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Article

Comparative Analysis of Components Involved in the Synthesis of Cellulose in Agave Species

by
María José García-Castillo
1,
Yahaira de Jesús Tamayo-Ordóñez
2,
María Concepción Tamayo-Ordóñez
3,
Felipe Barredo-Pool
1,
Luis Carlos Rodríguez-Zapata
1,
Benjamin Abraham Ayíl-Gutiérrez
4,
María Teresa Pulido-Salas
5 and
Lorenzo Felipe Sánchez-Teyer
1,*
1
Biotechnology Unit, Scientific Research of Yucatan, Calle 43 No. 130, Colonia Chuburná de Hidalgo, Mérida 97200, Mexico
2
Environmental Biotechnology Laboratory, Genomic Biotechnology Centre, National Polytechnical Institute, Boulevard del Maestro, s/n, Esquina Elías Piña, Reynosa 88710, Mexico
3
Genetic Engineering Laboratory, Biotechnology Department, Chemical Science Faculty, Coahuila University, Ing. J. Cardenas Valdez, s/n, Saltillo 25280, Mexico
4
Plant Biotechnology Laboratory, Genomic Biotechnology Centre, National Polytechnical Institute (Secretariat of Science, Humanities, Technology and Innovation—SECIHTI), Boulevard del Maestro, s/n, Esq. Elías Piña, Reynosa 88710, Mexico
5
Germoplasm Bank, Scientific Research of Yucatan, Parque Científico y Tecnológico de Yucatán, km 5.5 Carretera Sierra Papacal—Chuburná Puerto, Sierra Papacal, Mérida 97303, Mexico
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(6), 1435; https://doi.org/10.3390/agronomy15061435
Submission received: 25 April 2025 / Revised: 4 June 2025 / Accepted: 9 June 2025 / Published: 12 June 2025
(This article belongs to the Section Crop Breeding and Genetics)
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Versions Notes

Abstract

The process of obtaining Agave L. fibers dates back to pre-Hispanic times, and although humans have obtained different products from this crop, to date, the impact of humans (artificial selection, domestication and intensive cultivation) on these species is unknown. In this study, the expression of the CesA gene was evaluated in three species, namely, Agave L, A. sisalana Perrine and A. fourcroydes Lem. (Sac ki), both of which are used for fiber production, and Agave tequilana Weber. The results revealed that, compared with A. fourcroydes and A. tequilana, A. sisalana had a greater leaf area, a significantly greater cellulose content and a greater number of cellulose fibrils. In terms of cell organization, the number and size of sclerenchyma fibers were similar between A. sisalana and A. fourcroydes. However, the relative expression of the CesA gene was five times greater in A. fourcroydes than in A. sisalana and A. tequilana, in contrast with the number of copies in those genomes. In addition, the tertiary structure of the CESA protein in fiber-producing species was modeled, placing agaves in a group along with Populus, Linum, Corchorus and Boehmeria. The haplotype network analysis revealed that A. tequilana is closely grouped with species of the order Poales, unlike the rest of the fiber-producing agaves, which formed a unique cluster. These findings suggest that artificial selection by humans, for various purposes, has contributed to the specialization of genes associated with traits such as fiber production.

1. Introduction

Cellulose synthase is a membrane protein encoded by genes belonging to the superfamily of CesA that participate in the synthesis of (1,4)-β-glucan chains that polymerize in a microfibril [1,2].
Morgan et al., 2013 [3] revealed the architecture of cellulose synthase, demonstrated how the catalytic subunit A forms a cellulose-conducting channel and suggested a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is spread over a glucose molecule at a time.
In recent years, knowledge of their expression and regulation has gained relevance [4,5,6]. In the genomes of Gossypium hirsutum and Gossypium barbadense L., four isoforms of the CesA gene (GhCesA5–GhCesA10) that are differentially expressed were identified [5]. Similar data related to the differential expression of certain isoforms of CesA have been reported for Eucalyptus grandis L. [7] and for the hybrid aspen (Populus tremula (L.) × P. tremuloides (Michx.)) [8].
Recently, ref. [9] analyzed the CesA superfamily from 198 plant genomes and reported considerable variation in gene copy number across species and gene families, in contrast with the remarkable conservation of gene architecture across species.
In Agave, ref. [10] reported the expression of CesA4, a gene associated with secondary cell wall biosynthesis, which was directly correlated with a high CesA4 expression profile and increased plant height, leaf length, and cellulose content, suggesting a functional relationship between CesA4 activity and biomass accumulation. In contrast, the expression of CesA3 and CesA5, genes typically involved in primary cell wall formation, was not significantly correlated with these phenotypic traits.
Nonetheless, other studies have highlighted the potential of manipulating primary wall-related CesA genes to influence overall cell wall architecture; for example, ref. [11] reported that the overexpression of genes related to primary cell wall biosynthesis in Oryza improved primary wall formation, which facilitated more efficient secondary wall deposition, potentially enabling spatial and temporal control over cellulose microfibril twisting and bending during wall formation in transgenic lines.
Tamayo-Ordoñez et al. [12] modeled the tertiary structure of CESAs in plant species used in the production of fibers and observed two groups with different tertiary structures. Group I included genera such as Macleaya, Populus, Linum, Corchorus, Phycollastachys, Arabidopsis, and Gossypium, and Group II included genera such Solanum, Cynara, Pinus, and Boehmeria. These authors suggested that structural differences in the conformation of the CESA enzyme could result in a positive alteration of its enzymatic activity. Interestingly, the genera Corchorus and Gossypium presented two types of tertiary structures that could be related to isoforms of the CesA gene, and more recently, the same feature was reported in Glycine, Linum and Agave L. [4,5,13,14].
Recently, ref. [4] identified at least 38 CesA sequences from Agave H11648; in Agave sisalana and Agave, fourcroydes identified 14 CesA orthologs; and in Agave tequilana, 6 CesA orthologs were identified [15,16]. These studies were based on transcriptomes; however, more information on the cellulose synthase genes in Agave fiber-producing species still needs to be generated.
Lignocellulosic fibers have attracted considerable interest in recent years as promising alternatives to reduce the use of synthetic fibers because of their environmentally friendly cycle of life and potential for reuse [17,18].
The fiber from Agave L. has been used since pre-Hispanic times for the manufacture of different products, such as clothing, rope, bags, and footwear [19], considering that several species of the Agave genus, such as A. sisalana, A. americana, A. fourcroydes and Agave Hybrid H11648 (A. amaniensis Trel. and Nowell X A. angustifolia Haw.) X A. amaniensis) [4,12,20].
Some genetic research on selection has been conducted in biological models widely used to produce fiber, such as flax [13], cotton [21] and jute [22], which has shown that the quality and yield of fiber can be improved by selection on the basis of knowledge of the genetic and biochemical factors that regulate plant biopolymer production [14].
The aim of this research was to compare interspecific A. tequilana, A. sisalana and A. fourcroydes in terms of cellulose content, cellular organization, copy number and the expression profile of subunit A of the CesA gene, which could provide useful information for understanding the role of these genes to drive genetic improvement to achieve selection of highly fiber-producing individuals in Agave.

