HETEROPHYLLY IN PLANTS - O.N. NEDUKHA - 2011

CHAPTER TWO. MECHANISMS OF HETEROPHYLLY EXPRESSION IN PLANTS

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2.2. ENDOGENOUS MECHANISMS OF HETEROPHYLLY EXPRESSION IN PLANTS

2.2.4. The role of gene expression in the regulation of cell division and elongation

Molecular studies on the induction of heterophylly began in 2001 using the model plant Marsilea quadrifolia (Hsu et al., 2001). It was established that M. quadrifolia produces different leaf types in response to changes in natural or laboratory growth conditions. Under adequate conditions, exogenous application of ABA induced The formation of aerial-type leaves. The Cells responsible for ABA synthesis were localized in the SHOOT apical meristem and associated with the apex primordium (Hsu et al., 2001). These researchers identified two sets of ABA-regulated genes in the apical meristem, comprising 7 primary and 17 secondary genes. These genes were designated as ABRH (ABA-response heterophylly) genes, which are responsible for ABA-dependent heterophylly. Transcriptional changes in ABRH genes began 30-60 min after adding ABA to the culture medium. Some of these changes were transient, while others were sustained. Several regulatory genes showed Homology with genes involved in Cell growth and plastid biogenesis, specifically encoding METABOLISM/31.html">Transcription factors, protein Kinases, membrane transporters, metabolic Enzymes, structural Proteins, and proteins encoded by the Chloroplast Genome.

Thus, 24 genes were identified, seven of which are primary response genes, whereas the remaining genes involved in Transcriptional Regulation, signal Transduction, membrane transport, and metabolism were not directly linked to the ABA response (Hsu et al., 2001; Minorsky, 2003).

The molecular mechanisms underlying changes in leaf shape and Anatomical Structure during heterophylly remain largely unexplored. However, numerous studies have investigated genes involved in determining characteristics such as leaf shape, presence or absence of a petiole, leaf index (The ratio of blade length to width), margin shape, and leaf size in various Arabidopsis thaliana mutants (Tsuge et al., 1996; Tsukaya, 2006). It has been established that leaf shape is under Genetic control, regulated by the KNOX (KNOTTED-like homeobox) Gene (Hake et al., 2004) and auxin distribution within the apical meristem, whereas dorsiventrality is regulated by a microRNA-mediated pathway (Bowman, 2004).

In the A. thaliana double mutant phenotype with wing-like petioles, the expression of two genes is observed: LEP (LEAFY PETIOLE) and bop1-1 (blade-on petiole1-1). The first gene encodes the DNA-binding domain of a transcription factor, while the second encodes an NPP-like protein (NONEXPRESSOR of PR GENES1) within the leaf primordium (Bailey-Serres, Voesenek, 2008). The authors suggest that these specific genes control the GROWTH AND DEVELOPMENT of the leaf petiole.

The involvement of genes in the polar growth of the leaf blade has been demonstrated in dwarf mutants (Tsuge et al., 1996) and Arabidopsis mutants with narrow leaves of normal length (Tsukaya et al., 1994; Tsuge et al., 1996). Although polarized leaf growth is controlled by microtubules, phytohormones, and Cell wall Polysaccharides, the participation of genes in these processes is also well established. It has been shown that the longitudinal and lateral axes of cell elongation and proliferation in The Cell primordium are regulated by specific genes: polarized leaf cell growth along the long axis is regulated by the ROT3 (ROTUNDIFOLIA 3) gene (Tsuge et al., 1996), and cell number in the same direction by ROT4 (ROTUNDIFOLIA 4). The ROT3 gene is involved in steroid Biosynthesis (Kim et al., 1998; 2005a) in dwarf mutants, which exhibit defects in both cell number and cell elongation. The ROT4 gene induces the cessation of leaf cell growth (Bailey-Serres, Voesenek, 2008). Meanwhile, the polarized growth of the leaf blade along the width (short axis), including Cell Division, is regulated by the AN3 (ANGUSTIFOLIA3) gene, which affects meristematic cell division during morphogenesis. The AN3 gene encodes a transcription homolog of the SYT (synovial Sarcoma translocation) co-activator (Horiguchi et al., 2005); this cofactor is identical to GRF-INTERACTION FACTOR 1 (AtGIF1) (where GRF stands for Growth Hormone-releasing factor) (Kim, Kende, 2004), which participates in the Cell Cycle of the leaf primordium meristem.

