similar enzyme (ApeLiP) from a fungus of the order Agaricales (with ~13,000 described species), the soil-inhabiting mushroom Agrocybe pediades. X-ray crystallography revealed that ApeLiP is structurally related to Polyporales LiPs, with a conserved heme-pocket and a solvent-exposed tryptophan. Its biochemical characterization shows that ApeLiP can oxidize both phenolic and non-phenolic lignin model-compounds, as well as different dyes. Moreover, using stopped-flow rapid spectrophotometry and 2D-NMR, we demonstrate that ApeLiP can also act on real lignin. Characterization of a variant lacking the above tryptophan residue shows that this is the oxidation site for lignin and other high redox-potential substrates, and also plays a role in phenolic substrate oxidation. The reduction potentials of the catalytic-cycle intermediates were estimated by stopped-flow in equilibrium reactions, showing similar activation by H2O2, but a lower potential for the rate-limiting step (compound-II reduction) compared to other LiPs. Unexpectedly, ApeLiP was stable from acidic to basic pH, a relevant feature for application considering its different optima for oxidation of phenolic and nonphenolic compounds., This research was funded by the GENOBIOREF (BIO2017-86559-R) project of the Spanish Ministry of Science & Innovation (co-financed by FEDER funds) to F.J.R.-D. and A.T.M.; and by the Consejo Superior de Investigaciones Científicas PIE-202120E019 to A.T.M., and the SusPlast pltform to F.J.R.-D., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Rice (Oryza sativa L.) is a major cereal crop used for human nutrition worldwide. Harvesting and processing of rice generates huge amounts of lignocellulosic by-products such as rice husks and straw, which present important lignin contents that can be used to produce chemicals and materials. In this work, the structural characteristics of the lignins from rice husks and straw have been studied in detail. For this, whole cell walls of rice husks and straw and their isolated lignin preparations were thoroughly analyzed by an array of analytical techniques, including pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS), nuclear magnetic resonance (NMR), and derivatization followed by reductive cleavage (DFRC). The analyses revealed that both lignins, particularly the lignin from rice husks, were highly enriched in guaiacyl (G) units, and depleted in p-hydroxyphenyl (H) and syringyl (S) units, with H:G:S compositions of 7:81:12 (for rice husks) and 5:71:24 (for rice straw). These compositions were reflected in the relative abundances of the different interunit linkages. Hence, the lignin from rice husks were depleted in β–O–4′ alkyl-aryl ether units (representing 65% of all inter-unit linkages), but presented important amounts of β–5′ (phenylcoumarans, 23%) and other condensed units. On the other hand, the lignin from rice straw presented higher levels of β–O–4′ alkyl-aryl ethers (78%) but lower levels of phenylcoumarans (β–5′, 12%) and other condensed linkages, consistent with a lignin with a slightly higher S/G ratio. In addition, both lignins were partially acylated at the γ-OH of the side-chain (ca. 10–12% acylation degree) with p-coumarates, which overwhelmingly occurred over S-units. Finally, important amounts of the flavone tricin were also found incorporated into these lignins, being particularly abundant in the lignin of rice straw.

13 páginas.- 8 figuras.- 3 tablas.- 63 referencias, This perspective summarizes and compares computational results for the thermodynamics of bond dissociation, coupling, and rearomatization for a number of noncanonical lignin monomer–lignol combinations that have been found to occur experimentally. The noncanonical lignin monomers discussed are tricin, caffeyl alcohol, 5-hydroxyconiferyl alcohol, and piceatannol. Among dimeric combinations, the results for bond dissociation are generally similar, but in cases for which trimers have been reported (tricin-lignol adducts), this value can be quite variable, with stereochemical and structural preferences. Among the adducts examined thus far, the energies associated with quinone methide formation and rearomatization are not dissimilar and would not impede subsequent polymerization. These fundamental studies may help to elucidate how lignin monomers are incorporated into the lignin polymer, provide leads for targeted genetic modification, and be of use in deconstruction for the production of commodity chemicals., J. Re. and J.C.d.R. were funded by the Spanish Projects CTQ2014-60764-JIN and AGL2017-83036-R (financed by Agencia Estatal de Investigacion, AEI, and Fondo Europeo de Desarrollo Regional, FEDER).

