PLANT HETEROPHYLLTY - O.M. NEDUKHA - 2011

CHAPTER TWO. MECHANISMS OF HETEROPHYLLTY EXPRESSION IN PLANTS

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2.2. ENDOGENOUS MECHANISMS OF HETEROPHYLLTY EXPRESSION IN PLANTS

2.2.2. The role of cellulose

Cellulose is the primary structural polysaccharide of Cell walls, providing them with rigidity and mechanical strength. It consists of long, unbranched glucopyranose residues linked together by |0-(1-4)-glucosidic bonds. D-glucopyranose chains form parallel structures stabilized by Hydrogen Bonds (Brown, 1996). It is known that during cell growth and differentiation, alongside changes in other wall components, cellulose undergoes modifications in both its Structure and content. This is manifested by an increase in total cellulose content and the partial crystallization of its molecules (Tarchevsky, Marchenko, 1985). The cellulose content in cell walls has been established to depend on the species, organ, and tissue type. The total cellulose content in the primary Cell wall ranges from 20 to 30%, whereas in the secondary cell wall, it accounts for 40-90% of the dry mass. Cellulose content varies among different species: in grass leaves, it is 32% of dry mass; in oat stems, 60.6%; wheat, 75.7%; barley, 71.74%; alfalfa, 26.7%; clover, 23.6%; sainfoin, 28.7%; and in Arabidopsis stems, it ranges from 10 to 36%. In plant seeds, it varies from 14.7 to 80.15% (Kataoka, Kondo, 1999; Taylor et al., 1999).

It is well known that cell wall cellulose can exist in crystalline and/or amorphous forms. The proportion of amorphous cellulose within the total polysaccharide content depends primarily on the ontogenetic stage, plant species, and Cell Differentiation. In growing Cells, the content of amorphous cellulose is relatively low and species-dependent: in yellow poplar leaves, it is 10-15%, with the remainder being crystalline cellulose (Atalla, 2004); in leaves of Phragmites australis Trin. ex Steud. (common reed), it reaches 95% of amorphous cellulose; whereas in mature cotton fiber cells, only traces of amorphous cellulose are found, and their cell walls consist almost entirely of crystalline cellulose (Baran et al., 2002).

Amorphous cellulose has the capacity to absorb Water, initially within a single molecular chain, and subsequently between two amorphous cellulose chains within a single microfibril (Nilsson, Martin, 2006).

Prolonged exposure to moderate water deficit is known to inhibit plant growth, leading to typical xeromorphic features such as a reduction in the size of leaves and their constituent cells (Kordium et al., 2003). The causes of cell growth inhibition may include alterations in turgor pressure, cellular osmotic potential, and the suppression of cell wall Polysaccharide synthesis, including cellulose.

Data concerning Changes in the polysaccharide composition of hydrophyte leaves upon emergence from water onto its surface remain fragmented (Little, 2003). Most studies focus on the lignification of cell walls and the investigation of their structure, predominantly using cultivated plants. Researchers have examined the Structural and functional modifications of cell walls by inducing water stress through polyethylene glycol (PEG) treatments, salinity (428 mM NaCl), or transferring plants to hydroponic culture (Iraki et al., 1989; Sakumi et al., 1987a, b).

Considering the literature data cited above, we hypothesize that the features distinguishing submerged leaves of heterophyllous plants from aerial leaves lie in their cellulose content and the presence of its amorphous form, which plays a crucial role in apoplastic water transport (Czihak et al., 1999).

Below, we present our findings from the cytochemical study of cellulose, its distribution within cell walls, and the biochemical determination of this polysaccharide in Leaves of Sium latifolium growing both submerged in water and in terrestrial habitats (Fig. 1.3.1; see insert II; Fig. 2.2.2.1; see insert XIII) (Nedukha, 2010a).

Amphibious form of Sium latifolium. The soil moisture at the collection site was 75.2 ± 4.1%; the photon flux density of solar radiation above the submerged leaves (in water) was 80-90 µmol photons · m^-2 s^-1; On the surface of leaves with petioles submerged and blades above water, it was 300-320 µmol photons · m^-2 s^-1; on The surface of aerial leaves, it was 350-400 µmol photons • m^-2 s^-1; the average air Temperature was +28° (± 1°) C, and the water temperature surrounding the submerged leaves was +17° C.

