Lignin biosynthesis: old roads revisited and new roads explored

Lignin is a major component of secondarily thickened plant cell walls and is considered to be the second most abundant biopolymer on the planet. At one point believed to be the product of a highly controlled polymerization procedure involving just three potential monomeric components (monolignols), it is becoming increasingly clear that the composition of lignin is quite flexible. Furthermore, the biosynthetic pathways to the major monolignols also appear to exhibit flexibility, particularly as regards the early reactions leading to the formation of caffeic acid from coumaric acid. The operation of parallel pathways to caffeic acid occurring at the level of shikimate esters or free acids may help provide robustness to the pathway under different physiological conditions. Several features of the pathway also appear to link monolignol biosynthesis to both generation and detoxification of hydrogen peroxide, one of the oxidants responsible for creating monolignol radicals for polymerization in the apoplast. Monolignol transport to the apoplast is not well understood. It may involve passive diffusion, although this may be targeted to sites of lignin initiation/polymerization by ordered complexes of both biosynthetic enzymes on the cytosolic side of the plasma membrane and structural anchoring of proteins for monolignol oxidation and polymerization on the apoplastic side. We present several hypothetical models to illustrate these ideas and stimulate further research. These are based primarily on studies in model systems, which may or may not reflect the major lignification process in forest trees.


Review form: Reviewer 3
Recommendation Major revision is needed (please make suggestions in comments)

Do you have any ethical concerns with this paper? No
Comments to the Author In the ms submitted, the authors have reviewed lignin biosynthesis, and propose several hypothetical models for still unknown questions. This is a nicely written review that gives fresh ideas for further investigation. The review is based mostly on studies of Arabidopsis, Medicago and grasses, with only some citations to the work conducted in woody plants (mostly Populus). Since woody plants contain most of the lignin due to the huge biomass, it would have been good to additionally discuss original research papers related to them. Especially studies about coniferous (gymnosperm) species are not cited at all. The reader should be informed about this in the beginning of the review.

