# Polysaccharide distribution and cell wall deposition in the endosperm and outer layers of developing sorghum grains

Camille Costes, Lucie Le Bot, Camille Alvarado, Sylviane Daniel, Sergi Navarro Sanz, Caroline Calatayud, Alexandre Robin-Soriano, Fabienne Guillon, Hamza Mameri, Nancy Terrier, et al.

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## To cite this version:

Camille Costes, Lucie Le Bot, Camille Alvarado, Sylviane Daniel, Sergi Navarro Sanz, et al. Polysaccharide distribution and cell wall deposition in the endosperm and outer layers of developing sorghum grains. Journal of Cereal Science, 2025, 124, pp.104239. 10.1016/j.jcs.2025.104239. hal-05179516

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Submitted on 6 Aug 2025

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# Polysaccharide distribution and cell wall deposition in the endosperm and outer layers of developing sorghum grains

Camille Costes<sup>a</sup>, Lucie Le Bot<sup>a</sup>, Camille Alvarado<sup>a</sup>, Sylviane Daniel<sup>a</sup>, Sergi Navarro Sanz<sup>c,d</sup>,
Caroline Calatayud<sup>c</sup>, Alexandre Soriano<sup>c</sup>, Fabienne Guillon<sup>a</sup>, Hamza Mameri<sup>b</sup>, Nancy Terrier<sup>c</sup>,
Luc Saulnier<sup>a</sup>, Mathilde Francin-Allami<sup>a,*</sup> [10]

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## ARTICLE INFO

### Keywords:

- Sorghum

- Cell wall

- Grain development

- Polysaccharides

## ABSTRACT

Despite its climate resilience and nutritional value, sorghum remains less studied than most major cereals. Cell walls are key to grain quality and processing. Yet, little is known about cell wall deposition during sorghum grain development. By immunofluorescence labeling and biochemical approaches, an overview of the spatiotemporal distribution of cell wall polysaccharides in sorghum grain has been obtained from the end of the cellularization step to maturity, both in the endosperm and outer layers. For the first time, the presence of minor polysaccharides, such as xyloglucans, pectins, and heteromannans were detected. By analysing the oligosaccharides released by xyloglucan-specific endoglucanase treatment using high-performance anion-exchange chromatography, we demonstrated that xyloglucans were galactoxyloglucans with a XXXG core motif, mainly including XLXG and XLLG units. Consistent with biochemical and immunolabeling results, an important proportion of the cell wall related genes identified by RNAseq analysis exhibited significant differential expression with higher expression at earlier developmental stages whatever the tissue. Many cell wall genes, including cellulose and hemicellulose biosynthesis genes, were more highly expressed in the outer layers than in the endosperm, except at the last stage. Overall, these results have improved our understanding of sorghum grain quality by providing new insights into cell wall formation.

## 1. Introduction

Sorghum (Sorghum bicolor [L.] Moench) is the world's fifth most important cereals in terms of production. It belongs to the Poaceae family, which also includes maize, wheat, and rice. Sorghum is especially relevant in the face of climate change and agroecological transition, thanks to its heat tolerance and minimal need for water and inputs. While whole plant sorghum is mainly used for animal feed and bioethanol, sorghum grain is widely used for human consumption in Africa and Asia. Sorghum cultivation is gaining ground in Europe, with production reaching 1.14 million tonnes in 2021 (FAO). Sorghum grain is also of growing interest for human consumption due to its gluten-free status and richness in health-promoting macro- and micro-nutrients, particularly dietary fibers. In grain, dietary fibers are mainly composed of cell wall polysaccharides, which are key factors to consider

when improving the nutritional quality of cereals. Moreover, intact cell walls act as a barrier to protect starch and protein from enzyme hydrolysis, and could be partly responsible for the low digestibility of sorghum proteins (Duodu et al., 2003).

The polysaccharide composition has been well described in dry sorghum grains as well as in whole flour, endosperm, or bran (Holmes et al., 2013; Jaworski et al., 2015). Starch is the most abundant polysaccharide in sorghum grains. This polysaccharide accumulates in the cytoplasm of endosperm cells and serves as a carbohydrate reserve for germination and seedling establishment (Holmes et al., 2013). It represents around 65-70 % of the dry grain whereas non-starch polysaccharides represent around 5-6 % (Holmes et al., 2013). Non-starch polysaccharides are found in the cell walls that surround the cells. Cell walls of sorghum grains are mainly composed of glucuronoarabinoxylans (GAXs), cellulose and mixed-linked β-glucans (MLG) (Jaworski

<footnote><sup>a</sup> INRAE, UR1268 BIA, Nantes, France</footnote>

<footnote><sup>b</sup> INRAE, Université de Montpellier, Montpellier SupAgro, UMR 1208 IATE, 34060, Montpellier, France</footnote>

<footnote><sup>c</sup> UMR AGAP Institut, INRAE, Univ Montpellier, CIRAD, Institut Agro, F-34398, Montpellier, France</footnote>

<footnote><sup>d</sup> Montpellier Ressources Imagerie, BioCampus, University of Montpellier, CNRS, INSERM, Montpellier, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: mathilde.francin-allami@inrae.fr (M. Francin-Allami).</footnote>
et al., 2015). In addition, GAXs have been reported to be acetylated and acylated by phenolic acids, mainly ferulic and p-coumaric acids (Verbruggen et al., 1998).

Cell wall polysaccharides are synthesized and remodeled by genes throughout the grain development. Numerous cell wall polysaccharide biosynthesis genes have been identified in cereals these last decades (Burton et al., 2014). For example, several genes responsible for AX formation in wheat have been identified, which include arabinosyl-transferases (AT) belonging to the GT61 family and xylan xylosyl-transferases (XT) which belongs to the Glycosyl Transferase 43 and 47 families (GT43 and GT47) (Anders et al., 2012; Zeng et al., 2016). Likewise, genes responsible for synthesizing MLG have been identified in various species. They belong to the cellulose synthase-like families CsIF and CsIH. Other Csl genes are known to be involved in xyloglucan and mannan biosynthesis (respectively CsIC and CsIA/D families) (Daras et al., 2021).

However, the cell wall composition and assembly during sorghum grain development are still poorly described. The evolution of non-starch polysaccharide composition has been described in the endosperm only at late stages of development (Kavitha and Chandrashekar, 1997). Likewise, MLG content has been reported in the pericarp at a single stage of grain development (Ermawar et al., 2015). Cell wall deposition during grain development has been more deeply described in other cereals such as wheat, rice, and barley through biochemical and immunolabeling investigations. Recent studies conducted from developing wheat and barley grains highlighted minor cell wall polysaccharides such as xyloglucans, heteromannans and pectins (homogalacturonan (HG), rhamnogalacturonan-I (RGI) and rhamnogalacturonan-II (RGII) (Chateigner-Boutin et al., 2014; Palmer et al., 2015).

There is a lack of knowledge about the establishment of cell wall polysaccharides during sorghum grain development, as well as about the genes involved in cell wall deposition and remodeling. These are essential for controlling grain quality, particularly through its fiber content and composition. To overcome these shortcomings, the objective of this work was to track the evolution of polysaccharide composition and distribution both in outer layers and endosperm of sorghum developing grains by biochemical and imaging approaches. Additionally, the expression of cell wall genes was analyzed at three developmental stages. We then aimed to rely on the observed changes in the transcriptomic profiles with the spatio-temporal distribution of cell wall polysaccharides during grain development.

