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Somatostatin receptor biology in neuroendocrine and pituitary tumours: part 1 – molecular pathways

Abstract Neuroendocrine tumours (NETs) may occur at many sites in the body although the majority occur within the gastroenteropancreatic axis. Non-gastroenteropancreatic NETs encompass phaeochromocytomas and paragangliomas, medullary thyroid carcinoma, anterior pituitary tumour, broncho-pulmonary NETs and parathyroid tumours. Like most endocrine tumours, NETs also express somatostatin (SST) receptors (subtypes 1–5) whose ligand SST is known to inhibit endocrine and exocrine secretions and have anti-tumour effects. In the light of this knowledge, the idea of using SST analogues in the treatment of NETs has become increasingly popular and new studies have centred upon the development of new SST analogues. We attempt to review SST receptor (SSTR) biology primarily in neuroendocrine tissues, focusing on pituitary tumours. A full data search was performed through PubMed over the years 2000–2009 with keywords ‘somatostatin, molecular biology, somatostatin receptors, somatostatin signalling, NET, pituitary’ and all relevant publications have been included, together with selected publications prior to that date. SSTR signalling in non-neuroendocrine solid tumours is beyond the scope of this review. SST is a potent anti-proliferative and anti-secretory agent for some NETs. The successful therapeutic use of SST analogues in the treatment of these tumours depends on a thorough understanding of the diverse effects of SSTR subtypes in different tissues and cell types. Further studies will focus on critical points of SSTR biology such as homo- and heterodimerization of SSTRs and the differences between post-receptor signalling pathways of SSTR subtypes.

Introduction
Somatostatin (SST) is a cyclic peptide and a notable physiological regulator of neuroendocrine function across multiple organ systems [1]. It is produced by the hypothalamus, throughout the central nervous system (CNS), and in different peripheral organs including gastrointestinal tract (GIT) and pancreas [1][2][3]. The SST gene is located in chromosome 3q28 and encodes a 116 amino acid preprohormone (preprosomatostatin) which contains the 92 amino acid SST prohormone (prosomatostatin) [4]. Prosomatostatin is the precursor peptide of the two biologically active SST forms: 14 amino acid long SST-14 and amino-terminus extended SST-28 [5]. The biological roles of the two SST isoforms strongly overlap and the relative proportions of SST-14 to SST-28 change between different tissues [6,7]. SST-14 is the predominant form in the brain (including the hypothalamus), whereas SST-28 is the major form in the GIT, especially the duodenum and jejunum [8].
SST has a broad range of biological actions including inhibition of exocrine secretions (gastric acid production, pancreatic enzyme, bile and colonic fluid secretion) and endocrine secretions in the pituitary (GH, TSH, prolactin), pancreas (insulin, gastrin, glucagon) and GIT (cholecystokinin, vasoactive intestinal peptide and secretin) [3,[9][10][11][12]. In the CNS, SST is highly expressed in the cortex, lateral septum, extended amygdala, reticular nucleus of the thalamus, hippocampus and many brain stem nuclei where it acts as a neurotransmitter and neuromodulator [8,13]. The hippocampal SST receptor (SSTR)4 modulates memory formation by diminishing hippocampus-based spatial function while enhancing striatum-dependent behaviour. SSTR4-mediated regulation of neuronal activity in the hippocampus appears to depend on both competitive and cooperative interactions with SSTR2 [14]. In a study by Craft and colleagues [15], SST analogue infusion was shown to improve memory in patients with Alzheimer disease. Moreover, SST is inhibitory on cell survival and angiogenesis and has anti-proliferative activity on normal and neoplastic pituitary cells [16,17], cancer cell lines [18,19] and tumours in experimental animals [20].
There are five SSTR subtypes, each encoded by five separate genes on five separate chromosomes: 14, 17, 22, 20 and 16, respectively [4,10,12]. Sequence homology is 39-57% among the five subtypes [12]. SSTRs belong to the G-protein coupled receptor (GPCR) superfamily and are extensively distributed throughout many tissues ranging from the CNS to the pancreas and gut, and also in pituitary, kidney, thyroid, lung and immune cells [19], besides their presence in various cancer cells. The majority of tumours (and also normal tissues) express SSTR2, followed by SSTR1, SSTR5 and SSTR3, whereas SSTR4 is the least expressed subtype [19,21,22]. There are two different isoforms of SSTR2 (SSTR2A and SSTR2B) produced via alternative splicing [23,24] and SSTR2B is almost unexpressed in human beings [9]. SSTRs bind natural peptides, SST-14 and SST-28, with similar high affinity (nM range) [10]. However, SSTR5 has a 10-fold higher affinity for SST-28 compared to SSTR2A or SSTR3, which have a higher affinity to .
Investigations on SSTR signalling and determining the specific function of each SSTR pose difficulty due to various factors in experimental settings. First, the different SSTR subtypes can coexist in the same tissue and even on the same cell at different densities, and which type of signalling becomes predominant in the given cell depends on the cell-specific distribution of SSTR subtypes and signalling elements [4]. Second, experimental studies on SSTRs are generally performed in cell lines from different species and the significance of the findings of these studies for normal and malignant human tissues remains to be determined. Fortunately, a remarkable degree of structural conservation across species has been reported for SSTRs [13]. SSTR1 is the most highly conserved, with 97% identity between human beings and rat, whereas the sequence of SSTR5 is the most divergent, with 81% identity between human beings and rat [13]. There is 92%, 86% and 89% identity between human and rat SSTR2, SSTR3 and SSTR4, respectively [6,26]. The