2. Materials and Methods

Plant material
The species Agave tequilana, Agave sisalana and Agave fourcroydes were collected from the Germplasm Bank of the Scientific-Technological Park of Yucatán (GB-PCTY), located in Sierra Papacal, 20 km northwest of Merida (21°07′20″ N, 89°43′41″ W). Three individuals per species were included. All the plants selected were approximately 4 years old and cultivated under the same environmental conditions.
Determination of cellulose content
Leaves of the different accessions selected (in triplicate) were dried at 100 °C. The moisture content was determined following the method of ASTM D4442-92, so dry leaves were mashed in a mill, and the powder was sieved in mesh between 40 and 60 mm. The organic solvents and water extracts were determined via the TAPPI methods T-204 cm-07 and T-99-207, respectively. The lignin content was measured according to the Klason method (insoluble lignin T-222 of TAPPI), whereas the cellulose and hemicellulose contents were determined according to the procedure described by Kumar et al. (2009) [23]. The collected data were subjected to analysis of variance (ANOVA) via the SAS statistical software package ver. 9.0 (2000) and Origin 9.1 Software (Data Analysis and Graphing Software). Statistical F tests were evaluated at ≤0.05, and the means were compared via Student’s t tests (p ≤ 0.05).
Isolation of the sequence of partial subunit A of CesA
The sequences were isolated via the GenBank database of transcriptomes reported in the genus Agave, and the partial subunits of CesA in A. tequilana, A. sisalana and A. fourcroydes were identified via primers with the following sequences: FW CAGGCTACTTCCGAAAGAG, REV TGTTATTGCGTAGTGCAA. PCRs were carried out in a volume of 50 μL containing 25 ng of genomic DNA, 130 μM dNTPs, 15 μM of each primer, 2.5 units of Taq polymerase, and 1X PCR buffer (Life Technologies, Rockville, MD, USA) with 1.5 mM MgCl2. The PCR conditions included one cycle of 3 min at 94 °C for initial denaturation, followed by 35 cycles of 1 min at 94 °C, 1 min at alignment temperature (50 °C) according to each set of primers used, 1 min at 72 °C, and, finally, 7 min at 72 °C. The PCR products were separated via electrophoresis in 1.2% agarose gels. The purification, cloning and sequencing of the PCR products (151 bp fragment) were carried out according to the methods of [24].
Analysis of the relationship of cellulose synthase catalytic subunit A in plants
From the GenBank database, representative sequences of the order Poales, Rosales, Curcubitales, Malvales, and Malghigiales plants were obtained. Cellulose synthase A (CesA) clones of different species of Agave were sequenced via the NGS method of the ABI 3730 XL sequencer in Macrogen Korea via the M13 universal primers. The amino acid partial sequences were aligned and compared with the GenBank database using the programs BioEdit Sequence Alignment Editor 5.0.6 [25] and CLUSTAL W software 1.82 [26]. Phylogeny was reconstructed by maximum likelihood, and cluster confidence was tested by 1000 bootstrap iterations. Phylogenetic analysis was performed via MEGA version 6.0 software [27]. Domains of interest in the subunit of the CESA protein were identified with CDD [28]. The significant mutational changes in the amino acid sequences of CesA were evaluated by constructing a minimum distance network of mutations via software from NETWORK v.4.6 (http://www.fluxus-engineering.com, accessed on 25 October 2024) [29] and the median joining method, which assumes an epsilon of 0 and a ratio of transversion/transition of 1:2.
Tertiary structure analysis of the CESA was performed with SWISS-MODEL [30]. To model the tertiary structure of Agave L., a dimer was generated between the control sequence of Rhodobacter sphaeroides and the consensus sequence of Agave L. (33 aa region). The values of sequence identity (%), global model quality estimation and qualitative model energy analysis were considered. As references, sequences corresponding to Rhodobacter sphaeroides were included [3].
Determination of the copy number of the CesA gene
The region of the catalytic subunit A of CesA was amplified and used for the calibration curve according to [31]. The standard gene curve was built from the clones of the CesA gene in a serial dilution series (100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001 and 0.0000001 ng/mL). By sequencing, it was verified that each clone contained only one copy of the gene. Each concentration of plasmid was converted to a copy number according to the formula proposed by Whelan et al. (2003) [32] and Lu et al. (2012) [7]: DNA (copy) = 6.02 × 1023 (copies mol−1) × DNA amount (g)/DNA length(bp) × 660 (g mol−1 bp−1). CesA amplification was conducted via primers with the following sequences: FW CAGGCTACTTCCGAAAGAG and REV TGTTATTGCGTAGTGCAA. PCRs were carried out in a volume of 50 μL containing 25 ng of genomic DNA, 130 μM dNTPs, 15 μM of each primer, 2.5 units of Taq polymerase, and 1X PCR buffer (Life Technologies, Rockville, MD, USA) with 1.5 mM MgCl2. The PCR conditions included one cycle of 3 min at 94 °C for initial denaturation, followed by 35 cycles of 1 min at 94 °C, 1 min at alignment temperature (50 °C) according to each set of primers used, 1 min at 72 °C, and, finally, 7 min at 72 °C. The PCR products were separated via electrophoresis in 1.2% agarose gels. The purification, cloning and sequencing of the PCR products were carried out according to [33]. The number of PCR cycles for the fluorescence signal to reach a value above that of the background fluorescence (CT cycle threshold value) was determined according to the fluorescence threshold intensity by using StepOne Software v2.0 (Applied Biosystems, Waltham, MA, USA). The Ct values were plotted against the logarithm of their initial template copy concentrations. The standard curve was generated via linear regression of the plotted points. Following [34], a regression analysis was run for the standard curve. The regression procedure in SAS was first executed to model the Ct value against the log-transformed input DNA concentration. For data quality control purposes, the test statement establishing the Ct equivalence to −1 helps to estimate if the slope of the simple linear regression is −1 and indicates an amplification efficiency of 100%. Additionally, the PCR amplification efficiency (E) was calculated from the slope of each curve via the formula E = 10−1/slope − 1.
Relative quantification of the CesA gene
RNA isolation and cDNA synthesis were conducted according to previous methods [35]. RNA was obtained from a 200 mg sample of basal leaf from each analyzed Agave species via the TRIZOL method (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. RNA integrity and quantification were verified in 1.3% agarose gels after electrophoresis and by spectrometry via the A260/A280 and A260/A230 ratios in NANODROP (NANODROP-1000, Thermo Scientific, Waltham, MA, USA). One microgram of total RNA was digested with 1 U of RQ1 RNase-Free DNase I (Promega, Wilmington, NC, USA) and DNase 1X reaction buffer in a final volume of 10 µL. The samples were incubated at 37 °C for 30 min. Subsequently, DNase I was inactivated by adding 1 µL of RQ1 DNase Stop Solution, and the mixture was incubated at 65 °C for 10 min. The absence of DNA contamination was verified by agarose gel electrophoresis (1.3%) and negative PCR, with 50 ng/µL of RNA treated with DNase I used as a template. For PCR, a sample of genomic DNA was included as a positive control.
cDNA was synthesized via the GoScript Reverse Transcription System (Promega, Wilmington, NC, USA) following the manufacturer’s instructions. A positive control (1.2 kb kanamycin positive control RNA) provided in the kit was used to estimate the yield of cDNA synthesis. The concentrations of cDNA were verified by NANODROP measurements. The cDNAs obtained were stored at −60 °C until use.
With the aim of discarding fluorescence originating from nonspecific PCR products and primer concatenation, a melt curve analysis was performed. To eliminate DNA contamination, negative controls for the gene were always included in the experiments. The SYBR green signal was standardized to a passive reference dye (ROX) included in the SYBR Green PCR Master Mix. For the analysis of the expression of the CesA gene, we used the same conditions described above.
The relative expression of a gene was determined by the ∆∆Cq method between the target and reference (18S rDNA) genes by the following equation: Relative expression = (Eref)Ctref/(Etarget)Ctarget [36]. The 18S rDNA gene was selected as a reference because, in Agave plants, it has a conserved sequence, and in previous qPCR analyses, it has proven to be a reliable reference, as it is ubiquitously expressed across different field conditions [35].
Analysis of agave fibers by scanning electron microscopy
Microscopical studies of A. tequilana, A. sisalana and A. fourcroydes fibers were performed via SEM (scanning electron microscopy). The fibers were obtained manually by tearing or separating the fibers from the leaves. The manual process was performed with traditional instruments, such as a knife without an edge, bench or wooden mallet [37].
The samples were then mounted on metallic stubs with carbon conductive adhesive tape (Electron Microscopy Science) and sputter coated with a 150 Å gold layer (Denton Vaccum Desk II, Willingboro, NJ, USA). The thickness of the cellulose fibrils, the number of cellulose microfibrils that make up each fibril and the opening area of the cellulose microfibrils were analyzed via AutoCAD 2017 software. Measurements were made in ten fields per sample for each species. Sample analysis and image recording were performed via a scanning electron microscope (JEOL, JSM-6360LV, Singapore, Singapore). The gathered data were subjected to statistical analysis via Student’s t test at p ≤ 0.05.
Histological analysis of agave fibers
Leaf segments were fixed in FAA solution for 4 days in the absence of light. The samples were dehydrated with ethanol at different concentrations (30%, 50%, 70%, 85% and 96%) and embedded in a Glycol methacrylate plastic resin (JB-4 Embedding Solution Polysciences. Los Angeles, CA, USA), the tissues were sectioned at a thickness of 2–4 µm in a rotational microtome (HM 325 Microm, Jinhua, China) and stained with toluidine blue (0.05%, 0.2 M, in sodium acetate) to visualize the cellular organization, as described by [38], and were analyzed via an Axio Scope A1 Epifluorescence light microscope (Zeiss, Oberkochen, Germany) with an attached electronic CSsc1 camera. The images were photographed at 10× and 40× under a bright field.
Statistical analysis
All trials were conducted in triplicate. Statistical analyses were performed via analysis of variance (ANOVA) via the SAS statistical package. 9.0 (2000) and the Origin 9.1 program. The means were compared using Student’s t test (p ≤ 0.05).