# 2. Materials and methods

## 2.1. Materials

The MACIA variety, characterized by white grains without tannin, was chosen for this study. The sorghum plants were sown and grown in greenhouses at CIRAD Montpellier in 2022 as previously described (Costes et al., 2024). From the first panicle flowering, samples were annotated, and grains were harvested in triplicate at 7, 13, 21, 30, and 40 days after flowering (DAF) and directly frozen in liquid nitrogen.

## 2.2. Methods

### 2.2.1. Sample preparation for biochemical analysis

The harvested grains were thawed in 70 % ethanol, and at least 40 grains were manually dehulled for each stage, to isolate the outer layers and endosperm. The samples were freeze-dried and cryo-ground in liquid nitrogen and then placed in 80 % ethanol and heated in a boiling water bath for 10 min. After cooling, the supernatant was removed by centrifugation and the procedure was repeated 4–6 times. Samples were finally washed with 95 % ethanol, centrifuged and the alcohol-insoluble materials (AIMs) were collected after drying as described previously (Chateigner-Boutin et al., 2018). At least two experimental replicates

were performed for each sample.

### 2.2.2. Neutral sugar quantification

The AIM samples were treated with H₂SO₄ 12 M at 25 °C for 30 min to prehydrolyze the cellulose, followed by hydrolysis with 1M H₂SO₄ for 2h at 100 °C. Neutral sugars released by acid hydrolysis were individually quantified after derivatization into alditol acetate using gas chromatography (Chateigner-Boutin et al., 2018).

### 2.2.3. Starch and mixed linked-β-glucans content determination

Starch content was determined from the AIM samples according to AOAC (method 996.11) with modifications as described in (Chateigner-Boutin et al., 2018). Amylolysis was performed using a thermostable alpha-amylase (E-BSTAA, Megazyme, Ireland) and an amyloglucosidase (E-AMGDF, Megazyme, Ireland).

To determine mixed-linked-β-glucans (MLG) content, 5 mg of AIM samples were degraded using 10 U of lichenase (Megazyme, Wicklow, Ireland) for 8 h at 40 °C, followed by a further 10 U of lichenase overnight at 40 °C to generate MLG derived oligosaccharides. Maltopentaose was used as an internal standard. The main oligosaccharides cellotriosyl (DP3) and cellotetraosyl (DP4) were considered to estimate the amount of MLG.

Glucose from starch and DP3 and DP4 cello-oligosaccharides from MLG were then quantified by High-Performance Anion Exchange Chromatography (PA1 column 250 × 4 mm, Dionex, Sunnyvale, CA, USA) (Francin-Allami et al., 2019).

### 2.2.4. Quantification of ester-linked hydroxycinnamic acids

The AIMs were subjected to mild alkaline hydrolysis for 4 h at 40 °C, using a 1M NaOH solution to release ferulic and coumaric acids. Before adjusting the pH to 2 with 200 µL of 6M HCl, an internal standard consisting of o-coumaric acid (2 mg/mL) (Sigma, Darmstadt, Germany) was added. After centrifugation (13400 rpm, 10 min), 0.5 mL of the supernatant was deposited on a solid phase extraction cartridge (Waters Sep-Pack t18, Ireland) pre-conditioned before use (washed with 1 × 1 mL MeOH, then with 2 × 1 mL water containing 0.01 % HCOOH). The sample-loaded cartridge was washed with 1 mL of water containing 0.01 % HCOOH and eluted with 1 mL of HPLC-grade methanol. The samples were filtered through a 0.45 µm PVDF filter. After dilution, the samples were subjected to High-Performance Liquid Chromatography analysis (C18 column, NUCLEOSHELL RP 18plus 50 × 2 mm, Macherey Nagel) as previously described (Chateigner-Boutin et al., 2018).

### 2.2.5. Enzymatic fingerprinting of xyloglucans

Five mg of AIMs were subjected to digestion by 0.5U of endocellulase (E-CELTR, Megazyme, Ireland) in deionized water overnight at 40 °C with 20 rpm rotary agitation. After centrifugation for 10 min at 10000 rpm, the supernatants were heated at 100 °C for 10 min to inhibit the enzyme, and centrifuged for 10 min at 10000 rpm. Finally, the supernatants were filtered and injected via an auto-sampler (Ultimate 3000 Autosampler, Dionex, Sunnyvale, CA, USA). They were then subjected to HPAEC analysis using a PA200 250 × 3 mm column (Dionex, Sunnyvale, CA, USA), eluted at room temperature at a flow rate of 0.4 mL/min for 30 min with a gradient composed of 4.5 % of 1 M sodium acetate, 20 % of 0.5 M sodium hydroxide, and the remainder being water. A xyloglucan control from tamarind seeds was used to determine the retention times of the main xyloglucan oligomer types (XXXG XXLG, XLXG, XLLG according to the established nomenclature) (Steck et al., 2021).

### 2.2.6. Acetyl content determination

Five mg of AIMs were subjected to deacetylation by adding 0.5 mL water and 0.1 N NaOH for 30 min at room temperature. The reaction was stopped by adding 0.5 mL 0.1 N HCl. The acetyl assay was then carried out using the Megazyme K-ACET Kit, using a 96-well plate.
### 2.2.7. Preparation of grain sections for histological staining and immunolabeling

Grains were cut in half longitudinally for each developmental stage and then fixed in 0.5 % glutaraldehyde +3 % paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4 overnight at 4 °C. Samples were then dehydrated in an ethanol series and embedded in LR-White resin as described by (Chateigner-Boutin et al., 2014). Longitudinal sections (1 µm) were obtained using an ultracut (UC7, Leica).

### 2.2.8. Grain section staining

Grain sections were stained for 10 min at room temperature with Amido Black 10B (protein staining) diluted at 5 % in a 7 % acetic acid solution, followed by several rinses with deionized water. Next, grain sections were incubated in a 1 % solution of Acridine Orange (cell wall staining) in deionized water for 5 min, followed by rinses with deionized water. Finally, a 5 % Lugol solution (starch staining) diluted in deionized water was applied for 2 min and rinsed with deionized water. Stained sections were observed using a brightfield microscope (LEICA DM 2500 LED).

### 2.2.9. Immunolabeling

To observe the presence of cellulose, pectin, mannan, and xyloglucan, the grain sections were pre-treated overnight at 40 °C with lichenase (endo-1,3(4)-β-Glucanase, Clostridium thermocellum, Nzytech, Portugal, 1.7U/50 µL) and desalted β-Xylanase M1 (E-XYTR1, Megazyme, Ireland, 0.8U/50 µL) to remove mixed-linked β-glucans (MLG) and arabinoxylans (AXs), respectively. Finally, the sections were rinsed with deionized water. To observe the presence of AXs, the sections were pre-treated only with lichenase, then washed with water, and incubated with NaOH (50 mM) for 30 min at room temperature to remove acetyl groups and facilitate access to AX epitopes. To observe the presence of MLG, grain sections were not pre-treated. Immunolabeling was then carried out as described in (Francin-Allami et al., 2019). Primary antibodies were diluted in PBS buffer containing 1 % BSA +0.05 % Tween20 as follow: 1:20 for AX1, 1:1500 for Fer-Ara, 1:200 for anti-MLG, 1:975 for CBM3a, 1:5 for LM25 and LM21, 1:10 for LM20 and 1:3 for INRA-RU1.