I have following comments:
Thickening of the secondary cell wall is very prominent in tracheary elements, tracheids and fibers, the main lignifying cell types in xylem. A Casparian strip in the root -an excellent model for lignification studies -lacks this cell wall thickening. Thickening of the wall is an important point that needs to be taken into account when designing models. Fig. 5 proposes a model where peroxidase is in a module with dirigent protein and MASP, close to NADPH oxidase. This is based on an idea that has been observed in the Casparian strip. It is a nice idea, but can it be somehow realistic in a thickening secondary cell wall of tracheary elements and fibers during development? From the literature, it is known that lignification follows polysaccharide synthesis. Polysaccharides are produced first, making cell wall thick, and after polysaccharide synthesis has proceeded to a certain stage, lignification initiates farthest away from the membrane (in cell corners and the middle lamella), and the proceeds inwards, but always behind polysaccharide synthesis. If an oxidative enzyme, e.g. peroxidase, is located next to plasma membrane, it could participate only when lignification is almost finished, and not in the main lignification of the thick secondary wall. Similarly, if NADPH oxidase is the only source of apoplastic H2O2, could that diffuse around the apoplast to the site where that is needed without being reacted closer to the site of its generation? In addition to NADPH oxidase, there are other potential enzymatic sources for apoplastic H2O2 (e.g. peroxidases, several types of oxidases, e.g. Kärkönen and Kuchitsu, 2015, Phytochemistry). Some of these could equally well be involved in H2O2 production needed for lignification (even in addition to NADPH oxidase) -with more broad distribution in the wall.
l. 408-409. There are several older papers on trees that lignification is initiated in cell corners and middle lamella -please, cite some older papers. It is interesting to notice that ectopic lignin formation is also initiated in cell corners. It would be good to have a figure of developmental lignification next to this in Fig. 5 (or citation to a figure showing this).
l. 331-332 and Figure 4 legend: "Hydrogen peroxide is generated in nearly all intracellular compartments, with a major source being the plasma membrane-associated NADPH oxidase (NOX) with its catalytic site in the apoplast." NADPH oxidases may be a major source for apoplastic ROS production, but since ROS are generated e.g. in chloroplasts and peroxisomes, NADPH oxidase may not be the main cellular source for H2O2 generation in photosynthesizing cells. NADPH oxidases transfer electrons across the plasma membrane from cytoplasmic NADPH to molecular oxygen to produce superoxide in the apoplastic side of the membrane. Thus, product generation occurs in the apoplast, but electrons originate from NADPH that is in the cytoplasmic side, and are transferred via the heme groups in the transmembrane areas (Sagi and Fluhr 2006). Hence, catalytic site locates in the cytoplasmic side and through the membrane, and product is generated in the apoplastic side.
l. 329-344. This chapter discusses the need to transport H2O2 from the apoplast to cytosol to activate lysophospholipase C/CSE. Authors need to get familiar with papers about cytosolic ROS generation and antioxidants. There are multiple sources of cytoplasmic H2O2 generation, and efficient antioxidant systems (additionally to APX) that keep the redox balance of the cytosol highly reduced (e.g. Noctor and Foyer, 2016, Plant Cell Environm;Smirnoff and Arnaud, 2019, New Phytol). However, ROS signalling between different organs and compartments is known to be present (e.g. Waszczak et al., 2018, Annu Rev Plant Biol). It has to be kept in mind that there needs to be a concentration gradient between the sides of plasma membrane with higher concentration in the apoplast so that diffusion inwards is occurring. This gradient probably occurs in the defense response with RBOHs activated. Is the gradient enough during developmental lignification when H2O2 is consumed by peroxidases in the apoplastic space? Please, consider this knowledge and rewrite this chapter.
It is not uniform how plant species' names are written (sometimes only in English, sometimes only in latin). Please, uniform the naming.
Minor comments: l. 26 … highly controlled polymerization procedure… The idea of "random polymerization" is not equal to highly controlled procedure -please rephrase.
l. 65. Extracellular -Please change to "apoplastic" to be uniform with the rest of the text. l. 956: ("oxidation/polymerization via peroxidases"): Peroxidase oxidizes monolignol to monolignol radical -polymerization does not require H2O2 or the peroxidase enzyme anymore -> remove "polymerization".  We are pleased to inform you that your manuscript RSOB-19-0215 entitled "Lignin biosynthesisold roads revisited and new roads explored" has been accepted by the Editor for publication in Open Biology. The reviewer(s) have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, we invite you to respond to the reviewer(s)' comments and revise your manuscript.
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Data accessibility section To ensure archived data are available to readers, authors should include a 'data accessibility' section immediately after the acknowledgements section. This should list the database and accession number for all data from the article that has been made publicly available, for instance: Comments to the Author(s) Dixon and Barros have written a wonderful review of the complexities of lignin biosynthesis. Biochemical deciphering of the pathway has a long history, where ingenious discoveries have many times rearranged the thinking of how the pathway works. For example, the hiding activity of C3H was found to be buried in a transesterification reaction (the shikimate shunt), central for the lignin monomer biosynthesis were its inhibition (by down regulation of HCT) caused severe defects. After absorbing this fact, the scientific community was given a new surprise by reports of two alternative and direct reactions for the 3-hydroxylation, by a C4H-C3H membrane associated complex, or by a soluble C3H that turned out to be a peroxidase.
The history, present state and unsolved questions of lignin biosynthesis are described here in an exciting and insightful way.

Referee: 2
Comments to the Author(s) This is a well-written, comprehensive and inspiring perspective paper concerning the biochemistry and cell biology aspects of lignin biosynthesis. The authors are one of the most active research groups in this particular research area, and discuss timely and important topics within the current manuscript. Given that the topics covered in this manuscript are highly relevant to recent biotechnology efforts aiming to improve the quality of plant biomass for sustainable utilizations, I believe this manuscript will receive much attention from both plant biology and biotechnology communities. I have some minor suggestions that may help to improve the present version.
Referee: 3 Comments to the Author(s) In the ms submitted, the authors have reviewed lignin biosynthesis, and propose several hypothetical models for still unknown questions. This is a nicely written review that gives fresh ideas for further investigation. The review is based mostly on studies of Arabidopsis, Medicago and grasses, with only some citations to the work conducted in woody plants (mostly Populus). Since woody plants contain most of the lignin due to the huge biomass, it would have been good to additionally discuss original research papers related to them. Especially studies about coniferous (gymnosperm) species are not cited at all. The reader should be informed about this in the beginning of the review.

