# Secondary cell wall molecular determinism in sorghum is not a simple copy paste of arabidopsis story

Lauriane Hennet, Angélique Berger, Noemi Trabanco, Emeline Ricciuti, Jean-François Dufayard, Stéphanie Sidibé-Bocs, Laura Rossini, Delphine Luquet, Nancy Terrier, David Pot

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## To cite this version:

Lauriane Hennet, Angélique Berger, Noemi Trabanco, Emeline Ricciuti, Jean-François Dufayard, et al. Secondary cell wall molecular determinism in sorghum is not a simple copy paste of arabidopsis story. International Plant Cell Wall Meeting, Jul 2019, Cambridge, United Kingdom. CAP Partner, 1 p., 2019. [hal-05178894](https://hal.science/hal-05178894v1)

HAL Id: hal-05178894

https://hal.science/hal-05178894v1

Submitted on 23 Jul 2025

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Distributed under a Creative Commons Attribution 4.0 International License
# SECONDARY CELL WALL MOLECULAR DETERMINISM IN SORGHUM IS NOT A SIMPLE COPY PASTE OF ARABIDOPSIS STORY

Hennet Lauriane¹,², Berger Angélique¹,², Trabanco Noemi⁴, Ricciuti Emeline¹,², Jean-François Dufayard¹,², Stéphanie Sidibe-Bocs¹,², Laura Rossini⁴,⁵, Delphine Luquet¹,², Terrier Nancy³, Pot David¹,²

1 CIRAD, UMR AGAP, F-34398 Montpellier, France
2 Univ. Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier, France
3 AGAP, Univ. Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier, France
4 Parco Tecnologico Padano, Via Einstein, Loc. Cascina Codazza, Lodi (Italy)
5 Department of Agriculture and Environmental Sciences - Production, Landscape, Agroenergy (DiSAA), Università degli Studi di Milano, Via Celoria 2, Milano (Italy)

lauriane.hennet@cirad.fr & david.pot@cirad.fr

## Secondary cell wall composition is a key trait for sorghum breeding

Sorghum bicolor L. Moench is the world fifth crop for grain production. It is used for human and animal nutrition (grain and vegetative parts) and it is also increasingly mobilized for energy and natural fiber-reinforced polymers production. Sorghum biomass is mainly accumulated in stems and especially in secondary cell walls (SCW) and MYB and NAC transcription factors are expected to be key drivers of SCW composition (Zhong et al., 2008). Understanding how is regulated SCW deposition in sorghum would allow fine tuning of SCW deposition and stem quality to improve selection of sorghum varieties that match farmers and value chains needs.

In order to identify candidate genes we used **comparative genomics** and **co-expression gene network** analyses. We were able to identify regulatory networks involved in SCW deposition and highlight the roles of NAC and MYB members that are the more likely to control SCW deposition in sorghum.

### 1 - TWO CLUSTERS OF GENES INVOLVED IN SCW HAVE BEEN IDENTIFIED

* 45 modules of co-expressed genes were identified, each of them is named with a color randomly picked by WGCNA R package.

* 6 modules are enriched in Gene Ontologies (GO) related to SCW and CW and in CW enzymes (Fig. 2).

* Yellow and pink modules genes are likely to contain SCW genes because their expression pattern is synchronous with lignin accumulation (Fig. 3A and 3B).

<figure><img src="image_3.png" /><figcaption>**Figure 2:** Modules enrichment in CW enzymes and GO terms related to cell walls.</figcaption></figure>

The number of genes considered per module is indicated by the bar heights (principal Y-axis) and the number of CW related GO significantly enriched is indicated by the line plot (secondary Y-axis). Modules significantly enriched in CW enzymes are indicated in blue. Grey modules are not significantly enriched in CW enzymes.

<figure><img src="image_4.png" /><figcaption>**Figure 3:** A- Expression patterns of the 6 modules with CW related GO and CW enzymes enrichment detected (centered and normalized average expression of genes is represented) and B- Accumulation pattern of cell wall components (xylan, cellulose and lignin content expressed as percentage of dry matter (DM)) for IN 12 in the well-water treatment for 2013 trial (stages D1 to D5).</figcaption></figure>

### 2 - MOST OF At MASTER REGULATORS ARE ALSO KEY GENES IN SORGHUM

<figure><img src="image_5.png" /><figcaption>**Figure 4:** MYB, NAC and other regulators of SCW biosynthesis functionally validated in arabidopsis, maize, rice and sorghum.</figcaption></figure>

* NAC and MYB sorghum orthologs of At functionally characterized genes have been identified thanks to published work (Fig. 4) and a phylogenetic study of MYB and NAC families (Fig. 5).

* 122 SbNAC and 135 SbMYB in total were identified.

* SbNAC and SbMYB top co-expressed genes lists were extracted.