12 páginas.- 5 figuras.- 4 tablas.- 47 referencias., Lignins from different tree barks, including Norway spruce (Picea abies), eucalyptus (Eucalyptus globulus), mimosa (Acacia dealbata) and blackwood acacia (A. melanoxylon), are thoroughly characterized. The lignin from E. globulus bark is found to be enriched in syringyl (S) units, with lower amounts of guaiacyl (G) and p-hydroxyphenyl (H) units (H/G/S ratio of 1:26:73), which produces a lignin that is highly enriched in beta-ether linkages (83 %), whereas those from the two Acacia barks have similar compositions (H/G/S ratio of approximate to 5:50:45), with a predominance of beta-ethers (73-75 %) and lower amounts of condensed carbon-carbon linkages; the lignin from A. dealbata bark also includes some resorcinol-related compounds, that appear to be incorporated or intimately associated to the polymer. The lignin from P. abies bark is enriched in G units, with lower amounts of H units (H/G ratio of 14:86); this lignin is thus depleted in beta-O-4 ' alkyl-aryl ether linkages (44 %) and enriched in condensed linkages. Interestingly, this lignin contains large amounts of hydroxystilbene glucosides that seem to be integrally incorporated into the lignin structure. This study indicates that lignins from tree barks can be seen as an interesting source of valuable phenolic compounds. Moreover, this study is useful for tailoring conversion technologies for bark deconstruction and valorization., This study was funded by the Spanish project AGL2017-83036-R (co-financed by Agencia Estatal de Investigacion, AEI, and Fondo Europeo de Desarrollo Regional, FEDER). Fundacao para a Ciencia e a Tecnologia (FCT) funded both CEF (Centro de Estudos Florestais) through UID/AGR/00239/2013, and Duarte Neiva PhD scholarship (PD/BD/52697/2014) under the SUSFOR doctoral program.

16 páginas.- 6 figuras.- 131 referencias, Lignin is conventionally defined as being formed by the oxidative polymerization of three main monolignols, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, that are derived from the general phenylpropanoid biosynthetic pathway. Many other phenolic compounds that are also derived from the phenylpropanoid pathway are also known to perform as genuine lignin monomers in many plants, as is the case of the monolignol ester conjugates, phenolic compounds arising from the truncated biosynthesis of monolignols, or ferulate esters. Recent investigations, however, have indicated that phenolic compounds arising from beyond the canonical phenylpropanoid pathway, namely flavonoids, hydroxystilbenes, and hydroxycinnamic amides, may also behave as authentic lignin monomers and are incorporated into the lignin in some plants, further challenging the traditional definition of lignin. This is the case of the flavone tricin that is incorporated into the lignin of grasses and other monocots, the hydroxystilbene piceatannol (together with resveratrol and isorhapontigenin, at lower levels) that has been found in the lignins of palm fruit shells, their respective O-glucosides (astringin, piceid, and isorhapontin) that are present in the lignin of Norway spruce bark, or the ferulic amides feruloyltyramine, incorporated into the lignin of tobacco and potato tubers, and diferuloylputrescine, which appears to be incorporated into maize kernel lignin. These valuable compounds are potentially available in high amounts and at low cost and may be obtained from the waste products from the processing of agricultural or forest biomass., J. C. del Rıo, J. Rencoret, and A. Gutiérrez were funded by the Spanish Project AGL2017-83036-R (financed by Agencia Estatal de Investigacion, AEI, and Fondo Europeo de Desarrollo ́ Regional, FEDER). J. Ralph and H. Kim were funded by the DOE Great Lakes Bioenergy Research Center (DOE Office of Science DE-SC0018409). T. Elder was funded by the National Science Foundation (grant number ACI-1548562).