### 2.2.10. Transcriptomic analyses

Transcriptomic analyses were carried out on three developmental stages (7, 13, and 30 days after flowering (DAF)) ranging from early development to the end of grain filling. All the following experiments were carried out under RNAse-free conditions. Grains were embedded as blocks in O.C.T. (VWR® Q Path® O.C.T. Compound Mounting Medium, PA, USA). From these blocks, sections of 18 µm were cut using a Cryostat microtome (MICROM HM 520, Leica, IL, USA) before being adhered to frame slides (steel frame, PET-membrane 1.4 µm; RNAse-free.4 µm; RNAse-free, Leica, Germany), and the sections were fixed as described in (Costes et al., 2024). Once the slides had dried, outer layers and endosperms were separated by laser microdissection using a Leica LMD7000 laser (Laser parameters: Power 50, Speed 6, Aperture 14, Pulse Frequency 1260, Specimen Balance 12) as previously described (Costes et al., 2024). Total RNA was extracted and RNA libraries were prepared as described in (Costes et al., 2024). Raw sequence files were available in the short reads archive under bio project PRJNA1029354 [https://identifiers.org/ncbi/insdc.sra:SRP466948]. Low-quality sequences, with a cut-off of 30, as well as remaining adaptors and resulting reads of less than 35 bases, were filtered. The reads were then counted and aligned to 34129 transcripts from 34496 genes in the sorghum genome (Sbicolor_453_v3.2), as described by (Costes et al., 2024). The DIANE interactive workflow was used to normalize and process the data using the TMM (Trimmed Mean of M values) method. Only transcripts for which the sum of reads in all conditions is equal to or greater than 100 were retained. The differential expression analysis was based on the EdgeR package with parameters: LogFoldChange>1 and adjusted p-value<0.05. A list of 655 sorghum cell wall genes has been established

previously and inventoried in (Hennet et al., 2020). Of these, 384 were detected in our data filtered from our different samples with their associated quantifications for each biological stage, tissue, and replicate (Costes et al., 2024), Data File 5).

## 3. Results and discussion

### 3.1. Histology of developing sorghum grain

Histological analyses were performed from sorghum grains collected at four developmental stages covering cell differentiation till grain maturity. Weights of outer layers and endosperm varied throughout the development of sorghum grain (Fig. 1). The outer layers are the most important part of grain at the very beginning of grain development and act as protective maternal tissues. The endosperm, which accumulates the reserves for germination and seedling growth, became more predominant as the grain mature. Histological changes of grain during its development were monitored by a triple staining (Fig. 1). The nucellar epidermis possessed large cells at 7 DAF but rapidly compacted to form a thin layer from the 13 DAF stage. Inversely, aleurone cells progressively differentiated from 13 DAF. In addition, cell walls thickness increased during grain development particularly in the outer layers (Fig. 1). In the same way, the density of starch granules clearly increased in the endosperm all along grain development. Granule size also seemed to increase with grain development (Fig. 1). It was interesting to note the presence of small starch granules in the pericarp, even at a late stage (30 DAF) contrary to other cereals such as wheat, where no starch was observed beyond 25 days after anthesis (Zhuo et al., 2023). The intensity of protein labeling increased with grain growth, particularly in the aleurone from 13 DAF (Fig. 1). In central endosperm cells, proteins preferentially accumulated close to the cell walls as previously observed (Duodu et al., 2003) (Fig. 1).

### 3.2. Carbohydrate composition in the endosperm and outer layers of developing sorghum grains

The carbohydrate composition was determined for both the endosperm and outer layers at the stages representative to the grain development described above (Table 1). Glucose was the most abundant sugar both in the outer layers and in the endosperm, originating from cellulose, mixed-linked β-glucans (MLG), and starch. In the endosperm, total sugar content increased during grain development, aligning with the progressive accumulation of starch in this storage tissue (Fig. 1). In contrast, sugar content did not increase in the outer layers of developing grain. Glucose was accumulated in the grain endosperm till 30 DAF. In the outer layers, accumulation stopped as soon as the 13 DAF stage and then glucose amount decreased to reach a proportion of around 45 % of dry matter at later stages.

Beside glucose, arabinose and xylose are the two most common sugars found in the endosperm and outer layers of sorghum grains (Table 1). In cereal grains, these two sugars are mainly representative of arabinoxylans (AXs). While their proportion was less than 1 % in the endosperm, they ranged from 2.9 % at 7 DAF to over 10 % at 30 DAF in the outer layers. When expressed by grain, those values revealed that xylose and arabinose exhibited a rapid accumulation in the grain till 21 DAF and then tended to stabilize, both in the endosperm and in the outer layers. In wheat, the amount of arabinose and xylose also showed an increased proportion throughout grain development in the outer layers (Chateigner-Boutin et al., 2018).

Because acetylation may occur on hemicelluloses and pectins, we determined the amount of acetyl groups in tissues during grain development (Supplementary Table S1). Acetyls were detected in high amounts from stage 7 DAF in the outer layers. When expressed as % of dry matter, an increase was observed past 21 DAF, which may be related to the increase in cell wall amount as grain develops. In the endosperm, the proportion of acetyls decreased between 7 DAF and 30 DAF, whereas
A

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B

<figure><img src="image_6.png" /><figcaption>**Fig. 1.** A. Average weight (dry matter) of whole grains, endosperm and outer layers during development (7, 13, 21, 30 and 40 days after flowering). Data are the mean ± standard deviation of three biological replicates. B. Longitudinal section (1 µm) of developing sorghum grains labelled with a triple staining: acridine orange (yellow/orange) for cell walls, naphthol blue black (green) for proteins and iodine (blue/purple) for starch. 7DAF: Stage 7 Days After Flowering, 13DAF: Stage 13 Days After Flowering, 21DAF: Stage 21 Days After Flowering, 30DAF: Stage 30 Days After Flowering, 40DAF: Stage 40 Days After Flowering, Ep: Epicarp, End: Endosperm, Pe: pericarp, Ne: nucellar epidermis, Al: Aleurone, Sg: Starch granules. Bar scale: 30 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

most of the carbohydrates, including arabinose and xylose, increased during the same developmental stages (Supplementary Table S1; Table 1). This could result from a lower extent of polysaccharide acetylation in the endosperm as previously observed in wheat grain. Although the biological function of cell wall polysaccharide acetylation is not fully understood, it likely plays a significant role in modulating polymer chain interactions and influencing the mechanical and physicochemical properties of cell wall during plant development (Fanuel et al., 2022). Finally, minor sugars such as rhamnose, galacturonic, and glucuronic acid were identified in all tissues along the development, whereas others, mannose and galactose, were only identified in the outer layers (Table 1). These sugars were detected at very low levels, preventing the observation of variations across developmental stages. In the developing peripheral tissues of wheat grains, a low proportion of galactose, mannose, glucuronic acid, and galacturonic acid were also observed, with no variation in their proportion during grain development (Chateigner-Boutin et al., 2018). The detection of these sugars most likely indicates the presence of pectins and mannans in developing sorghum grains.