The somatostatin receptor as a GPCR
GPCRs are characterized by a core of seven transmembrane ␣helices connected by three intra-and three extracellular loops [4]. Binding of SST to its corresponding GPCR (SSTRs) triggers a cyclical activation and inactivation process in the G protein whereas the signal is transduced intracellularly. GPCRs are components of multiprotein networks, called 'receptosomes', which are organized around scaffolding proteins [28]. In this regard, GPCR-interacting proteins (GIPs), regulators of G-protein signalling (RGS) and GPCR kinases (GRKs) are the main proteins that are known to effect GPCR signalling after ligand binding. GIPs are transmembrane or cytosolic proteins which may alter either binding or functional responses of GPCR resulting in an abundance of potential receptor-protein connections [52]. RGS proteins have been found to regulate GPCR responses by binding to and stimulating the GTPase activity of the receptor-activated GTP-bound G␣ [53]. Although GIPs and RGS have been found to interact with distinct recombinant SSTRs [54,55], the real functional role of these complexes in native systems with respect to signal transduction is still not very well documented. SST binding is known to be rapidly followed by phosphorylation of SSTR1, SSTR2A and SSTR3 by GRKs [56][57][58]. The GRK family consists of six serine-threonine kinases that specifically bind to and phosphorylate agonist-activated GPCRs [59,60]. GRKs play key roles in the fundamental pathways leading to phosphorylation-dependent GPCR desensitization, endocytosis, intracellular trafficking and resensitization, as well as in the modulation of important intracellular signalling cascades by GPCR [61]. Receptor phosphorylation by GRKs results in the recruitment of cytoplasmic proteins called arrestins in a receptor subtype-specific manner [62][63][64]. Arrestins are proteins involved in intracellular vesicle trafficking. In general, binding of arrestins to an activated, phosphorylated GPCR blocks further interaction between the receptors and G proteins, and thus results in the desensitization of G-protein mediated signalling. In addition, arrestins play a key role in directing GPCRs to clathrin-coated vesicles and thus preparing them for endocytosis [65]. The con-sequent receptor internalization removes GPCRs from the cell surface so that they are no longer available for agonist stimulation. This is a second mechanism for GPCR desensitization, and the concentration of different arrestins as well as GRK subtypes influences the extent of receptor internalization [66]. However, not all SSTRs internalize equally after agonist binding [67,68]. SSTR2, SSTR3 and SSTR5 are internalized to a much higher extent than SSTR1 or SSTR4 after stimulation [25]. However, SSTR2 is rapidly recycled to the plasma membrane and does not enter any degradative pathway [25]. Desensitization and internalization are not induced by SSTR2 antagonists [69], but there is a growing body of evidence on agonist-induced desensitization and/or internalization of the SSTR2 subtypes, which have been assessed in several cell lines and tissues [21,67,70]. After activation and internalization through a clathrin-dependent pathway, SSTR3 and SSTR5 rapidly dissociate from arrestin and undergo ubiquitindependent lysosomal degradation, preventing plasma membrane recycling [64]. Arrestins have also been shown to play an important role in G-protein independent GPCR signalling by recruiting cytosolic molecules to the receptor-arrestin complex [71]. The precise role of receptor phosphorylation and arrestin binding in the desensitization, trafficking and signalling of different SSTR subtypes has not been fully elucidated as yet, but they probably play a critical role in SSTR function [65]. As desensitization is known to occur a few minutes after ligand binding, it is expected to have a strong impact in experimental settings in which second messenger systems are being evaluated [56,72,73].
Not only the nature of the SSTR subtypes present in a particular cellular environment but also the nature of the agonist is a critical determinant of the tissue response to SST [65]. In GPCRs, including SSTR signalling, a classical two-state model was accepted for a long time. This model proposed that the binding of an agonist shifted an equilibrium from 'the inactive', basal conformation of a receptor to 'the active' conformation by stabilizing the latter [74,75]. According to this model the relative potencies of agonists for inducing any two biological effects from a single receptor would be the same [74,75]. In studies evaluating receptor dynamics, analogue activities were generally retrieved from only one or two measurements; such as inhibition of hormone secretion or modulation of second messenger production [65]. Then these measurements were used to indicate the overall potency and efficacy of the analogues under investigation [65]. However, with this model it was not possible to explain the effect of many agonists which might induce only some of the possible responses following receptor activation, or to activate two effectors with different relative potencies [65]. New terms including 'functionally selective agonism' have been used to describe the idea that agonists can selectively activate different signalling pathways and responses via a single GPCR [65]. Described in detail in an elegant review by Schonbrunn, such selective signalling is explained by a model in which agonists not only exhibit different affinities for a receptor, but they also stabilize different active receptor conformations [65]. These differentially activated receptor structures determine the interaction of the receptor with the cytoplasmic proteins, including G proteins, kinases and phosphatases, and additionally its regulation by GRKs or arrestins [65]. Thus, this model predicts that in any given cell type, the relative potencies or efficacies of agonists for various biological effects may differ even though their actions are triggered by a single receptor subtype [65]. Meanwhile, it is clear that signalling by, and regulation of, SSTR2, SSTR4 and SSTR5, are sensitive to the nature of the specific activating agonist [65]. Thus, a basic and simple assumption that all agonists are functionally equivalent and produce the same spectrum of effects at individual SSTRs can not be accepted any longer [65].
Receptor homodimerization and oligomerization of SSTRs are other critical issues, as these processes have been shown to modify some properties of the receptors, such as ligand binding affinity, signal transduction, desensitization and up-regulation [30, [76][77][78]. It has been demonstrated that (excepting SSTR1) SSTRs transfected in different cell lines form homodimers and some form heterodimers with other SSTR subtypes and with different families of GPCR [29,30,79] (Table 1). Furthermore, GPCR dimerization is also regulated by ligand binding, yet not all receptors function similarly [80,81]. Even in the same family, the behaviour of receptors can be opposite, as reported for SSTR2 and SSTR5, whose dimerization decreases or increases, respectively, after ligand binding [82,83]. Interestingly, interactions among SSTRs, in other words heterodimerization between each other, seem to be highly selective, as not all the potential combinations occur. Nevertheless, not all the possibilities of SSTR heterodimers have been explored, because the interactions characterized so far are between murine SSTR2 and SSTR3 [76], human SSTR1 and SSTR5 [30], human SSTR4 and SSTR5 [84] and human SSTR2 and SSTR5, some being demonstrated under certain conditions [85]. Heterodimerization among these SSTR subtypes causes both distinct effects on SSTR functioning and modifies the endocytic process of these receptors [33]. An example for this may be the interaction between SSTR2 and SSTR3, which results in both inactivation and impaired internalization of SSTR3 [76]. Cotransfection of human SSTR1 and SSTR5 in Chinese hamster ovary (CHO)-K1 cells results in an increased ligand affinity for SST [30]. On the other hand, internalization of this heterodimer is better in response to a specific SSTR1 ligand and smaller in response to a specific SSTR5 ligand compared to monomer forms of these receptors [30]. Stabilization of human Somvanshi et al. [84] Heterodimerization between human SSTR4/human SSTR5, but not between human SSTR4/human SSTR1.
Grant et al. [85] SSTR2 and SSTR5 heterodimerize. Stabilization of human SSTR2 and SSTR5 heterodimers was shown to occur following selective activation of SSTR2 but not human SSTR5 or their concurrent stimulation. Heterodimerization increases the recycling rate of internalized SSTR2 by destabilizing its interaction with ‚-arrestin.
Sharif et al. [86] Increased recycling rate and a greater propensity of SSTR2 to signal and induce growth inhibition following its heterodimerization with SSTR5.
Baragli et al. [87] Interaction between the D2R and the human SSTR2 does not seem to occur under basal conditions, but is induced by ligand binding. SSTR2 pharmacology and signalling are not altered by heterodimerization with the D2R, but its endocytic rate is increased as a consequence of this interaction. Ligand interaction results in a heterodimer with an increased affinity for dopamine and increased signalling via the D2R.
SSTR2 and SSTR5 heterodimers was shown to occur following selective activation of SSTR2 but not human SSTR5 or their concurrent stimulation [85]. Moreover, an increased recycling rate and a greater propensity of SSTR2 to signal and induce growth inhibition following its heterodimerization with SSTR5 were observed [86]. An interaction between the D2R and the human SSTR2 has been also reported, which does not seem to occur under basal conditions, but is induced by ligand binding [87]. Their interaction results in a heterodimer with an increased affinity for dopamine and increased signalling via the D2R receptor [87]. However, SSTR2 pharmacology and signalling are not altered by heterodimerization with the D2R, but its endocytic rate is increased as a consequence of this interaction [87].