3. Results

3.1. Cellulose Content

To characterize the interspecific variability in fiber-producing species, leaves from A. tequilana, A. sisalana and A. fourcroydes were collected and compared, resulting in 2.5 and 1.8 times greater foliar areas than those of A. sisalana with A. tequilana and A. sisalana with A. fourcroydes, whereas the cellulose content of A. sisalana revealed the highest content of fiber (62%), followed by A. fourcroydes, with 53%, and the lowest cellulose content was observed in A. tequilana, with 38%. All differences were significant (Student test; p ≤ 0.05) (Table 1).

3.2. Absolute and Relative Quantification of the CesA Gene

The amplification profile of a fragment of subunit A of the CesA gene among species was obtained via specific primers for Agave, which amplified a 151 bp fragment, and the copy number was calculated. The copy number of the CesA gene (Figure 1A) was two times greater in A. tequilana (Ct 11.42) than in A. sisalana (Ct 16.56) and A. fourcroydes (Ct 15.24), and the copy number among A. sisalana and A. fourcroydes was 0.8 times lower in A. sisalana.
In contrast, the results of the relative expression analysis (Figure 1B) using the 18’S gene as a reference indicated that A. fourcroydes presented greater relative expression of CesA, with fivefold greater expression than did A. sisalana and A. tequilana. All species presented significant differences.

3.3. Fiber Structure and Organization

SEM analysis of the fibers of A. tequilana, A. sisalana and A. fourcroydes revealed that the A. sisalana fibrils were relatively thick (252 µm ± 4), whereas 128 µm ± 3 A. fourcroydes and 129 µm ± 5 A. tequilana fibrils were present (Figure 2A,D,G).
Compared with A. fourcroydes (21 ± 3) and A. sisalana (23 ± 2), A. tequilana presented more cellulose microfibrils (28 ± 2) (Figure 2B,E,H). The opening area of the microfibrils (23 to 26 µm) did not differ among the three Agave species analyzed (Figure 2C,F,I).
A histological analysis was performed to observe the structure and organization of the cells that make up the fibers. The analysis of the cellular components associated with the Agave species is shown in Figure 3. In longitudinal sections, 100 µm long A. sisalana (Figure 3A), A. fourcroydes (Figure 3D) and A. tequilana (Figure 3G) exhibited differences in parenchyma cells; in the case of A. sisalana, the parenchyma cells were larger than those of A. fourcroydes and A. tequilana.
The vascular bundles in A. sisalana, A. fourcroydes and A. tequilana were completely differentiated and defined. On the basis of the longitudinal sections at 20 µm (Figure 3B,E,H), the cellular organization of spiral-type tracheids, parenchymal cells and sclerenchyma fibers revealed that A. sisalana and A. fourcroydes were similar in terms of the thickness of each cell. Transversal sections at 20 µm revealed that A. sisalana (Figure 3C) and A. fourcroydes (Figure 3F) were similar in size in the vascular bundles and that the sclerenchyma fibers and the phloem maintained a similar organization. In A. tequilana (Figure 3I), the sclerenchyma fibers are smaller and present in a minor number.

3.4. Analysis of Subunit A of Protein CESA in Agave

To identify the structural features of cellulose synthase, we modeled the tertiary structure consensus sequence of CESA in Agave and compared it with the tertiary structure from genes of several species used to obtain fibers (Figure 4). Our analysis revealed that the protein accessions annotated via CESA had a sequence identity greater than 21% with the CESA subunit A of Rhodobacter sphaeroides, and two groups with different tertiary structures were defined (designated Groups I and II): Group I included Agave, Populus tremuloides, Populus tomentosa, Linum usitatissimun, Corchorus capsularis, Phycollastachys edulis, and Gossypium hirsitum, and Group II included Corchorus olitorius, Gossypium herbaceum and Boehmeria nívea (Figure 4).
Corchorus and Gossypium are present in both groups and present two types of tertiary structures that could be related to isoforms of the CesA genes described in these genera and were recently described in soybean and Agave. Analysis of amino acid sequence changes via CESA revealed that the sequence of Agave is similar to that of Populus (Table S1).
The haplotype network (Figure 5) was modeled to identify microevolutionary changes between Agave L. and other species of Asparagales, Malphigiales, Poales, Malvales, Curcubitales and Rosales. In general, the construction of the minimum haplotype network of the partial region (alpha helix CESA) of Agave species and members of the order of plants Poales, Rosales, Curcubitales, Malvales, and Malghigiales was supported by 146 total mutations (52 discrete and 94 divergent) and the formation of 29 haplotypes.
Analysis of the evolutionary relationship of alpha-helix CESA (33 aa) (Figure 6) revealed conservation of the amino acid residues between the species of Agave (A. fourcroydes, A. sisalana and A. hybrid H11648), except for A. tequilana. The results revealed the formation of 3 haplotype groups, among which were grouped: A. sisalana and A. fourcroydes ‘Xix ki’, followed by A. hybrid AH11648, A. fourcroydes ‘Kitam ki’, A. fourcroydes ‘Sac ki’ and A. fourcroydes ‘Yaax ki’. Distinguishing an intraspecific relationship between A. hybrid H11648, A. fourcroydes ‘Kitam ki’, A. fourcroydes ‘Sac ki’ and A. fourcroydes ‘Yaax ki’. Interestingly, the formation of an exclusive group between A. tequilana ‘Azul’ and species of the order Poales was observed as a result of certain mutations shared between the accessions Bambusa emeiensis (AFG25777.1) and Bambusa oldhamii (AAY43222.1, AAY43220.1).
This analysis allowed the discrimination of 7 haplotypes of the order Rosales (Fragaria vesca XP004296326.1, Cicer arietinum XP004498142.1, Morus notabilis EXB363045.1), of which the accessions of Prunus (XP003635328.1, XP008218356.1, ONH99326.1) are close (2-- to 3-point amino acid changes), and 2 haplotypes of the cubital order were derived (Cucumis melo XP010644596.1 [Cucumis sativus KGN44038.1], Momordica charantia XP022139561.1].
Interestingly, this analysis revealed slow genetic dynamism in Agave but high genetic specialization in the region of the alpha helix CESA. Thus, the Agave species have an evolutionary path shared with the accessions Populus tremuloides (AAL23710.2), Populus trichocarpa (XP024443523.1), Populus trementosa (AKE81080.1), Jatropha curcas (XP020539381.1), Gossypium herbaceum (ADZ16120. 1), Gossypium hirsutum (NP001314638.1), Phyllostachys edulis (ACT16001.1), Boehmeria nivea (AGC97433.2) and the specific haplotype Linum usitatissimum (AHL45029.1). This evolutionary pattern is supported by the amino acid similarity analysis of CESA data from different orders of plants (Asparagales, Poales, Rosales, Curcubitales, Malvales and Malghigiales) (Figure 6).
Analysis of amino acid sequence change in CESA revealed that a subgroup was obtained, including accessions of the genera Agave, Linum, Gossypium, Boehmeria, and Populus, which are used for textile production.

4. Discussion

Globally, interest in and demand for natural fibers and their components have increased because of the benefits they offer to the environment, notably, the high cellulose/lignin ratio of different applications for biofuels and bioenergy owing to their high yield of biomass [39]. Researchers are interested in the potential for the exploitation of natural fibers [40,41]. Different percentages of cellulose have been reported in species such as Acer monspessulanum (12%), Pinus pinaster (27%), Miscanthus lutarioriparius (42%), Corchurus capsularis (63%), Linum usitatissimum (70%), Boehmeria nívea (71%) and Gossypium hirsutum (93%) [42,43,44,45,46,47,48].
The Agave species most commonly used for fiber production include hybrid 11648, A. americana Lem, A. fourcroydes Lem, A. lechuguilla Torr and A. sisalana Perrine, among others [4,37,49,50]. The fiber yields obtained from each Agave species vary with the method used for extraction [49,50,51].
The percentage of cellulose in several Agave species can be between 43% and 80% [41,52,53,54,55]. The highest percentage of cellulose content was observed in A. sisalana.
The results obtained in this work indicated that A. sisalana, A. fourcroydes and A. tequilana presented significant differences in their cellulose contents. Beside A. sisalana has a longer leaf length, wider leaf area, and larger leaf area than A. fourcroydes. Compared with those of A. sisalana, the cellulose content significantly differs, as the thickness of the leaf and leaf area are distinctive characteristics for obtaining outstanding fiber-producing plants. In other Agave species (A. salmiana, A. Lechuguilla, A. Americana, A. mapisaga, A. angustifolia Haw., A. tequilana), the cellulose content is related to greater leaf width and greater leaf area and fiber content, as described by [56].
A. fourcroydes and A. tequilana have been domesticated for many years, and the interest in comparing these species led us to carry out expression analyses to determine whether there was any trend that would indicate a relationship in the cellulose content.