20 páginas.- 8 figuras.- 4 tablas.- 77 referencias.- Additional supporting information may be found online in the Supporting Information section at the end of this article or in https://onlinelibrary.wiley.com/action/downloadSupplement?doi=10.1111%2Fpce.13805&file=pce13805-sup-0001-supinfo.docx, Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in‐depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size‐exclusion chromatography, 2D‐NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt‐stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D‐NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC–MS‐based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3‐ and 4‐O‐feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity., Conselho Nacional de Desenvolvimento Científico e Tecnológico, Grant/Award Number: GM/GD ‐ 141076/2016‐0; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Grant/Award Numbers: PDSE ‐ 88881.188627/2018‐01, PDSE ‐ 88881.188639/2018‐01; Czech Science Foundation, Grant/Award Number: 18‐07563S; European Regional Development Fund; Ministerio de Economía y Competitividad, Agencia Estatal de Investigación, Grant/Award Number: AGL2017‐83036‐R; Ministry of Education, Youth and Sports of the Czech Republic, Grant/Award Number: CZ.02.1.01/0.0/0.0/16_019/0000827

8 páginas.- 5 figuras.- 1 tabla.- 40 referencias, Maize (Zea mays L.) fibers and rice (Oryza sativa L.) husks are abundant and low cost by-products generated during grain milling. In this work, the composition of the lipophilic compounds present in these materials, which accounted for 4.1 % in maize fibers and 2.2 % in rice husks, was thoroughly studied by GC–MS. The most abundant lipophilic compounds identified in these residues were n-fatty acids and acylglycerols (mono-, di-, and triglycerides), that altogether accounts for up to 88 % of all extractives in maize fibers and up to 95 % in rice husks. Steroid compounds, including free and esterified sterols, hydrocarbons, and ketones were identified in both samples, being present in significant amounts in maize fibers. Tocopherols were also present in both cereal by-products, albeit in lower amounts, with α-tocopherol being the most abundant one. Minor amounts of n-fatty alcohols and high molecular esters were also found in rice husks. Maize fibers and rice husks can, therefore, be potential feedstocks to obtain valuable phytochemicals of diverse industrial interest. © 2020 The Author(s), This work has been funded by the Spanish project AGL2017-83036-R (financed by FEDER/Ministerio de Economía, Industria y Competitividad – Agencia Estatal de Investigación ).

42 páginas..- 4 figuras.- 2 tablas.- 63 referencias.- Material suplementario en http://dx.doi.org/10.1021/acssuschemeng.0c01764, Cereal brans constitute an unexploited source of valuable cell wall polysaccharides and phenolics. Sequential subcritical water extraction (SWE) of wheat, barley and rye bran enables the isolation of cell wall polysaccharides with targeted molecular structures. The extracts switched from glucan-rich to higher contents of feruloylated arabinoxylan (F-AX) with extraction time, resulting in significant radical scavenging activity. Structurally, wheat and rye F-AXs exhibited mostly single C(O)-3 arabinose substitutions, whereas barley bran F-AX showed a complex substitution pattern. The recalcitrance of the insoluble residues after SWE was revealed by the enrichment of F-AX populations with di-substituted arabinose units, and the presence of hydroxycinnamic acids, lignin and proteins. This study demonstrates the versatility of SWE to extract cell wall polysaccharides from cereal brans with tailored molecular structures and radical scavenging activity, with potential use in food and biomedical applications., This work was funded by the Lantmännen Research Foundation (project 2014F1-0001) and the Swedish Research Council Formas (project 942-2016-119). JCdR and JR were funded by the Spanish project AGL2017-83036R (financed by FEDER/Agencia Estatal de Investigación). We acknowledge Dr. Annelie Moldin (Lantmännen) for her valuable comments to the manuscript.

10 páginas.- 5 figuras.- 1 tabla.- 54 referencias.- The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssuschemeng.0c02023, Eucalyptus globulus (E. globulus) wood samples were treated in media containing acidified water and methyl isobutyl ketone (MIBK) in order to achieve the one-pot conversion of the hemicellulosic and cellulosic fractions into furfural and levulinic and formic and acetic acids, respectively. The reactions were performed under diverse operational conditions (180 or 190 °C, reaction times up to 70 min, liquid-to-solid ratios of 10 or 20 g aqueous phase/g wood, sulfuric acid concentrations of 0.5 or 1 g/100 g aqueous phase). Near-quantitative molar conversion of pentosans into furfural was achieved under conditions leading to limited generation of organic acids. In one-pot experiments performed under selected conditions, the overall yield of furfural and organic acids reached 45.2 g/100 g of wood. MIBK-soluble lignin was recovered from the organic phase by water precipitation at a yield of 17.4 g/100 g of wood, defining an effective method for the simultaneous delignification and polysaccharide conversion of E. globulus wood. The recovered lignin was characterized for structure (using pyrolysis-gas chromatography-mass spectrometry and heteronuclear single quantum coherence) and molar mass distribution. © 2020 American Chemical Society., The authors are grateful to the Spanish “Ministry of Economy and Competitiveness” for supporting this study in the framework of the project “Modified aqueous media for wood biorefineries” (reference no. CTQ2017-82962-R), partially funded by the FEDER program of the European Union. M.L. thanks “Xunta de Galicia” and the European Union (European Social Fund, ESF) for her predoctoral grant (reference no. ED481A-2017/316).