### 3.3. Polysaccharide structure and distribution during grain development

#### 3.3.1. Starch

Our results indicated that most of the glucose arose from starch in the endosperm throughout grain development and in the outer layers, especially during the early stages (Table 1). The accumulation of starch

in developing grains is a key process in grain ripening, as it serves as the main energy reserve component and is essential for germination and seedling growth in most cereals. Starch proportion reached more than 75 % of the endosperm dry matter by 21 DAF and starch amount in the grain increased till 30 DAF. Starch persisted in the outer layer throughout grain development, and still accounted for almost half of the glucose content in the latest stages, which was consistent with our previous histological observations (Fig. 1). In wheat pericarp, starch acts as a temporary reserve for starch biosynthesis in the endosperm during early grain development (Zhuo et al., 2023). The same process may occur in sorghum grain, except that, contrary to wheat, starch persists in the outer layers until grain maturity.

#### 3.3.2. *Arabinoxylans*

As indicated above, we considered that arabinose and xylose sugars were mainly derived from AXs. Both in the endosperm and the outer layers, A/X ratios were relatively high compared with other Poaceae such as wheat, barley, or *Brachypodium distachyon*, with ratios up to 1.6 in the endosperm and 1.2 in the outer layers (Table 1) (Francin-Allami et al., 2019).

In dry sorghum grains, AXs were shown to be substituted with glucuronic acids and acetylated by phenolic compounds such as p-coumaric or ferulic acids (Miafo et al., 2021). Here we described the spatio-temporal occurrence of these AX substitutions during grain development. In the outer layers, the proportion of glucuronic acid increased between 7 and 21 DAF stages and remained stable thereafter
<table><caption>**Table 1**

Sugar composition of the Alcohol Insoluble Material (AIM) of endosperm (A) and outer layers (B) in developing sorghum grains.</caption><thead><tr><th rowspan="2">A<br>Sugars</th><th colspan="2">Endosperm 7 DAF</th><th colspan="2">Endosperm 13 DAF</th><th colspan="2">Endosperm 21 DAF</th><th colspan="2">Endosperm 30 DAF</th><th colspan="2">Endosperm 40 DAF</th></tr><tr><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th></tr></thead><tbody><tr><td>Rhamnose</td><td>0,2 ±<br>0,1</td><td>2,6 ± 0,3</td><td>0,4 ±<br>0,1</td><td>51,8 ± 8,9</td><td>0,3 ±<br>0,1</td><td>91,4 ± 10,7</td><td>0,4 ±<br>0,1</td><td>109,6 ± 15,5</td><td>0,4 ±<br>0,1</td><td>121,4 ± 21,6</td></tr><tr><td>Arabinose</td><td>0,7 ±<br>0,1</td><td>6,2 ± 0,9</td><td>0,4 ±<br>0,1</td><td>55,8 ± 11,3</td><td>0,5 ±<br>0,2</td><td>156,2 ± 25,4</td><td>0,6 ±<br>0,1</td><td>178,9 ± 28,6</td><td>0,6 ±<br>0,1</td><td>187,5 ± 22,0</td></tr><tr><td>Xylose</td><td>0,4 ±<br>0,1</td><td>4,0 ± 0,5</td><td>0,3 ±<br>0,1</td><td>36,7 ± 4,9</td><td>0,3 ±<br>0,0</td><td>76,2 ± 11,3</td><td>0,4 ±<br>0,1</td><td>131,1 ± 30,4</td><td>0,4 ±<br>0,1</td><td>111,0 ± 24,3</td></tr><tr><td>Glucuronic acid</td><td>&gt;0,01</td><td>0,0 ± 0,0</td><td>&gt;0,01</td><td>0,8 ± 0,2</td><td>&gt;0,01</td><td>1,4 ± 0,3</td><td>&gt;0,01</td><td>2,0 ± 0,4</td><td>&gt;0,01</td><td>1,7 ± 0,3</td></tr><tr><td>Galacturonic acid</td><td>0,03 ±<br>0,01</td><td>0,3 ± 0,0</td><td>0,02 ±<br>0,00</td><td>2,8 ± 0,4</td><td>0,01 ±<br>0,00</td><td>3,7 ± 1,0</td><td>0,02 ±<br>0,00</td><td>5,9 ± 1,6</td><td>0,02 ±<br>0,00</td><td>7,0 ± 0,8</td></tr><tr><td>Glucose (total)</td><td>58,2 ±<br>5,1</td><td>547,7 ±<br>48,2</td><td>81,6 ±<br>8,5</td><td>10718,8 ±<br>1112,6</td><td>85,5 ±<br>3,8</td><td>21078,2 ±<br>929,7</td><td>86,4 ±<br>5,2</td><td>24676,2 ±<br>1477,3</td><td>85,1 ±<br>6,2</td><td>25716,1 ±<br>1866,9</td></tr><tr><td>Starch</td><td>51,1 ±<br>2,1</td><td>480,6 ±<br>20,2</td><td>70,8 ±<br>2,3</td><td>9307,6 ±<br>306,2</td><td>77,0 ±<br>1,0</td><td>18970,1 ±<br>238,1</td><td>78,3 ±<br>1,2</td><td>22373,0 ±<br>352,5</td><td>76,2 ±<br>1,6</td><td>23044,7 ±<br>476,1</td></tr><tr><td>β-glucans</td><td>0,2 ±<br>0,04</td><td>1,9 ± 0,4</td><td>0,05 ±<br>0,00</td><td>6,0 ± 0,8</td><td>0,06 ±<br>0,00</td><td>14,7 ± 2,6</td><td>0,09 ±<br>0,02</td><td>25,6 ± 5,3</td><td>0,10 ±<br>0,01</td><td>30,3 ± 3,2</td></tr><tr><td>Cellulose</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td>Total sugars</td><td>59,6 ±<br>5,2</td><td>560,0 ±<br>48,5</td><td>82,6 ±<br>8,6</td><td>10854,7 ±<br>1126,8</td><td>86,7 ±<br>3,8</td><td>21364,6 ±<br>934,8</td><td>87,7 ±<br>5,0</td><td>25052,8 ±<br>1437,7</td><td>86,4 ±<br>6,3</td><td>26126,6 ±<br>1910,2</td></tr><tr><td>Ratio A/X</td><td colspan="2">1,6</td><td colspan="2">1,6</td><td colspan="2">1,6</td><td colspan="2">1,4</td><td colspan="2">1,4</td></tr><tr><td>BG3/BG4</td><td colspan="2">0,7</td><td colspan="2">1,5</td><td colspan="2">1,6</td><td colspan="2">1,9</td><td colspan="2">1,8</td></tr><tr><th rowspan="2">B<br>Sugars</th><th colspan="2">Outer Layers 7 DAF</th><th colspan="2">Outer Layers 13 DAF</th><th colspan="2">Outer Layers 21 DAF</th><th colspan="2">Outer Layers 30 DAF</th><th colspan="2">Outer Layers 40 DAF</th></tr><tr><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th></tr><tr><td>Rhamnose</td><td>0,3 ± 0,1</td><td>9,9 ± 1,5</td><td>0,2 ± 0,1</td><td>16,4 ± 3,2</td><td>0,3 ± 0,1</td><td>11,5 ± 1,3</td><td>0,3 ± 0,1</td><td>10,0 ± 0,4</td><td>0,3 ± 0,0</td><td>11,4 ± 1,2</td></tr><tr><td>Arabinose</td><td>2,9 ± 0,2</td><td>86,6 ± 6,3</td><td>4,1 ± 0,3</td><td>233,1 ±<br>16,8</td><td>7,9 ± 1,0</td><td>353,4 ±<br>29,3</td><td>12,3 ±<br>1,2</td><td>408,9 ±<br>35,9</td><td>11,7 ±<br>0,7</td><td>409,0 ±<br>24,7</td></tr><tr><td>Xylose</td><td>2,9 ± 0,2</td><td>86,8 ± 5,7</td><td>3,7 ± 0,2</td><td>206,8 ± 7,4</td><td>6,8 ± 0,9</td><td>306,3 ±<br>22,8</td><td>10,4 ±<br>0,9</td><td>351,6 ±<br>22,0</td><td>9,9 ± 0,5</td><td>350,0 ±<br>16,0</td></tr><tr><td>Glucuronic Acid</td><td>0,07 ±<br>0,04</td><td>2,1 ± 1,1</td><td>0,11 ±<br>0,02</td><td>6,4 ± 1,4</td><td>0,16 ±<br>0,04</td><td>6,7 ± 1,9</td><td>0,12 ±<br>0,04</td><td>4,1 ± 1,5</td><td>0,13 ±<br>0,01</td><td>4,56 ± 0,3</td></tr><tr><td>Galacturonic Acid</td><td>0,25 ±<br>0,03</td><td>7,4 ± 0,8</td><td>0,32 ±<br>0,03</td><td>17,8 ± 1,4</td><td>0,36 ±<br>0,07</td><td>14,9 ± 3,1</td><td>0,43 ±<br>0,03</td><td>14,5 ± 0,9</td><td>0,45 ±<br>0,08</td><td>15,9 ± 2,8</td></tr><tr><td>Mannose</td><td>0,6 ± 0,2</td><td>20,3 ± 3,5</td><td>0,5 ± 0,2</td><td>34,6 ± 5,3</td><td>0,7 ± 0,2</td><td>29,7 ± 4,2</td><td>0,8 ± 0,2</td><td>30,7 ± 4,0</td><td>0,7 ± 0,2</td><td>29,3 ± 4,0</td></tr><tr><td>Galactose</td><td>0,8 ± 0,4</td><td>29,1 ± 3,2</td><td>0,8 ± 0,3</td><td>57,2 ± 12,8</td><td>0,9 ± 0,2</td><td>43,1 ± 5,6</td><td>1,2 ± 0,2</td><td>46,2 ± 2,6</td><td>1,1 ± 0,2</td><td>41,1 ± 3,9</td></tr><tr><td>Glucose (total)</td><td>60,2 ±<br>2,3</td><td>1774,5 ±<br>70,5</td><td>53,8 ±<br>1,5</td><td>3027,5 ±<br>84,5</td><td>47,0 ±<br>1,1</td><td>1975,5 ±<br>46,9</td><td>44,8 ±<br>2,7</td><td>1497,2 ±<br>91,0</td><td>45,7 ±<br>2,5</td><td>1611,3 ±<br>86,2</td></tr><tr><td>Starch</td><td>45,3 ±<br>1,7</td><td>1337,3 ±<br>49,4</td><td>41,1 ±<br>2,0</td><td>2314,6 ±<br>110,4</td><td>29,8 ±<br>1,4</td><td>1249,8 ±<br>59,9</td><td>22,0 ±<br>1,2</td><td>734,6 ±<br>40,3</td><td>24,2 ±<br>0,9</td><td>850,1 ±<br>30,3</td></tr><tr><td>β-glucans</td><td>4,6 ± 0,2</td><td>134,5 ± 5,9</td><td>3,7 ± 0,2</td><td>208,4 ±<br>10,3</td><td>6,0 ± 0,6</td><td>253,4 ±<br>26,2</td><td>6,7 ± 0,2</td><td>224,3 ± 8,1</td><td>6,9 ± 0,2</td><td>243,2 ±<br>21,8</td></tr><tr><td>Cellulose</td><td>10,1 ±<br>1,3</td><td>296,5 ±<br>37,7</td><td>9,0 ± 2,1</td><td>504,6 ±<br>105,4</td><td>11,2 ±<br>1,5</td><td>472,4 ±<br>80,2</td><td>16,3 ±<br>1,0</td><td>545,0 ±<br>71,0</td><td>14,4 ±<br>1,9</td><td>504,7 ±<br>50,4</td></tr><tr><td>Total sugars</td><td>67,7 ±<br>2,9</td><td>1996,4 ±<br>86,0</td><td>63,2 ±<br>1,6</td><td>3555,8 ±<br>91,3</td><td>63,7 ±<br>2,3</td><td>2673 ±<br>95,3</td><td>69,9 ±<br>4,1</td><td>2333,7 ±<br>138,6</td><td>69,4 ±<br>1,6</td><td>2444,8 ±<br>57,8</td></tr><tr><td>Ratio A/X</td><td colspan="2">1,0</td><td colspan="2">1,1</td><td colspan="2">1,2</td><td colspan="2">1,2</td><td colspan="2">1,2</td></tr><tr><td>Ratio GluA/A</td><td colspan="2">0,04</td><td colspan="2">0,04</td><td colspan="2">0,02</td><td colspan="2">0,02</td><td colspan="2">0,02</td></tr><tr><td>BG3/BG4</td><td colspan="2">2,0</td><td colspan="2">2,0</td><td colspan="2">1,9</td><td colspan="2">2,0</td><td colspan="2">2,0</td></tr></tbody></table>