4.1. Expression of the CesA Gene in Agave and Its Relationship with Quantification

In recent years, with the rapid development of genetic engineering and bioinformatics, new CesA genes that may participate in cell wall biosynthesis have been discovered in Miscanthus × giganteus (MgCesA) [57]. The CesA1, CesA3 and CesA6 genes of sorghum are involved in the formation of primary cell walls and play the same role as those of Arabidopsis. The CesA4, CesA7 and CesA9 genes are highly homologous to the formation of secondary cell walls in rice and the CesA4, CesA7 and CesA8 genes in Arabidopsis [58]. The MlCesA7 gene may be involved in the formation of the primary cell wall in Miscanthus lutarioriparius, whereas the MlCesA4 and MlCesA9 genes may be involved in the formation of the secondary cell wall [59].
The relationship observed between gene expression levels and gene copy number in fiber-producing species in Agave appears to be enhanced in those selected specifically for fiber production. In contrast, A. tequilana, which is cultivated for other purposes, exhibited a distinct cellular organization characterized by a greater number of smaller cells, fewer microfibrils, and shorter fibers. These findings suggest coordinated regulation between primary and secondary cell wall biosynthesis. This hypothesis is supported by previous studies in Oryza and Arabidopsis, where the overexpression of genes involved in primary cell wall formation not only improved the structure of the primary wall but also facilitated more efficient deposition of the secondary wall [11,60].
In Agave species, genes involved in cellulose biosynthesis have been reported; in A. tequilana, six CesA genes have been elucidated; in A. hybrid, 11,648 five genes have been elucidated; and in A, sisalana and A. fourcroydes, 14 CesA genes have been identified [4,15,16].
In our previous work, García-Castillo et al. (2022) [10] reported a direct correlation between a high CesA4 expression profile and increased plant height, leaf length, and cellulose content, suggesting a functional relationship between CesA4 activity and biomass accumulation; in contrast, the expression of CesA3 and CesA5, genes typically involved in primary cell wall formation, was not significantly correlated with these phenotypic traits.
The analysis of the absolute quantification of the number of copies of the CesA gene (Figure 1) revealed a relationship that could be associated with the genetic background and differences in the genome size of each accession analyzed. Polyploidy is a little explored fundamental biological phenomenon in Agave L. The maintenance of multiple copies in each species is an effective mechanism to resist the risk of mutation. In addition, the homologous CesAs between the subgenomes of polyploid plants may have produced functional differentiation, which is related to domestication [61].
The increase in the number of genomes of these species could cause a deregulation of genes as a result of genetic changes that originate from polyploidy [62,63].
Compared with the other species analyzed, A. tequilana presented a greater CesA gene copy number, which contrasts with their expression levels. This discrepancy can be explained by the findings of [9], who analyzed the CesA superfamily from 242 genomes, obtaining considerable variation in gene copy number across the species and gene families, and reported two main patterns in chromosomal organization: singletons and tandem arrays.
The results of the relative quantification analysis indicated that the A. fourcroydes species presented 5-fold greater CesA expression. On the other hand, A. sisalana and A. tequilana are very similar, possibly because A. sisalana and A. tequilana are diploid species.
Synteny patterns were associated with the subfunctionalization of genes into primary and secondary CesA genes [9], similar to our observations in A. sisalana and A. fourcroydes, which are species selected for fiber production. In contrast, A. tequilana, which has been selected for sugar accumulation, reflects a divergent evolutionary path driven by anthropocentric selection pressures.

4.2. Impact, Use of Cellulose Fibrils and Histological Analysis of Agave spp.

Understanding the formation and organization of microfibrils in the cell wall is essential for studying fundamental biological processes, such as plant growth and morphogenesis [64].
In terms of the number of cellulose microfibrils, A. tequilana presented more cellulose microfibrils than did A. sisalana and A. fourcroydes; however, the opening area did not differ among the three Agave accessions analyzed.
Different models have been described for the structure of the organization of cellulose microfibrils in other species, such as spruce, celery, Mung vea, A. thaliana, Brachupodium and Cladophora [65,66,67,68]. Consequently, while an average structure can be represented and the number of cellulose chains may not vary, it is not clear to what extent there may be variation between the structures of individual microfibrils or even different parts of them [69].
Cellulose microfibrils and their orientation are the major determinants of cell morphogenesis [70,71]. The cylindrical shape of many elongated plant cells is likely achieved through the deposition of transversely oriented cellulose microfibrils [72]. This notion was validated by observing the formation of spiral xylem vessel walls, in which organized CSC trajectories have been observed [73].
Our analysis of the cellular components in the Agave species revealed differences in the case of A. sisalana, as the parenchyma cells were larger than those in the varieties A. fourcroydes Sac ki and A. tequilana, which could be especially relevant in expanding tissues, such as young leaves, where larger parenchyma cells could provide greater capacity for expansion, storage and growth [47]. The vascular bundles in A. sisalana, A. fourcroydes Sac ki and A. tequilana are completely differentiated and defined as reported by [74]. However, a decrease in the vascular bundle was observed mainly in A. tequilana, which represented the majority of the varieties used for fiber (A. sisalana and A. fourcroydes). These data agree with the SEM analyses of cellulose fibrils in A. tequilana, which are likely to have a reduced transport capacity and can negatively affect the growth and development of the plant.
In A. sisalana and A. fourcroydes, wider sieve tubes, which have greater amounts of sclerenchymal fibers and parenchyma, are found in the phloem. These findings suggest subfunctionalization of the phloem in these plants, specializing in conduction and storage [75].
In general, the larger size of parenchyma and sclerenchyma cells in plants can contribute to their structure, function and adaptability and advantages in terms of support, resistance, storage and transport of substances.

4.3. Genetic Specialization of CesA in the Agave Crop

The study of the mechanism regulating the synthesis and modification of the plant cell wall has been one of the most important fields of plant development biology [76].
In Arabidopsis, a loss of function of AtCesA3 and AtCesA6 led to a decrease in cellulose [77,78,79]. The functional loss mutants of AtCesA4, AtCesA7 and AtCesA8 presented a reduced cellulose content in the secondary cell wall, which was usually accompanied by changes in the structure of the xylem. Further analysis revealed that AtCesA4, AtCesA7 and AtCesA8 are coexpressed and interact together [80,81,82].
On the other hand, owing to the selection of specific characteristics in Agave L. crops and in exploited crops for fiber production, certain genes related to cell wall biosynthesis may have undergone genetic changes that could affect (positive or negative) enzyme activity. The objective is to determine whether amino acid and genetic changes are related to the artificial selection that humans have imposed since prehispanic times on cultivars of Agave L. [19,83,84,85]. We modeled the tertiary structure of CESA in the consensus sequence of Agave and compared it with that of accessions of plants used to obtain fibers. Corchorus and Gossypium are present in both groups and present two types of tertiary structures that could be related to isoforms of the CesA genes described in these genera and were recently described in soybean and Agave L. The functionality of each protein could contribute significantly to variations in cellulose production, which might be related to plant fiber quality and biopolymer yield [12].
These previous results suggest that evolutionarily, it is possible that the cultivars used for fiber production present genetic differences that make them ideal for this purpose.
These findings indicate that the artificial selection that man has exercised on these cultivars for their different uses has contributed to the specialization of genes related to a certain characteristic. In Agave, the presence of 3 groups of haplotypes could reflect the differences in the use that man has exercised on these evaluated accessions. While A. tequilana has been used to obtain sugars, its haplotype has been shown to be related to that of Poales, and the other varieties analyzed have been used to obtain fiber, presenting differential haplotypes. In a genomic context, a shift in a group of cotton secondary CesA genes was associated with peculiar properties of cotton fiber synthesis [9].

5. Conclusions

The artificial selection that man has exercised on these species (A. sisalana and A. fourcroydes) has contributed to the specialization of genes related to a certain characteristic, fibers. In this research, according to methodologies evaluated in fibers, the species A. sisalana and A. fourcroydes are promising for the extraction of cellulose fibrils; currently, the species used for this industry is A. sisalana; however, genetic studies on CesA A. fourcroydes are promising for the production of fibers, and even more studies are lacking, such as studies evaluating the functionality of each CESA protein, which could contribute significantly to variations in cellulose production. We also consider that genetic improvement in these species could allow the selection of highly fiber-producing Agave individuals.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy15061435/s1, Table S1: Amino acid substitutions detected in 33 aa of cellulose synthase subunit A in plants; Figure S1: Modeling of the tertiary structure of cellulose synthase in different species of fiber-producing plants.