The phytohormone Ethylene serves as a signal for rapid stem elongation underwater (Malone, Ridge, 1983; Ridge, 1987; Voesenek et al., 1999; 2004). A correlation has been established between ethylene synthesis and the growth of Cells and Tissues. The rate of ethylene synthesis depends on its oxidation and efflux from cells: in submerged Organs, the outward diffusion of ethylene occurs very slowly, causing it to accumulate within the cells. The endogenous concentration of ethylene increases in parallel with rising endogenous oxygen concentrations. The enzyme ACC synthase is involved in ethylene synthesis, whereas ACC oxidase is responsible for its oxidation (Rieu et al., 2005; Van der Straeten et al., 2001; Vriezen et al., 1999). It has been revealed that intracellular oxygen levels also regulate the expression of ethylene receptor genes, including RpERS1 (ERS1 - ethylene response sensor 1 in Ranunculus palustris) (Vriezen et al., 1997), OsERL1 in deepwater rice Oryza sativa (ERL1 - ethylene response 2 like 1), and ETR2 in A. thaliana (Klok et al., 2002; Loreti et al., 2003; Branco-Price et al., 2005; Liu et al., 2005).

Ethylene is known to signal the rapid elongation of vegetative organs underwater through several pathways. Under complete submergence, accumulated endogenous ethylene downregulates ABA biosynthesis, thereby inhibiting the expression of 9-cis-epoxycarotenoid dioxygenase (NCED) (Kende et al., 1998; Benschop et al., 2005; Saika et al., 2007). The reduction in endogenous ABA concentration stimulates the expression of gibberellin 3-oxidase, which catalyzes The conversion of active gibberellin (Benschop et al., 2006) into a simpler gibberellin (Kende et al., 1998). The decrease in gibberellin content is mediated by genes induced in The Stem as it elongates underwater:

1) genes encoding proteins involved in cell wall loosening;

2) genes that promote cell cycle progression;

3) genes that influence starch Hydrolysis.

Let us examine the aforementioned gene groups in greater detail.

Genes encoding proteins involved in cell wall loosening. The formation of a rigid cell wall is directed toward cell growth driven by turgor pressure. A significant acceleration of acid-induced cell wall elongation underwater has been observed in the Water/115.html">Vegetative organs of Oryza sativa (Cho, Kende, 1997a), Rumex palustris (Vreeburg et al., 2005), Regnellidium diphyllum Lindm., and Marsilea quadrifolia (Kim et al., 2000). It has been established that cell wall expansion is associated with expansins (EXPs) and xyloglucan endotransglucosylases/Hydrolases (XTHs) (Darley et al., 2001). Furthermore, cell wall elongation underwater directly correlates with an increase in the content and activity of $\alpha$- and $\beta$-expansins (EXPA, EXPB) (Cho, Kende, 1997b; Kim et al., 2000; Lee, Kende, 2001; Ookawara et al., 2005; Vreeburg et al., 2005). The regulation of expansin proteins involves not only gibberellin but also ethylene (Kim et al., 2000; Vreeburg et al., 2005). In the petioles of the submerged plant Ranunculus palustris, ethylene not only increased expansin expression but also stimulated proton transport into the apoplast (Vriezen et al., 2000), which is essential for the functioning of these proteins.

Genes encoding proteins involved in the cell cycle. This group comprises gibberellin-dependent genes that participate in Cell cycle regulation. For instance, in young petioles of Nymphoides peltata and young internodes of deepwater rice (Oryza sativa), ethylene stimulated not only cell elongation but also cell division. Progression through the Phases of the cell cycle is known to depend on The activity of cyclins (CYC2Os1), histone H3, and Replication protein A1 (OsRPA1) (Sauter, 1997; 2000; Vander Knaap et al., 1997).