The values were calculated based on the percentage of dry matter (% DM) and per grain (µg/grain). Estimated cellulose as followed: Glucose (total)- Starch + mixed β-glucans; ratio A/X: ratio arabinose/xylose; GluA/A = Glucuronic acid/Arabinose; nd = not determined. 7DAF: stage 7 Days After Flowering; 13DAF: stage 13 Days After Flowering; 21DAF: stage 21 Days After Flowering; 30DAF: stage 30 Days After Flowering; 40DAF: stage 40 Days After Flowering. Data are the mean ± standard deviation of three biological replicates.

(Table 1). This corresponds to an accumulation in the grain outer layers till 13 DAF. The ratio of glucuronic acid to arabinose was stable in the outer layers throughout grain development and glucuronic acid accumulated in this part of the grain till 30 DAF (Table 1). Ferulic acid was the predominant phenolic compound in the outer layers and the endosperm of sorghum grains (Supplementary Table S1). In the endosperm, ferulic acid proportion was low, between 0.01 % and 0.03 % of dry matter, although higher than wheat endosperm (Pellny et al. 2012). In the outer layers, the percentage of phenolic acids increased between 7 DAF and 21DAF (Supplementary Table S1). The ratio between ferulic acid and AX increased by a factor of 2 between 7 and 13 DAF in the outer layers and between 13 and 21 DAF in the endosperm, suggesting that feruloylation of AX occurred relatively early during grain development

(Supplementary Table S1). A high decrease was observed between 21 DAF and 40 DAF in the outer layers, likely due to increased cross-linking of ferulic acid which occurs in most grass tissues as they mature.