Anti-tumour effects of somatostatin
There are a number of mechanisms responsible for the antitumour actions of SST [88]. These are the direct blockade of cell cycle progression through the activation of phosphotyrosine phosphatases (PTPs), the indirect influence on tumour growth mediated by the inhibition of the production of growth factors that sustain tumour development, and an anti-angiogenic effect that involves the regulation of the activity of both endothelial cells and monocytes. Some of the key points in direct and indirect antitumour actions of SST will be summarized in this section. In general, SSTR1, SSTR2, SSTR4 and SSTR5 produce cytostatic effects through similar downstream effector pathways, whereas SSTR2 and SSTR3 induces pro-apoptotic (cytotoxic) signals when activated [79,89,90] (Table 2) (for detailed information see next section). SST suppresses insulin-like growth factor (IGF)-I serum levels through a direct inhibition of its gene expression or through the inhibition of GH secretion from pituitary and the consequent reduction of the GH-stimulated IGF-1 production in liver. Moreover, SST analogues inhibit the secretion of autocrine/paracrine effectors of tumour cell survival such as the IGF-1 and -2, EGF, interleukin-6 and the transforming growth factor family. The attenuation of secretion of such survival factors in the tumour microenvironment accordingly establishes an autocrine/paracrine anti-proliferative effect [4].
Stable transfection of pancreatic cancer cell lines with human SSTR2A was reported to induce an overexpression of the connexins 26 and 43, which resulted in formation of functional intercellular gap junctions and hence restored contact inhibition of cell proliferation [91].
During the initial stages of metastatic development, malignant cells must enter the lymphatic and systemic circulation by detaching from adjacent cells and then attaching to and disrupting the endothelial basement membrane [92,93]. SST has been shown to reduce the adhesion of carcino-sarcoma cells to blood vessels and thus attenuate the metastatic potential of these tumours [94]. For the anti-angiogenic activity of SSTRs, three signal pathways were identified: inhibition of endothelial cell activity (proliferation, migration and invasion), inhibition of the synthesis and secretion of pro-angiogenic factors such as vascular endothelial growth factor and basic fibroblast growth factor, and inhibition of monocyte activation. Very recently, thrombospondin-1 was defined as a critical effector of the inhibitory role of SSTR2 on the neoangiogenesis and oncogenesis induced by pancreatic cancer cells [95].

Post-receptor signalling pathways
Studies in the signal transduction field have demonstrated that native SST inhibits the secretion and proliferation of both normal and neoplastic pituitary cells by inducing several intracellular pathways, depending on receptor subtype and target tissue [96].  [6,68,79,97]. Although there are a number of specific receptor agonists, some agonists used in experimental settings can activate two receptors at the same time with different levels of affinities, which makes it difficult to clarify receptor-specific effects. [98].

Phosphotyrosine phosphatases and their action on MAPK and PI3K/Akt pathways
In the human genome around 107 PTPs have been identified, which includes 38 so-called 'classical PTPs' with an elevated specificity for phosphotyrosines [99]. (Fig. 1), and control of the activity of another important downstream signalling pathway, PI3K/Akt (Fig. 2) [101]. The inhibition of the ERK 1/2 pathway can occur either indirectly via the inhibition of the growth factor tyrosine kinase receptors or directly by dephosphorylating ERK 1/2 or by hyperactivation of ERK 1/2 (see below) [102,103] [104][105][106][107], for DEP-1 with PDGF and the vascular endothelial growth factor receptor [108,109] and for PTP1B with the EGF receptor [110].

. Such altered growth factor signalling through the selective dephosphorylation and inactivation of their receptors have been reported for SHP-1 with the insulin receptor, for SHP-2 with the insulin, EGF and platelet-derived growth factor (PDGF) receptor
SSTR activation of SHP-1 was reported to induce arrest of cell proliferation in human pituitary adenomas [111], GH3 rat pituitary tumour cells [112], in different tumour cell lines derived from pancreatic cancers (MIA-PaCa-2, PANC-1, PC-1) and from thyroid medullary carcinoma, among others [79,101,[113][114][115]. SHP-1 activation is the critical step for SSTR2-mediated anti-proliferative signalling [101,116]. Tyrosine phosphorylated SSTR2 interacts with and activates SHP-2 and Src, a cytosolic tyrosine kinase, inducing consequent SHP-1 recruitment and activation [117] (Fig. 1) [122]. Of interest in this regard is the finding that SSTR2 also affects the apoptosis induced by death receptors, sensitizing the cells to tumour necrosis factor (TNF)-␣-and TRAIL-mediated responses through the up-regulation of their receptors: death receptor 4 and TNFR1 [119]. In essence, SHP-1 activity was also involved in SSTR3dependent apoptosis in transfected CHO cells, but this time by involving the induction of p53 and the subsequent activation of the pro-apoptotic protein Bax [123]. In GH3 cells, SSTR2-induced activation of SHP-1 causes the dephosphorylation of p85 and hence the inhibition of PI3K activity [101]. As would be expected, inhibition of PI3K activity causes inhibition of PDK1 and Akt activities, which in turn results in the activation of the glycogen synthase kinase-3␤ (GSK-3␤) (Fig. 2). Theodoropoulou and colleagues have reported that the enhanced GSK-3␤ activity up-regulated the expression of Zac1 gene, which ultimately induced growth arrest [101]. Zac1 is highly expressed in normal pituitary, mammary and ovarian glands but is down-regulated in pituitary, breast and ovarian tumours, suggesting that it might act as a tumour suppressor gene [101]. Their further study investigating the immunoreactivity of Zac1 protein in tumour tissues of patients treated with SST analogues prior to surgery has also revealed a significantly positive correlation between strong Zac1 immunoreactivity and IGF-I normalization and the presence of tumour shrinkage after SST analogue treatment [124].

