The Cell Wall-Related Gene Families of Wheat (Triticum aestivum)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This bioinformatic study of cell wall genes in wheat can be a useful resource for adding to the functional annotation of wheat genes. The author has used the very latest wheat genome reference IWGSC 2.1 adding to the value as there are still relatively few resources or publications for this reference. 

Emphasis is placed on expanded number of genes in wheat compared to the expectation of 3x as many as rice. However gene models in rice and maize are generally supported by transcript evidence whereas the approach here will find non-expressed pseudogenes. It would be possible to check if genes described here are expressed using public RNAseq mapped to IWGSC 2.1 but this may be beyond the scope of this paper. However there needs to be at least a sentence acknowledging that some of the genes uncovered may be pseudogenes (which are common in wheat). 

Some of the sections relating to arabinoxylan are not quite up-to-date. GT61s (line 109-110) not only add Ara to arabinoxylan (AX) but also thought to add feruloyl-Ara (Feijao et al., 2022). Feruloylation of AX is not mentioned here but is important for wheat quality as it allows cross-linking of AX determining solubility in wheat grain (Pellny et al., 2020). Peroxidases are mentioned here but not their role in cross-linking feruloyl-AX ; a recent preprint describes a peroxidase controlling wheat dietary fibre solubility via this mechanism doi.org/10.1101/2023.03.08.531735. I suspect the role of the GT61 and peroxidase families in AX feruloylation may be behind the expansion of these families reported here (line 251). Some mention of importance of feruloyl-AX and relevant citations need to be added. 

line 670: "xyloglucan" should be "xylan"

Author Response

Emphasis is placed on expanded number of genes in wheat compared to the expectation of 3x as many as rice. However gene models in rice and maize are generally supported by transcript evidence whereas the approach here will find non-expressed pseudogenes. It would be possible to check if genes described here are expressed using public RNAseq mapped to IWGSC 2.1 but this may be beyond the scope of this paper. However there needs to be at least a sentence acknowledging that some of the genes uncovered may be pseudogenes (which are common in wheat). 

We thank the reviewer for their comment. Lines 260-261 did touch on this subject as “Wheat gene names followed by LC indicate low confidence genes with the appropriate gene family protein motifs but unknown expression [72]”.

However, it may have been a little unclear and the sentence was re-written. Lines 260-263 now read: “High-confidence wheat genes as defined by the IWGSC have known gene models from other plants and expression in wheat tissues. Low-confidence wheat genes which have LC following their names contain the appropriate gene family protein motifs but unknown expression from the IWGSC [72].”

Some of the sections relating to arabinoxylan are not quite up-to-date. GT61s (line 109-110) not only add Ara to arabinoxylan (AX) but also thought to add feruloyl-Ara (Feijao et al., 2022). Feruloylation of AX is not mentioned here but is important for wheat quality as it allows cross-linking of AX determining solubility in wheat grain (Pellny et al., 2020). Peroxidases are mentioned here but not their role in cross-linking feruloyl-AX ; a recent preprint describes a peroxidase controlling wheat dietary fibre solubility via this mechanism doi.org/10.1101/2023.03.08.531735. I suspect the role of the GT61 and peroxidase families in AX feruloylation may be behind the expansion of these families reported here (line 251). Some mention of importance of feruloyl-AX and relevant citations need to be added. 

We thank the reviewer for pointing out this detail.  We have added the two published references as requested and changed the following sentences to address feruloyl-AX and its importance:

Lines 111-112 were: “The GT61 family are arabinosyl and xylosyl transferases that add sugars to the xylan backbone [30, 31].”

And have been changed to:

Lines 111-112: “The GT61 family are arabinosyl, arabinofuranosyl, and xylosyl transferases involved in producing arabinoxylan and GAX from a xylan backbone [30, 31, 32].” Where 32 is Feijao et al., 2022.

With the new reference listed as 32, references 32 to 86 have been incremented by 1 in the text. 

Instead of Line 251, the expansion of the GT61 family is covered in the section describing the GT61 family.

The following new lines have been added:

Lines 537-542: “In addition to adding arabinose to arabinoxylans, a rice mutant XAX1 and GT61 family member was observed to have reduced hydroxycinnamic acid-modified arabinosyl-substituted xylose [32]. With GT61 family members TaXAT1 and TaXAT2 having abrabinofuranosyltransferase activity in wheat, it is possible the role of arabinoxylan feruloylation may partly explain the expansion of the GT61 family [30, 32].” Where 32 is Feijao et al., 2022.

The following Reference has been added to lines 804-806:

32. Feijao, C.; Morreel, K.; Anders, A.; Tryfona, T.; Busse-Wicher, M.; Kotake, T.; et al. Hydroxycinnamic acid-modified xylan side chains and their cross-linking products in rice cell walls are reduced in the Xylosyl arabinosyl substitution of xylan 1 Plant J. 109 2022, 1152-1167. DOI: 10.1111/tpj.15620

 To cover the importance of AX solubility towards human health and the role played by crosslinking of feruloylation of arabinoxylans the following lines were added or modified:

 The line “Alteration of TaXAT1 also changed the fraction of soluble to insoluble fiber in wheat flour with impact to human health [30].”