The distribution of AXs has been observed throughout grain development by immunofluorescent labeling using AX1 antibodies that recognized (1/4)-β-linked linear xylosyl regions of low-substituted AXs (Fig. 2) (Francin-Allami et al., 2019). Throughout the development, high labeling was observed in the outer layers (epicarp, pericarp, and nucellar epidermis) and the aleurone cells from 21 DAF (Fig. 2). By contrast, no labeling was detected in the starchy endosperm. This could be explained by thinner cell walls and/or lesser accessibility of the antibodies due to a higher AX substitution by arabinose (Table 1). In rice and wheat, a high labeling was observed in the aleurone layer and also,
<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Distribution of cell wall polysaccharides during sorghum grain development. Immunofluorescent labeling of developing grain (from 7 to 33 DAF) using AX1 antibodies (arabinoxylans), Fer-Ara antibodies, anti-(1-3) (1-4)-β-D-glucans antibodies (Anti-MLG) and CBM3a (cellulose); LM25 antibodies (xyloglucans); RU1 antibodies (Rhamnogalacturonan I); LM20 antibodies (highly methylated homogalacturonans); and LM21 (heteromannans). Bars represent 100 µm. 7DAF: stage 7 Days After Flowering, 13DAF: stage 13 Days After Flowering, 21DAF: stage 21 Days After Flowering, 30DAF: stage 30 Days After Flowering, 40DAF: stage 40 Days After Flowering, Ep = Epicarp, Pe = pericarp, Ne = Nucellar epidermis, Al = Aleurone, End = Endosperm.</figcaption></figure>
although to a lesser extent, in the endosperm, unlike sorghum (Palmer et al., 2015; Saulnier et al., 2012).

Immunofluorescent labeling using Fer-Ara antibodies has been per-
formed to detect ferulic acid ester-linked to arabinose in AX (Fig. 2). A
labeling was observed in the outer layers at all developmental stages,
and more intensively at the 21 DAF stage. Aleurone and peripheral
endosperm cells were immunolabelled with Fer-Ara from 13 DAF, but
less intensively. The results indicated the presence of feruloylated AXs in
the developing grain, especially in the outer layers, which is in line with
our biochemical analysis (Supplementary Table S1). The same trends
were reported in maize, with feruloylated AXs detection earlier and
higher in the pericarp than in the endosperm and aleurone. They
mentioned a lower intensity of immunolabeling in the endosperm due to
thin cell walls in this grain compartment (Chateigner-Boutin et al.,
2016).

3.3.3. *Mixed-linked* β-glucans

Mixed-linked β-glucans (MLG) are polysaccharides typically found in
cereals. A low amount of MLG was detected in the endosperm of sor-
ghum developing grains (0.1 %-0.2 % of dry matter), whereas up to 4.6
% (±0.2) of dry matter was observed in the outer layers at 7 DAF and up
to 7 % at 40 DAF (Table 1). MLG accumulation ceased around 21 DAF in
the grain outer layers, while it continued until 30 DAF in the endosperm.
In barley endosperm, MLG content increased throughout grain devel-
opment (Wilson et al., 2012). Inversely in wheat endosperm, MLG
content slightly decreases during grain development (Pellny et al.,
2012). The BG3/BG4 ratio, which served as an indicator of MLG struc-
ture and linked to their solubility and extractability properties (Wilson
et al., 2012). While the BG3/BG4 ratio was stable in the outer layers
(around 2), it progressively increased in the endosperm during grain
development (0.7-1.9) (Table 1). These ratios remained lower than
those reported for wheat dry grains (3-4.5) but were closer to those of
barley or oat grains (respectively 2.3 to 3.4 and 1.5 to 2.3). As reported
for barley, BG3/BG4 ratio was higher in the outer layers than in the
endosperm (Wilson et al., 2012). By immunofluorescent labeling with
specific antibodies, MLG were observed in the pericarp, epicarp, and
nucellar epidermis of developing grains as early as stage 7 DAF. In
contrast, MLG were hardly detected in the cell walls of the endosperm,
and not at all in the aleurone cells of developing sorghum grain (Fig. 2).
MLG has previously been investigated in the outer layers of sorghum
grains but only at late stages of development (Ermawar et al., 2015).

3.3.4. Cellulose

The estimated amount of cellulose tended to slightly increased at the last developmental stages in the outer layers of sorghum grains (Table 1B). In the endosperm, a reliable estimation of the cellulose content was not possible due to the high starch content relative to the low proportion of cell wall polysaccharides. Nevertheless, we looked for the presence of cellulose in grain sections by fluorescent labeling with the carbohydrate-binding module CBM3a. Cellulose labeling was more intense in the outer layers (pericarp and nucellar epidermis) from the 7 DAF stage. On the contrary, the cellulose was hardly detected in the cell walls of the endosperm. A slight labeling was visible in the aleurone cell wall from 21 DAF. Our results corroborate those observed in wheat, where cellulose was primarily detected in the outer layers at early developmental stages, as well as in the aleurone cells (Saulnier et al., 2012).

3.3.5. *Xyloglucans*

Immunofluorescent labeling with the LM25 antibody was used to
detected xyloglucans in developing sorghum grains (Fig. 2). The outer
layers showed only slight labeling at 7 DAF, but the fluorescence signal
increased as grain development progressed (Fig. 2). In the endosperm
and aleurone cells, xyloglucan immunolabeling was visible at 21 DAF
and 30 DAF stages. As far as we know, xyloglucans have not been re-
ported to occur in sorghum. It was surprising to observe such a strong

xyloglucan immunolabeling since this polysaccharide is usually poorly
represented in cereal grains (Francin-Allami et al., 2019; Pellny et al.,
2012; Saulnier et al., 2012; Wilson et al., 2012). Fingerprinting analysis
using a specific xyloglucan enzyme confirmed not negligible presence of
xyloglucans in developing sorghum grains (Table 2 A). Oligomers
derived from xyloglucans were detected at all developmental stages,
with a higher release of xylo-oligosaccharides in the outer layers than in
the endosperm. In the endosperm, the estimated proportion of xylo-
glucans was higher at 7 DAF but decreased at later stage. However, the
total xyloglucan content per grain increased significantly with grain
development due to the endosperm growth. In contrast, the proportion
of xyloglucans in the outer layers remained constant throughout the
development. Both in the endosperm and outer layers, the predominant
oligomer detected by fingerprinting is XLXG at all developmental stages,
except in the mature stage of endosperm where it was replaced by the
XLLG which contains an additional galactose residue (Table 2 B).
Interestingly, no galactose residue was detected in the endosperm, even
at the late developmental stage (Table 1), likely due to the overall low
level of cell wall polysaccharides in this tissue. While the XXG<sub>n</sub> type was
predominantly found in Poaceae, XLXG and XLLG oligomers have also
been detected in maize, oats, and barley (Hsieh and Harris, 2009).

3.3.6. Pectins

To monitor pectin distribution during sorghum grain development, LM20 and RU1 antibodies were used to detect highly methylated homogalacturonans (HG) and rhamnogalacturonan-I (RGI), respectively. RGI epitopes were detected in the endosperm and aleurone cell walls at all stages of grain development (Fig. 2). In contrast, only weak immunolabeling was observed in the outer layers. A similar RGI distribution has been reported in wheat, whereas rice RGI was highly detected in the developing pericarp (Chateigner-Boutin et al., 2014; Palmer et al., 2015). Contrary to RGI, highly methylated homogalacturonans were present in the outer layers of sorghum grain at all developmental stages, but they were not detected in the endosperm or aleurone cells (Fig. 2). Similarly, in developing wheat and rice grains, highly methylated homogalacturonans were mainly observed in the outer layers (Palmer et al., 2015). Immunolabeling of pectins throughout development corroborated the detection of galacturonic acids both in the outer layers and endosperms (Table 1).