. In CHO cells, SST-activated SHP-1 rapidly associates to the insulin receptor causing a tyrosine dephosphorylation of both the receptor itself and its substrates (i.e. IRS-1, Shc) leading to a negative modulation of insulin mitogenic signalling [106]. Additionally, SHP-1 inhibits the ERK 1/2 pathway by directly or indirectly dephosphorylating ERK 1/2 [4], causes overexpression of the cyclin-dependent kinase inhibitor p 27kip1 and an increase in hypophosphorylated retinoblastoma gene product (Rb) which, taken together, leads to inhibition of the entry in the S phase of the cell cycle and accumulation of the cells in G1 [118]. In pancreatic and pituitary tumour cells SSTR2 activation of SHP-1 was found to be involved in the p53-independent induction of apoptosis [119-121]. Additionally, a completely novel mechanism was identified in NIH3T3 (NIH Swiss mouse embryonic fibroblast cell line) cells in which the activation of SHP-1 by SSTR2 activates the transcription factor nuclear factor-B causing an inhibition of the antiapoptotic effects of the MAP kinase, Jun N-terminal kinase and, in turn, hyperactivation of caspase 8 and apoptosis
The other PTP, SHP-2, was involved in the anti-proliferative activity of SST following SSTR1 [102,125], SSTR2, SSTR3 and SSTR4 activation [126]. Besides dephosphorylating and inactivating the tyrosine kinase receptors for insulin and EGF [104,107], SHP-2 is involved in cell growth arrest [125] (Fig. 1). It is known that the effects of ERK 1/2 on cell proliferation are also related to the duration and intensity of ERK 1/2 activation, and hyperactivation of the ERK 1/2 pathway can also cause cell cycle arrest [127]. In light of this knowledge, it is now known that the activation of both SSTR1 and SSTR2 induces cell cycle arrest via the hyperactivation of ERK 1/2 and the up-regulation of p21 cip1/waf1 and p27 kip1 , respectively [102,103]. In particular, the activation of SSTR1-induced ERK 1/2 pathway involved Src/SHP-2/PI3K/ras/Raf-1/MEK (mitogen-activated protein) [102], the  [103] while, on the contrary, SSTR3 activation caused Raf-1 inactivation and blockade of the ERK 1/2 cascade [128,129].

SSTR2-regulated pathway involved SHP-1/SHP-2/PI3K/rap1 and ras/B-Raf/MEK
In addition, besides SHP-1 and SHP-2 activity, another delayed and long-lasting PTP activity was also induced following SST treatment [125]. As opposed to SHP-1 and SHP-2 which are cytosolic, non-membrane PTPs, one of these PTPs was identified in a receptor like PTP named DEP-1 in human beings (PTP in rats), whose tumour suppressor role had already been established [130]. It was suggested that, in endothelial cells, DEP-1/PTP represents a possible effector of SSTR to inhibit tumoral angiogenesis and the activity of DEP-1 seems fundamental in blocking endothelial cell migration and proliferation [109,131,132]; the in vivo and in vitro anti-angiogenic activity of SST was dependent on the activation of PTPs [133,134]. SSTR1-activated DEP-1 was shown to inhibit ERK 1/2 in PC C13 thyroid cells and SSTR1-, SSTR2-and SSTR5-activated DEP-1 was shown to inhibit ERK 1/2 in glioma cells [135]. In CHO-K1 cells expressing SSTR1, a large multimeric protein aggregation composed of the G protein, Jak2,  [136]. DEP-1 activation required the sequential activation of Jak2 (G-protein mediated), which phosphoryated SHP-2. When phosphorylated, SHP-2 shows increased activity, dissociates from the receptor and dephosphorylates the inhibitory tyrosine on Src which, activated this way, in turn, phosphorylate DEP-1 causing the sustained activity of this PTP to inactivate ERK 1/2 [136]. As activation of similar multieffector cascades by SSTR1 and SSTR2 involving a similar interaction of kinases and PTPs (Jak2, SHP-2, Src) leads to final PTP activation (SHP-1 or DEP-1), it has been suggested that this multieffector pathway could represent a common modular mechanism by which cytostatic mechanisms are induced by SST [117,136].

Anti-secretory action of SST through cAMP, K ϩ and Ca ϩϩ channels
The inhibitory effects of SST on cAMP production, and its effects on K ϩ and Ca ϩϩ currents, are mainly involved in the regulation of hormone secretion in different endocrine cells [19]. Decreased intracellular cAMP and Ca ϩϩ levels and increased outward K ϩ currents all lead to decreased hormone secretion [79]. Starting with cAMP, the negative modulation of AC activity leads to the regulation of cAMP production. This also has a negative impact on various downstream elements, in particular, protein kinase A, which is the major target for cAMP in the cell. The inhibition of AC by SST has been demonstrated in many different systems, including human pituitary adenomas [137]. All SSTR subtypes inhibit AC via the pertussis toxin-sensitive G-protein family, Gi/Go [65]. Specifically, SSTR2 decreased AC activity/cAMP accumulation in BON-1 (pancreatic NET cell line) and SH-SY5Y (neuroblastoma cell line) cells [138,139] and in pituitary cells [140]. The stimulation of SSTR3 in CHO and HEK-293 cells decreases AC activity [141,142]. SSTR5 decreased cAMP levels in human SH-SY5Y and BON-1 cells [138,139] and in AtT-20 mouse pituitary corticotroph cells [34,[143][144][145].
SSTRs have been shown to directly (via G-protein activation of the K ϩ channel) or indirectly (via G-protein activation of second messenger pathways, which ultimately lead to activation of the K ϩ channel) activate a number of different K ϩ channels and increase K ϩ currents, leading to cell membrane hyperpolarization and a reduction in action potential frequency and duration [13,68,146,147]. Specifically, SSTR2 (more efficiently than the other subtypes), SSTR3, SSTR4 and SSTR5 were shown to activate the Gprotein gated inward-rectifier K ϩ channels [148]. However, in rat GH3 cells, SSTR1, SSTR2, SSTR4 and SSTR5 are all involved in the activation of delayed rectifying and transient outward K ϩ channels, although SSTR2 and SSTR4 were suggested to mediate the full activation of K ϩ current caused by SST [149].

