PREreview of Differing structures of galactoglucomannan in eudicots and non-eudicot angiosperms

The current study focuses on evaluating the presence of β-galactoglucomanans (β-GGM), a specific type of galactoglucomannan characterized by β-(1,2)-Gal-α-(1,6)-Gal disaccharide branches on some mannosyl residues of a strictly alternating Glc-Man backbone. Previously, this type of polysaccharide had only been described in eudicotyledonous plants. The research aimed to investigate the mannan structure in 21 species of monocotyledonous and ANA-grade plants to ascertain the distribution of β-GGM in angiosperms. Additionally, the study sought to identify potential mannan β-galactosyltransferases (MBGT) in non-eudicot angiosperms.

The study's primary findings indicate the absence of evidence for β-GGM in non-eudicot angiosperms. The results suggest that these plants possess galactoglucomannan with an unpatterned backbone, which does not align with the strictly alternating Glc-Man backbone characteristic of β-GGM. Furthermore, β-(1,2)-Gal decoration appears to be absent, even when considering the possibility of attachment to a different type of mannan in non-eudicots. The authors conclude these plants may lack one or two key enzymes, namely patterned backbone synthase and/or MBGT, responsible for β-(1,2)-Gal transfer. Efforts to identify orthologous genes with MBGT activity in monocots and ANA-grade plants did not yield positive results, as the selected genes were associated with xyloglucan galactosyltransferase activity. In conclusion, the study suggests that β-GGM is likely specific to eudicots. However, it acknowledges certain limitations, such as the limited number of species studied, the potential existence of β-GGM in unsampled tissues, and limitations associated with the techniques used.

This type of research is particularly relevant as mannans are a group of polysaccharides that have been relatively understudied compared to other classes. A better understanding of the structural diversity of mannans and their functional implications is essential for advancing our knowledge of the physiological roles of these polysaccharides.

Major issues

• 1.       Abstract Enhancement: To improve the preprint's quality, we recommend enhancing the abstract by incorporating the main findings of the study, particularly the investigation of β-GGM in non-eudicots, which is currently missing from the abstract.

  2.       Enzyme Sources Clarification: The authors conclude that the enzymes CjMan26 and AnGH5 have the same specificity for the branch present in mannan. However, it's noteworthy that these enzymes have different origins, originating from bacteria and fungi, respectively. Therefore, it cannot be assumed that these enzymes exhibit identical selectivity for the same branching in the polysaccharide. It's essential to specify these enzyme sources in the methodology section. Additionally, the methodology should describe how the authors obtained these enzymes. Furthermore, sources of other enzymes used in the study, such as β-galactosidase (from Aspergillus niger, GH35), α-galactosidase (from Cellvibrio mixtus, GH27), β-glucosidase (from Aspergillus niger, GH3), or β-mannosidase (from Cellvibrio mixtus, GH5), should also be clearly stated.

  3.       Choice of Positive Control: The positive control, a tomato fruit, significantly differs from most of the samples, primarily leaves. It is advisable to use a corresponding tissue type to ensure a more valid comparison. The reasons for selecting different tissue types in various species should be explained. Additionally, adding the positive control to the second image in Figure 2A would enhance the results by subjecting the control to the same analysis conditions and potential interferences as each assay.

  4.       Definition of GAGM, GBGM, GAGAGM, and GBGAGM: The terms GAGM, GBGM, GAGAGM, and GBGAGM should be clearly defined in the study, as they play a crucial role in data interpretation. Although they are included in Figure 1A, their presentation should be more prominent to facilitate identification and correlation.

  5.       Identification of Bands in Positive Control Results: In the positive control results (Figure 2a), two bands, GBGAGM and GAGAG, appear very close to each other. However, the discussion does not describe how the positions of these bands were determined for band identification. The 20-minute boiling process may cause oligosaccharide disruption. To strengthen the results, it would be beneficial to include a negative control for the entire boiling process of oligosaccharides (specifically, for 20-minute boiling aimed at enzyme denaturation). This would allow the analysis of sugar heating during each heating step, demonstrating that the branching process is primarily enzymatic and not due to sugar degradation.

  6.       Graphical Hypothesis: To aid readers in visualizing potential galactoglucomannan structures in the evaluated samples, it would be valuable to provide a graphical representation of these hypotheses.

  7.       Consideration of Additional Techniques: In future confirmations of the study, it could enhance robustness to consider using an additional technique, in addition to the PACE technique, for the detection and structure analysis of these different oligosaccharides.

  8.       Inclusion of a Concluding Section: Given the volume of data and the complexity of the work, it would benefit the paper to include a concluding section summarizing key findings and insights.

  9.       Presentation of OsGT47A-VII Investigation: The investigation of OsGT47A-VII function could be summarized in the preceding section and its results presented as supplementary material. This would streamline the paper and improve reader comprehension since it is not directly related to the central focus of the study.

Minor issues

• 1.       In Figure 3C, a typographical error was identified in the name of the açaí palm. The document lists it as "Euterpe olerecea," but the correct classification should be "Euterpe oleracea." Also, Euterpe oleracea is a monocotyledon and not an ANA-grade plant. It is wrongly classified in this Figure.

  2.       Due to the numerous abbreviations used, the readability of the text has been compromised. Therefore, we strongly suggest including a glossary to help readers identify and understand these abbreviations.

  3.       Improve figure titles by making them more descriptive and complete, giving readers a clearer understanding of each figure's content and context.

Competing interests

The author declares that they have no competing interests.