Partitioning ···························································································································

Carbohydrate partitioning in plants is the distribution of carbohydrates in various organs or tissues as the results of carbohydrate transport and metabolic processes, controlling the flow of assimilates from source organs to sink organs (Marcelis, 1996). A source can be defined as an organ or tissue that is a net exporter of carbon assimilates. In contrast, a sink is a net importer. The definitions of source and sink strength of organs are usually used to discuss the capability of plant organs to export or import photoassimilates, specifically carbohydrates (Wilson, 1972; Wareing & Patrick, 1975; Marcelis, 1996; Herbers & Sonnewald, 1998; McCormick et al., 2006; Mohapatra et al., 2009); however, the validation of the definition/calculation/measurement of source and sink strength has also been argued by some authors (Farrar, 1993; Minchin & Thorpe, 1993; Herbers & Sonnewald, 1998). Source strength was early defined as source size × source activity, i.e. leaf area (m2) × net assimilation (g m-2 d-1) (Wilson, 1967, 1972). This calculation apparently refers to the net rate at which carbon assimilates are produced rather than exported. For example, while young developing leaves may have a positive net assimilation rate, and export some photosynthate, they are initially net importers of photosynphate due to growth, usually until about 50% expanded. Similarly, sink strength was defined as sink size × sink activity, i.e. dry weight (g) × relative growth rate (g g-1 d-1) (Wilson, 1967, 1972). This calculation actually refers to the net accumulation of dry matter and does not account for the imported assimilates used for respiration and remobilization. This definition of sink strength is also questioned as the import of assimilates is not determined by the sink strength alone, but also source strength and transport in phloem (Farrar, 1993; Minchin & Thorpe, 1993; Marcelis, 1996). Parameters that are independent of the rest of plant are, therefore, needed to describe sink strength

more precisely. Biochemical and molecular studies have found the correlation between sink strength and enzyme activities such as invertases, sucrose synthase and hexokinases, and these findings have brought new promising approaches to determine the sink strength (Black, 1993; Herbers & Sonnewald, 1998).

The complex processes of carbohydrate partitioning are associated with carbohydrate synthesis, transfer, compartmentalisation, storage, hydrolysis, remobilization and, finally, utilization (Wardlaw, 1990). Plant growth and development during the growth cycle are accompanied by sink-source transitions influenced by source strength, sink strength, and transport between sources and sinks (Roitsch, 1999; Blanke, 2009). Within this thesis therefore, quantifying the distribution and seasonal fluctuation of the NSCs aimed to provide potential insight to understanding the physiological function of carbohydrates in terms of crop yield and quality.

2.5.2.1 Production

The initial source of NSCs entering a plant system is photosynthesis in leaves (Farrar, 1999). Some NSCs, for example starch and fructan, are synthesized indirectly in storage organs after translocation and metabolism of assimilate from leaves or elsewhere in the plant. The biochemistry of NSCs based on sucrose and starch has been well characterized, including localization of key enzymes for synthesis and degradation (Winter & Huber, 2000; Koch, 2004). However, other NSCs of interest in this thesis, such as gentianose and gentiobiose, have not been studied extensively. In contrast to the basic biochemistry noted above, the mechanism of regulation for production of NSCs is not completely understood (Roitsch, 1999).

It is accepted that photosynthesis is influenced by environmental and physical factors like light-intensity, temperature, CO2 concentration, chlorophyll content, etc. Photosynthesis

occurring within the leaves determines the availability of carbohydrates for export to other organs (i.e. source strength). In addition, however, evidence increasingly supports the concept that the photosynthetic rate, and rate of carbohydrate export in leaves, is not only controlled by environmental factors, but also carbohydrate metabolism in sinks; even remote organs such as roots (McCormick et al., 2006; Cheng et al., 2008; Kaschuk et al., 2009). Given that gentian utilise an under-ground crown for storage of carbohydrates, i.e. a sink, it was considered plausible that this sink might also regulate the photosynthetic rate

Chapter 2 – Literature review

and carbohydrate export in leaves. Despite the obvious importance of photosynthesis on carbohydrate acquisition in gentians, limited time resources did not permit development of experiments to investigate within this thesis.

