The majority of species studied had above 60% of crystalline cellulose and the distribution of cellulose was in unlignified parenchyma, VE, and WBT, both with secondary walls as helical and annular thickenings.
The low crystallinity indexes (47–58%) obtained for some species of cacti were similar to those reported for most gymnosperms and some angiosperms. The species with crystallinity indexes greater than 60% such as Coryphantha pallida and Echinocereus pentalophus were similar to most angiosperms reported by Agarwal et al. (2013) and to the bark of the cactus Cereus forbesii, which had 82% crystalline cellulose (Orrabalis et al. 2019).
Opuntia ficus-indica is the species with reports of crystalline cellulose in different parts of the plant: cladodes 27.2% (Yang et al. 2015) and 79% (Maceda et al. 2020), spines 33.81-70% (Vignon et al. 2004; Marin-Bustamante et al. 2017), vascular tissue 22–28% (Greco and Maffezzoli 2015), fruit epidermis 38% (Habibi et al. 2009) and 60% in seeds (Habibi et al. 2008). Maceda et al. (2020) reported for Opuntia streptacantha and O. robusta, percentages of crystalline cellulose of 76% and 74%, respectively, percentages similar to those reported in this study for some fibrous species and similar to the values of O. stenopetala and O. stricta.
Cellulose distribution and chemical composition
The tonalities observed in the fluorescence emission for cellulose and lignin correspond to that reported in other similar studies with safranin O / fast green staining technique and the three excitation bands (De Micco and Aronne 2007; Maceda et al. 2019, 2021; Donaldson 2020; Kitin et al. 2020). This technique allowed the detection of differences in the distribution of both structural compounds (cellulose and lignin) between non-fibrous and fibrous species. The safranin O dye allows the detection of lignin autofluorescence and fluorescence in analyzed tissues (De Micco and Aronne 2007; Kitin et al. 2020), with tonalities blue to green (Donaldson 2020; Baldacci-Cresp et al. 2020). In the case of celluloses and hemicelluloses that do not have autofluorescence as lignin (Donaldson 2020), with safranin O /fast green staining, the fluorescence emission of cellulose can be detected at 570–620 nm and its tonalities were reddish similar to the results of Maceda et al. (2019, 2021). However, subsequent studies with immunohistochemistry or specific fluorophores could help confirm the distribution of cellulose (Donaldson 2020; Bidhendi et al. 2020).
When comparing the results with that reported by Maceda et al. (2019) for the primary xylem, cellulose accumulated in the primary walls and lignin in the secondary ones of the helical and annular thickenings of the protoxylem and metaxylem of fibrous species, similar to that observed in non-fibrous adult plants. Only in the metaxylem of Leuenbergeria lychnidiflora, there was a decrease in the accumulation of cellulose in the primary walls and a greater accumulation in the alternate pits, similar to that obtained in the secondary xylem of the fibrous species.
The presence of high percentages of crystalline cellulose in most of the studied cacti, mainly in non-fibrous species, could be related to the abundant unlignified parenchyma and the lower accumulation of lignin in the xylem as seen in Fig. 4 and Fig. 5b. In fibrous species of Cylindropuntia and Opuntia, there was a higher percentage of crystalline cellulose in vascular tissue compared to non-fibrous species, possibly because xylem has a greater accumulation of lignin in the cell walls of the VE, F, and P (Maceda et al. 2018). This high percentage of lignin may contribute as a physical (Hamann 2012) and chemical barrier against the attack of pathogens (Durkovič et al. 2014). Zhao and Dixon (2014)) and Bacete and Hamann (2020) mentioned that the cell wall is a dynamic barrier in conditions of abiotic stress. Therefore, in some cells, the presence of increased lignin accumulation can be observed (Polo et al. 2020), while in others, similar to the gelatinous layer (G), it mainly accumulates cellulose (Festucci-Buselli et al. 2007).