Other post-receptor pathways
Following the evidence showing SST-induced increase of inositol 1,4, in bovine adrenal medullary cells [153], it is now widely accepted that SSTRs may affect the activity of phospholipase C (PLC) in native systems and, hence, modulate intracellular levels of IP3 and/or the activity of PLC-dependent protein kinase (PKC). SSTR1 was shown to mediate phospholipase PLC activation and IP3 production in CHO cells [154,155]. SSTR2 mediates the activation of PLC in GH4C1 (rat pituitary tumour cell line) and F4C1 (rat pituitary cell line) cells [150,154,156]. Activation of PLC and IP3 production by SSTR4 was also observed in transfected COS (monkey kidney cell line) cells [157].
Phospholipase A2 (PLA2) is a cytoplasmic enzyme that hydrolyses triglycerol to form arachidonic acid, the metabolites of which have been shown to regulate different physiological and pathological functions [158,159]. SST coupling negatively to arachidonate release has been shown in the rat anterior pituitary gland [160]. In contrast, SST has been suggested to generate arachidonic acid metabolites in rat GH4C1 cells, through pertussis toxin-sensitive G proteins [161]. Nevertheless, in a few cases SST was reported to induce cell proliferation through SSTR4 activation, which was observed to increase ERK 1/2 activity and, in turn, regulate PLA2 activation [162].
Moreover, also other MAPKs, more frequently associated to the induction of cell growth arrest, are regulated by SST: p38 is activated by SSTR2 and SSTR4 in CHO-K1 cells [98] and Jun Nterminal kinase is activated by SSTR5 [163].
SST was reported to regulate nitric oxide generation through the activation of both the endothelial and neuronal nitric oxide synthases (eNOS and nNOS, respectively). However, SST has been shown to have a dual effect on the release of nitric oxide. In different paradigms it has been shown to increase or inhibit nitric oxide levels via the involvement of different receptor subtypes and intracellular pathways [164]. SSTR2 caused a SHP-1-dependent dephosphorylation and activation of nNOS in mouse pancreas acini cells, which leads to increased nitric oxide production [165]. Nitric oxide is a major regulator of soluble guanylyl cyclase [28]. Guanylyl cyclase converts GTP to cyclic nucleotide guanosine monophosphate (cGMP), which is a regulatory mediator of cell proliferation [166]. The increased nitric oxide production in these cells results in activation of cGMP levels which is believed to be instrumental in growth arrest [164,165]. cGMP exerts its effects via modulation of cGMP-dependent protein kinase (PKG), cGMPgated ionic channels and cyclic nucleotide phosphodiesterases [167,168]. Conversely, in a study by Cordelier and colleagues, inhibition of nNOS activity and a reduction in intracellular cGMP formation which resulted in cell growth arrest was observed in CHO cells expressing SSTR5 [169]. It was noted in this study that SSTR5 inhibits cell proliferation through SSTR5-mediated Src activation and subsequent nNOS tyrosine phosphorylation and inactivation, leading to a decrease of cGMP production and subsequent MAPK inhibition [166,169]. SST-induced negative regulation of nitric oxide was also shown to be implicated in the inhibition of tumour angiogenesis and growth via the SSTR3-mediated negative regulation of eNOS [170].
The inhibition of Na ϩ -H ϩ (NHE1) exchanger activity is responsible for anti-proliferative effects of SST in enteric endocrine cells and hepatic cells [79]. These effects are mediated by SSTR1, SSTR3 and SSTR4. Interestingly, the inhibition of NHE1 is the only intracellular signalling regulated by SSTRs that was reported to be pertussis toxin insensitive [171].
Involvement of serine-threonine phophatase in SST action was also implied in a study by Grozinsky-Glasberg and colleagues. In this study in INS-1 rat insulinoma cell line, both octreotide and the mTOR inhibitor everolimus (RAD001, Novartis, Basel, Switzerland) were shown inhibit cell proliferation and dephosphorylate tuberous sclerosis 2 (TSC2), which could imply activation of a serinethreonine phosphatase by these agents [172]. We


extfieldsofstudy: Chemistry Biology Medicine

text:
Tricarboxylic acid cycle activity regulates tomato root growth via effects on secondary cell wall production.

Transgenic tomato (Solanum lycopersicum ‘Moneymaker’) plants independently expressing fragments of various genes encoding enzymes of the tricarboxylic acid cycle in antisense orientation have previously been characterized as exhibiting altered root growth. In this study, we evaluate the rates of respiration of roots from these lines in addition to determining their total dry weight accumulation. Given that these features were highly correlated, we decided to carry out an evaluation of the cell wall composition in the transformants that revealed a substantial reduction in cellulose. Since the bulk of cellulose is associated with the secondary cell walls in roots, we reasoned that the transformants most likely were deficient in secondary wall cellulose production. Consistent with these findings, cross-sections of the root collar (approximately 15 mm from the junction between root and stem) displayed reduced lignified secondary cell walls for the transformants. In contrast, cell and cell wall patterning displayed no differences in elongating cells close to the root tip. To further characterize the modified cell wall metabolism, we performed feeding experiments in which we incubated excised root tips in [U-14C]glucose in the presence or absence of phosphonate inhibitors of the reaction catalyzed by 2-oxoglutarate dehydrogenase. Taken together, the combined results suggest that restriction of root respiration leads to a deficit in secondary cell wall synthesis. These data are discussed in the context of current models of biomass partitioning and plant growth.

Metabolism is arguably the best characterized of all molecular interaction networks in biology. From pioneering studies defining key metabolic pathways, such as the Calvin cycle, through decades of enzymology and more recent genetic studies of metabolism, there is an unprecedented density of both mechanistic and descriptive data relating to metabolic behavior . However, many pathways and processes have been studied in isolation, and the relative importance of primary plant metabolism to the sustenance and regulation of either anabolic polymer production or secondary metabolism has re-ceived relatively scant attention. Indeed, publications directly addressing such questions are few and instead tend to concentrate on the direct (primary) precursors of specific pathways (Amor et al., 1995;Cronin and Hay, 1996;German et al., 2003;Ruan et al., 2003;Reiter, 2008). For example, coordination of primary metabolism and cell wall synthesis has largely focused on the immediate precursors, such as different nucleotide sugars (Amor et al., 1995;Ruan et al., 2003;Reiter, 2008).
The importance of a basic understanding for cell wall synthesis is reflected in the recent financial incentives to maximize the conversion of solar energy into ethanol-based biofuels (Somerville, 2006;Carpita and McCann, 2008;Heaton et al., 2008;Lopez-Casado et al., 2008;Pauly and Keegstra, 2008;Yuan et al., 2008;Carroll and Somerville, 2009). Current efforts are largely focused on the conversion of the lignocellulosic matrix to ethanol by improving the extraction of sugarbased molecules or through reductions of the polyphenolic polymer lignin (Carroll and Somerville, 2009). However, it appears that only limited success will be obtained through genetic manipulations of enzymes directly associated with specific cell wall polymers. For example, substantial reductions in one polymer typically result in unwanted growth phenotypes or compensation by other polymers (His et al., 2001;Pena et al., 2007;Persson et al., 2007;Schilmiller et al., 2009). Such outcomes are usually unwanted traits and moreover are difficult to predict. An alternative route is to alter whole cassettes of genes or perturb the ratio of precursor levels for the different cell wall polymers. Such changes may be achieved, for example, by alterations in nucleotide-sugar ratios (Seifert, 2004;Sharples and Fry, 2007). That said, it can be envisaged that these types of changes could also be obtained by changes in upstream metabolic fluxes.
While it may be difficult to directly link changes in biomass with single primary metabolites, it is evident that certain metabolic signatures can signify changes in plant biomass (Meyer et al., 2007;Sulpice et al., 2009). However, no large-scale study has assessed the role of cell wall composition in this context. Several reverse genetic studies have revealed clear correlations between energy metabolism and plant growth. For example, manipulations of apoplastic and cytosolic apyrase and plastidial adenylate kinase activities have all recently been documented to yield clear growth phenotypes (Regierer et al., 2002;Riewe et al., 2008aRiewe et al., , 2008b. In addition, suppression of several enzymes associated with the tricarboxylic acid (TCA) cycle also affects aerial plant growth (Carrari et al., 2003;Studart-Guimarães et al., 2007;Sienkiewicz-Porzucek et al., 2008;van der Merwe et al., 2009). These data, and those coming from plants exhibiting deficiencies in photosynthesis per se (Gifford et al., 1984;Stitt and Schulze, 1994;Henkes et al., 2001) as well as data documenting conditional growth deficiencies of mutants of photorespiration (Somerville and Ogren, 1980;Wingler et al., 2000;Voll et al., 2006;Timm et al., 2008;Foyer et al., 2009), suggest that plant growth is restricted by cellular energy availability. Interestingly, tuber-specific down-regulation of plastidial adenylate kinase (Regierer et al., 2002) or cytosolic UMP synthase (Geigenberger et al., 2005) in potato (Solanum tuberosum) both resulted in increased tuber growth. In the latter case, the reduction in UMP synthase also led to elevated cell wall constituents. Consequently, a possible explanation for reduced root growth in TCA cycle-deficient tomato (Solanum lycopersicum) plants may be that the root cannot produce enough cell wall to sustain growth. To test this, we took two independent approaches. First, we evaluated a range of previously characterized transgenic lines Studart-Guimarães et al., 2007;Sienkiewicz-Porzucek et al., 2008) as well as one novel set of transgenics that affect different enzymes of the TCA cycle. Second, we utilized a phosphonate analog of 2-oxoglutarate, which has been demonstrated to produce a rapid inhibition of the TCA cycle through a specific effect on the reaction catalyzed by the 2-oxoglutarate dehydrogenase complex (Bunik et al., 2005;Araú jo et al., 2008), in order to ascertain which altered secondary cell wall phenotype could directly be ascribed to the inhibition of the TCA cycle itself. The results are discussed in terms of current models linking plant energy metabolism with biosynthesis and growth.