I have following comments:
Thickening of the secondary cell wall is very prominent in tracheary elements, tracheids and fibers, the main lignifying cell types in xylem. A Casparian strip in the root -an excellent model for lignification studies -lacks this cell wall thickening. Thickening of the wall is an important point that needs to be taken into account when designing models. Fig. 5 proposes a model where peroxidase is in a module with dirigent protein and MASP, close to NADPH oxidase. This is based on an idea that has been observed in the Casparian strip. It is a nice idea, but can it be somehow realistic in a thickening secondary cell wall of tracheary elements and fibers during development? From the literature, it is known that lignification follows polysaccharide synthesis. Polysaccharides are produced first, making cell wall thick, and after polysaccharide synthesis has proceeded to a certain stage, lignification initiates farthest away from the membrane (in cell corners and the middle lamella), and the proceeds inwards, but always behind polysaccharide synthesis. If an oxidative enzyme, e.g. peroxidase, is located next to plasma membrane, it could participate only when lignification is almost finished, and not in the main lignification of the thick secondary wall. Similarly, if NADPH oxidase is the only source of apoplastic H2O2, could that diffuse around the apoplast to the site where that is needed without being reacted closer to the site of its generation? In addition to NADPH oxidase, there are other potential enzymatic sources for apoplastic H2O2 (e.g. peroxidases, several types of oxidases, e.g. Kärkönen and Kuchitsu, 2015, Phytochemistry). Some of these could equally well be involved in H2O2 production needed for lignification (even in addition to NADPH oxidase) -with more broad distribution in the wall.
l. 408-409. There are several older papers on trees that lignification is initiated in cell corners and middle lamella -please, cite some older papers. It is interesting to notice that ectopic lignin formation is also initiated in cell corners. It would be good to have a figure of developmental lignification next to this in Fig. 5 (or citation to a figure showing this).
l. 331-332 and Figure 4 legend: "Hydrogen peroxide is generated in nearly all intracellular compartments, with a major source being the plasma membrane-associated NADPH oxidase (NOX) with its catalytic site in the apoplast." NADPH oxidases may be a major source for apoplastic ROS production, but since ROS are generated e.g. in chloroplasts and peroxisomes, NADPH oxidase may not be the main cellular source for H2O2 generation in photosynthesizing cells. NADPH oxidases transfer electrons across the plasma membrane from cytoplasmic NADPH to molecular oxygen to produce superoxide in the apoplastic side of the membrane. Thus, product generation occurs in the apoplast, but electrons originate from NADPH that is in the cytoplasmic side, and are transferred via the heme groups in the transmembrane areas (Sagi and Fluhr 2006). Hence, catalytic site locates in the cytoplasmic side and through the membrane, and product is generated in the apoplastic side.
l. 329-344. This chapter discusses the need to transport H2O2 from the apoplast to cytosol to activate lysophospholipase C/CSE. Authors need to get familiar with papers about cytosolic ROS generation and antioxidants. There are multiple sources of cytoplasmic H2O2 generation, and efficient antioxidant systems (additionally to APX) that keep the redox balance of the cytosol highly reduced (e.g. Noctor and Foyer, 2016, Plant Cell Environm;Smirnoff and Arnaud, 2019, New Phytol). However, ROS signalling between different organs and compartments is known to be present (e.g. Waszczak et al., 2018, Annu Rev Plant Biol). It has to be kept in mind that there needs to be a concentration gradient between the sides of plasma membrane with higher concentration in the apoplast so that diffusion inwards is occurring. This gradient probably occurs in the defense response with RBOHs activated. Is the gradient enough during developmental lignification when H2O2 is consumed by peroxidases in the apoplastic space? Please, consider this knowledge and rewrite this chapter.
It is not uniform how plant species' names are written (sometimes only in English, sometimes only in latin). Please, uniform the naming.
Minor comments: l. 26 … highly controlled polymerization procedure… The idea of "random polymerization" is not equal to highly controlled procedure -please rephrase.
l. 65. Extracellular -Please change to "apoplastic" to be uniform with the rest of the text. l. 956: ("oxidation/polymerization via peroxidases"): Peroxidase oxidizes monolignol to monolignol radical -polymerization does not require H2O2 or the peroxidase enzyme anymore -> remove "polymerization".  We are pleased to inform you that your manuscript entitled "Lignin biosynthesis -old roads revisited and new roads explored" has been accepted by the Editor for publication in Open Biology.
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The Open Biology Team mailto: openbiology@royalsociety.org Response to reviews Referee: 1 Comments to the Author(s) Dixon and Barros have written a wonderful review of the complexities of lignin biosynthesis. Biochemical deciphering of the pathway has a long history, where ingenious discoveries have many times rearranged the thinking of how the pathway works. For example, the hiding activity of C3H was found to be buried in a transesterification reaction (the shikimate shunt), central for the lignin monomer biosynthesis were its inhibition (by down regulation of HCT) caused severe defects. After absorbing this fact, the scientific community was given a new surprise by reports of two alternative and direct reactions for the 3-hydroxylation, by a C4H-C3H membrane associated complex, or by a soluble C3H that turned out to be a peroxidase.
The history, present state and unsolved questions of lignin biosynthesis are described here in an exciting and insightful way.
Response. Thank you for the positive response.

