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280 Biochemical Society Transactions (2005) Volume 33, part 1 The role of trehalose-6-phosphate synthase in Arabidopsis embryo development L.D. Gómez, S. Baud and I.A. Graham1 CNAP, Department of Biology, University of York, Heslington, York YO10 5YW, U.K. Abstract We previously showed that trehalose-6-phosphate synthase 1 (TPS1), which catalyses the first step in trehalose synthesis, is essential for embryo maturation in Arabidopsis [Eastmond, van Dijken, Spielman, Kerr, Tissier, Dickinson, Jones, Smeekens and Graham (2002) Plant J. 29, 225–235]. The tps1 mutant embryos develop more slowly than wild type. Patterning in the tps1 embryos appears normal but they do not progress past the torpedo stage to cotyledon stage, which is when storage reserves start to accumulate in the expanding cotyledons. Our initial data led to the hypothesis that trehalose metabolism plays a key role in regulating storage reserve accumulation by allowing the embryo to respond to the dramatic increase in sucrose levels that occurs at the torpedo stage of embryo development. More recent data demonstrate that while the tps1 mutant is blocked in the developmental progression of embryos from torpedo to cotyledon stage the expression of genes involved in the accumulation of storage reserves proceeds in a similar fashion to wild type. Thus it appears that induction of metabolic processes required for accumulation of storage reserves in tps1 occurs independently of the developmental stage and instead follows a temporal programme similar to wild-type seeds in the same silique. Introduction Seed development in Arabidopsis is generally divided into three phases. In the first, known as embryo morphogenesis, a series of programmed cell divisions results in the basic cell patterning of the mature plant being established. The torpedo stage embryo represents the end of this phase at which stage the root and shoot meristems are formed and small unexpanded cotyledons are evident [1]. The second phase is dominated by the expansion of the cotyledons and hypocotyl and the associated accumulation of storage reserve compounds. A significant amount of cell division as well as cell expansion occurs during this phase with the number of cotyledon cells increasing substantially from torpedo through to mid-late cotyledon stage [2]. During the final phase, the mature seed is subjected to desiccation after which the embryo reaches a stage of quiescence [3]. This developmental programme is underpinned by coordinate regulation of gene expression including for example those involved in storage reserve accumulation during the second phase of development [4]. A large number of embryo-lethal Arabidopsis mutants that fail to produce viable seed due to disruption in essential embryo development (EMB) genes have been identified [5]. These mutants range from those disrupted in basic housekeeping functions to specific signalling processes essential for embryo development. Until recently trehalose metabolism was not thought to play any significant role in development in higher plants but a number of lines of evidence have led Key words: Arabidopsis, embryo development, reserve accumulation, trehalose-6-phosphate, TPS1. Abbreviations used: TPP, trehalose phosphate phosphatase; TPS, trehalose-6-P synthase; T-6-P, trehalose-6-P. 1 To whom correspondence should be addressed (email [email protected]). C 2005 Biochemical Society to a revision of this view [6,7]. Key among these was the discovery that trehalose-6-P synthase 1 (TPS1) is required for progression past the torpedo stage of embryo development [7] leading to it being classified as an EMB gene [5]. The non-reducing disaccharide trehalose is widely distributed in nature, playing a role in stress protection and carbohydrate storage. Since the demonstration that manipulation of the trehalose biosynthetic pathway results in profound effects on plant growth and development in mature plants [6], the function of the trehalose pathway in higher plants has been a matter of intense research. Although in a few desiccation-tolerant plants trehalose is present in concentrations compatible with a role in stress protection, in most plants the amounts (0.15 mg g−1 dry weight) are too low to support such a role [8]. The biosynthesis of trehalose occurs via a phosphorylated intermediate, trehalose-6-P (T-6-P), with two steps catalysed by the enzymes trehalose6-P synthase (TPS) and trehalose phosphate phosphatase (TPP). In Saccharomyces cereviseae the TPS1 protein and T-6-P play a key role in regulating the flux of carbon into glycolysis through the regulation of hexokinase [9,10]. The demonstration that the E. coli otsA gene, which encodes a protein with the same catalytic activity as TPS1 but