Author Contributions

Conceptualization, M.J.G.-C. and L.F.S.-T.; methodology, M.J.G.-C., F.B.-P., B.A.A.-G., M.C.T.-O. and Y.d.J.T.-O.; formal analysis, M.J.G.-C., L.F.S.-T., Y.d.J.T.-O., L.C.R.-Z. and B.A.A.-G.; visualization, B.A.A.-G.; resources, L.F.S.-T.; data curation, M.T.P.-S., Y.d.J.T.-O., F.B.-P. and B.A.A.-G.; investigation, Y.d.J.T.-O., M.C.T.-O. and M.T.P.-S.; supervision, Y.d.J.T.-O., M.C.T.-O., F.B.-P., L.C.R.-Z. and L.F.S.-T.; project administration, M.C.T.-O., F.B.-P. and M.T.P.-S.; validation, M.C.T.-O., F.B.-P. and L.C.R.-Z.; writing—original draft preparation, M.J.G.-C. and L.F.S.-T.; writing—review and editing, L.C.R.-Z. and M.C.T.-O.; funding acquisition, L.F.S.-T. All authors have read and agreed to the published version of the manuscript.

Funding

The authors are grateful for the support of CONACyT for the doctoral fellowship for MJGC (591024).

Data Availability Statement

Data reported in this study is available in the article and Supplementary Materials.

Acknowledgments

The authors express their gratitude to Miriam Monforte González MC for her technical assistance and fellowship 591024 for M.J.G.C. for PhD and degree support.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CESACellulose synthase
TAPPITechnical association of pulp and paper industries
cDNAComplementary Deoxyribonucleic acid