Referee: 2
Comments to the Author(s) This is a well-written, comprehensive and inspiring perspective paper concerning the biochemistry and cell biology aspects of lignin biosynthesis. The authors are one of the most active research groups in this particular research area, and discuss timely and important topics within the current manuscript. Given that the topics covered in this manuscript are highly relevant to recent biotechnology efforts aiming to improve the quality of plant biomass for sustainable utilizations, I believe this manuscript will receive much attention from both plant biology and biotechnology communities. I have some minor suggestions that may help to improve the present version.
Response 1. Thank you for the positive response, and for the suggestions below, especially those concerning relevant papers that we had missed. We hope we have fully addressed the omissions in the revised MS.
Response 13. Done  Figure 2 (legend): "5, hydroxycinnamate CoA ligase;" -> "5, 4-hydroxycinnamate (or 4-coumarate) CoA ligase (4CL);", "9, ferulic acid/coniferaldehyde 5-hydroxylase" -> "9, ferulic acid/coniferaldehyde 5hydroxylase (F5H)" Response 16. Thanks for spotting this. Abbreviations added. Comments to the Author(s) In the ms submitted, the authors have reviewed lignin biosynthesis, and propose several hypothetical models for still unknown questions. This is a nicely written review that gives fresh ideas for further investigation. The review is based mostly on studies of Arabidopsis, Medicago and grasses, with only some citations to the work conducted in woody plants (mostly Populus). Since woody plants contain most of the lignin due to the huge biomass, it would have been good to additionally discuss original research papers related to them. Especially studies about coniferous (gymnosperm) species are not cited at all. The reader should be informed about this in the beginning of the review.
Response 1. We agree that this review mainly deals with studies in model species, and have now made this fact clear in the summary. I have following comments: Thickening of the secondary cell wall is very prominent in tracheary elements, tracheids and fibers, the main lignifying cell types in xylem. A Casparian strip in the root -an excellent model for lignification studies -lacks this cell wall thickening. Thickening of the wall is an important point that needs to be taken into account when designing models. Fig. 5 proposes a model where peroxidase is in a module with dirigent protein and MASP, close to NADPH oxidase. This is based on an idea that has been observed in the Casparian strip. It is a nice idea, but can it be somehow realistic in a thickening secondary cell wall of tracheary elements and fibers during development? From the literature, it is known that lignification follows polysaccharide synthesis. Polysaccharides are produced first, making cell wall thick, and after polysaccharide synthesis has proceeded to a certain stage, lignification initiates farthest away from the membrane (in cell corners and the middle lamella), and the proceeds inwards, but always behind polysaccharide synthesis. If an oxidative enzyme, e.g. peroxidase, is located next to plasma membrane, it could participate only when lignification is almost finished, and not in the main lignification of the thick secondary wall. Similarly, if NADPH oxidase is the only source of apoplastic H2O2, could that diffuse around the apoplast to the site where that is needed without being reacted closer to the site of its generation? In addition to NADPH oxidase, there are other potential enzymatic sources for apoplastic H2O2 (e.g. peroxidases, several types of oxidases, e.g. Kärkönen and Kuchitsu, 2015, Phytochemistry). Some of these could equally well be involved in H2O2 production needed for lignification (even in addition to NADPH oxidase) -with more broad distribution in the wall.
Response 2. We do not disagree with any of these comments. Figure 5 does indeed show the initiation of lignification in the cell corners (in both the pictures and the model). We have added sentences to the text to point out the sequence of events in developmentally-controlled lignification in xylem and fiber cells, and cited the possibility of other sources of hydrogen peroxide. We have also added a caveat to the legend to Figure 5. l. 408-409. There are several older papers on trees that lignification is initiated in cell corners and middle lamella -please, cite some older papers. It is interesting to notice that ectopic lignin formation is also initiated in cell corners. It would be good to have a figure of developmental lignification next to this in Fig. 5 (or citation to a figure showing this).
Response 3. The legend to Figure 5 has been supplemented to address this point. We don't feel that adding an extra Figure would be helpful, as we have defined the scope of the article in the introduction. Furthermore, we believe we have been careful to not over-extend the potential applicability of the models we have presented. We believe that the following section of the text makes this clear "It has been proposed that monolignols are exported out of the cell into all areas of the apoplast where they are highly mobile [99]. However, there may be different mechanisms of monolignol transport in living tissues such as the Casparian strip (CS) versus terminally dying cell types (e.g xylem elements) during development, and in plant cells responding defensively to pathogen attack (e.g. programmed cell death and cooperative lignification in living cells (reviewed in [100]). We deliberately avoid making these