Genes encoding proteins involved in starch hydrolysis. This group includes gibberellin-dependent genes. In Ranunculus palustris, a decrease in soluble sugar and starch content was noted upon submergence (Groenveld et al., 2003). During rapid organ growth, CARBOHYDRATES are known to be utilized for energy production and the synthesis of new cell wall components (Sauter, 2000; Voesenek et al., 2006).

Carbohydrate demands can be met through Photosynthesis and the hydrolysis of stored starch via the activation of $\alpha$-amylase (Sauter, 2000). Fukao et al. (2006) reported that the expression of the $\alpha$-amylase gene (OsAmy3D) in the leaves of submerged rice is regulated by the ERF domain of an ethylene-responsive factor. This gene is strongly regulated by gibberellic acid. These results indicate that carbohydrate levels in submerged plants are under hormonal control and are activated through the expression of specific genes.

The culmination of hormonal action on cell growth and the involvement of specific genes in the division and elongation of cells in submerged plant stems is illustrated in the diagram (Fig. 2.2.4.1; see insert XVI) presented by Bailey-Serres and Voesenek (2008).

A group of scientists established the presence of DNA polymorphism in the stem buds of Trapa natans during a cytochemical study (using random amplified polymorphic DNA) of nuclei within the meristem of floating and submersed buds, which give rise to floating and submersed leaves (Bitonti et al., 1996). The researchers revealed a nuclear Chromatin Structure with prominent chromocenters in both floating and submersed buds. The authors suggest that the unique nuclear features in the meristem of floating buds are associated with a high adenine-thymine (A-T) base content and a higher level of nuclear genome DNA Methylation compared to the meristem of submersed buds, which facilitates more efficient functioning of these organs in varying environmental conditions (Bitonti et al., 1996).

Genetic diversity has also been observed in species that elongate underwater. For instance, in Rumex acetosa L. and R. palustris, ethylene Treatment was found to either inhibit or stimulate petiole elongation. However, data obtained from studying the submersed organs of one species (R. acetosa) demonstrated a loss of ABA-regulated growth elongation (Banga et al., 1996; Benschop et al., 2005), whereas enhanced elongation rates were observed in this species under The Influence of gibberellic acid (Rijnders et al., 1997) and expansin expression (Vriezen et al., 1999; 2000). It was also established that, unlike R. palustris, ethylene in R. acetosa cannot override the inhibitory effect of ABA to promote elongation. Factors that diminish ethylene action while enhancing ABA/GA responses account for the differences in ethylene-induced elongation between the two studied species.

Genetic variations in several ERF (ethylene response factor) proteins regarding their capacity for underwater elongation have also been investigated in rice plants. Specifically, the submergence-tolerant rice cultivars harbor the SUB1A gene (where SUB stands for tolerant to submergence), which is induced by ethylene. Meanwhile, other rice cultivars possess the SUB1C gene, the induction of which is regulated by a different phytohormone, gibberellic acid (Fukao et al., 2006; Xu et al., 2006). It is believed that the expression of the SUB1A gene coincides with the repression of expansin transcript accumulation as well as a decrease in SUB1C Gene Expression (Fukao et al., 2006). This led the researchers to hypothesize that the SUB1A gene acts as a negative regulator of pathways dependent on gibberellic acid, expansins, and SUB1C proteins (Bailey-Serres, Voesenek, 2008).

Recently, Japanese researchers (Iida et al., 2009), studying heterophyllous and homophyllous species of the genus Potamogeton, established that species exhibiting heterophylly are characterized by a specific STRUCTURE OF THE rbcL gene (ribulose-1,5-bisphosphate carboxylase/oxygenase), which features twelve Amino Acid Substitutions compared to homophyllous Potamogeton species (Iida et al., 2009). The rbcL gene encodes the Rubisco protein, the primary enzyme of photosynthesis.