References

  1. Richmond, T.A.; Somerville, C.R. The cellulose synthase superfamily. Plant Physiol. 2000, 124, 495–498. [Google Scholar] [CrossRef] [PubMed]
  2. Ruan, Y.L.; Llewellyn, D.J.; Furbank, R.T. Suppression of sucrose synthase gene expression represses cotton fiber cell initiation, elongation, and seed development. Plant Cell 2003, 15, 952–964. [Google Scholar] [CrossRef] [PubMed]
  3. Morgan, J.L.W.; Strumillo, J.; Zimmer, J. Crystallographic snapshot of cellulose synthesis and membrane translocation. Nature 2013, 493, 181–186. [Google Scholar] [CrossRef] [PubMed]
  4. Huang, X.; Xiao, M.; Xi, J.G.; He, C.P.; Zheng, J.L.; Chen, H.L.; Gao, J.M.; Zhang, S.Q.; Wu, W.H.; Liang, Y.Q.; et al. De Novo Transcriptome Assembly of Agave H11648 by Illumina Sequencing and Identification of Cellulose Synthase Genes in Agave Species. Genes 2019, 10, 103. [Google Scholar] [CrossRef]
  5. Li, A.; Xia, T.; Xu, W.; Chen, T.T.; Li, X.L.; Fan, J.; Wang, R.Y.; Feng, S.Q.; Wang, Y.T.; Wang, B.R.; et al. An integrative analysis of four CESA isoforms specific for fiber cellulose production between Gossypium hirsutum and Gossypium barbadense. Planta 2013, 237, 1585–1597. [Google Scholar] [CrossRef] [PubMed]
  6. Liu, X.; Zhao, B.; Zheng, H.J.; Hu, Y.; Lu, G.; Yang, C.Q.; Chen, J.D.; Chen, J.J.; Chen, D.Y.; Zhang, L.; et al. Gossypium barbadense genome sequence provides insight into the evolution of extra-long staple fiber and specialized metabolites. Sci. Rep. 2015, 5, 14139. [Google Scholar] [CrossRef]
  7. Lu, S.F.; Li, L.G.; Yi, X.P.; Joshi, C.P.; Chiang, V.L. Differential expression of three eucalyptus secondary cell wall-related cellulose synthase genes in response to tension stress. J. Exp. Bot. 2008, 59, 681–695. [Google Scholar] [CrossRef]
  8. Djerbi, S.; Aspeborg, H.; Nilsson, P.; Sundberg, B.; Mellerowicz, E.; Blomqvist, K.; Teeri, T.T. Identification and expression analysis of genes encoding putative cellulose synthases (CesA) in the hybrid aspen, Populus tremula (L.) × P. tremuloides (Michx.). Cellulose 2004, 11, 301–312. [Google Scholar] [CrossRef]
  9. Pancaldi, F.; van Loo, E.N.; Schranz, M.E.; Trindade, L.M. Genomic Architecture and Evolution of the Cellulose synthase Gene Superfamily as Revealed by Phylogenomic Analysis. Front. Plant Sci. 2022, 13, 870818. [Google Scholar] [CrossRef]
  10. García-Castillo, M.J.; Rodríguez-Zapata, L.C.; Teyer, L.F.S. Differential expression of CesA genes and the relationship with fiber content in henequen. Trop. Subtrop. Agroecosyst. 2022, 25, #129. [Google Scholar] [CrossRef]
  11. Ai, Y.; Wang, H.; Liu, P.; Yu, H.; Sun, M.; Zhang, R.; Tang, J.; Wang, Y.; Feng, S.; Peng, L. Insights into contrastive cellulose nanofibrils assembly and nanocrystals catalysis from dual regulations of plant cell walls. Sci. Bull. 2024, 69, 3815–3819. [Google Scholar] [CrossRef] [PubMed]
  12. Tamayo-Ordonez, M.C.; Ayil-Gutierrez, B.A.; Tamayo-Ordonez, Y.J.; Rodriguez-Zapata, L.C.; Monforte-Gonzalez, M.; De la Cruz-Arguijo, E.A.; Garcia-Castillo, M.J.; Sanchez-Teyer, L.F. Review and in silico analysis of fermentation, bioenergy, fiber, and biopolymer genes of biotechnological interest in Agave L. for genetic improvement and biocatalysis. Biotechnol. Prog. 2018, 34, 1314–1334. [Google Scholar] [CrossRef] [PubMed]
  13. Galinousky, D.V.; Anisimova, N.V.; Raiski, A.P.; Leontiev, V.N.; Titok, V.V.; Khotyleva, L.V. Cellulose synthase genes that control the fiber formation of flax (Linum usitatissimum L.). Russ. J. Genet. 2014, 50, 20–27. [Google Scholar] [CrossRef]
  14. Gou, J.Y.; Wang, L.J.; Chen, S.P.; Hu, W.L.; Chen, X.Y. Gene expression and metabolite profiles of cotton fiber during cell elongation and secondary cell wall synthesis. Cell Res. 2007, 17, 422–434. [Google Scholar] [CrossRef]
  15. Maceda, A.; Soto-Hernandez, M.; Terrazas, T. Chemical-Anatomical Characterization of Stems of Asparagaceae Species with Potential Use for Lignocellulosic Fibers and Biofuels. Forests 2022, 13, 1853. [Google Scholar] [CrossRef]
  16. Raya, F.T.; Marone, M.P.; Carvalho, L.M.; Rabelo, S.C.; de Paula, M.S.; Campanari, M.F.Z.; Freschi, L.; Mayer, J.L.S.; Silva, O.R.R.F.; Mieczkowski, P.; et al. Extreme physiology: Biomass and transcriptional profiling of three abandoned Agave cultivars. Ind. Crop Prod. 2021, 172, 114043. [Google Scholar] [CrossRef]
  17. Francucci, G.; Vazquez, A.; Rodriguez, E.S. Key differences on the compaction response of natural and glass fiber preforms in liquid composite molding. Text. Res. J. 2012, 82, 1774–1785. [Google Scholar] [CrossRef]
  18. Munasinghe, H.T.; Winterstein-Beckmann, A.; Schiele, C.; Manzani, D.; Wondraczek, L.; Afshar, V.S.; Monro, T.M.; Ebendorff-Heidepriem, H. Lead-germanate glasses and fibers: A practical alternative to tellurite for nonlinear fiber applications. Opt. Mater. Express 2013, 3, 1488–1503. [Google Scholar] [CrossRef]
  19. Zizumbo-Villarreal, D.; Vargas-Ponce, O.; Rosales-Adame, J.J.; Colunga-GarciaMarin, P. Sustainability of the traditional management of Agave genetic resources in the elaboration of mezcal and tequila spirits in western Mexico. Genet. Resour. Crop Evol. 2013, 60, 33–47. [Google Scholar] [CrossRef]
  20. Megiatto, J.D.; Silva, C.G.; Rosa, D.S.; Frollini, E. Sisal chemically modified with lignins: Correlation between fibers and phenolic composites properties. Polym. Degrad. Stabil. 2008, 93, 1109–1121. [Google Scholar] [CrossRef]
  21. Abdurakhmonov, I.Y.; Buriev, Z.T.; Saha, S.; Jenkins, J.N.; Abdukarimov, A.; Pepper, A.E. Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L. Nat. Commun. 2014, 5, 3062. [Google Scholar] [CrossRef]
  22. Samanta, P.; Sadhukhan, S.; Basu, A. Identification of differentially expressed transcripts associated with bast fibre development in Corchorus capsularis by suppression subtractive hybridization. Planta 2015, 241, 371–385. [Google Scholar] [CrossRef] [PubMed]
  23. Kumar, A.; Singh, L.K.; Ghosh, S. Bioconversion of lignocellulosic fraction of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to ethanol by Pichia stipitis. Bioresour. Technol. 2009, 100, 3293–3297. [Google Scholar] [CrossRef]
  24. Ta Espinosa-Barrera, L.; Sánchez-Teyer, L.; Quiroz-Moreno, A.; Narváez-Zapata, J. Identification and characterization of a new satellite-like DNA family in three Agave species. Plant Gene 2018, 16, 8–18. [Google Scholar] [CrossRef]
  25. Hall, T.A. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 1999, 41, 95–98. [Google Scholar]
  26. Thompson, J.D.; Higgins, D.G.; Gibson, T.J. Clustal-W—Improving the Sensitivity of Progressive Multiple Sequence Alignment through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice. Nucleic Acids Res. 1994, 22, 4673–4680. [Google Scholar] [CrossRef]
  27. Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [Google Scholar] [CrossRef]
  28. Derbyshire, M.K.; Gonzales, N.R.; Lu, S.; He, J.; Marchler, G.H.; Wang, Z.; Marchler-Bauer, A. Improving the consistency of domain annotation within the Conserved Domain Database. Database 2015, 2015, bav012. [Google Scholar] [CrossRef] [PubMed]
  29. Bandelt, H.J.; Forster, P.; Rohl, A. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 1999, 16, 37–48. [Google Scholar] [CrossRef]
  30. Biasini, M.; Bienert, S.; Waterhouse, A.; Arnold, K.; Studer, G.; Schmidt, T.; Kiefer, F.; Cassarino, T.G.; Bertoni, M.; Bordoli, L.; et al. SWISS-MODEL: Modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 2014, 42, W252–W258. [Google Scholar] [CrossRef]
  31. Rubio-Pina, J.; Quiroz-Moreno, A.; Felipe Sanchez-Teyer, L. A quantitative PCR approach for determining the ribosomal DNA copy number in the genome of Agave tequila Weber. Electron. J. Biotechnol. 2016, 22, 9–15. [Google Scholar] [CrossRef]
  32. Whelan, J.A.; Russell, N.B.; Whelan, M.A. A method for the absolute quantification of cDNA using real-time PCR. J. Immunol. Methods 2003, 278, 261–269. [Google Scholar] [CrossRef]
  33. Tamayo-Ordonez, M.C.; Rodriguez-Zapata, L.C.; Narvaez-Zapata, J.A.; Tamayo-Ordonez, Y.J.; Ayil-Gutierrez, B.A.; Barredo-Pool, F.; Sanchez-Teyer, L.F. Morphological features of different polyploids for adaptation and molecular characterization of CC-NBS-LRR and LEA gene families in Agave L. J. Plant Physiol. 2016, 195, 80–94. [Google Scholar] [CrossRef]
  34. Yuan, J.S.; Burris, J.; Stewart, N.R.; Mentewab, A.; Stewart, C.N. Statistical tools for transgene copy number estimation based on real-time PCR. BMC Bioinform. 2007, 8, S6. [Google Scholar] [CrossRef]
  35. Tamayo-Ordonez, Y.J.; Narvaez-Zapata, J.A.; Sanchez-Teyer, L.F. Comparative Characterization of Ribosomal DNA Regions in Different Agave Accessions with Economical Importance. Plant Mol. Biol. Report. 2015, 33, 2014–2029. [Google Scholar] [CrossRef]
  36. Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29, e45. [Google Scholar] [CrossRef] [PubMed]
  37. Mayorga-Hernández, E.; Rössel-Kipping, D.; Ortiz-Laurel, H.; Quero-Carrillo, A.R.; Amante-Orozco, A. Análisis comparativo en la calidad de fibra de Agave lecheguilla Torr., procesada manual y mecánicamente. Agrociencia 2004, 38, 219–225. [Google Scholar]
  38. Perez-Nunez, M.T.; Chan, J.L.; Saenz, L.; Gonzalez, T.; Verdeil, J.L.; Oropeza, C. Improved somatic embryogenesis from Cocos nucifera (L.) plumule explants. Cell. Dev. Biol.–Plant 2006, 42, 37–43. [Google Scholar] [CrossRef]
  39. Corbin, K.R.; Byrt, C.S.; Bauer, S.; DeBolt, S.; Chambers, D.; Holtum, J.A.M.; Karem, G.; Henderson, M.; Lahnstein, J.; Beahan, C.T.; et al. Prospecting for Energy-Rich Renewable Raw Materials: Agave Leaf Case Study. PLoS ONE 2015, 10, e0135382. [Google Scholar] [CrossRef]
  40. Ben Mlik, Y.; Jaoudi, M.; Khoffi, F.; Slah, M.; Durand, B. Study the Effect of Chemical and Enzymatic Extraction Methods on the Kenaf Fibers Properties. J. Nat. Fibers 2020, 19, 1168–1177. [Google Scholar] [CrossRef]
  41. Mylsamy, K.; Rajendran, I. Influence of alkali treatment and fibre length on mechanical properties of short Agave fibre reinforced epoxy composites. Mater. Des. 2011, 32, 4629–4640. [Google Scholar] [CrossRef]
  42. Kern, Z.; Kimak, A.; Hatvani, I.G.; Llanos Campana, D.M.; Leuenberger, M. Cellulose in Foliage and Changes during Seasonal Leaf Development of Broadleaf and Conifer Species. Plants 2022, 11, 2412. [Google Scholar] [CrossRef] [PubMed]
  43. Burrola-Núñez, H.; Herrera-Franco, P.; Soto-Valdez, H.; Rodríguez-Félix, D.E.; Meléndrez-Amavizca, R.; Madera-Santana, T.J. Production of biocomposites using different pre-treated cut jute fibre and polylactic acid matrix and their properties. J. Nat. Fibers 2021, 18, 1604–1617. [Google Scholar] [CrossRef]
  44. Li, X.; Tabil, L.G.; Panigrahi, S. Chemical treatments of natural fiber for use in natural fiber-reinforced composites: A review. J. Polym. Environ. 2007, 15, 25–33. [Google Scholar] [CrossRef]
  45. Marinho, N.P.; de Muñiz, G.I.B.; Nisgoski, S.; Venson, I.; de Cademartori, P.H.G.; de Andrade, A.S. Histochemical analysis of stem and fiber of ramie (Boehmeria nivea (L.) Gaud var. Murakami). Acta Sci. Biol. Sci. 2018, 40, e37207. [Google Scholar] [CrossRef]
  46. Aripov, K.; Ioelovich, M. Comparative study of supramolecular structure of cellulose in cotton fibers of Gossypium hirsutum and Gossypium barbadense. Cellul. Chem. Technol. 2020, 54, 635–641. [Google Scholar] [CrossRef]
  47. Guillemin, F.; Devaux, M.-F.; Guillon, F. Evaluation of plant histology by automatic clustering based on individual cell morphological features. Image Anal. Stereol. 2004, 23, 13–22. [Google Scholar] [CrossRef]
  48. Qin, J.; Yang, Y.; Jiang, J.; Yi, Z.; Xiao, L.; Ai, X.; Chen, Z. Comparison of lignocellulose composition in four major species of Miscanthus. Afr. J. Biotechnol. 2012, 11, 12529–12537. [Google Scholar]
  49. Hulle, A.; Kadole, P.; Katkar, P. Agave Americana Leaf Fibers. Fibers 2015, 3, 64–75. [Google Scholar] [CrossRef]
  50. Thamae, T.; Baillie, C. Influence of fibre extraction method, alkali and silane treatment on the interface of Agave americana waste HDPE composites as possible roof ceilings in Lesotho. Compos. Interface 2007, 14, 821–836. [Google Scholar] [CrossRef]
  51. Ben Sghaier, A.E.; Chaabouni, Y.; Msahli, S.; Sakli, F. Morphological and crystalline characterization of NaOH and NaOCl treated Agave americana L. fiber. Ind. Crop Prod. 2012, 36, 257–266. [Google Scholar] [CrossRef]
  52. Garcia-Reyes, R.B.; Rangel-Mendez, J.R. Contribution of agro-waste material main components (hemicelluloses, cellulose, and lignin) to the removal of chromium (III) from aqueous solution. J. Chem. Technol. Biot. 2009, 84, 1533–1538. [Google Scholar] [CrossRef]