12 páginas.- 5 figuras.- 4 tablas.- 103 referencias.- Supplementary data to this article can be found online at https://doi.org/10.1016/j.apenergy.2020.114493.0.00.51.01.52.0, Novel laccases have promising and valuable applications in biorefineries. This investigation documents, for the first time, the potential of depolymerising and repolymerising lignin by the secretome, rich in laccases, from a newly isolated white-rot basidiomycete Marasmiellus palmivorus VE111, for further saccharification and ethanolic fermentation steps. Proteomic analyses of the secretome of M. palmivorus show that laccases are the most predominant enzyme released by this fungus. The whole crude enzymatic broth is used for the delignification of lignin in Eucalyptus globulus wood, with the aim of enhancing the saccharification by cellulolytic and xylanolytic enzymes from Penicillium echinulatum S1M29. In addition, two different strategies, namely, laccase treatment before and after enzymatic hydrolysis, are employed to detoxify steam-exploded E. globulus wood. The objective is to increase the fermentative performance by removing substances formed during the feedstock pretreatment that can inhibit microbial fermentation. The E. globulus wood delignification results in a 31% decrease in the lignin content and a 10% increase in the glucose yield after hydrolysis. An important finding of the present work is the successful wood delignification in the absence of laccase mediators. This laccase-rich preparation also demonstrates its potential in removing the phenolic inhibitors present in steam-exploded E. globulus wood, increasing the ethanol yield by an additional 10%. Furthermore, it is important to highlight that these findings are achieved in the absence of commercial enzymes, making M. palmivorus laccases a potential candidate not only for the production of biofuels but also for the generation of lignin-derived aromatic compounds for different applications in the biotechnology industry. © 2020 Elsevier Ltd, Funding from University of Caxias do Sul (UCS) Coordination of Improvement of Higher Education Personnel (CAPES) (Process 3255/2013), Research Support Foundation of the State of Rio Grande do Sul (Process 16/2551-0000 246-0), Brazilian Agricultural Research Corporation (EMBRAPA) (34/2014), National Council for Scientific and Technological Development (CNPq) (Process 201925/2018-4), the Biobased Industries Joint Undertaking (www.bbi-europe.eu) project EnzOx2 (H2020-BBI-PPP-2015-2-720297, www.enzox2.eu), the projects RETOPROSOST-2-CM [S2018/EMT-4459] financed by the Comunidad de Madrid, HIDROFUN [BIO2015-68387-R], and GLYSUS [RTI2018-093683-B-I00] from MINECO/FEDER, and the CSIC Project (201740E07)

