Function of NSCs····················································································································

As noted in the preceding sections, NSCs are the primary energy and substrate resource for metabolic activities in plants, and these contribute to determining the yield and quality of crops. Simultaneously, they also play significant physiological functions in regulating plant growth, development, and responses to environmental factors. Acting as osmotic agents, some sugars convey resistance against periods of adverse environment, such as drought, salinity and cold (Jacobsen et al., 2007; Martinelli, 2008; Perez-Lopez et al., 2010). Evidence indicates an additional role of sugars as signalling molecules in plants (Smeekens, 2000; Teng et al., 2005). As detailed in the following sections, NSCs and their physiological function and metabolism are documented.

2.5.4.1 Energy and metabolites

In higher plants, there are two fates for the metabolism of NSCs: one is incorporation into newly synthesized carbon compounds including amino acids, phytohormones, cell wall, more complex carbohydrates (e.g. starch) and lipids; the other is to produce energy for plant metabolism by respiration (i.e. the tricarboxylic acid (TCA) cycle, glycolysis and the oxidative pentose phosphate pathway), in which energy is released as chemical energy in the form of ATP from sugar (glucose) by a series of chemical reactions controlled by enzymes (Farrar, 1999). During respiration, glucose is the favoured substrate for respiration. Other carbohydrates are usually first converted into glucose by various enzymes before they are used for respiration (Lewis, 1984b). Hence in the current research programme, the properties and activity of carbohydrate-associated hydrolases were proposed for preliminary investigation.

2.5.4.2 Osmoregulation

Carbohydrates can also be related to the tolerance of adverse conditions, such as drought, coldness and salinity due to their roles as osmotic protectants (Jacobsen et al., 2007; Patton et al., 2007; Martinelli, 2008; Perez-Lopez et al., 2010). For example, the frost resistance of Quinoa (Chenopodium quinoa Willd.) was correlated to a high soluble sugar content, which may result in a lowering of the freezing point of plant tissue. The content of sugars has, therefore, been suggested as a useful indicator in future breeding of Quinoa (Jacobsen et al., 2007), indicating the potential value of such a research strategy. Similarly in gentian, Keller et al (1982) suggested that the high concentration of carbohydrates in storage-roots may serve as a cryoprotectant, allowing the crown to tolerate the cold winter. Considerable variation exists in the ability of different gentian genotypes to survive winter in Japan (J. Eason, personal communication, 2008). For instance, gentian ‘Showtime Spotlight’ survived the cold winter conditions well in Japan, but ‘Showtime Starlet’ did not. It has been hypothesised that the poor tolerance to winter by ‘Showtime Starlet’, may be attributed to the lack of an alpha/beta-hydrolase fold-protein (Hikage et al., 2007), however, whether the carbohydrates in the storage-roots, rhizome and crown buds of gentian are responsible for cold tolerance deserves further study.

The content of various carbohydrates may change in response to differing strengths of stress. For example, under mid-range stress in the leaves of Sporobolus stapfianus Gand.,

Chapter 2 – Literature review

i.e. 95% and 56% relative water content (RWC) of plants, significant increases in glucose, fructose and sucrose were observed; when severe desiccation stress was applied, i.e. lower than 56% RWC, glucose and fructose content decreased to very low levels, but sucrose content increased to the maximum level (Martinelli, 2008). It was possible therefore that seasonal changes of carbohydrate composition in gentian might also be able to be interpreted similarly.

Carbohydrates not only provided carbon and energy, but also acted as an osmotic agent influencing morphogenesis via affecting cell pressure potential and expansion (Brown et al., 1979; Milani et al., 2013). With gentian treated in vitro to increasing sucrose concentration of the medium (i.e. from 1.5% to 6%), when subsequently grown in vivo, the increasing sucrose concentration resulted in an increased frequency of initiation of crown buds (i.e. 30% to 95%), and more crown buds per plant (i.e. 1.3 to 2.1) (Sato, 1988a). Within this thesis, it was proposed that the role of carbohydrates in the formation of crown buds of gentian would be further examined via control of the carbohydrate supply in vitro and in vivo.

2.5.4.3 Signalling function

It has been identified that carbohydrates (mainly small molecular sugars) play a signalling function (Smeekens, 2000). For example, different carbohydrates play different roles in the shoot development of tobacco (Nicotiana tabacum L.) in vitro, wherein the presence of maltose increased the number of developed shoots, whereas, fructose enhanced shoot length (Gemas & Bessa, 2006). Many studies indicated that sugars, as signalling compounds, have a phytohormone-like function involving all stages of the plant’s life cycle (Smeekens, 2000; Rolland et al., 2006; Cho & Yoo, 2011). Recent research in sugar signalling in plants has found that a wide variety of genes are sugar-regulated at the transcriptional level (Sheen, 2001).

Carbohydrates are the products of photosynthesis and, simultaneously, carbohydrates regulate photosynthesis and the source-sink relationship. This regulation ultimately determines the pattern of carbon partitioning among the various plant organs, tissue and cells (Roitsch, 1999). Feedback inhibition is a long-known mechanism in photosynthetic regulation (Abdin et al., 1998), in which sugars can play a key role by inhibiting enzymatic activity or repressing the expression of photosynthetic genes (Koch, 1996;

Roitsch, 1999; Blanke, 2009). Sugars as a link between source and sink also induce a number of sink-related enzymes involved in sugar degradation and synthesis of storage products (Roitsch & Gonzalez, 2004). Extracellular invertase has been suggested to be a key enzyme in source-sink regulation, with the function of supplying carbohydrates to sink tissue, regulation of source-sink transition, amplification of signals, and integration of signals (Roitsch, 1999).

In gentians, little information has been published about the effects of sugars on crown bud development. As a potential parallel, it has been suggested that sugar-signalling may play an important role in regulating the growth and development in underground adventitious buds in Leafy Spurge (Euphorbia esula L.), by enhancing the gene expression of hexokinase and sucrose synthase, which increase the influx of sugar into underground buds (Anderson et al., 2005). However, whether or how the sugars in gentian influence crown bud initiation and development and, in turn, affect the yield and quality of flowering shoots, needs to be investigated. In the current study, it was the first intention to investigate the effect of carbohydrate supply on crown bud formation, winter survival, and re-growth, before any future investigations could be undertaken to understand whether carbohydrates act as a signal and regulate relevant gene expression.