  53. Mcdougall, G.J.; Morrison, I.M.; Stewart, D.; Weyers, J.D.B.; Hillman, J.R. Plant Fibers—Botany, Chemistry and Processing for Industrial Use. J. Sci. Food Agric. 1993, 62, 1–20. [Google Scholar] [CrossRef]
  54. Vieira, M.C.; Heinze, T.; Antonio-Cruz, R.; Mendoza-Martinez, A.M. Cellulose derivatives from cellulosic material isolated from Agave lechuguilla and fourcroydes. Cellulose 2002, 9, 203–212. [Google Scholar] [CrossRef]
  55. Cruz, M.C. Tequila production from agave: Historical influences and contemporary processes. In The Alcohol Textbook; Nottingham University Press: Nottingham, UK, 2003; Volume 223. [Google Scholar]
  56. Parra-Negrete, L.; Villar-Quiñones, P.; Prieto-Rodríguez, A. Extraction of agave fibers to make paper and crafts. Acta Univ. 2010, 20, 77–83. [Google Scholar]
  57. Zeng, X.F.; Sheng, J.J.; Zhu, F.L.; Wei, T.Z.; Zhao, L.L.; Hu, X.H.; Zheng, X.F.; Zhou, F.S.; Hu, Z.L.; Diao, Y.; et al. Genetic, transcriptional, and regulatory landscape of monolignol biosynthesis pathway in Miscanthus x giganteus. Biotechnol. Biofuels 2020, 13, 179. [Google Scholar] [CrossRef]
  58. McKinley, B.; Rooney, W.; Wilkerson, C.; Mullet, J. Dynamics of biomass partitioning, stem gene expression, cell wall biosynthesis, and sucrose accumulation during development of Sorghum bicolor. Plant J. 2016, 88, 662–680. [Google Scholar] [CrossRef]
  59. Li, Y.; He, Y.; Lin, Y.; Wan, T.; Li, M.; Chen, Z. Bioinformatics Analysis of Cellulose Synthase CesA Gene from Miscanthus lutarioriparius. Mol. Plant Breed. 2021, 12, 1–8. [Google Scholar] [CrossRef]
  60. Hu, H.; Zhang, R.; Feng, S.; Wang, Y.; Wang, Y.; Fan, C.; Li, Y.; Liu, Z.; Schneider, R.; Xia, T.; et al. Three AtCesA6-like members enhance biomass production by distinctively promoting cell growth in Arabidopsis. Plant Biotechnol. J. 2018, 16, 976–988. [Google Scholar] [CrossRef]
  61. Zhang, L.; Ming, R.; Zhang, J.; Tao, A.; Fang, P.; Qi, J. De novo transcriptome sequence and identification of major bast-related genes involved in cellulose biosynthesis in jute (Corchorus capsularis L.). BMC Genom. 2015, 16, 1062. [Google Scholar] [CrossRef]
  62. Baduel, P.; Bray, S.; Vallejo-Marin, M.; Kolar, F.; Yant, L. The “Polyploid Hop”: Shifting Challenges and Opportunities over the Evolutionary Lifespan of Genome Duplications. Front. Ecol. Evol. 2018, 6, 117. [Google Scholar] [CrossRef]
  63. Cheng, F.; Wu, J.; Cai, X.; Liang, J.; Freeling, M.; Wang, X. Gene retention, fractionation and subgenome differences in polyploid plants. Nat. Plants 2018, 4, 258–268. [Google Scholar] [CrossRef]
  64. Zhang, B.; Gao, Y.; Zhang, L.; Zhou, Y. The plant cell wall: Biosynthesis, construction, and functions. J. Integr. Plant Biol. 2021, 63, 251–272. [Google Scholar] [CrossRef]
  65. Fernandes, A.N.; Thomas, L.H.; Altaner, C.M.; Callow, P.; Forsyth, V.T.; Apperley, D.C.; Kennedy, C.J.; Jarvis, M.C. Nanostructure of cellulose microfibrils in spruce wood. Proc. Natl. Acad. Sci. USA 2011, 108, E1195–E1203. [Google Scholar] [CrossRef]
  66. Newman, R.H.; Hill, S.J.; Harris, P.J. Wide-Angle X-Ray Scattering and Solid-State Nuclear Magnetic Resonance Data Combined to Test Models for Cellulose Microfibrils in Mung Bean Cell Walls. Plant Physiol. 2013, 163, 1558–1567. [Google Scholar] [CrossRef] [PubMed]
  67. Thomas, L.H.; Forsyth, V.T.; Sturcova, A.; Kennedy, C.J.; May, R.P.; Altaner, C.M.; Apperley, D.C.; Wess, T.J.; Jarvis, M.C. Structure of Cellulose Microfibrils in Primary Cell Walls from Collenchyma. Plant Physiol. 2013, 161, 465–476. [Google Scholar] [CrossRef]
  68. Wang, T.; Hong, M. Solid-state NMR investigations of cellulose structure and interactions with matrix polysaccharides in plant primary cell walls. J. Exp. Bot. 2016, 67, 503–514. [Google Scholar] [CrossRef]
  69. Turner, S.; Kumar, M. Cellulose synthase complex organization and cellulose microfibril structure. Philos. Trans. R. Soc. A 2018, 376, 20170048. [Google Scholar] [CrossRef] [PubMed]
  70. Cosgrove, D.J. Wall Structure and Wall Loosening. A Look Backwards and Forwards1. Plant Physiol. 2001, 125, 131–134. [Google Scholar] [CrossRef]
  71. Cosgrove, D.J. Diffuse Growth of Plant Cell Walls. Plant Physiol. 2017, 176, 16–27. [Google Scholar] [CrossRef]
  72. Zhang, D.; Xu, Z.; Cao, S.; Chen, K.; Li, S.; Liu, X.; Gao, C.; Zhang, B.; Zhou, Y. An Uncanonical CCCH-Tandem Zinc-Finger Protein Represses Secondary Wall Synthesis and Controls Mechanical Strength in Rice. Mol. Plant 2018, 11, 163–174. [Google Scholar] [CrossRef] [PubMed]
  73. Watanabe, Y.; Meents, M.J.; McDonnell, L.M.; Barkwill, S.; Sampathkumar, A.; Cartwright, H.N.; Demura, T.; Ehrhardt, D.W.; Samuels, A.L.; Mansfield, S.D. Visualization of cellulose synthases in Arabidopsis secondary cell walls. Science 2015, 350, 198–203. [Google Scholar] [CrossRef] [PubMed]
  74. De la Torre-Espinosa, Z.Y.; Barredo-Pool, F.; de la Serna, E.C.; Sanchez-Teyer, L.F. Active telomerase during leaf growth and increase of age in plants from Agave tequilana var. Azul. Physiol. Mol. Biol. Plants 2020, 26, 639–647. [Google Scholar] [CrossRef]
  75. Pace, M.R.; Lohmann, L.G.; Angyalossy, V. Evolution of disparity between the regular and variant phloem in Bignonieae (Bignoniaceae). Am. J. Bot. 2011, 98, 602–618. [Google Scholar] [CrossRef]
  76. Cao, S.; Cheng, H.; Zhang, J.; Aslam, M.; Yan, M.; Hu, A.; Lin, L.; Ojolo, S.P.; Zhao, H.; Priyadarshani, S.V.G.N.; et al. Genome-Wide Identification, Expression Pattern Analysis and Evolution of the Ces/Csl Gene Superfamily in Pineapple (Ananas comosus). Plants 2019, 8, 275. [Google Scholar] [CrossRef]
  77. Beeckman, T.; Przemeck, G.K.H.; Stamatiou, G.; Lau, R.; Terryn, N.; De Rycke, R.; Inze, D.; Berleth, T. Genetic complexity of cellulose synthase A gene function in Arabidopsis embryogenesis. Plant Physiol. 2002, 130, 1883–1893. [Google Scholar] [CrossRef]
  78. Cano-Delgado, A.; Penfield, S.; Smith, C.; Catley, M.; Bevan, M. Reduced cellulose synthesis invokes lignification and defense responses in Arabidopsis thaliana. Plant J. 2003, 34, 351–362. [Google Scholar] [CrossRef]
  79. Fagard, M.; Desnos, T.; Desprez, T.; Goubet, F.; Refregier, G.; Mouille, G.; McCann, M.; Rayon, C.; Vernhettes, S.; Hofte, H. PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of arabidopsis. Plant Cell 2000, 12, 2409–2423. [Google Scholar] [CrossRef] [PubMed]
  80. Brown, D.M.; Zeef, L.A.H.; Ellis, J.; Goodacre, R.; Turner, S.R. Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics. Plant Cell 2005, 17, 2281–2295. [Google Scholar] [CrossRef]
  81. Persson, S.; Paredez, A.; Carroll, A.; Palsdottir, H.; Doblin, M.; Poindexter, P.; Khitrov, N.; Auer, M.; Somerville, C.R. Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis. Proc. Natl. Acad. Sci. USA 2007, 104, 15566–15571. [Google Scholar] [CrossRef]
  82. Timmers, J.; Vernhettes, S.; Desprez, T.; Vincken, J.P.; Visser, R.G.F.; Trindade, L.M. Interactions between membrane-bound cellulose synthases involved in the synthesis of the secondary cell wall. FEBS Lett. 2009, 583, 978–982. [Google Scholar] [CrossRef] [PubMed]
  83. Casas, A.; Blancas, J.; Otero-Arnaiz, A.; Cruse-Sanders, J.; Lira, R.; Avendaño, A.; Parra, F.; Guillén, S.; Figueredo, C.J.; Torres, I.; et al. Evolutionary Ethnobotanical Studies of Incipient Domestication of Plants in Mesoamerica. In Ethnobotany of Mexico: Interactions of People and Plants in Mesoamerica; Lira, R., Casas, A., Blancas, J., Eds.; Springer: New York, NY, USA, 2016; pp. 257–285. [Google Scholar]
  84. Robertson, I.; Cortes, M.O.C. Teotihuacan pottery as evidence for subsistence practices involving maguey sap. Archaeol. Anthropol. Sci. 2017, 9, 11–27. [Google Scholar] [CrossRef]
  85. Huang, X.; Wang, B.; Xi, J.; Zhang, Y.; He, C.; Zheng, J.; Gao, J.; Chen, H.; Zhang, S.; Wu, W.; et al. Transcriptome Comparison Reveals Distinct Selection Patterns in Domesticated and Wild Agave Species, the Important CAM Plants. Int. J. Genom. 2018, 2018, 5716518. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Number of copies of the CesA gene (A) and relative expression analysis of the CesA gene (B) evaluated in different species of Agave, A. tequilana, A. sisalana and A. fourcroydes. The bars represent the SDs. Different letters represent significant differences according to Student’s t test; p ≤ 0.05.
Figure 1. Number of copies of the CesA gene (A) and relative expression analysis of the CesA gene (B) evaluated in different species of Agave, A. tequilana, A. sisalana and A. fourcroydes. The bars represent the SDs. Different letters represent significant differences according to Student’s t test; p ≤ 0.05.
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Figure 2. Longitudinal analysis of cellulose fibrils (A,D,G) at 50 µm and transverse (B,E,H) at 10 µm and (C,F,I) at 5 µm in different species of Agave, A. tequilana (G–I), A. sisalana (A–C) and A. fourcroydes (D–F).
Figure 2. Longitudinal analysis of cellulose fibrils (A,D,G) at 50 µm and transverse (B,E,H) at 10 µm and (C,F,I) at 5 µm in different species of Agave, A. tequilana (G–I), A. sisalana (A–C) and A. fourcroydes (D–F).
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Figure 3. Structure and organization of the cellular components of the fibers, longitudinal sections at 100 μm (A,D,G), and 20 μm (B,E,H) and transverse sections at 20 μm (C,F,I), in different species of Agave, A. tequilana (G,H,I), A. sisalana (A,B,C) and A. fourcroydes (D,E,F). The black arrows correspond to vascular bundles, the yellow arrows correspond to spiral tracheids, the orange arrows correspond to parenchyma cells, the white arrows correspond to sclerenchyma fibers, and the red arrows correspond to phloem.
Figure 3. Structure and organization of the cellular components of the fibers, longitudinal sections at 100 μm (A,D,G), and 20 μm (B,E,H) and transverse sections at 20 μm (C,F,I), in different species of Agave, A. tequilana (G,H,I), A. sisalana (A,B,C) and A. fourcroydes (D,E,F). The black arrows correspond to vascular bundles, the yellow arrows correspond to spiral tracheids, the orange arrows correspond to parenchyma cells, the white arrows correspond to sclerenchyma fibers, and the red arrows correspond to phloem.
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Figure 4. Modeling of the tertiary structure of the catalytic subunits A and B of cellulose synthase in different plant species. (A) Tertiary structure of the subunits, (B) tertiary structure in the different fiber-producing species.
Figure 4. Modeling of the tertiary structure of the catalytic subunits A and B of cellulose synthase in different plant species. (A) Tertiary structure of the subunits, (B) tertiary structure in the different fiber-producing species.
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Figure 5. Haplotype network comparing Agave species with different families: Asparagales, Malphigiales, Poales, Malvales, Curcubitales and Rosales.
Figure 5. Haplotype network comparing Agave species with different families: Asparagales, Malphigiales, Poales, Malvales, Curcubitales and Rosales.
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Figure 6. Evolutionary relationship of alpha-helix CESA amino acid similarity of CESA data from different orders of plants (Asparagales, Poales, Rosales, Curcubitales, Malvales and Malghigiales).
Figure 6. Evolutionary relationship of alpha-helix CESA amino acid similarity of CESA data from different orders of plants (Asparagales, Poales, Rosales, Curcubitales, Malvales and Malghigiales).
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Table 1. Parameters evaluated for each species. L and W were measured in the middle of the leaf (three leaves per plant, three plants per species). Different letters represent significant differences according to Student’s t test; p ≤ 0.05.
Table 1. Parameters evaluated for each species. L and W were measured in the middle of the leaf (three leaves per plant, three plants per species). Different letters represent significant differences according to Student’s t test; p ≤ 0.05.
A. tequilana (2×)A. sisalana (5×)A. fourcroydes (5×)
Length of leaf (L) (cm)70.3 ± 2 a81.2 ± 1.3 c72.7 ± 4 b
Width of leaf (W) (cm)4.1 ± 0.5 a9.0 ± 0.6 c5.5 ± 0.7 b
Foliar area (L × W) (cm2)288.0 ± 11.6 a730.5 ± 33.8 c397.7 ± 21.2 b
Dimeter of bole Plant (cm)112.3 ± 6.3 a177.3 ± 7.1 c133.7 ± 10.3 b
Cellulose content (%)38 ± 0.6 a62 ± 2 c53 ± 2.6 b
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García-Castillo, M.J.; Tamayo-Ordóñez, Y.d.J.; Tamayo-Ordóñez, M.C.; Barredo-Pool, F.; Rodríguez-Zapata, L.C.; Ayíl-Gutiérrez, B.A.; Pulido-Salas, M.T.; Sánchez-Teyer, L.F. Comparative Analysis of Components Involved in the Synthesis of Cellulose in Agave Species. Agronomy 2025, 15, 1435. https://doi.org/10.3390/agronomy15061435