9 páginas.-- 8 figuras.-- 2 tablas.-- 35 referencias.-- The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b02721, Catalytic depolymerization of organosolv lignin to phenolic monomers with zeolites was investigated under various operating conditions. H-USY (Si/Al molar ratio = 5) outperformed H-USY with Si/Al ratios of 50 and 250, H-BEA, H-ZSM5, and fumed SiO2 to produce the highest phenolic monomer yield from a commercial organosolv lignin in methanol at 300 °C for 1 h. It was then further investigated in the presence of acetone, ethyl acetate, methanol, and methyl isobutyl ketone (MIBK) on the depolymerization of organosolv bagasse lignin (BGL). The total highest phenolic monomer yield of 10.6 wt % was achieved with MIBK at 350 °C for 1 h with a catalyst loading of 10 wt %. A final total phenolic monomer yield of 19.4 wt % was obtained with an initial H2 pressure of 2 MPa under similar processing conditions. The main phenolic monomers obtained are guaiacol (7.9 wt %), 4-ethylphenol (6.0 wt %), and phenol (3.4 wt %). The solvent properties were used to account for the differences in phenolic monomer yields obtained with different organic solvents., The authors express their sincere appreciation to the Joint raduate School of Energy and Environment (JGSEE), Petchra Pra Jom Klao Doctoral Scholarship, King Mongkut’s University of Technology Thonburi (KMUTT), Thailand Research Fund (RTA5980006), and the Queensland University of Technology (QUT), Australia, for financing this project. The authors thank Dr. Jorge Rencoret and Dr. Ana Gutierrez, who were partly funded by the Spanish projects AGL2014-53730-R and CTQ2014-60764-JIN (cofinanced by FEDER funds), for PyGC/MS analysis of the lignins.

11 páginas.- 6 figuras.- 5 tablas.- 57 referencias, Papyrus (Cyperus papyrus L.) is a plant with high productivity rate that is considered an interesting raw material for obtaining biofuels and biomaterials. In the present work, the composition and structural features of the lignins present in the rind and pith of papyrus culms have been studied. The analyses revealed that the lignins from both parts of papyrus culms differed greatly not only in their contents but also in composition and structure. In the rind, the lignin represented up to 27.0% (dry-matter) and was composed mainly of G-units (H:G:S composition of 4:63:33), while the lignin in the pith (15.7% dry-matter) presented a slightly higher amount of S-units (H:G:S composition of 8:43:49). These lignins also presented considerable amounts of cinnamates (p-coumarates and ferulates) incorporated. The differences in composition influenced the content of the lignin inter-unit linkages. Thus, the rind lignin presented lower amounts of β–O–4′ ether linkages (77% of all identified linkages) and higher content of condensed structures, such as phenylcoumarans (15%), resinols (2%), dibenzodioxocins (3%), and spirodienones (2%), while the pith lignin contained more β–O–4′ ethers (85% of all identified linkages) and lower amounts of phenylcoumarans (11%), tetrahydrofurans (2%), and spirodienones (2%). Moreover, 2D NMR analyses revealed that the lignins from papyrus were highly acylated at the γ-OH (40% acylation in the rind, and 60% acylation in the pith). DFRC analyses proved that p-coumarates were the main acylating groups, and that they were predominantly acylating the S-lignin units. Finally, the analyses also showed the presence of important amounts of the flavanone tricin in the rind lignin and, at lower amounts, in the pith lignin. © 2021 The Authors, This work was partially funded by the Spanish Project AGL2017-83036-R (financed by Agencia Estatal de Investigación, AEI and Fondo Europeo de Desarrollo Regional, FEDER) and by Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Junta de Andalucía, Spain (project P20_00017). MJR thanks the Spanish Ministry of Science, Innovation and Universities for a FPI fellowship (PRE2018-083267). FB, TR, and AP were funded by the Austrian Biorefinery Center Tulln (ABCT).

ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes., The lignins from different anatomical parts of macaúba (Acrocomia aculeata) palm fruit, namely stalks, epicarp, and endocarp, were studied. The lignin from stalks was enriched in S-lignin units (S/G 1.2) and β-ether linkages (84% of the total) and was partially acylated at the γ-OH of the lignin side-chains (26% lignin acylation), predominantly with p-hydroxybenzoates and acetates. The epicarp lignin was highly enriched in G-lignin units (S/G 0.2) and consequently depleted in β-ethers (65%) and enriched in condensed structures such as phenylcoumarans (24%) and dibenzodioxocins (3%). The endocarp lignin was strikingly different from the rest and presented large amounts of piceatannol units incorporated into the polymer. This resulted in a lignin polymer depleted in β-ethers but enriched in condensed structures and linked piceatannol moieties. The incorporation of piceatannol into the lignin polymer seems to have a role in seed protection., This study has been funded by the Spanish Projects AGL2014-53730-R, CTQ2014-60764-JIN, and AGL2017-83036-R (cofinancedby FEDER funds) and the CSIC project 2014-40E097. John Ralph and Hoon Kim were funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494)., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).