PLANT HETEROPHYLLY - O.M. NEDUKHA - 2011

CHAPTER ONE. MORPHOLOGICAL AND STRUCTURAL-FUNCTIONAL CHARACTERISTICS OF VEGETATIVE ORGANS IN HETEROPHYLLOUS PLANTS

1.6. ANATOMICAL STRUCTURE OF LEAVES AND STEMS IN HETEROPHYLLOUS PLANTS

When the Water level in lakes and rivers drops naturally, PARTS OF THE stems of aquatic plants become exposed above the water surface. Under these conditions, aerial leaves can form on the short emergent stems, differing significantly from submersed leaves in their morphological and anatomical parameters. For instance, in Potamogeton anguillantus, P. perfoliatus, P. malaianus, and hybrids of P. anguillantus, after the stems 'emerged' from the water, the aerial leaves differed from the submersed ones by the following features: The Development of a leaf petiole, a multi-fold decrease in leaf blade length, an almost twofold increase in leaf blade thickness, and the appearance of Stomata on both the adaxial and abaxial surfaces (Iida et al., 2009).

Thus, it was established that phenotypic changes in leaf shape and Structure during the transition from water to air are mediated by environmental factors. The differences in the Anatomical Structure of submersed and aerial leaves, as well as stems of heterophyllous plants, share certain features typical of many higher aquatic plant species. Heterophily is accompanied by alterations in leaf anatomical structure; furthermore, it has been noted that the anatomical structure of submersed and aerial shoots also differs in plants characterized by heterophily.

LEAVES. In aquatic heterophyllous plants, aerial, floating, and submersed leaves are characterized by distinct types of mesophyll and epidermal structure. As the degree (depth) of leaf submersion in water increases, a transition in mesophyll types occurs: isopalisade → dorsiventral → homogeneous. The isopalisade mesophyll type is characterized by the presence of palisade parenchyma beneath both the upper and lower epidermis, with spongy parenchyma located between the palisade layers. The type of mesophyll structure and its quantitative characteristics are determined by environmental conditions, primarily the light factor (Ronzhina, Pyankov, 2001). Aerial and floating leaves of hydrophytes growing under sufficient and high illumination typically exhibit a dorsiventral anatomical structure with two to three layers of palisade tissue, as seen, for example, in the aerial leaves of arrowhead and yellow pond-lily. The isopalisade anatomical structure, in which palisade parenchyma is not differentiated and the mesophyll is represented by uniform rounded Cells, was found in two species (Typha latifolia, T. angustifolia). Plants of this group may experience moisture deficits during periods of water body desiccation, and the isopalisade parenchyma can serve as a beneficial ecological adaptation during such times (Ronzhina, Pyankov, 2001).

In most submersed leaves of heterophyllous plants, the leaf blades feature an isolateral anatomical type with an undifferentiated mesophyll, a reduced blade thickness, and an absence of stomata compared to aerial leaves. These characteristics are typical of such plants as Nuphar lutea, Sagittaria sagittifolia, Potamogeton natans, P. fluitans, and others.

In certain species, notably Ludwigia repens and Sium latifolium, the thickness of submersed and aerial leaf blades shows little difference, maintaining a uniform blade anatomy (Little, 2003; Nedukha, 2004). At the same time, other traits—such as stomatal density on the upper leaf surface and the number of starch grains in leaf METABOLISM/14.html">Chloroplasts—were reliably higher in aerial leaves (Table 1.6.1).

It should be noted that the heterophily index (HI), defined as The ratio of leaf length to its width (Young, Horton, 1985), is significantly greater in submersed leaves than in aerial ones. The HI of submersed leaves continuously increases during their growth. For example, in submersed leaves of Ranunculus flabellaris (Woodvine, 1999; Woodvine, Dengler, 1999), this index increased during development: in aerial leaves from the twelfth to the twenty-fourth day, the HI value changed from five to ten, whereas in submersed leaves over the same period, it increased from five to twenty-five.

