Intracellular sucrose communicates metabolic demand to sucrose transporters in developing pea cotyledons

Yuchan Zhou 1 Katie Chan 1 Trevor L. Wang 0 Cliff L. Hedley 0 Christina E. Offler 1 John W. Patrick 1 0 Metabolic Biology, John Innes Centre, Norwich Research Park , Colney, Norwich NR4 7UH, UK 1 School of Environmental and Life Sciences, The University of Newcastle , Callaghan, NSW 2308, Australia Mechanistic inter-relationships in sinks between sucrose compartmentation/metabolism and phloem unloading/ translocation are poorly understood. Developing grain legume seeds provide tractable experimental systems to explore this question. Metabolic demand by cotyledons is communicated to phloem unloading and ultimately import by sucrose withdrawal from the seed apoplasmic space via a turgor-homeostat mechanism. What is unknown is how metabolic demand is communicated to cotyledon sucrose transporters responsible for withdrawing sucrose from the apoplasmic space. This question was explored here using a pea rugosus mutant (rrRbRb) compromised in starch biosynthesis compared with its wild-type counterpart (RRRbRb). Sucrose influx into cotyledons was found to account for 90% of developmental variations in their absolute growth and hence starch biosynthetic rates. Furthermore, rr and RR cotyledons shared identical response surfaces, indicating that control of transporter activity was likely to be similar for both lines. In this context, sucrose influx was correlated positively with expression of a sucrose/H+ symporter (PsSUT1) and negatively with two sucrose facilitators (PsSUF1 and PsSUF4). Sucrose influx exhibited a negative curvilinear relationship with cotyledon concentrations of sucrose and hexoses. In contrast, the impact of intracellular sugars on transporter expression was transporter dependent, with expression of PsSUT1 inhibited, PsSUF1 unaffected, and PsSUF4 enhanced by sugars. Sugar supply to, and sugar concentrations of, RR cotyledons were manipulated using in vitro pod and cotyledon culture. Collectively the results obtained showed that intracellular sucrose was the physiologically active sugar signal that communicated metabolic demand to sucrose influx and this transport function was primarily determined by PsSUT1 regulated at the transcriptional level. - Most nutrients are imported into maternal seed tissues through the phloem and reach the symplasmically isolated filial tissues (endosperm/embryo) following their release to the seed apoplasm (Zhang et al., 2007). Import of sucrose, together with a spectrum of amino acids and amides, largely accounts for biomass gain of seed filial tissues (Patrick and Offler, 2001). Sugar and amino acid transporters, localized to filial cells that are juxtaposed to maternal tissues, take up these compounds from the seed apoplasm for subsequent symplasmic delivery to filial storage sites (Zhang et al., 2007). Demand for sugars and amino nitrogen compounds is set by biosynthetic capacities of processes responsible for their sequestration into fats, proteins, and starch (Borras et al., 2004). How this metabolic demand is communicated from filial storage sites to phloem import of nutrients into seed maternal tissues is poorly understood. At least for developing seeds of grain legumes, nutrient demand by filial tissues appears to be sensed by osmotically driven alterations in turgor pressures of nutrient release cells located in their coats (Zhang et al., 2007; but see Wang and Fisher, 1994; van Dongen et al., 2001). Here enhanced nutrient uptake by filial tissues decreases the osmolality of the seed * To whom correspondence should be addressed. E-mail: 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. apoplasmic sap with a consequent rise in seed coat cell turgor. If seed coat turgor exceeds a set point, activities of transporters responsible for nutrient release increase to meet filial demand. Under conditions of sustained nutrient demand, the turgor set point decreases to drive higher rates of phloem import (Zhang et al., 2007). A key element missing from the above turgor-homeostat model (Patrick, 1994) is the underlying mechanism that integrates storage product biosynthesis with activities of cotyledon transporters retrieving substrates from the seed apoplasmic space. The model predicts that nutrient uptake by cotyledons is regulated by rates of their intracellular consumption; a phenomenon consistent with observed sinklimited gains in seed biomass (Borras et al., 2004). Here it is envisaged that intracellular pool sizes of nutrients inversely reflect activities of biosynthetic enzymes. These pools function as signals to regulate activities of cotyledon transporters at the transcriptional level through substrate derepression of transporter gene expression. Tentative support for such a mechanism comes from the finding that expression of a sucrose/symporter (VfSUT1) was repressed by culturing Faba bean cotyledons for 3 d on medium containing elevated sucrose or glucose concentrations (Weber et al., 1997). However, the results from these studies (Weber et al., 1997) do not indicate whether the sugar signal was sucrose or its hydrolysis products and whether its action was mediated at the cotyledon plasma membranes or intracellularly. Moreover, the observed response may not be representative of in planta regulatory mechanisms. An opportunity to discover in planta sugar regulation of transporter activity is offered by the near-isoline of a pea mutant (rrRbRb) with a lesion at the rugosus (r) locus encoding starch branching enzyme 1 (SBEI; Bhattacharyya et al., 1990). This insertion results in wrinkled seeds containing cotyledons with reduced starch (;56%) and elevated sucrose levels (180%) compared with those of the round wild-type cotyledons (RRRbRb; Wang and Hedley, 1991). Indeed, rates of sucrose uptake by cotyledons from wrinkled and round seeded cultivars of pea are consistent with starch biosynthesis regulating sucrose transporter activity (Edwards and ap Rees, 1986). Net rates of in vitro sucrose uptake were found to be less for cotyledons of wrinkled seeds (Edwards and ap Rees, 1986) when excised cotyledons were incubated in media concentrations of sucrose at, or below, those of the seed apoplasmic space (i.e. <180 mM; Rosche et al., 2005). Whether this relationship is causal and mediated by de-repression of sucrose transporter gene expression awaits further study. Using differences in starch biosynthetic capacities of rrRbRb and RRRbRb cotyledons of pea (Wang and Hedley, 1991), the hypothesis that intracellular sugars function as signals to co-ordinate sink demand with sucrose uptake by regulating sucrose transporter activities was explored. The weight of current evidence points to regulation of sucrose transporter activity at the transcriptional (...truncated)