* Co-expressed genes lists of sorghum orthologs of most At master regulators are enriched in CW related GO (e.g. SbMYB46 in Fig. 6A, SbNST1 in Fig. 6B).

* 5 out of 8 VND family sorghum homologs are not expressed in our sampling conditions and only one sorghum VND7 ortholog co-expressed gene list is enriched in CW related GO.

<figure><img src="image_6.png" /><figcaption>**Figure 5:** MYB family tree including 9 species with a focus on SbMYB13a</figcaption></figure>

located in the pink clade along (but not closely related to) other MYB SCW regulators. Colors represent clades, classification inspired by Li et al., 2016.

Aggregated GO per color :
* SCW
* Lignin
* Xylan
* Cellulose
* CW
* Pectin
* Vessels dvp
* GO not related to walls

<figure><img src="image_7.png" /><figcaption>**Figure 6:** Number of significantly enriched CW related GO in co-expressed genes with A-SbMYB46 B-SbNST1 C-SbMYB13a D-SbNAC25.</figcaption></figure>

## MATERIAL & METHODS

### Identification of gene networks :

* 3 trials (2013, 2014, 2015) on a commercial biomass hybrid.

* Between 2 and 4 internodes (IN) sampled at 2 to 5 stages in 2 water availabilities (Fig. 1).

* Biochemical characterization of the IN composition.

* 357 RNAseq libraries sequenced.

* Gene co-expression network built through WGCNA signed analysis (Langfelder & Horvath, 2008).

* High co-expression correlation between genes considered (>0.82).

### Enrichment analysis:

* TopGO R package with PLAZA 4.0 GO database.

* Fisher t-test using a list of cell wall (CW) involved enzymes (Rai et al. 2016, McKinley et al. 2016, Petti et al., 2015).

### Comparative genomic analyses of the MYB and NAC families:

* Use of 9 different dicotyledon and monocotyledon species.

* 1.170 NAC and 1.328 MYB protein used.

* Use of HMM signature, trimal, ML method to build the phylogenetic analysis and RAP-Green to root the tree.

<figure><img src="image_8.png" /><figcaption>**Figure 1:** 2013 trial sampling scheme (2 IN x 5 stages x 2 water treatments x 4 biological replicates).</figcaption></figure>

### 3 - NEW MYB AND NAC POTENTIALLY INVOLVED IN SCW HAVE BEEN IDENTIFIED

* We identified SbMYB and SbNAC genes whose orthologs are not yet identified as key SCW regulators but their co-expressed genes lists are enriched in CW related GO (e.g. SbMYB13a in Fig.5, 6C and 7 and SbNAC25 in Fig. 6D and 7).

* There are strong connections between co-expressed genes with SbMYB and SbNAC in the SCW yellow module.

* In this subnetwork, there are highly connected genes not annotated with CW related GO.

<figure><img src="image_9.png" /><figcaption>**Figure 7:** SbMYB and SbNAC (white rounded) within yellow module and their top coexpressed genes (purple gradient for correlation strength) that are enriched in CW related GO genes (blue). SbMYB and SbNAC with no ortholog SCW or CW annotated are red colored.</figcaption></figure>

## CONCLUSION AND DISCUSSION

* **Genes in the 6 modules** (Fig. 3A) identified are likely to be good candidate for a role in CW biosynthesis and especially SCW biosynthesis for pink and yellow module genes.

* **Most orthologs of arabidopsis master regulators of SCW are likely to be involved in SCW regulation in sorghum.** However, there are weaker evidences for involvement of some orthologs of At key SCW regulation genes such as VND7. As VND family is responsible for SCW regulation in vessels, this could be due to vessels and fibers pattern differences in the stem between monocotyledons and dicotyledons.

* **New MYB and NAC candidates for SCW regulation in sorghum are identified.**

* **SbMYB and SbNAC SCW putative regulators top co-expressed genes are also top candidate genes for a role in SCW biosynthesis.** It could be interesting to see how they overlap with other candidate genes lists such as differentially expressed genes between stem composition varying sorghum varieties or genes detected in stem composition association studies.

This work was supported by the BIOSORG project funded by Agropolis (AF 1301-010) and Cariplo (FC 2013-1890) foundations and Biomass For the Future project (ANR-11-BTBR-0006-BFF) funded by the French National Research Agency (ANR). Lauriane Hennet received support from the Ministry of Higher Education and Research and from Dufrenoy fundation.

## REFERENCES:

Langfelder and Horvath, 2008, BMC Bioinformatics

Li et al., 2016, Plant and Cell Physiology

McKinley et al., 2016, The Plant Journal: For Cell and Molecular Biology

Zhong et al., 2011, Plant and Cell Physiology

Zhong et al., 2008, Plant Cell

Petti et al., 2015, Plant Physiology

Rai et al., 2016, SpringerPlus