Submerged leaves. Laser scanning confocal Microscopy of S. latifolium leaves using calcofluor white, a cellulose-specific fluorophore, revealed that cellulose in the epidermal and mesophyll cell walls fluoresced bright green regardless of the leaf type (Fig. 2.2.2.2, a-d, g, h). The fluorescence intensity levels of cellulose are presented in Table 2.2.2.1. The fluorescence intensity varied significantly between the outer and anticlinal walls of the upper and lower epidermis. In The Cell walls of the spongy parenchyma, fluorescence was lower than in the palisade parenchyma. The luminescence intensity of cellulose in the cell walls of the upper and lower epidermis of aerial leaves was significantly higher compared to submerged leaves (Table 2.2.2.1).

According to biochemical analyses of the three leaf types in amphibious broad-leaved water parsnip individuals, the total cellulose content was relatively low (Table 2.2.2.2) and was detected in both amorphous and crystalline forms. Regardless of the leaf type, the amorphous cellulose content was relatively high, ranging from 61 to 67% of the total polysaccharide content, whereas the crystalline cellulose content was nearly half as much. The ratio of amorphous to crystalline cellulose was 2.06 in submerged leaves, 1.7 in leaves with blades emerged above water, and 1.93 in aerial leaves (Table 2.2.2.2).

TABLE 2.2.2.1. Luminescence intensity of the calcofluor-cellulose complex in cell walls of leaves from the amphibious form of Sium latifolium collected in May

Notes: ^a - semi-submerged leaves had petioles in water and leaf blades above water;

* - P ≤ 0.05 (significantly different from values for submerged leaf cell walls)

TABLE 2.2.2.2. Cellulose content in leaf blades of the amphibious form of Sium latifolium collected in May

Notes: ^a - semi-submerged leaves had petioles in water and leaf blades above water;

* - P ≤ 0.05 (significantly different from values for submerged leaf cell walls)

The terrestrial form of Sium latifolium also exhibited features of heterophylly (Fig. 1.3.6; see insert VI and subsection 1.3). Soil moisture at the site where the terrestrial water parsnip plants grew was nearly half that of the amphibious form, registering at 39.5 ± 3.1%; the photon flux density of solar radiation above the upper leaf surface was 450-500 µmol photons m^-2 s^-1; the average air temperature was +28° (± 1°) C. During the vegetative growth phase, the leaves of the terrestrial broad-leaved water parsnip exhibited two blade forms: the first three leaves possessed an entire, acute-ovate blade, whereas leaves of higher nodes were pinnately dissected (Fig. 2.2.2.1, b).

TABLE 2.2.2.3. Luminescence intensity of the calcofluor-cellulose complex in cell walls of leaves from the terrestrial form of Sium latifolium collected in May

Note: * - P ≤ 0.05 (statistically significant difference compared to cell walls of leaves with entire lamina)

TABLE 2.2.2.4. Cellulose content in the leaves of the terrestrial form of Sium latifolium collected in May

Note: * - P ≤ 0.05 (statistically significant difference compared to cell walls of leaves with an entire lamina)

The entire leaf blades were acute-ovate in shape, with an average length of 2.2 ± 0.5 cm and an average width of 2.0 ± 0.2 cm; their margins were broadly crenate. At the time of material fixation (May), each plant specimen possessed three entire leaflets and two dissected ones, which consisted of entire oblong-ovate lobes (leaflets). The pinnately dissected leaves were composed of four to five pairs of lateral lobes and a terminal lobe, with average dimensions of 10 ± 1.2 cm in length and 2.9 ± 0.5 cm in width. The paired leaf lobes were arranged oppositely.

Cytochemical analysis of cellulose localization in both leaf types of the terrestrial form of S. latifolium demonstrated that cellulose in the cell walls of all Tissues exhibited green fluorescence (Fig. 2.2.2.2, e, є). The fluorescence intensity levels of cellulose are presented in Table 2.2.2.3. While fluorescence intensity showed no variation in the cell walls of the upper epidermis and spongy parenchyma between the two leaf types, the luminescence intensity in the cell walls of the lower epidermis and palisade tissue of the primary leaves with entire laminae was nearly twice as high as that in the corresponding cell walls of young pinnately dissected leaves.

According to biochemical analysis, the total cellulose content in the two leaf types of the terrestrial form of the broadleaf waterparsnip was 1.3–1.5 times higher than that in submerged leaves, and 1.6–1.8 times higher than that in emergent leaves of the amphibious form (Table 2.2.2.4). The leaves of terrestrial forms of broadleaf waterparsnip contained both amorphous and crystalline forms of cellulose. The ratio of amorphous to crystalline forms was 1.02 and 0.92 in leaves with entire and pinnately dissected laminae, respectively; these values were nearly half as high as those recorded in the submerged and emergent leaves of amphibious S. latifolium specimens.