AMA Style

García-Castillo MJ, Tamayo-Ordóñez YdJ, Tamayo-Ordóñez MC, Barredo-Pool F, Rodríguez-Zapata LC, Ayíl-Gutiérrez BA, Pulido-Salas MT, Sánchez-Teyer LF. Comparative Analysis of Components Involved in the Synthesis of Cellulose in Agave Species. Agronomy. 2025; 15(6):1435. https://doi.org/10.3390/agronomy15061435

Chicago/Turabian Style

García-Castillo, María José, Yahaira de Jesús Tamayo-Ordóñez, María Concepción Tamayo-Ordóñez, Felipe Barredo-Pool, Luis Carlos Rodríguez-Zapata, Benjamin Abraham Ayíl-Gutiérrez, María Teresa Pulido-Salas, and Lorenzo Felipe Sánchez-Teyer. 2025. "Comparative Analysis of Components Involved in the Synthesis of Cellulose in Agave Species" Agronomy 15, no. 6: 1435. https://doi.org/10.3390/agronomy15061435

APA Style

García-Castillo, M. J., Tamayo-Ordóñez, Y. d. J., Tamayo-Ordóñez, M. C., Barredo-Pool, F., Rodríguez-Zapata, L. C., Ayíl-Gutiérrez, B. A., Pulido-Salas, M. T., & Sánchez-Teyer, L. F. (2025). Comparative Analysis of Components Involved in the Synthesis of Cellulose in Agave Species. Agronomy, 15(6), 1435. https://doi.org/10.3390/agronomy15061435

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