Generation of Aconitase Antisense Lines
Although we have previously shown that the aco1 mutant of the wild species tomato Solanum pennellii  displays reduced root growth (Carrari et al., 2003), which was similar to the subsequent phenotypes observed by reducing the activities of malate dehydrogenase, fumarase, succinyl-CoA ligase, and citrate synthase in cultivated tomato Studart-Guimarães et al., 2007;Sienkiewicz-Porzucek et al., 2008), we decided to create aconitasedeficient transgenics of the cultivated tomato to afford a more uniform comparison. For this purpose, we cloned an 800-bp fragment of the S. lycopersicum aconitase antisense orientation into the vector pBINAR (Liu et al., 1999) between the cauliflower mosaic virus 35S promoter and the octopinsynthase (ocs) terminator and transformed this into plants using an Agrobacterium tumefaciens-mediated protocol. Primary transformants were selected on kanamycin and transferred to the greenhouse, where they were screened at the level of leaf aconitase activity, with 32 lines being chosen for further studies, and propagated in tissue culture (Fig.  1). Having generated these additional lines, we then grew them and the previously generated transformants in the greenhouse alongside wild-type controls across a series of four different plantings. Characterization of the roots of the aconitase lines (ACO) and the antisense lines for succinyl-CoA ligase (SCoAL), fumarase (FL), mitochondrial citrate synthase (CS), and mitochondrial malate dehydrogenase (mMDH), confirmed a reduction in the maximal catalytic activities of the enzyme of interest within the tissue ( Table I).

Characterization of Root Respiration and Root Growth
Having established that the transgenic material was appropriate for our biological questions, we next characterized the rate of respiration in the roots. For this purpose, we took two complementary approaches. First, we directly characterized the rate of oxygen consumption in excised root tips (the terminal 10 mm of the root tips) of 5-week-old plants in a Clarke-type electrode. These experiments demonstrated that oxygen consumption was significantly reduced in all transgenic lines, with the exception of Respiration experiments were carried out as described by Palmieri et al. (2008). Values marked with different letters differ significantly from each other (P , 0.05). ANOVA was done between transgenic lines and their corresponding wild type (WT). FW, Fresh weight. lines FL11 and SCoAL25, as assessed using ANOVA across the entire data set (Fig. 2). In a complementary approach, we performed tracer experiments following the incubation of similarly aged root tips in unilabeled [ 13 C]Glc. Following quenching of metabolism and extraction, the enrichment of individual metabolites was determined by the quantification of 13 C enrichment following incubation in [ 13 C]Glc for 5 h using gas chromatography-mass spectrometry (GC-MS). These studies revealed a significant decrease in citrate labeling in the lines FL11 and FL41, mMDH21, and ACO38, while a decrease in the fractional enrichment of malate was additionally observed in FL11 and FL41 (Table II). Perhaps surprisingly, these were the only observable changes in the level of enrichment of organic acids. However, this may be due to the well-known extensive compartmentation of these metabolites (Stitt et al., 1989;ap Rees and Hill, 1994).
To characterize the effect of the down-regulated TCA cycle on root growth, we evaluated the root biomass ( Fig. 3; Supplemental Fig. S1). The data revealed a clear decrease in total root biomass for the transgenics compared with controls when assessed by ANOVA across the entire data set. Interestingly, the variance in root respiration and root biomass strongly correlated when plotted against each other (i.e. the reduction in root biomass appears to be directly related to the rate of respiration; Fig. 4).

Metabolite Levels and Cell Wall Composition of the Transformants
We next decided to evaluate the content of carbohydrates in the roots from 5-week-old plants. Soluble sugars and starch were extracted and measured (Table  III). These studies revealed that the aconitase lines had increased levels of Fru, Suc, and Glc (significantly only in lines ACO19 and ACO38), and succinyl-CoA ligase lines had increased levels of Glc (significantly in lines SCoAL25 and SCoAL47). By contrast, the mitochondrial citrate synthase lines were invariant in all these parameters, with only a single significant difference being observed. However, these changes were not consistent across the lines and therefore cannot be the cause for the reduction in growth, since we have, by contrast, previously reported that mitochondrial malate dehydrogenase and fumarase transgenic lines  Interestingly, the level of cellulose was substantially reduced in the fumarase and mitochondrial malate dehydrogenase lines both when assessed by ANOVA across the entire data set (Fig. 5). Similar trends were also observed for all the transgenic lines (Fig. 5). In addition, the neutral sugar composition was unaltered in the mutant lines as assessed by alditol acetate analyses using GC-MS (Table IV), suggesting no changes in hemicellulose polymer.
The substantial decrease in cellulose suggests a dramatic reduction in cell wall synthesis. To assess whether these reductions altered the cell morphology or cell wall integrity, we made root tips and root collar crosssections of the transgenic tomato lines and compared these with the wild type. We used the dye phloroglucinol to visualize polyphenolic polymer lignin, which is a major component of the secondary cell wall ( Fig. 6; Ros- Barcelo, 1997). Interestingly, the wall for the mMDH and FL lines appeared to have a reduction in lignin content, particularly prominent in the case of lines MDH8 and FL11 (Fig. 6). In addition, several transgenic root cross-sections displayed irregular xylem vessels, which have previously been described in secondary cell wall-deficient mutants (Brown et al., 2005;Fig. 6). However, no, or very minimal, effects were observed in the CS, ACO, and SCoAL lines (data not shown). It is important to know that the mMDH and FL enzymes comprise the last steps of the TCA cycle and are responsible for the regeneration of acetyl-CoA for subsequent use in gluconeogenesis. In contrast, the CS, ACO, and SCoAL enzymes represent the first three steps in the cycle and therefore draw on the acetyl-CoA pool. It is evident, therefore, that despite the reduced root biomass and the underdeveloped xylem, the root is still able to sustain growth.