2.5.2.2 Transportation

The phloem is the primary pathway for carbohydrate transport, particularly long distance transport from the site of synthesis in the leaves to the sites of utilization or storage (Thorne & Giaquinta, 1984). The process of sugar transport between source and sink regions of plants includes the movement of sugars to the phloem, loading into the phloem, transport within the phloem, and unloading to the sink tissue and consuming cells (Thorne & Giaquinta, 1984).

Sucrose is the main carbohydrate transported in most plants, but other oligosaccharides and polyalcohols (sugar alcohols) may also be transported (Reidel et al., 2009; Rennie & Turgeon, 2009). Conventionally in most plant species, the carbohydrates transported in the phloem are non-reducing carbohydrates, such as sucrose, raffinose or polyalcohol; however, van Bel (2008), using EDTA exudation, found a widespread occurrence of hexoses, i.e. a reducing monosaccharide, in the phloem sap. This may indicate that hexose translocation may also be a normal mode of transfer of carbohydrate by the phloem, being equivalent to that of sucrose, raffinose-family-carbohydrates, or polyalcohol. While this result was later denied by Chao and Turgeon (2012), as using EDTA exudation they detected only sucrose in phloem of the same plant species used by van Bel.

To investigate phloem transport, it is essential to analyse the phloem sap to know the chemical composition and solute concentrations, so as to understand the partitioning of NSCs and sink-source interactions. However, research into phloem transport is currently restricted by the methodologies available to obtain pure phloem sap. Phloem sap may be collected by incision and cutting (Hall & Baker, 1972; Kallarackal & Komom, 1989), EDTA-induced exudation (King & Zeevaart, 1974; Urquhart & Joy, 1981; van Bel & Hess, 2008) and, via the stylets of insects (Hayashi & Chino, 1986; Oshima et al., 1990; Gould et al., 2005). For differing plant species each method has advantages and disadvantages. Despite the presence of unusual carbohydrates, i.e. gentianose and gentiobiose, the forms of carbohydrate(s) transported in the phloem of gentians have not been reported. While time consuming and requiring special equipment, either the stylet technique or EDTA- induced exudation were considered worthy of exploration within this thesis.

2.5.2.3 Storage and remobilization

Depending on the ability to export or import carbohydrates, plant organs can be divided into sources (mature leaves and exporting storage organs) and sinks (immature leaves, stems, seeds, rhizome/stem, storage roots, tubers, etc). Typically each plant species stores one type of carbohydrate (for instance, sucrose, starch, raffinose, etc.) over others (Brocklebank & Hendry, 1989). Any one species, however, often contains different types of carbohydrates, so providing a flexible means of regulating carbon flux and mobilization (Farrar, 1999).

The changes in concentration and nature of pools of NSCs in various plant organs and tissues, or during different seasons, e.g. the turnover of NSCs in storage pools, can provide important information as to the dynamics of supply and demand for carbohydrates (Boldingh et al., 2000; Gesch et al., 2007), and the possible activities of regulating enzymes such as invertase, sucrose synthase, hexokinase, fructokinase and sucrose- phosphate synthase (Klann et al., 1993; Irving et al., 1997). In storage roots of G. lutea, the changes of gentianose, gentiobiose and sucrose in the storage roots of G. lutea were measured, and showed a similar trend of accumulation of these NSCs in autumn as evident in other perennial plants (Bridel, 1911), however, data covering winter and early spring, or for other organs, was not presented. Hence, in the current study, it was proposed that the seasonal changes of the concentration of NSCs should be quantified, so as to explore the physiological function of the storage and utilization of NSCs in the growth cycle of gentians, and their importance to the commercial production of their flowering shoots.