which is significantly divergent at the amino acid level, indicates that the Arabidopsis tps1 mutant phenotype is due to the lack of T-6-P rather than an inherent property of the TPS1 protein. A reverse genetics approach was used for the isolation of transposon insertions in the first and second exons of the AtTPS1 gene (tps1-2 and tps1-1 mutants, respectively) [7]. A third allele with a T-DNA insertion in the last exon of the gene has also recently been identified in the SAIL collection [11]. All of these tps1 alleles are recessive and show Nutrient Sensing through the Plasma Membrane of Eukaryotic Cells Figure 1 Comparison of embryo development in tps1 and wild type The graph shows the length of wild-type and tps1 embryos over 18 days after flowering (DAF). The images show progressive development of wild-type embryos from torpedo stage at 6 DAF through early, mid and late-cotyledon stages. Tps1 embryo development is delayed, reaching late globular 6 DAF and torpedo stage 12–15 DAF. Images are not to scale. of the TPS and TPP genes occurs in a wide range of tissues and is controlled by stimuli such as nutritional status, abiotic stress, circadian cycle, and in stomatal guard cells following ABA (abscisic acid) treatment [12,13]. Different members of the TPS family are upregulated under specific conditions, with for example TPS5 being induced by sucrose feeding [14] and TPS1 being induced during embryo development [7,11]. The lack of complementation of the tps1 mutant by any of the other TPS genes and the absence of any reports to date of similar phenotypes caused by mutation of any of the other TPS genes is consistent with TPS1 playing a unique role in plant development. Since the metabolic targets of T-6-P are unknown, a transcriptomic analysis of the changes produced by alterations in trehalose metabolism represents a valuable approach to identify the processes controlled by this metabolite. Recent analysis of the transcriptional changes induced by altered levels of T-6-P in plants overexpressing TPS, TPP and trehalase or treatments involving feeding different carbon sources were studied using the Affymetrix 8200 gene chip [14]. Clusters of genes that correlated with alterations in levels of T-6-P were identified and the majority of these were associated with stress signalling, with only two genes from the clusters being associated with primary metabolism, namely aldose 1-epimerase and a SNF-related kinase, AKIN11 [14]. The effect of altered T-6-P levels thus appears to extend well beyond primary metabolism. Storage reserve accumulation in tps1 mutants a wrinkled seed phenotype. The development of the tps1 embryo follows a normal growth and patterning process, but the rate of growth is decreased. Consequently, at the time when the wild-type embryos reach the end of the maturation phase, the tps1 embryos have only reached torpedo or early cotyledon stage in the same siliques. This delay in progression of tps1 relative to wild type is observed from early stages of seed development, becoming more marked as development proceeds (Figure 1). AtTPS1 is also expressed in a variety of mature plant tissues [7]. Recently, a dexamethasone-inducible transcription system has been used to drive expression of a TPS1 transgene in the tps1 mutant during seed development, allowing mature tps1 plants to be recovered [11]. Tps1 plants obtained in this way remain in the vegetative phase and do not produce inflorescences unless the TPS1 transgene is again induced. Interestingly, the growth region in roots of rescued tps1 plants appears to be reduced in the absence of TPS1 transgene expression [11]. The Arabidopsis genome contains approximately 11 TPS and 10 TPP homologues, while there is one gene encoding a trehalase enzyme. Microarray databases show that expression In wild-type embryos storage reserve gene expression and reserve accumulation are massively induced at the transition from torpedo to early cotyledon phase. Interestingly, storage reserve gene induction and reserve accumulation have been reported to still occur in a number of embryo lethal mutants that are arrested as early as the globular stage of embryo development [15,16]. Thus it appears that induction of metabolic processes required for accumulation of storage reserves occurs independently of the developmental stage and instead follows a temporal programme similar to wild-type seeds in the same silique. Such a temporal programme could be regulated for example by phytohormones such as ABA and metabolites such as soluble sugars [17]. On the basis of RTPCR experiments we previously reported that two storage reserve-related gene transcripts, namely oleosin and napin, were not induced in tps1 torpedo stage embryos. We have subsequently performed a more detailed analysis