Identification of sugar-modulated genes and evidence for in vivo sugar sensing in Arabidopsis
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- Gonzali, S., Loreti, E., Solfanelli, C. et al. J Plant Res (2006) 119: 115. doi:10.1007/s10265-005-0251-1
Sugar status regulates mechanisms controlling growth and development of plants. We studied the effects of sucrose at a genome-wide level in dark-grown 4-day-old Arabidopsis thaliana seedlings, identifying 797 genes strongly responsive to sucrose. Starting from the microarray analysis data, four up-regulated (At5g41670, At1g20950, At1g61800, and At2g28900) and four down-regulated (DIN6, At4g37220, At1g28330, and At1g74670) genes were chosen for further characterisation and as sugar sensing markers for in vivo analysis. The sugar modulation pattern of all eight genes was confirmed by real time RT-PCR analysis, revealing different concentration thresholds for sugar modulation. Finally, sugar-regulation of gene expression was demonstrated in vivo by using the starchless pgm mutant, which is unable to produce transitory starch. Sucrose-inducible genes are upregulated in pgm leaves at the end of a light treatment, when soluble sugars levels are higher than in the wild type. Conversely, sucrose-repressible genes show a higher expression at the end of the dark period in the mutant, when the levels of sugars in the leaf are lower. The results obtained indicate that the transcriptional response to exogenous sucrose allows the identification of genes displaying a pattern of expression in leaves compatible with their sugar-modulation in vivo.
KeywordsArabidopsis thalianaPhosphoglucomutase mutantSucroseSugar sensing
The ability of plants to sense sugars plays an important role in carbon partitioning and allocation in source and sink tissues. These processes are modulated as a consequence of the sugar status of the plant, and sugar signals act both at the transcriptional and translational levels in tight coordination with light and other environmental stimuli (Koch 1996; Roitsch 1999; Coruzzi and Zhou 2001). Besides sugar regulation of metabolic activities, sugar sensing and signalling are involved in the control of growth and development throughout the life of the plant, from germination to floral transition and senescence (Gibson 2005). Furthermore, sugar modulation of gene expression appears to be involved in responses to many biotic and abiotic stresses, often cross-talking with hormones (Gazzarrini and McCourt 2003; Gibson 2004). At least three different sugar-sensing systems have been proposed to operate in plants. One is a sucrose-specific signalling mechanism, the others are involved in hexose perception, which can be sensed via systems that are hexokinase-independent, not requiring hexose metabolism, or hexokinase-dependent (see Smeekens 2000; Loreti et al. 2001; Rolland et al. 2002; Halford and Paul 2003; Gibson 2005).
Several different approaches have been used to uncover and study sugar-regulated genes. Many such approaches employed increasing concentrations (often in the range of 60–180 mM) of either sucrose or glucose, exogenously fed to plants, to test the resulting changes in gene expression (Koch et al. 1992; Mita et al. 1995; Dijkwel et al. 1996; Umemura et al. 1997; Oliveira and Coruzzi 1999). Less frequently, lower (1–50 mM) concentrations of sugars were used (Jang and Sheen 1994; Loreti et al. 2000; Wingler et al. 2000; Villadsen and Smith 2004).
DNA microarray analysis was recently used to evaluate the effect of sugars (alone or interacting with other signals) on gene expression in Arabidopsis thaliana seedlings. Price et al. (2004) and Thum et al. (2004) showed that sugars modulate a broad range of genes involved in all the main cellular processes, from carbohydrate and nitrogen metabolism to signal transduction, metabolite transport and stress responses.
Little is known about the in vivo occurrence of sugar sensing, but Thimm et al. (2004) recently described the response of Arabidopsis to sugar starvation, claiming that exogenous sugars can revert the gene modulation resulting from an extended night period. This suggests that genes that are modulated by exogenous sugars are similarly modulated in vivo by the sugar status of the plant tissues. Lloyd and Zakhleniuk (2004) studied the genomic response to sugar accumulation in leaves of the mutant pho3, where sugar levels are higher than in wild type because of a defective copy of the SUC2 gene, supplying evidence of some consequent primary and secondary metabolic responses.