The acclimatization of leaves to submersed living conditions can be traced in true hydrophytes (Bruni et al., 1996; Wells, Pigliucci, 2000; Frost-

Christensen et al., 2003), particularly in the leaves of Rumex palustris (Mommer et al., 2005). Leaves of this plant developing underwater are 20% thinner than aerial leaves, contain 10 times less reserve starch, and are characterized by an increased specific leaf area (area-to-fresh-mass ratio, m²g^-1), indicating a large surface area relative to leaf mass. The aforementioned features facilitate acclimatization and support Plant resistance to submersed existence, during which CO₂ is transported directly into photosynthetic epidermal cells via diffusion through the outer epidermal Cell walls. The rate of CO₂ diffusion in submersed leaves of Rumex palustris differs markedly from that in aerial leaves (Mommer et al., 2005); CO₂ assimilation in submersed leaves of this plant and other hydrophytes is very high compared to aerial leaves (He et al., 1999; Boeger, Poulson, 2003; Mommer et al., 2006, a; 2006, b).

The Study of anatomical and functional features in aerial, floating, and submersed leaves of numerous hydrophyte species has enabled researchers to identify the dependence of certain traits on the species and its growing conditions, as well as common and distinctive species-specific traits (Nekrasova et al., 2003). Investigating plants from the Sysert and Iset rivers in the Sverdlovsk Region of Russia, the authors demonstrated that aerial leaves during the flowering phase (Ranunculus gmelini DC, Ranunculus sceleratus, Typha angustifolia L., Typha latifolia L., etc.) were characterized by a dorsiventral anatomical structure and a high number of chloroplasts per unit of leaf area (17.4 ± 0.28) (Nekrasova et al., 2003).

TABLE 1.6.1. Anatomical Characteristics of submersed and aerial leaves of Ludwigia repens (Little, 2003)

In hydrophytes with floating leaves (Nuphar lutea, N. pumila (Timm.) DC, Potamogeton natans, Ranunculus sceleratus, etc.), the anatomical structure type of the

blade was both dorsiventral and homogeneous, and the mean number of chloroplasts per unit of leaf area was lower (10.20 ± 0.18) compared to aerial leaves (Nekrasova et al., 2003).

At the same time, in submersed leaves of hydrophytes (both heterophyllous and homophyllous higher aquatic plants), particularly in Nuphar lutea, Potamogeton alpinus Balb., P. compresus L., P. crispus L., P. lucens, P. pectinatus L., P. perfoliatus L., P. pusillus L., and others, the mean number of chloroplasts per unit area of the submersed leaf was 3.10 ± 0.05 (which is nearly five times lower than in aerial leaves), whereas the mean chlorophyll content per chloroplast was higher than in aerial and floating leaves (Nekrasova et al., 2003).

Investigating the anatomy of terrestrial and submersed leaf blades of the spikerush (Eleocharis vivipara Link) using Abscisic acid Treatment, O. Ueno and co-workers (Ueno, 1996, a; b; Ueno et al., 1998) described The structure of vascular bundle sheaths and mesophyll cells. As a result, it was found that the leaves of terrestrial forms possessed Kranz anatomy and Three types of vascular bundles. The outer sheath of parenchymatous cells contained small chloroplasts that functioned like mesophyll chloroplasts. The middle bundle and its sheath lacked chloroplasts. The inner bundle contained Kranz cells with well-developed granal chloroplasts and large Mitochondria. Thus, the Structural Features of the leaf cells reflected the Biochemical characteristics of the C₄ plant type (Ueno, 1996, a).