3.3.7. *Heteromannans*

LM21 antibodies were used to detect heteromannans (Fig. 2). Labeling was mainly observed in the endosperm and aleurone cells of sorghum grain, with a higher fluorescence intensity detected at 30 DAF and 40 DAF stages. These results were unexpected in light of the carbohydrate composition as mannose was detected exclusively in the outer layers (Fig. 2 and Table 1). Mannans has been detected by immunolabeling in the endosperm but not in the aleurone of Brachypodium and wheat grains (Chateigner-Boutin et al., 2014; Francin-Allami et al., 2019; Palmer et al., 2015). Their structure has recently been elucidated in wheat endosperm, but their role in cereal grains remains to be determined (Verhertbruggen et al., 2021).

3.4. *Identification of genes linked to cell wall formation during grain*
*development*

To identify the genes involved in cell wall deposition during sorghum
grain development, we performed a transcriptomic analysis at the three
developmental stages (7, 13, and 30 DAF) where we observed the most
significant variations in cell wall polysaccharide composition and dis-
tribution (Supplementary Table S2). The main steps of our tran-
scriptomic analysis have been previously described in (Costes et al.,
2024). After data filtering and normalization, 20790 transcripts
remained in the analysis. The developmental stages were compared
between them for each tissue, as well as each tissue according to
developmental stage, to identify differentially expressed genes (p-value
<caption>Table 2</caption>

A) Estimated xyloglucan content expressed as a percentage of dry matter (% DM) and by weight per grain(µg/grain) in the outer layers and endosperm cell walls of developing sorghum grains based on HPAEC analysis. B) Proportion of each xyloglucan oligomers (%) in the outer layers and endosperms of developing sorghum. In bold is indicated the proportion of oligomers the most represented. 7DAF: stage 7 Days after Flowering, 13DAF: stage 13 Days after Flowering, 21DAF: stage 21 Days after Flowering, 30DAF: stage 30 Days after Flowering, 40DAF: stage 40 Days after Flowering. Data are the mean ± standard deviation of three biological replicates.

<table><thead><tr><th colspan="2">A</th><th colspan="2">13DAF</th><th colspan="2">21DAF</th><th colspan="2">30DAF</th><th colspan="2">40DAF</th></tr><tr><th>Xyloglucan content</th><th>7DAF</th><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th></tr></thead><tbody><tr><td>Endosperm</td><td>0,11 ± 0,02</td><td>1,06 ± 0,22</td><td>0,02 ± 0,01</td><td>3,10 ± 1,00</td><td>0,04 ± 0,01</td><td>10,12 ± 2,45</td><td>0,06 ± 0,01</td><td>17,94 ± 3,22</td><td>0,06 ± 0,02</td><td>18,54 ± 5,65</td></tr><tr><td>Outer layers</td><td>1,45 ± 0,18</td><td>42,89 ± 5,33</td><td>1,58 ± 0,20</td><td>88,74 ± 11,00</td><td>1,90 ± 0,16</td><td>79,66 ± 6,59</td><td>2,10 ± 0,30</td><td>70,01 ± 10,07</td><td>1,67 ± 0,03</td><td>58,75 ± 1,07</td></tr><tr><th colspan="2">B</th><th colspan="2">13DAF</th><th colspan="2">21DAF</th><th colspan="2">30DAF</th><th colspan="2">40DAF</th></tr><tr><th>Xyloglucan-oligomers (%)</th><th>7DAF</th><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th><th>% DM</th><th>µg/grain</th></tr><tr><td>Endosperm</td><td>8,9</td><td>3,4</td><td>85,5</td><td>2,2</td><td>18,0</td><td>5,2</td><td>75,3</td><td>1,4</td><td>75,9</td><td>24,1</td><td>4,5</td></tr><tr><td>Outer layers</td><td>5,0</td><td>1,7</td><td>92,0</td><td>1,4</td><td>4,8</td><td>1,0</td><td>90,4</td><td>3,8</td><td>88,0</td><td>5,6</td><td>7,4</td><td>1,2</td></tr></tbody></table>

In bold is indicated the proportion of oligomers the most represented. 7DAF: stage 7 Days after Flowering, 13DAF: stage 13 Days after Flowering, 21DAF: stage 21 Days after Flowering, 30DAF: stage 30 Days after Flowering, 40DAF: stage 40 Days after Flowering. Data are the mean ± standard deviation of three biological replicates.

<0.05, Log Fold change >1) (Costes et al., 2024). Next, we performed a gene ontology (GO) analysis to examine the biological processes of genes differentially regulated between stages 7-13 DAF and stage 30 DAF for both tissues (Supplementary Fig. S1).

The two most common metabolisms represented were the carbohydrate metabolic process and the cell wall organization (p values < 0.01 and 200 genes involved in this ontology). Previous studies focusing on cell wall biosynthesis in sorghum stem led to the identification of a list of 655 genes (Hennet et al., 2020), and 384 of them were detected in our samples, putatively involved in the synthesis or remodeling of the grain cell wall (Supplementary Table S2). This set of genes was analyzed in relation to the biochemical and imaging results described above. Among them, 361 transcripts showed significant differential expression at least in one condition when compared to other stages or tissue (Supplementary Fig. S2 and Table S2). In accordance with our biochemical analysis and imaging results, an important proportion of the identified cell wall related genes exhibited significant differential expression in favour of a higher expression at the earlier developmental stages 7 and 13 DAF versus the later stage (30 DAF) whatever the tissue (Supplementary Fig. S2 and Table S2). This is the case for genes potentially involved in the biosynthesis of cell wall polysaccharides such as Cellulose synthases (CesA) and related genes (Cellulose synthase-like, Csl). For the CesA gene family, ten genes were identified in our transcriptomic analysis and the expression level of their transcripts was higher at 7 and 13 DAF compared to 30 DAF (Fig. 3A). In the endosperm, several CesA genes showed higher expression level at 13 DAF than at 7 DAF, which likely explains the delayed immunolabeling detection of cellulose at 21 DAF (Fig. 2).

