Pronounced Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Sucrose Synthase May Reveal a Novel Sugar Signaling Pathway

Soluble sugars not only serve as nutrients, but also act as signals for plant growth and development, but how sugar signals are perceived and translated into physiological responses in plants remains unclear. We manipulated sugar levels in transgenic plants by overexpressing sucrose synthase (SuSy), which is a key enzyme believed to have reversible sucrose synthesis and sucrose degradation functions. The ectopically expressed SuSy protein exhibited sucrose-degrading activity, which may change the flux of sucrose demand from photosynthetic to non-photosynthetic cells, and trigger an unknown sucrose signaling pathway that lead to increased sucrose content in the transgenic plants. An experiment on the transition from heterotrophic to autotrophic growth demonstrated the existence of a novel sucrose signaling pathway, which stimulated photosynthesis, and enhanced photosynthetic synthesis of sucrose, which was the direct cause or the sucrose increase. In addition, a light/dark time treatment experiment, using different day length ranges for photosynthesis/respiration showed the carbohydrate pattern within a 24-h day and consolidated the role of sucrose signaling pathway as a way to maintain sucrose demand, and indicated the relationships between increased sucrose and upregulation of genes controlling development of the shoot apical meristem (SAM). As a result, transgenic plants featured a higher biomass and a shorter time required to switch to reproduction compared to those of control plants, indicating altered phylotaxis and more rapid advancement of developmental stages in the transgenic plants.

Introduction

The transition from heterotrophic to autotrophic growth is one of the most important processes during the plant life cycle, as plants survive and develop independently from the quantities of carbohydrate and nutrients that accumulate in seeds (Koornneef et al., 2002; Finch-Savage and Leubner-Metzger, 2006). Light-induced biomass production during autotrophic growth occurs through chlorophyll in chloroplasts to produce photosynthetically fixed carbon compounds (such as triose phosphates—TP), which are later released into the cytosol (Bédard and Jarvis, 2005; Philippar et al., 2007). Sucrose is a primary sugar synthesized mainly from TP through the catalytic action of sucrose-phosphate synthase (SPS) and sucrose-phosphatase (SPP) in the cytosol. As a disaccharide formed by the combination of a glucosyl and fructosyl moiety, sucrose is a major transport carbohydrate, transported from photosynthetic to non-photosynthetic cells (Geigenberger and Stitt, 2000; Salerno and Curatti, 2003; Rolland et al., 2006; Wind et al., 2010). Thus, sucrose acts as the primary energy source and as a plant growth and development signal (Eveland and Jackson, 2012; Lastdrager et al., 2014). However, only invertase (INV; IC 3.2.1.16) and sucrose synthase (SuSy; IC 2.4.1.13), have sucrose-catalyzing ability in plants. It is generally believed that INV hydrolyzes sucrose to glucose and fructose in the cell wall, vacuolar, and cytosolic fractions, whereas SuSy is localized in sink tissues and has reversible functions of both sucrose synthesis and degradation (Geigenberger and Stitt, 1991, 1993; Fernie et al., 2002; Koch, 2004; Rolland et al., 2006; Bieniawska et al., 2007; Angeles-Núñez and Tiessen, 2012; Eveland and Jackson, 2012). The structure of At.SuSy1 provides insight into its functions (Zheng et al., 2011), but it is unclear how SuSy actually affects sucrose metabolism.