In the present paper, the effects of sucrose were studied at a genome-wide level in dark-grown 4-day-old Arabidopsis seedlings. Starting from microarray analysis data, a set of sucrose-modulated genes was chosen and their expression in the presence of sucrose and other sugars was thoroughly characterised by real time RT-PCR analysis. Finally, sugar-regulation of gene expression was demonstrated in vivo using the starchless pgmArabidopsis mutant (Caspar et al. 1985).
Materials and methods
Arabidopsis thaliana ecotype Columbia glabra (gl1-1) was used in this study. Seeds were sterilised with diluted bleach (10 min incubation in 1.7% sodium hypochlorite, rinsing and washing thoroughly in sterile water), and incubated in 2.5 ml liquid growing medium [Murashige-Skoog (MS) half-strength solution] in six-well plates. Plates were incubated in the dark at 4°C for 2 days and finally transferred to 23°C for 4 days before the sugar treatments. Two independent, replicated sucrose treatment experiments were performed. Each independent experiment consisted of four replicated seedlings cultures, pooled after RNA extraction.
Seeds of the phosphoglucomutase (pgm) Arabidopsis mutant were obtained from the Nottingham Arabidopsis Stock Centre, NASC (Scholl et al. 2000). Experiments performed using the pgm mutant used Arabidopsis ecotype Columbia 0 (Col 0) as the wild type.
RNA isolation, cRNA synthesis, and hybridisation to Affymetrix GeneChips
Total RNA was extracted from seedling samples using an Ambion RNAqueous extraction kit (Ambion, Austin, TX). RNA quality was assessed by agarose gel electrophoresis and spectrophotometry. RNA was processed for use on Affymetrix Arabidopsis ATH1 GeneChip arrays (Affymetrix, Santa Clara, CA) as previously described (Loreti et al. 2005). Hybridisation, washing, staining, and scanning procedures were performed by Biopolo (University of Milano Bicocca, Italy) as described in the Affymetrix technical manual. Expression analysis via the Affymetrix Microarray Suite software (version 5.0) was performed with standard parameters. Two independent, replicated experiments were performed for the sucrose treatment, and the output of the Affymetrix Microarray Suite software for each independent experiment was subjected to further analysis using Microsoft Excel (Microsoft, Redmond, WA). Signal values (indicating the relative abundance of a particular transcript) and detection call values (indicating the probability that a particular transcript is present) were generated by Microarray Analysis Suite 5.0 software. Probe pair sets (genes) called “Absent” in both the “control” and “treated” samples were removed from subsequent analyses. Furthermore, genes with “Absent” for the detection value in the baseline data and “Decrease” for the change call were excluded from the list. Similarly, genes with “Absent” for the detection call in the experimental data and “Increase” for the change value were also excluded from the list. Differences in transcript abundance, expressed as fold change, were calculated using the Microarray Analysis Suite 5.0 software change algorithm. Fold change was assumed to be correct only if the corresponding “change call” indicated a significant change (“I” increase; “D” decrease, generated by Microarray Analysis Suite 5.0 software). Expression data were filtered to select only those genes showing a coinciding change call in the two biological replicates samples for each experimental condition. Comparing the datasets by using the Microarray Analysis Suite 5.0 software algorithm identified genes significantly affected by exogenous sucrose. Only genes showing a significant change in both biological replicates were selected.
Real time RT-PCR
RNA was extracted from 4-day-old seedlings grown on MS 0.5× solution (control) or in the same medium supplemented with different sugars at the concentrations indicated in the figure legends. Total RNA, extracted with an RNAqueous kit (Ambion) according to the manufacturer’s instructions, was subjected to a DNase treatment using the TURBO DNA free Kit (Ambion). Two micrograms of each sample was reverse transcribed into cDNA with a High capacity cDNA archive kit (Applied Biosystems, Foster City, CA). Real time PCR amplification was carried out with the ABI Prism 7000 Sequence Detection System (Applied Biosystems), using the primers described in Supplemental Table S1. UBQ10 was used as an endogenous control. TaqMan probes specific for each gene were used. Probe sequences are reported in Supplemental Table S1. PCR reactions were carried out using 50 ng cDNA and TaqMan Universal PCR Master Mix (Applied Biosystems) following the manufacturer’s protocol. Relative quantitation of each single gene expression was performed using the comparative CT method as described in the ABI PRISM 7700 Sequence Detection System User Bulletin #2 (Applied Biosystems).