Analysis of the Effects of Chemical Inhibition of the TCA Cycle on Cellular Metabolism in Roots
To further characterize metabolism in the transgenics, we next performed feeding experiments in which we incubated excised root tips in a buffered solution containing 10 mM [U-14 C]Glc in the presence and absence of phosphonate inhibitors of the reaction catalyzed by 2-oxoglutarate dehydrogenase for a period of 2 h. The rationale of this experiment was that the phosphonate inhibitors succinyl phosphonate and carbonyl ester of succinyl phosphonate have been documented to be potent and specific inhibitors of 2-oxoglutarate dehydrogenase in widely divergent species (Bunik et al., 2005;Bunik and Fernie, 2009). Moreover, inhibition of this activity has previously been reported to have dramatic consequences on plant respiration, even following short-term application (Araujo et al., 2008), which would not be complicated by secondary effects of a long-term inhibition of energy metabolism. In order to assess whether a sudden block of the metabolite flux through the TCA cycle would result in reduced cell wall material, we applied   (Fig. 7A), metabolism (Fig. 7B), and 14 CO 2 evolution (Fig. 7C), which were more severe in incubations in which a higher concentration of inhibitor was used. Moreover, despite unaltered accumulation in organic acids (Fig. 7D), similar reductions of radiolabel redistribution to protein ( Fig. 7E) and cell wall (Fig. 7F) and increase of radiolabel redistribution to phosphoesters (Fig. 7G) to those observed in potato tuber were apparent. In this study, we estimated absolute fluxes following calculation of the specific activity of the hexose phosphate pool (Fig. 7H). The results of these calculations revealed that the rates of starch (Fig. 7I) and Suc synthesis (Fig. 7J) were essentially unaltered, as was the rate of glycolysis (Fig. 7K). The rate of cell wall synthesis, however, was significantly reduced (Fig. 7L), thus suggesting that inhibitors of the TCA cycle have a direct and immediate effect on cell wall biosynthesis.

Deficiency in Expression of Genes Encoding Enzymes of the TCA Cycle Results in Dramatic Decreases in Respiration and in Root Growth
Unsurprisingly, reduction in the activity of TCA cycle in the root has a far greater impact on respiration in the root than in the leaf, which is able to compensate for deficiencies in the TCA cycle by means of both photosynthetic and photorespiratory processes (Krö mer et al., 1993;Krömer, 1995). It is possible that alternative electron donors of the mitochondrial electron transport chain exist in heterotrophic tissues, such as the reactions catalyzed by galactolactone dehydrogenase and the electron transfer flavoprotein complex. However, evidence of their operation in plants is, as yet, confined to photosynthetic tissues (Ishizaki et al., 2005(Ishizaki et al., , 2006Nunes-Nesi et al., 2005;Alhagdow et al., 2007). That said, it would appear likely that the respiratory bypass pathways function in heterotrophic tissues even if the pathways of photosynthesis and photorespiration do not. The results from the direct measurement of oxygen consumption were largely, although not entirely, in keeping with flux determinations, which suggested a decreased redistribution of label to both citrate and malate. However, evaluation of such data is extraordinarily difficult, given the complex subcellular distribution of the organic acid pools of plants (Stitt et al., 1989;ap Rees and Hill, 1994). The reduced rate of respiration observed in the transformants was closely coupled to a decreased root biomass when assessed on a dry weight basis, a fact that we believe is most likely due to the decreased energy status and production of precursor metabolites to support biosynthesis.

Deficiency in Expression of Genes Encoding Enzymes of the TCA Cycle Results in Metabolic Shifts That Influence Cell Wall Accumulation and Composition
We document here that the restriction in TCA cycle activity in the transformants provokes considerable changes in the levels of cellulose suggestive of a markedly altered secondary cell wall. In keeping with this hypothesis, cross-sections of the root collar of the mMDH and FL lines, MDH8 and FL11, clearly contained less lignin as assessed by phloroglucinol staining. However, differences were neither observed in cell organization nor in cell wall patterning of cells in the root elongation zone. It is conceivable that these reductions in cellulose and lignin lead to a decreased root dry biomass, such as that previously described in Arabidopsis cellulose synthase mutants (Taylor et al., 1999(Taylor et al., , 2000(Taylor et al., , 2003; however, further experiments would be required to categorically prove this hypothesis. In order to further investigate this observation, we followed the metabolic fate of [U-14 C]Glc supplied exogenously to excised root tips of the wild-type tomato in the presence or absence of potent inhibitors of the reaction catalyzed by 2-oxoglutarate dehydrogenase. The short-term nature of this inhibition effectively allows us to discriminate between the primary and secondary features of the transgenics, since it removes the aspect of long-term adaptations. As was previously observed in potato tubers (Araujo et al., 2008), the inhibitor clearly results in a dramatic reduction of respiration. Intriguingly, estimating absolute fluxes in the root system revealed a depressed rate of cell wall synthesis but unaltered rates of glycolysis, starch, and Suc biosynthesis. This finding suggests that at times when energy is scarce, the root cell prioritizes maintenance of primary metabolic pathways that support energy generation and storage above those that support other processes. This finding is not without precedence, since studies of the metabolic architecture of both tomato and Arabidopsis cell suspension cultures came up with similar conclusions (Rontein et al., 2002;Williams et al., 2008). Our results thus provide strong evidence that the inhibition of secondary cell wall biosynthesis in the transgenics is a direct consequence of the reduced TCA cycle activity and support our initial contention that this process is energy limited in heterotrophic plant tissues. Further- Figure 7. Effect of SP or CESP on metabolism of [U-14 C]Glc by root tips. Root tips were preincubated in 10 mM MES-KOH buffer, pH 6.5, containing 2 mM Glc in the absence (control) or presence of varying concentrations of succinyl phosphonate (SP) or carbonyl ester of succinyl phosphonate (CESP) for 1 h, then [U-14 C]Glc (specific activity, 8.11 MBq mmol 21 ) was added and incubated for 2 h. Each sample was extracted with boiling ethanol, and the amount of radioactivity in each metabolic fraction was determined as described in "Materials and Methods." Values are expressed as percentages of the total radiolabel metabolized and are means 6 SE of four biological replicates. Asterisks demarcate values that were judged to be significantly different from the control (P , 0.05). FW, Fresh weight.
van der Merwe et al. more, we believe that this work highlights a novel link between primary metabolism and secondary cell wall synthesis that nicely complements prior work focusing on nucleotide sugar partitioning. This fact notwithstanding, it is clear that teasing out the precise mechanisms linking these processes will be of high future interest at both fundamental and applied levels.

Plant Material
Previously characterized tomato (Solanum lycopersicum 'Moneymaker') seeds, exhibiting reduced expression of mitochondrial malate dehydrogenase, fumarase, succinyl-CoA ligase, and citrate synthase Studart-Guimarães et al., 2007;Sienkiewicz-Porzucek et al., 2008), were sterilized and germinated on MS medium (Murashige and Skoog, 1962). Plants were grown in a long-day regime at day/night temperatures of 22°C/ 20°C and a relative humidity of 50% on a vermiculite medium supplemented with slow-release fertilizer (Lewatit HD 50; Beyer). The presence of the transgene was verified by PCR using standard protocols, prior to analysis of enzyme activities of the protein in question.