Has been changed to lines 542-547

Wheat grain endosperm that is milled to flour contains arabinoxylan, a source of fiber. Alteration of TaXAT1 and XAX1 changed the amount of arabinoxylan feruloylation which changed the fraction of soluble to insoluble fiber in wheat flour. The feruloylation of arabinoxylan increases cross-linking of molecules leading to more insoluble fiber. However, wheat grain is an important source of soluble fiber for human health. [30, 32, 87]. Where 87 is now Pellny et al., 2020. 

The following reference has been added to lines 923-925:

87. Pellny, T.K.; Patil, A.; Wood, A.J.; Freeman, J.; Halsey, K.; Plummer, A.; et al. Loss of TaIRX9b gene function in wheat decreases chain length and amount of arabinoxylan in grain but increases cross-linking. Plant Biotech. J. 18 2020, 2316-2327. DOI: 10.1111/pbi.13393

With the new reference listed as 87, references 86 to 98 have been incremented by 2 in the text (one for the new 32 and one for the new 87). 

line 670: "xyloglucan" should be "xylan"

Thank you for pointing this out.  This was fixed in line 680 where it moved after the additions above.

Note to Editor: The addition of the two references changed all the reference numbers in the text after #31. References 32 to 85 have been incremented by 1 and references 86 to 98 have been incremented by 2 to match the updated references section. These are all highlighted in yellow and occur on lines: 113, 115, 116, 117, 119, 121, 122, 125, 126, 130, 131, 132, 133, 135, 137, 141, 143, 147, 150, 156, 157, 161, 163, 164, 167, 172, 175, 193, 196, 210, 211, 215, 217, 220, 221, 223, 225, 233, 263, 277, 280, 283, 539, 542, 547, 551, 554, 562, 563, 565, 568, 569, 571, 575, 577, 579, 594, 600, 601, 614, 615, 619, 621, 624, 627, 640, 643, 647, 648, 654, 656, 661, 665, 681, and 684.

Reviewer 2 Report

Comments and Suggestions for Authors

The author presents an interesting summary of bioinformatic data handling, demonstrating identification and the potential categorization of wheat cell wall-related genes. The approach is well-crafted and very nicely described. My doubt is that this seems to be more of a demonstration of a method rather than presenting new scientific results (some new insight in wheat cell wall related gene family is still based on publicly available data). There is a clear utility for such data and a method as the author shows. Therefore, my suggestion is maybe to classify this article as a method/protocol communication rather than an original research article?

Author Response

Thank you for the suggestion.  However, a large portion of this research article is the over 4,000 wheat genes classified into their respective families and the trees developed from wheat genetic sequences compared to known maize, rice, and Arabidopsis sequences for over 80 families. Reviewer 1 did not appear to take this work into account as reviewer 2 does, calling the manuscript “This wheat research presents a set of more complete curated cell wall gene families…”.  The author believes this manuscript qualifies as an original research article based on the work to develop the wheat cell wall gene families and compare them to three other species. The single paragraph in L653 to L681 is meant only as an illustration of the utility of the research generated in this manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

This wheat research presents a set of more complete curated cell wall gene families compared to maìz, Arath, and rice homologous sequences. 81 gene families with a total of 4,086 cell wall-related genes were captured. Several wheat gene families were more expanded than expected in hexaploidy than diploids (maize, rice, and Arath). This work is helpful for cell wall omics research.

 

Minor observation

L75: Add examples of conserved functions.

L120: correction of “galctosyl”.

L145: italicize the “in vitro” word.

L246: I did not find the use of “AC”. Check it or delete it.

L615-620: move to the materials and methods section.

L656: change the “,” for “;” before Scotty.

Author Response

Author Response:

We thank the reviewer for their observations and have made the changes below which are highlighted yellow in the newly uploaded manuscript.

L75: Add examples of conserved functions: L75-76, added “including production of cellulose, hemicelluloses, and lignin”

 L120: correction of “galctosyl”: “galctosyl” changed to “galactosyl”

L145: italicize the “in vitro” word: italicized.

L246: I did not find the use of “AC”. Check it or delete it.: There are 26 Maize genes that start with AC in the following gene families: CCR, COMT, AGP, CE13 _ PAE, 2 in Expansins, GH16 – XTHs, GH17, GH18 – Yieldins, GH9, GT2 – CslC, GT31, GT34, 2 in GT8- D, 4 in NST, 4 in Peroxidases, PL1 – Pectin Lyases, and 2 in Proteases.

L615-620: move to the materials and methods section. The text asked to be moved is : “Trees for all 81 cell wall gene families of wheat, maize, rice, and Arabidopsis are available as Supplemental File 1. An Excel sheet which lists the gene family, gene name, and species for each gene used in this study in the first through third columns, respectively is available as Supplemental File 2. Gene families not described were small or did not show drastically different gene numbers of wheat compared to maize or rice and have been previously described in many publications.” 

The trees and files are results of the research and the last sentence describes why specific gene families were chosen for discussion.  To clarify this, we have re-written the paragraph as: “Results of this research are trees for all 81 cell wall gene families of wheat, maize, rice, and Arabidopsis available as Supplemental File 1. An Excel sheet lists the wheat gene family classification resulting from this research, gene name, and species for each gene in the study in the first through third columns, respectively and is available as Supplemental File 2. Gene families not described were small or did not show drastically different gene numbers of wheat compared to maize or rice and have been previously described in many publications.”

L656: change the “,” for “;” before Scotty: Changed, now in line 657 due to change in L615-620