In this type of cell where only the primary wall is present, the accumulation of crystalline cellulose packed by hydrogen chain β-(1→4)-D-Glcp bonds (Malinovsky et al. 2014) makes cellulose hydrophobic (Festucci-Buselli et al. 2007), confers structural support (Malinovsky et al. 2014) and causes a decrease in the efficiency of cellulase enzymes (Thomas et al. 2013) by not presenting sites binding with enzymes, making it difficult to degrade (Rytioja et al. 2014). In contrast, amorphous cellulose is slightly hydrophilic (Thomas et al. 2013), susceptible, and degrades rapidly due to the action of cellulase enzymes (Suchy et al. 2011) and pH changes by pathogens (Kubicek et al. 2014).
Infection with some pathogenic fungi occurs when the hyphae invade the roots and subsequently the vascular tissue (Kesten et al. 2019). In the fibrous species of Cactaceae, the accumulation of lignin works as a physical barrier in VEs (Liu et al. 2018). However, in non-fibrous species with little accumulation of lignin in helical or annular thickenings, the presence of crystalline cellulose in the primary wall of the tracheary elements and the unlignified parenchyma, would function as a physical barrier to prevent the spread of the fungus by reducing the effectiveness of cellulase enzymes (Thomas et al. 2013). In subsequent studies using transmission electron microscopy techniques (Ruel et al. 2012), the presence of crystalline cellulose in the primary wall of the tracheal elements can be analyzed and characterized, which will support and confirm this assertion.
For Leuenbergeria lychnidiflora, whose accumulation of lignin in vascular tissue is 36% (Reyes-Rivera et al. 2015) to 42.52% (Maceda et al. 2018), the percentage of crystalline cellulose is 80% an,d a cellulose arrangement of 83% similar to that observed in non-fibrous species. These results suggest a possible relationship with the sites where this species is distributed, which is found in areas of higher humidity (Edwards and Donoghue 2006) compared to the other fibrous species (Cylindropuntia and Opuntia) analyzed whose distribution is in arid and semi-arid climates (Gómez-Hinostrosa and Hernández 2000). Further analyses would be necessary to determine whether this percentage of crystalline cellulose in L. lychnidiflora prevails in other Leuenbergeria and Pereskia species, early-diversified taxa of Cactaceae as well as other Opuntia species.
The resistance of plants to stressful conditions is energetically expensive, in addition to the constitutive expression of defense mechanisms, such as the accumulation of callose (Srivastava et al. 2017; Lampugnani et al. 2018), pectins (Bacete and Hamann 2020), or secondary metabolites (Meraj et al. 2020). This is not always the best strategy against colonization of pathogens or diseases, because it can restrict physiological processes and have negative impacts on the plant, such as reduced seed production and biomass (Bacete et al. 2018). Therefore, the presence of primary physical barriers, such as lignin (Miedes et al. 2014) and crystalline cellulose (Malinovsky et al. 2014) that inhibit the spread of pathogens, would decrease the expression of these defensive systems energetically expensive (Bacete et al. 2018). The heterogeneity in the composition of cell walls between species reflects the diversity of defensive mechanisms against degrading enzymes that pathogens have developed to break plant cell walls, such as numerous cell wall-degrading enzymes (CWDEs), polygalacturonases, or xylanases (Annis and Goodwin 1997).
In these Cactaceae species, as mentioned before, cellulose would work to confer structural support on the primary wall without losing flexibility (Moura et al. 2010) and thus maintain the cell structure during periods of water stress and rain (Garrett et al. 2010). Furthermore, the increased amount of crystalline cellulose could function as a defense against pathogens (Thomas et al. 2013), by providing resistance to degradation by glycosyl hydrolase enzymes (Malinovsky et al. 2014) and enzymes produced by pathogenic fungi (Kesten et al. 2019). Therefore, that non-fibrous cacti species would accumulate a greater amount of crystalline cellulose as a primary defense in the xylem against possible pathogen attacks. Further analysis will be necessary to confirm the presence of percentages of crystalline cellulose and its possible polymorphisms by X-ray diffraction (French 2014). In addition to analyzing more species of dimorphic and non-fibrous cacti together with other families of plants, that present succulent stems to determine if the high percentages of crystalline cellulose persist due to the abundance of non-lignified parenchyma.