using several storage reserve promoter–reporter gene lines crossed into the tps1 mutant (Table 1). A line carrying the At2S3 promotor fused with the coding sequence of GFP [18] was crossed into the tps1 mutant in order to follow the temporal and spatial pattern of expression. In the wild-type background, At2S3::GFP fluorescence is first observed at late torpedo stage in the embryo axis and then spreads throughout the whole embryo as development proceeds to the cotyledon stage. At2S3-GFP is also expressed in torpedo stage tps1, such that C 2005 Biochemical Society 281 282 Biochemical Society Transactions (2005) Volume 33, part 1 Table 1 Reserve proteins promoter–reporter activity in tps1-arrested embryos Promoter–reporter activity At2S3::GFP β-conglycinin::GUS DC8::GUS WT torpedo ++ − − WT cotyledon tps1 +++ ++ +++ +++ +++ +++ at the end of silique development it gives a spatial pattern of expression similar to the late torpedo stage wild type. We have also introduced the GUS reporter driven by the promoter of the β subunit of β-conglycinin and the DC8 promoter [19,20] into tps1. In contrast to At2S3::GFP, the expression of DC8 and β-conglycinin promoters in tps1 shows levels similar to the wild type (Table 1). These data contrast with our initial observation on the expression of oleosin and napin transcripts by RT-PCR, but are consistent with other reports showing that cell differentiation proceeds separately from morphogenesis. These results using promoter reporter lines to monitor induction of storage reserve related genes in tps1 are consistent with transcriptomic, metabolite and electron microscopic based studies in our laboratory, which all show that the cellular differentiation in torpedo stage tps1 embryos is more typical of cotyledon stage wild-type embryos that are accumulating storage reserves than torpedo stage wild-type embryos (data not shown). Separate programmes therefore appear to operate during embryo development governing cell differentiation and morphogenesis in the tps1 mutant. Is T-6-P involved in the regulation of cell growth and division? The principal phenotypic defect of the tps1 mutant is the progressive delay in morphogenesis during phase 1 of embryo development rather than a strict block imposed at the torpedo stage (Figure 1). The fact that cellular differentiation in the morphogenically delayed tps1 mutant proceeds in a similar fashion to that in wild-type embryos suggests that cell growth and division are affected in this mutant. How then does T-6-P mediate this effect on embryo development? On the basis of the S. cerevisiae model of T-6-P regulation of carbon flux into glycolysis and the observation that sucrose levels increase significantly during the torpedo stage of embryo development one possible explanation is that T-6-P plays a similar role in plants to that in yeast. However, the biochemical analysis of a number of plant hexokinases shows that they are not inhibited by T-6-P unlike the S. cereviseae enzyme [7,21]. It is possible that other enzymes or proteins associated with primary carbon metabolism could be direct targets for regulation by T-6-P but to date none have been identified. Transcriptomic data from mature plant tissues discussed above [14] along C 2005 Biochemical Society with the fact that the tps1 mutant is able to undergo cellular differentiation including incorporation of soluble sugars and amino acids into storage reserve compounds suggest that the primary defect may not be associated directly with the regulation of metabolism. Little is known about the linkage between nutritional status and growth and division of plant cells. Work in Arabidopsis cell cultures has shown that two of the D-type cyclin genes are regulated by sugars suggesting that at least some of the control occurs at the transcriptional level [22]. It is possible that disruption of trehalose metabolism may either directly or indirectly affect the crosstalk that must occur to coordinate nutritional status with cell growth and division. This would account for the delayed embryo development in the tps1 mutant. Further genetic and biochemical studies are needed to test the validity of this hypothesis. References 1 Jurgens, G. and Mayer, U. (1994) in Embryos-Colour Atlas of Development (Bard, J.B.L., ed.), pp. 7–21, Genetics Unit of Western General Hospital, Edinburgh 2 Mansfield, S.G. and Briarty, L.G. (1993) in Arabidopsis, an Atlas of Morphology and Development (Bowman, J., ed.), pp. 376–384, Springer-Verlag, Berlin 3 Harasda, J.J. (1997) in Cellular and Molecular Biology of Plant Seed Development (Larkins, B.A. and Vasil, I.K., eds.), pp. 545–592, Kluwer Academic Publishers, Dordrecht 4 Ruuska, S.A., Girke, T., Benning, C. and Ohlrogge, J.B. 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