Samples were rapidly frozen in liquid nitrogen and ground to a powder. Samples were then extracted and assayed by coupled enzymatic assay methods measuring the increase in A340 as described by Guglielminetti et al. (1995).
Results and discussion
Analysis of Arabidopsis transcriptome in young seedlings grown in the presence of sucrose
Research into sugar sensing has taken great advantage of the use of exogenous sugars to identify physiological processes affected by carbohydrate levels. The present available knowledge about gene modulation by exogenous sugars is vast, but still suffers from the lack of evidence about the in vivo expression pattern of genes identified as sugar-modulated using an in vitro approach, usually based on the exogenous feeding of sugars to young seedlings.
Analysis of the data using MapMan software (Thimm et al. 2004) allows clustering of the modulated genes according to their putative function. We analysed the effects of sucrose on some aspects of plant primary metabolism to obtain clues as to the consequences of exogenous sucrose treatment (Fig. 1b). Pathways involved in amino acid synthesis, starch synthesis/degradation and glycolysis are substantially induced by sucrose, while amino acid and lipid degradation as well as the photosynthesis light reactions appear to be sucrose-repressed. The effects on the pentose-P pathway are less well defined, with a comparable percentage of genes induced as well as repressed by sucrose (Fig. 1b). Interestingly, Thimm et al. (2004) evaluated the effects of sugar starvation on genes involved in plant primary metabolism, observing that low leaf sugar levels result in induction of lipid and amino acid breakdown, and repression of starch and amino acid synthesis and glycolysis. Thus, the overall effect of sugar starvation in vivo (Thimm et al. 2004) is indirectly confirmed by the results obtained by us in vitro (Fig. 1b).
Choice of some candidate sucrose-induced or repressed genes
The microarray experiment allowed identification of a very high number of genes that exhibited a strong response to exogenous sucrose at the mRNA level. To verify a possible coherent response to endogenous fluctuations in sucrose levels, we decided to select a limited number of sucrose-modulated genes for complete molecular characterisation. Eight genes (Fig. 1a, red dots) were thus selected as candidates from the 797 genes identified as sucrose-responsive (Fig. 1a, yellow + red dots). Besides high induction or repression levels, two choice criteria were principally employed: (1) genes codifying for proteins involved in different metabolic processes (sugar metabolism, amino acid metabolism, stress responses, etc.), and (2) possibly acting in different cell compartments (cytosol, chloroplast, mitochondrion, etc.). Six of the eight genes have not yet been characterised. We also included in our analysis two genes already identified as sugar-responsive but only partially characterised. The four sucrose-induced genes are At5g41670, At1g20950, At1g61800, and At2g28900. Of these, only At1g61800, codifying for a putative chloroplast glucose-6-phosphate/phosphate translocator, has been previously studied; its expression was found to be induced in leaves of the mutant pho3, according to its high endogenous sucrose levels (Lloyd and Zakhleniuk 2004). This gene is usually expressed only in heterotrophic tissues. For this reason, Lloyd and Zakhleniuk (2004) hypothesised that a change in the nature of metabolite exchange between the plastid and the cytosol occurs in the pho3 mutant as a consequence of high sucrose levels. Concerning the other three sucrose-induced genes, both At5g41670 (a mitochondrial 6-phosphogluconate dehydrogenase family protein) and At1g20950 (a putative pyrophosphate-fructose-6-phosphate 1-phosphotransferase) are involved in glucose metabolism (pentose-phosphate pathway and glycolysis, respectively), a pathway activated by high sugar levels (see also Fig. 1b). The product of At2g28900 is a mitochondrial import inner membrane translocase subunit, which should be involved in protein transport; its induction concurs with a general increase in respiratory activity by high sucrose.