Sucrose is a major photosynthetic product that is actively transported by the phloem and affects cell growth and division; thus, sucrose has a pivotal role in plant growth and development. Plant growth is a highly energy-demanding process that requires optimal sugar balance, particularly that of sucrose, between photosynthetic and non-photosynthetic cells. Numerous sugar signaling pathways have been identified which involve in the maintaining the balance between the sugar production and consumption, which helps avoid energy stress (Tiessen and Padilla-Chacon, 2013; Lastdrager et al., 2014). Starch regulates sugar status through biosynthesis and degradation during day and night, respectively (Chourey et al., 1998; Smith and Stitt, 2007; Angeles-Núñez and Tiessen, 2010; Graf and Smith, 2011; Farré and Weise, 2012). A relationship between sugar status and cell growth and development has been demonstrated in Arabidopsis via the SnRKs and TOR signaling pathways. SnRK1 is activated when plants have low sugar status (Chiou and Bush, 1998; Halford et al., 2003; Rolland et al., 2006; Coello et al., 2011), whereas TOR is activated in the presence of high levels of sucrose (Deprost et al., 2007; Robaglia et al., 2012; Lastdrager et al., 2014). Sucrose induces the expression of phytochrome-interacting factors (PIFs; Leivar and Quail, 2011), whereas degradation of PIFs is promoted by light-activated phytochromes (Castillon et al., 2007). This finding has helped bridge the gap to determine how plants alter growth through different day length (Nagel and Kay, 2012; Shin et al., 2013). Such sugar signaling pathways help to explain how plants sense and adapt to their energy source to regulate growth. However, there are still gaps in our understanding of how plants regulate and respond to sucrose level and the demand of sucrose flux to maintain the sucrose balance between photosynthetic and non-photosynthetic cells. In addition, changes in the morphology and development of plants occur after directly adding exogenous sucrose to plant culture media (Rolland et al., 2006; Wind et al., 2010; Liu et al., 2011; Eveland and Jackson, 2012); however, how plants regulate sucrose production and consumption for responses and the effects of increased endogenous sucrose on plant metabolism are poorly understood.

Plants possess pluripotent stem cells located in specialized regions called meristems that are capable of producing new cells to drive organogenesis. Stem cells are located in the center zone (CZ) of shoot apical meristems (SAMs) and receive energy (i.e., sucrose from source cells) and signals (e.g., phytohormones) to stimulate the production of new cells, thus making important contributions to plant growth and organogenesis. The populations of stem cells and their progenitors are tightly controlled during proliferation by a negative feedback loop between the WUSCHELL (WUS) transcription factor and the CLAVATA (CLV) pathway (Schoof et al., 2000; Grandjean et al., 2004; Traas and Bohn-Courseau, 2005; Williams and Fletcher, 2005; Francis and Halford, 2006). WUS promotes an increase in the number of stem cells, whereas the CLV pathway limits the number of stem cells by inhibiting WUS. The exogenous sucrose supply promotes WUS expression by stimulating cell division (Wu et al., 2005) and CycD expression (Riou-Khamlichi et al., 2000), which can increase cell division and, consequently, increase the number of stem cells, and thus plant development.

Several studies have suggested that heterologous overexpression of the SuSy gene in plants promotes the production of biomass (Coleman et al., 2006, 2009; Baroja-Fernández et al., 2009; Jiang et al., 2012; Xu et al., 2012; Li et al., 2013). These studies focused on changes in soluble sugars and biomass in ectopically expressed SuSy transgenic plants; however, the mechanism of how the changes in soluble sugars affect plant growth and development is poorly understood. Herein, we present the following results after transforming six SuSy genes (S1–S6) into Nicotiana tabacum: (1) sucrose-degrading sucrose synthase (SuSy) activity increased significantly in transgenic plants compared to that in wild-type (WT) plants; (2) total soluble sugars (TSS), particularly sucrose and fructose, increased markedly in the transgenic plants; and (3) increased chlorophyll content, a higher rate of photosynthetic efficiency, and the upregulated expression levels of the genes involved in the photosynthetic sucrose synthesis were observed in the transgenic plants compared to those in WT plants. These results suggest the existence of a novel sucrose signaling pathway. This novel signaling pathway involves unknown factors that stimulate photosynthesis in the photosynthetic cells, thereby securing the sucrose flux demanded from photosynthetic to non-photosynthetic cells. Consequently, the increase in sucrose upregulated the transcription of genes controlling growth, division, and elongation of stem cells in the SAM, resulting in pronounced changes in the development and phenotype of the transgenic plants.

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