Identification, Characterization, and Expression Analysis of Spondin-Like and Fasciclin-Like Genes in Neopyropia yezoensis, A Marine Red Alga

Round 1

Reviewer 1 Report

This piece of research is interesting, given the lack of data on ECM proteins in seaweeds, and in red algae in particular. Yet I am afraid that the general reader will not be sufficiently attracted by these  transcriptomic data as their biological significance is not discussed enough. 

The Introduction section should be improved. F-spondin and fasciclin should introduced first (i.e., before the ASPO genes) and information should be provided on the nature of polysaccharide components, e.g. agars or porphyrans, which make the bulk of ECM and likely are different in the sporophytic and gametophytic generations. With the exception of a reference (which I could not find on Pubmed), one does not have information on the identity of ASPO genes and their relevance here is questionnable. Also, I suggest that the authors incorporate a figure on the life history of Neopyropia, where abbreviations such as VGA and VSP are shown. 

The Result section is adequate. Yet, the strikingly high level of the expression of the fasciclin genes NyFAL1 and NyFAL4 in the sporophytes should be highlighted a bit more.

The Discussion section is too generalistic, with no reference as to the potential function of ECM proteins in relation with the developemental morphology of the sporophytes and the gametophytes. Could it be that the very high expression of the AGP-like NyFAL1 and NyFAL4 genes in the sporophytes is related to the morphogenesis of apically growing, chonchocelis filaments? 

Author Response

We are grateful to your valuable suggestions. In major points, we revised as follows. In minor points, we revised in accordance with your instructions as mentioned by you.

Introduction:

We added the description concerning polysaccharide components in seaweeds and the figure shown the life cycle of Neopyropia with abbreviations such as VGA and VSP. As you pointed out, the function of ASPO is unknown in Neopyropia, thus, we deleted the description about ASPO from Introduction section.

Result:

We highlighted high level of the expression of the fasciclin genes NyFAL1 and NyFAL4 in the sporophytes.

Discussion:

As you pointed out, it is important to discuss the sporophyte specific characteristics of the growth and morphogenesis. Thus, we added the description concerning preferential expression of NyFAL4 in the sporophyte related to different cell wall composition, the morphology and the growth behavior from the gametophyte stage.

Reviewer 2 Report

See attached pdf file for details of review

Comments for author File: Comments.pdf

Author Response

We are grateful to your valuable suggestions. In major points, we revised as follows. In minor points, we revised in accordance with your instructions as mentioned by you.

Introduction:

We added the more detailed information the role of the SPL and FAL genes during ECM synthesis. Moreover, we added the findings on the difference of ECM structure during the life cycle and the fossil which closely resemble the extant Bangiophyceae to be clear the description on the life cycle and the experiments on expression analysis of ECM genes during life cycle.

Materials and Methods:

We added the description on BLAST, microscopy and RNA extraction.

We also added the reference to the bioinformatic tools.

Figures:

We combined Fig.1-Fig.3 to one Figure and changed the title.

We added the supplemental data on the expression analysis in gametophytes formed archeosporangia.

Discussion:

We moved the description on Spondin and fasciclin in Discussion to Introduction and added the discussion, for example, correlation of the expression pattern of NyFLA with sporophyte- and gametophyte- specific growth and development, and the expression pattern of NySPLs with sexual reproduction.

As you pointed out, we just found the potential N-linked glycosylation. Thus, we changed the description concerning N-glycan structure of ECM proteins.

“further research on N-glycan structures of NySPLs during sexual reproduction is required to elucidate the role of N-linked protein glycosylation in ECM protein activity.  “

Similarly, we changed the description concerning the activity of the ECM protein during the life cycle.

Reviewer 3 Report

The manuscript [Identification, characterization, and expression analysis of spondin- and fasciclin-like genes in Neopyropia yezoensis, a marine red alga] is important due to the fact that it acknowledges and discusses previously unknown findings about the homologous genes encoding ECM proteins that are known to associate with integrins in animal cell, in the red macroalga Neopyropia yezoensis. These findings facilitate future studies on ECM architecture in the life cycles of red macroalgae.

The comments on particular parts of the text are to be found below.

Abstract

The Abstract is sufficiently informative, correctly written and edited.

Introduction

The introduction briefly summarizes knowledge about term of extracellular matrix (ECM) from macroalgae. The Author/Authors described marine red algae Bangiophyceae such as Neopyropia, Porphyra and Pyropia one of the most important marine aquaculture crops harvested for human food. However, there is nothing about the ecology and morphology main species Neopyropia yezoensis (besides Figure 1 presenting of life cycle of marine red alga).

Materials and methods

How did the Authors identify the gametophytes and sporophytes of Neopyropia yezoensis? Did the Authors use identification keys? The group of Neopyropia are taxonomical complicated, how the identifications were carried out? N. yezoensis definitive identification requires molecular work. Do the Authors have photographic documentations of Neopyropia yezoensis? Whether the herbarium algological material have been prepared?

The species in the red algal family Bangiaceae (Bangiales) consists of more than ten genera including the recently established genus Neopyropia (Porphyra sensu lato), some species known as Nori in Japanese, have been cultivated in the temperate regions of Japan (Terada et al. Photosynthetic response of a cultivated red alga, Neopyropia yezoensis f. narawaensis (=Pyropia yezoensis f. narawaensis; Bangiales, Rhodophyta) to dehydration stress differs with between two heteromorphic life-history stages. Algal Research, 2021, 55, 102262).

Author Response

We are grateful to your valuable suggestions. In minor points, we revised in accordance with your instructions as mentioned by you.

Introduction

We included the description of the life history, ECMs and morphorogy in Bangiaphyceae, because the knowledge of Neopyropia yezoensis is still scare. We added the economy importance of Neopyropia yezoensis.

Materials and methods

>How did the Authors identify the gametophytes and sporophytes of Neopyropia yezoensis?

As shown Fig.1, Neopyropia has distinct morphology between gametophytes and sporophytes and it is easy to distinguish them.

As you pointed out, the group of Neopyropia are taxonomical complicated. In this study, we used the strain TU-1 of Neopyropia yezoensis that is a descendant used for whole genomic analysis in N. yezoensis (Nakamura et al. 2013). We added the reference on the strain used in this study (Kuwano et al. 1996)

Round 2

Reviewer 1 Report

Thank you for taking my recommendations into consideration. The ms has been substantially improved. English language can still be improved. 

Author Response

Thank  you four sparing your invaluable time in reviewing our manuscript.

Reviewer 2 Report

See attached file

Comments for author File: Comments.pdf

Author Response

We are grateful to your valuable suggestions. In major points, we revised as follows. In minor points, we revised in accordance with your instructions as mentioned by you.

Materials and Methods:

We added the detail description on BLAST search and the reference for the 2-ΔΔCt method.

Figures:

We move the original Fig.1 to a supplement Figure to fit on one page. Moreover, we changed the Fig.5 to fit on one page.

Discussion:

Tools for the function gene analysis have not been developed in red marine algae including N. yezoensis, so we can’t analyze the function of NySPLs and NyFALs. We described the function analysis as future task.