Chemicals
Unless stated otherwise, all chemicals, cofactors, and enzymes were purchased either from Sigma-Aldrich or Merck. Phosphate analogs were synthesized at the Chemical Department of Moscow Lomonosov State University according to Bunik et al., (2005).

cDNA Cloning and Expression
An 800-bp fragment of aconitase was cloned in antisense orientation into the vector pBINAR (Liu et al., 1999) between the cauliflower mosaic virus 35S promoter and the ocs terminator. This construct was introduced into plants by an Agrobacterium tumefaciens-mediated transformation protocol, and plants were selected and maintained as described in the literature (Tauberger et al., 2000). Initial screening of 32 lines was carried out using a combination of activity and mRNA measurements. These screens allowed the selection of three lines, which were chosen for subsequent physiological characterization.

Root Respiration
Root tips (25 mg) were cut with a sharp blade, washed, and incubated in 10 mM MES-KOH, pH 6.5, in a Clark-type oxygen electrode. The rate of oxygen consumption was calculated as described by Geigenberger et al. (2000).

Root Staining and Visualization
About 5-mm root collars (approximately 15 mm from the junction between root and stem) from 5-week-old plants were embedded in 4% (w/v) agarose. Cross-sections (50 mm thick) were cut by vibrotome (Leica VT 1000S) and stained with 0.1% (w/v) phloroglucinol (Sigma-Aldrich).
Serial cross-sections were observed with an epifluorescence motorized microscope (Olympus BX 61). Images were captured by Cell^P Software (Olympus).

Metabolism of [ 13 C]Glc or [ 14 C]Glc by Tomato Roots
Wild-type mature roots (approximately 500 mg) were cut directly from 5-week-old plants, washed three times with 10 mM MES-KOH buffer, pH 6.5, following a 1-h preincubation in the presence or absence of either 50 or 100 mM of the inhibitor succinyl phosphate or carboxyethyl ester of succinyl phosphate, and then incubated for 2 h in 10 mM MES-KOH buffer, pH 6.5, containing 2 mM Glc in a 100-mL Erlenmeyer flask shaken at 90 rpm containing 1.00 mCi of [ 14 C]Glc (specific activity of 8.11 MBq mmol -1 ). The 14 CO 2 liberated was captured (in hourly intervals) in a KOH trap, and the amount of radiolabel was subsequently quantified by liquid scintillation counting.
In the case of [ 13 C]Glc, the procedure was similar but roots were incubated with 10 mM unilabeled [ 13 C]Glc or unlabeled Glc.

Extraction and Fractionation of Radiolabeled Material
Tissue was fractionated exactly as described by Fernie et al. (2001b), with the exception that hexoses were fractionated enzymatically rather than utilizing thin-layer chromatography. Labeled Suc levels were determined after 4 h of incubation of 200 mL of total neutral fraction with 4 units mL -1 hexokinase in 50 mM Tris-HCl, pH 8.0, containing 13.3 mM MgCl 2 and 3.0 mM ATP at 25°C. For labeled Glc and Fru levels, 200 mL of neutral fraction was incubated with 1 unit mL -1 Glc oxidase and 32 units mL -1 peroxidase in 0.1 M potassium phosphate buffer, pH 6, for a period of 6 h at 25°C. After the incubation time, all reactions were stopped by heating at 95°C for 5 min. The label was separated by ion-exchange chromatography as described by Fernie et al. (2001a). The reliability of these fractionation techniques has been thoroughly documented previously (Runquist and Kruger, 1999;Fernie et al., 2001b;Araujo et al., 2008). Fluxes were calculated as described by Geigenberger et al. (2000).

Extraction and Fractionation of Stable Isotope-Labeled Material
Stable isotope-labeled material was extracted and prepared for GC-MSbased analysis as described previously (Roessner et al., 2001). The GC-MS was run using the exact same settings as detailed by Lisec et al. (2006). Fractional enrichment of metabolites was calculated by directly comparing replicate samples incubated in unilabeled [ 13 C]Glc with those incubated in Glc of natural abundance as previously described by Roessner-Tunali et al. (2004).

Cell Wall Analyses
Cell walls were isolated from about 5-mm root collars (approximately 15 mm from the junction between root and stem) from 5-week-old plants preceded by washing 500 mg of homogenized tissue with 1 mL of 70% ethanol. After vigorous centrifugation, the supernatant was mixed with an equal volume of a chloroform:methanol (1:1, v/v), the supernatant was decanted, and this procedure was repeated three times. Finally, the pellet was resuspended in 1 mL of acetone and centrifuged, and the pellet was dried under vacuum. Starch was removed by incubating with 40 units of a-amylase (from porcine pancreas; Sigma-Aldrich) and b-amylase (from sweet potato [Ipomoea batatas]; Sigma-Aldrich) in 20 mL of Tris-HCl, pH 7.0, for 24 h. After incubation, the cell wall material was precipitated in 70% ethanol by adding an appropriate volume of 96% ethanol to the reaction mixture and washed with acetone.
Plant cell walls were hydrolyzed for monosaccharide composition analysis (Selvendran et al., 1979). Cell wall material (1 mg) was incubated in 250 mL of trifluoroacetic acid (TFA) in a screw-capped borosilicate test tube for 1 h at 121°C. The suspension was diluted with 300 mL of 2-propanol and evaporated at 40°C. This step was repeated three times and subsequently resuspended in 200 mL of water, vortexed, and centrifuged, and the TFA-insoluble residues were separated from the solution to measure the cellulose content.
The hydrolysate was used for neutral sugar composition determined by GC according Blakeney et al. (1983) using 30 mg of myoinositol internal standard. The hydrolysate was evaporated in order to prepare the alditol acetates. Reduction to alditols was achieved by resuspending the pellet in reducing agent (250 mL of sodium borohydride in 1 M ammonium hydroxide) and incubating for 1 h at room temperature. The mixture was neutralized by the addition of 20 mL of glacial acetic acid and subsequently thoroughly washed with acetic acid:methanol (1:9, v/v) followed by methanol washes. The mixture was acetylated by the addition of 50 mL of acetic anhydride and 50 mL of pyridine and incubated for 20 min at 121°C. The mixture was washed with toluene. Cleanup proceeded by adding 500 mL of distilled water and 500 mL of methylene chloride and vortexing thoroughly. The apolar phase was removed, evaporated, and resuspended in 100 mL of acetone. The hydrolyzed monosaccharide composition was determined by GC-MS on an Agilent 6890 Series GC system equipped with a 5975B inert XL MSD and an SP-2380 fused silica capillary column (30 m 3 0.25 mm i.d. 3 20 mm film thickness; Supelco).
The preparation and hydrolysis of crystalline cellulose from the pellet left after TFA hydrolysis were performed as described (Updegraff, 1969). After hydrolysis, the cellulose-derived Glc was determined using the anthrone assay (Dische, 1962), with Glc as standard.

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Root biomass accumulation in a second independent trial.