The glutamine-dependent asparagine synthetase 1 (ASN1) gene (At3g47340, Lam et al. 1994) is one of the four sucrose-repressed genes examined. Its expression is repressed by light and by sucrose in both light- and dark-grown plants (Lam et al. 1995, 1998). At3g47340 also corresponds to DIN6, a dark-induced and sugar-repressed gene (Fujiki et al. 2001). Expression of this gene correlates with sugar starvation, a situation in which asparagine represents a very important metabolite for nitrogen storage or transport (Fujiki et al. 2001). The other sucrose-repressed genes chosen were At4g37220 (a putative stress responsive protein, similar to cold acclimatisation proteins targeted to membranes, Breton et al. 2003), At1g28330 (a putative dormancy/auxin-associated protein, also known as DRM1), and At1g74670 (a putative gibberellin-responsive protein). Their negative regulation by high sucrose is less obvious, and their tentative annotation does not allow speculation about their actual function; however, these genes were chosen because they represent useful markers for a general analysis of sugar sensing in vivo (e.g. they were not chosen on the basis of preexisting knowledge about their possible involvement in sugar-modulated pathways).
Analysis of gene expression in the presence of different sugars
Turanose turned out to be ineffective in modulating expression of sucrose-induced genes (Fig. 2). This could reflect a lack of specificity for sucrose-sensing or the existence of a sensing machinery located inside the cell, where turanose cannot be transported (Loreti et al. 2000). With regard to the effects of the other three sugars tested, the genes could be divided in two different groups. At5g41670 and At1g20950 showed low sugar specificity, being induced by sucrose, glucose, and fructose, but high sensitivity to low concentrations of sugars (Fig. 2). In contrast, At1g61800 and At2g28900 were principally induced by sucrose and only moderately by glucose, but were less responsive to low sucrose concentrations (Fig. 2). Concerning sucrose-repressed genes, a different behaviour was observed. DIN6, At4g37220 and DRM1 were almost completely repressed by sucrose or glucose at concentrations as low as 5–10 mM (Fig. 3). Fructose was effective at higher concentrations (25–50 mM) and turanose was able to slightly repress DIN6 expression in a concentration-dependent manner, although it was ineffective on At4g37220 and DRM1 expression (Fig. 3). At1g74670 was shown to be repressed by 50–200 mM sucrose, while it was slightly induced by 5 mM sucrose or glucose, and by 5–10 mM fructose (Fig. 3). Again, turanose had no effect on the expression of this gene (Fig. 3).
On the whole, five out of eight genes responded to concentrations of sucrose as low as 5 mM (At5g41670, At1g20950, DIN6, At4g37220, DRM1), while At1g61800, At2g28900 and At1g74670 required 50 mM sucrose to be modulated (see Figs. 2, 3). Sucrose and glucose appear to be the most effective modulators of gene expression, although fructose can modulate At5g41670 and At1g20950. It is tempting to speculate that distinct sensing mechanisms may operate, possibly with a low sugar-concentration sensor as well as a high sugar-concentration sensor. Interestingly, all five genes regulated by low sucrose (At5g41670, At1g20950, DIN6, At4g37220, DRM1) are also modulated by glucose and fructose, suggesting that the low-concentration sensor could also be a low-specificity sensor.
Time-course of gene expression
The time-course experiment (Fig. 4) reveals that genes that behave similarly in terms of sugar concentration response (e.g. DIN6 and At4g37220) may differ as far as the timing of the response is concerned (Fig. 4). This could reflect, for example, a different mechanism of regulation, with some genes that can be modulated directly by sugars being therefore earlier in their response, while others may be modulated indirectly. Further work is certainly needed to ascertain the existence of multiple sugar sensing mechanisms, the existence of which have been postulated (Smeekens 2000; Loreti et al. 2001; Rolland et al. 2002; Halford and Paul 2003; Gibson 2005) and, at least in part, suggested by the results presented here.
Analysis of in vivo gene expression in wild type and pgm mutant plants
In the present paper we describe the identification of a large set of sucrose-modulated genes using a transcriptome approach. Real time RT-PCR characterisation carried out on a restricted number of selected genes, confirmed the microarray data and demonstrated that not only sucrose, but in some cases also hexoses, although to only a minor extent, can play a role in the regulation of mRNA accumulation of specific genes. We further demonstrated that the eight selected genes, as well as the whole set of sucrose-modulated genes, can respond to fluctuations in sugar levels taking place in vivo. We conclude that the transcriptional response to exogenous sucrose allows identification of genes that display a pattern of expression in leaves suggesting that they are actually sugar modulated. Multiple sugar sensing mechanisms may operate in vivo, and further work is certainly required to identify the sensing and signalling pathways operating in A. thaliana leaves subjected to fluctuations in their sugar content.