Meanwhile, the submersed leaves of this spikerush species possessed large air cavities between the mesophyll and epidermis. Vascular bundles were very small and characterized by a less dense distribution compared to terrestrial leaf forms. Kranz anatomy was reduced in terms of cell number and size, and cells contained a small number of Organelles. Chloroplasts of the reduced Kranz cells were smaller than those in terrestrial spikerush. Such anatomical characteristics are typical of hydrophyte submersed leaves. When the submersed form of spikerush was placed in water supplemented with 5 micromol ABA, new leaves with a structure similar to that of aerial leaves (the terrestrial form) emerged within a week. The bundle sheaths of such newly formed leaves resembled those of terrestrial leaves. Kranz cells were well developed and contained numerous chloroplasts, mitochondria, and Peroxisomes structurally similar to the organelles of Kranz cells in terrestrial leaves. Chloroplasts were large, possessed grana, and exceeded the size of chloroplasts in Kranz cells of submersed leaves. The epidermis of these leaves contained non-functioning stomata; thus, in submersed leaves treated with ABA, changes occurred in cellular anatomical structure and ultrastructure. It should be noted that leaves formed prior to ABA treatment (older rather than newly formed ones) turned brown and died (Ueno, 1998).

Light-optical examination of aerial and submersed leaves of Trapa natans (Bercu, 2004) showed that their anatomical structures differed greatly. Aerial leaves possessed typical single-layered upper and lower epidermis. Stomata were present only on the upper epidermis of aerial leaves, while numerous trichomes were found on the lower epidermis. The mesophyll comprised palisade and spongy parenchyma: a 2-3-layered palisade and a 1-2-layered spongy parenchyma. Druses (calcium oxalate crystals) were detected in stomatal cavities (Batanouny, 1992). Large air cavities were present among the spongy parenchyma cells. The Vascular System was represented by small vascular bundles. The leaf petiole of aerial leaves is covered by a thin-walled epidermis containing chloroplasts within its cells. The hypodermis consists of 5-6 cell layers (in the narrow zone of the petiole) with compact cells, followed by aerenchyma containing calcium oxalate crystals. Cross-sections of submersed dissected leaves of Trapa natans exhibit a very simple anatomical structure: epidermis, cortex, and a vascular bundle. The cortex consists of 8-9 layers of parenchymatous cells. Small air cavities are present among the cortical parenchymatous cells; they are very small and resemble cavities found in Vallisneria spiralis L. (Bercu, Fagaras, 2002) and Myriophyllum verticillatum L. (Serbanescu-Jitariu, Torna, 2002). The cortex and endodermis consisted of only a single cell layer. The vascular system consists of xylem and phloem surrounded by the pericycle. The phloem was formed by metaphloem vessels directed towards the xylem, and protophloem vessels. Thus, the submersed leaves of Trapa natans resembled ROOT structure in their anatomy, where xylem elements were reduced and the pericycle had only a single cell layer (Bercu, 2004).

L. Mommer and co-workers (Mommer et al., 2007) also conducted studies using Rumex palustris to investigate its flood tolerance. Based on the response of hydrophytes to complete submergence, it can be hypothesized that organ tolerance to flooding in intolerant species may partly occur via mechanisms similar to those of aquatic heterophyllous plants. The researchers put forward the hypothesis that the response of leaves and stems to complete submergence depends on the species' tolerance to it. This is supported by the induction of certain traits—an increase in plant weight and aerenchyma, and a decrease in the specific leaf area (Mommer et al., 2006, a). L. Mommer and co-workers (Mommer et al., 2007) applied microelectrode oxygen measurements to assess submersed gas exchange during the flooding of nine species differing in flood tolerance. Internal oxygen content was measured in leaf petioles, as oxygen entered them from the surrounding water layer and via Photosynthesis in the light. The studies were conducted in conjunction with an investigation of morphological and anatomical adaptive traits recently identified through changes in submersed gas exchange (Mommer et al., 2005).

In these experiments, L. Mommer and co-workers (Mommer et al., 2007) used flood-tolerant and flood-sensitive plant species growing in meadows along the Rhine River (in the Netherlands). Plant species were selected from various dicotyledonous families. The researchers established a dependence of trait plasticity on plant genotype and phenotype. Tolerant species were defined as those flooded for more than 100–150 days per year (Voesenek et al., 2004), whereas sensitive species were those flooded for less than two days per year. All plants were grown from seeds, except for Potentilla reptans. Plants were cultivated in specialized chambers under an illumination of 250 µmol quanta m^-2 s^-1, a 16-h light / 8-h dark photoperiod, and a Temperature of +20 °C. After 32 days of growth, the plants were submerged in a hydroponic Hoagland solution for 21 days under a twofold reduction in light intensity.