In addition, we identified two genes potentially involved in MLG biosynthesis and more expressed at early stages: one Cellulose synthase-like F6 (CslF6) annotated gene (Sobic.007G050600) and one Cellulose synthase-like H (CslH) annotated gene (Sobic.010G008600) ((Holmes et al., 2013); Fig. 3C). These monocot-specific class genes exhibited higher expression at 7 DAF supporting an early deposition of MLG in sorghum grain (Fig. 2). Their expression level decreased all along the grain development to reach very low values at the 30 DAF, for both the outer layers and endosperm. In the same way, CslC genes, as well as genes annotated as xyloglucan xylosyl transferase (XXT) and potentially involved in xyloglucan synthesis, showed high transcript abundance at early stages of grain development, supporting the detection of xyloglucan in both endosperm and outer layers (Fig. 2, Table 2, Fig. 3D). The glycosyltransferase genes from the GT families previously described as potentially involved in the synthesis of arabinoxylan in wheat (that is GT43, GT47 and GT61 families (Pellny et al., 2012);) also exhibited a higher expression level at the early 7 and 13 DAF stages than 30 DAF in sorghum grain, except for the arabinosyl transferase (Sobic.003G390700) upregulated at 30 DAF (Supplementary Table S2, Fig. 3B). The Sobic.004G007600 GT61 gene ortholog of XAX1 was annotated as adding xylose to AX but more recent evidence suggested an alternative function of XAX1 in the transfer of hydroxycinnamic acid-modified arabinosyl substitutions to xylan. Additionally, we identified four genes annotated as HXXXD type acyl-transferase that could esterify ferulic acid to arabinoxylans (Chandrakanth et al., 2023) (Supplementary Table S2; Sobic.009G034300, Sobic.003G219700, Sobic.003G043600, Sobic.003G037800). These four genes were upregulated at 7 and 13 DAF stages compared to 30 DAF stage in endosperm and outer layers (Supplementary Table S2). Whereas most Csl genes were upregulated at 7 and 13 DAF, the CsIE class stands out for a higher expression at 30 DAF (Supplementary Table S2). One was particularly highly expressed at the mature stage (Sobic.002G237900). To date, the function of this seed plant-specific class has not yet been defined. Higher expression at the mature stage 30 DAF was also observed for some genes coding for hydrolases such as one from the GH9 family (Sobic.009G129800) or several genes from the GH17 family (Supplementary Table S2). These endoglucosidases could participate in the turnover of hemicelluloses including MLG. Particularly, two
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** A. Expression of Cellulose synthase-like A (CesA) genes potentially involved in the cellulose biosynthesis during grain development. B. Expression of genes potentially involved in the arabinoxylan biosynthesis during grain development. *Xylan Synthase (XS)* in orange; arabinosyl transferase (AT) in blue. C. Expression of genes potentially involved in the mixed-linked β-glucan biosynthesis during grain development. Cellulose synthase-like F6 (CslF6) in orange; Cellulose synthase-like H (CslH) in green. D. Expression of genes putatively involved in the xyloglucan biosynthesis during grain development. Cellulose synthase-like C (CslC) in blue; Xyloglucan XylosylTransferases (XXT) in yellow. **7End:** endosperm at 7 DAF (Days After Flowering); **13End:** endosperm at 13 DAF; **30End:** endosperm at 30 DAF; **7OL:** outer layers at 7 DAF, **13OL:** outer layers at 13 DAF, **30OL:** outer layers at 30 DAF. Data are the mean of the normalization data ±standard deviation of three biological replicates. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
populations of GH17 genes stand out, with one upregulated at 30 DAF and the other upregulated at the earlier stages 7 and 13 DAF (Supplementary Table S2). These GH17 genes likely play different roles throughout grain development.

Overall, more cell wall genes were detected as upregulated in the outer layers versus the endosperm except at the latest stage 30 DAF, including most of the genes involved in the biosynthesis of cellulose and hemicelluloses (Fig. 3; Supplementary Fig. S2 and Table S2). This finding was consistent with immunolabeling and biochemical data highlighting a high proportion of cell walls and sugar contents in outer layers. Some genes were more expressed in the endosperm than in the outer layers (Supplementary Table S2). This was the case for three CslA (Sobic.001G252700; Sobic.002G385800; Sobic.002G139900) and one CslD (Sobic.008G125700). These genes may be involved in hetero- mannan synthesis which primarily occurs in the endosperm (Verherbruggen et al., 2021) (Fig. 2). Two genes annotated as Sucrose synthase (SuSy) and belonging to the GT4 family also exhibited high expression in the endosperm at early developmental stages (Supplementary Table S2). SuSy plays a key role in sugar metabolism by converting sucrose into fructose and UDP-Glc and thus highly control- ling the channeling of sucrose into cell wall polysaccharides and starch (Brandon and Scheller, 2020).

Besides biosynthesis genes, those encoding for remodeling proteins were also more often upregulated in outer layers than endosperm (Supplementary Table S2). For instance, we identified 23 expansin genes, most of which were upregulated in the early stage of development and in the outer layers (Supplementary Table S2). Numerous expansins were previously found more abundant in the outer layers of Brachypo- dium and wheat grain at early developmental stages (Cherkaoui et al., 2019; Francin-Allami et al., 2015). Expansins play key roles in cell wall growth and may affect grain development and size. Hydrolase genes such as those of polygalacturonases (PGs) were also preferentially expressed in the outer layers and at early development. PGs belong to the GH28 family and are pectolytic glycanases that cleave the O-glycosyl bonds of polygalacturonans. The high level of PG transcripts in the outer layers is consistent with the immunolocalization of homogalacturonans in these tissues (Fig. 2). Curiously, in wheat, GH28 members were mainly found in the endosperm of a relatively advanced stage of development (Cherkaoui et al., 2019). Further functional validation would be necessary to substantiate the relationships between the expression of these genes and polysaccharide deposition during

sorghum grain development.

4. Conclusion

This study provided new insights into the dynamics and distribution of cell wall polysaccharides from early development to mature stages of developing sorghum grains, both in the endosperm and outer layers. Notably, we highlighted some interesting features of sorghum compared with other Poaceae. A schematic overview of the results was given in Fig. 4. This work revealed the presence of cell wall polysaccharides not previously observed in developing sorghum, such as xyloglucans, pec- tins, and heteromannans, providing information on their structure and distribution during the grain development. These minor polysaccharides may play important roles in the structure and properties of the cell walls that remain uncovered. The accumulation of cell wall compounds was higher in the outer layers and remained active till around 21 DAF, whereas it was less active in the endosperm but lasted till later stages. The expression of most of the genes involved in the cell wall metabolism pathways was consistent with these variations. Differences in cell wall composition between the outer layers and the endosperm indicated distinct and tissue-specific regulatory processes. Furthermore, tran- scriptomic analyses suggested that the regulation of polysaccharide biosynthesis and remodeling is a complex process involving numerous genes, the precise functions of which have yet to be fully elucidated. These findings opened new perspectives for understanding the cell wall formation in developing sorghum grains, an essential prerequisite for improving grain quality.

CRediT authorship contribution statement

Camille Costes: Writing – original draft, Methodology, Investiga-
tion, Formal analysis. Lucie Le Bot: Investigation, Formal analysis.
Camille Alvarado: Investigation, Formal analysis. Sylviane Daniel:
Investigation, Formal analysis. Sergi Navarro Sanz: Methodology,
Investigation. Caroline Calatayud: Investigation, Formal analysis.
Alexandre Soriano: Resources. Fabienne Guillon: Writing – review &
editing, Investigation. Hamza Mameri: Writing – review & editing,
Validation, Supervision, Funding acquisition, Conceptualization. Nancy
Terrier: Writing – review & editing, Validation, Supervision, Funding
acquisition, Conceptualization. Luc Saulnier: Writing – review & edit-
ing, Validation, Supervision. Mathilde Francin-Allami: Writing –

<figure><img src="image_9.png" /><figcaption>Fig. 4. Schematic representation of the immunolabeled detection of polysaccharides in the different tissues of the Sorghum grain during the development. On the left, is the distribution of polysaccharides into the albumen and aleurone; on the right, the distribution of polysaccharides into the main structures of the outer layers (pericarp, nucellar epidermis, and epicarp). The appreciative abundance of polysaccharides (for a given immunolabeling) is indicated by a thick, a thin or a dotted line.</figcaption></figure>
review & editing, Writing – original draft, Validation, Supervision, Funding acquisition, Conceptualization.

## Funding

This research was financially supported by the TRANSFORM Department of the National Research Institute for Agriculture food and Environment (INRAE) and by Region Pays de la Loire (AIC project "So-WallDigest" and thesis grant).

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

Sorghum plants were cultivated at the CIRAD of Montpellier. Imaging analyses were performed on the Platform BIBS (Biology and Structural Biochemistry). The authors wish to thank Axelle Bouder for her help in manual dehulling of sorghum grains.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcs.2025.104239.

## Data availability

Data will be made available on request.

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