During subsequent ontogenetic phases (budding and early fruiting) in both ecoforms of S. latifolium, a significant increase in the crystalline form of cellulose was observed in the leaves (Nedukha, 2009) compared to the vegetative phase. The author established that the crystalline cellulose content in the leaves of amphibious plants increased up to 62% during the budding phase and up to 76.2% during the fruiting phase, whereas in the leaves of terrestrial plants it increased to 68.6% and 86.1%, respectively.

Thus, cytochemical and biochemical studies of the distribution and composition of cellulose in the leaf cells of S. latifolium have demonstrated that cellulose is a sensitive cell wall polysaccharide, whose content and composition during vegetative growth vary depending on the environmental conditions of the given ecotype.

Cytochemical analysis of epidermal and mesophyll cells from various leaves of the amphibious form of broadleaf waterparsnip revealed a specific redistribution of cellulose depending on the aquatic environment and tissue type: the relative cellulose content was significantly lower in the epidermal cell walls of submerged leaves compared to emergent ones, yet higher in the mesophyll cells of submerged leaves compared to the corresponding cells of emergent leaves. A comparison of cytochemical data regarding the relative cellulose content in various cell walls of emergent leaves from amphibious plants and leaves from terrestrial waterparsnip plants (Tables 2.2.2.1 and 2.2.2.3) indicates that it was higher in the leaves with entire laminae of terrestrial plants than in the pinnately dissected leaves.

It is well established that the leaves and stems of submerged plants exhibit considerable flexibility (Kerstetter, Poethig, 1998; Mommer, Visser, 2005), the degree of which depends on the content and composition of cell wall Polysaccharides (Delmer, 1999). Furthermore, intensive gas exchange takes place across the cell walls of submerged plants (Frost-Christensen et al., 2003; Minorsky, 2003; Mommer, Visser, 2005; Mommer et al., 2006, b). It was previously established that the outer cell walls and cuticle layer in the submerged leaves of five amphibious plant species are one and a half times thinner than the epidermal cell walls of terrestrial individuals (Mommer et al., 2005). In terrestrial plants, the outer epidermal cell walls, which protect the plant against insolation and biotic factors, are several times thicker and contain twice as much cellulose as mesophyll cell walls (Carpita et al., 2001). Taking these data and our own findings into account, it can be noted that during the vegetative growth phase (in May), cellulose synthesis in the epidermis and mesophyll of the amphibious form of S. latifolium is uneven, depending on its submerged or emergent status. In contrast, in terrestrial specimens, cellulose is distributed between the epidermis and mesophyll in a manner similar to that observed in the leaves of Zea mays L. (Brown, 1996; Carpita et al., 2001).

Comparative analysis of cellulose content in submerged and emergent leaves of the amphibious form and leaves of the terrestrial form of waterparsnip showed that terrestrial plant development during the vegetative growth stage promotes more intensive cellulose accumulation in both entire and pinnately dissected leaves. It is known that the intensity of cellulose synthesis depends on the concentration of its precursors (glucose), The activity of cellulose synthases, and the activation of genes belonging to the CesA family (Richmond, Somerville, 2000). It can be hypothesized that the increased cellulose content in the leaves of the terrestrial form of broadleaf waterparsnip during the vegetative growth period is driven by enhanced glucose synthesis and the activation of specific genes.

Consequently, submergence in water increases the concentration of amorphous cellulose in S. latifolium leaves by 12–19% compared to specimens growing on land (Nedukha, 2010, a). It is known that amorphous cellulose zones absorb water, thereby facilitating apoplastic water transport, whereas crystalline cellulose domains lack this property (Czihak et al., 1999; Nilsson, Martin, 2006). Additionally, studies on wild-type petunia and a Petunia hybrida mutant (PhEXPl) revealed that the mutant P. hybrida plant possesses an allelic Gene (RSW1) responsible for amorphous cellulose synthesis (Zenoni et al., 2004). Considering the results of cellulose studies in S. latifolium and the aforementioned literature data, we can suggest that:

- the presence of amorphous cellulose in the cell walls of S. latifolium leaves facilitates plant survival in an aquatic environment, whereas the presence of crystalline cellulose contributes to plant adaptation to terrestrial conditions;

- the elevated content of the amorphous cellulose form in the submerged leaves of broadleaf waterparsnip is evidently caused by genetic differences.

These hypotheses require further experimental verification.