Particular attention is warranted for experiments comparing anatomical traits and physiological parameters in aquatic and terrestrial species growing on soils with varying moisture levels (Table 1.6.2). Oxygen measurements in petioles after 21 days of submergence showed that internal oxygen levels increased in plant species growing in water compared to terrestrial species. Anatomical traits and aerenchyma volume were studied in all species used in the experiments (Table 1.6.3).

TABLE 1.6.2. List of species used in the flooding tolerance experiment (Voesenek et al., 2004)

Note *: H - species sensitive (susceptible) to flooding; C - flood-tolerant (resistant) species

TABLE 1.6.3. Aerenchyma volume in the leaves of flood-tolerant and flood-sensitive species. Measurements taken following submergence (Mommer et al., 2007)

It was established that the petioles and laminae of aquatic plants were thinner, possessed thinner cell walls and cuticles, and exhibited a larger leaf area per unit biomass compared to aerial leaves.

In flood-sensitive species, the corresponding response was more pronounced, particularly regarding the thickness of epidermal cell walls. The authors concluded that flood-tolerant plants constitutively maintain thin cell walls in the leaf epidermis. One of the most flood-resistant species, Oenanthe aquatica, showed little plasticity in either leaf thickness or Cell wall thickness, which is presumably related to the species' adaptation to continuously submerged habitats. Oxygen measurements in the petioles of flood-tolerant species revealed that oxygen diffusion from water into the petiole was significantly lower than in flood-sensitive species. Aerenchyma content also increased upon submergence, scaling with the degree of stress tolerance (Table 1.6.3).

Thus, it was demonstrated that the flooding tolerance of leaves depends on internal oxygen supply entering the stems of the studied species. The capacity for plastic adjustments during submergence appears to induce plant resistance to this factor, correlating with shifts in internal oxygen levels. In sensitive species, oxygen content was lower in the upper portions of the petioles compared to flood-tolerant species.

Plant acclimatization also depended on petiole length. In tolerant species subjected to dark conditions (nighttime) during flooding, oxygen levels were higher than in flood-sensitive species, suggesting that leaves of tolerant species benefit from inherent genetic and phenotypic advantages. The lifespan of terrestrial leaves is shorter in flood-sensitive species than in flood-tolerant ones (Mommer et al., 2006a); however, the photosynthetic productivity of aquatic leaves is markedly lower in sensitive species compared to tolerant ones (Mommer, Visser, 2005). The plasticity of submersed leaves should be regarded as an evolutionary adaptation for aquatic life. An essential factor for surviving underwater is the internal aeration system present in flood-tolerant species, which eventually develops in sensitive ones as well. As previously noted, light intensity significantly affects the anatomical and structural traits of hydrophyte leaves (Wells, Pigliucci, 2000). A comparison of Anatomical Features between terrestrial and aquatic plants exhibiting heterophily versus typical terrestrial plants (non-heterophyllous) revealed both shared and distinct characteristics (Table 1.6.4).

TABLE 1.6.4. Response traits of terrestrial and heterophyllous aquatic plants to varying light intensity (Wells, Pigliucci, 2000)

It was established that current velocity also affects certain parameters of submersed leaves, including their area, biomass, and photosynthetic pigment content. Thin leaves are particularly suited to cope with limited CO₂ diffusion into the leaf lamina, where restricted carbon dioxide flux correlates with reduced photosynthetic performance (Black et al., 1981; Madsen, Breinholt, 1995). Increased water velocity leads to reduced thickness of submersed leaves, higher tissue density, decreased surface area, and an elevated chlorophyll content (Boeger, Poulson, 2003).