To investigate light and carbon signaling interactions at the genome-wide level, genes responding to either white light and/or sucrose were analyzed using microarrays. Affymetrix AG gene chips containing 8,000 unique genes from A. thaliana, approximately one-third of the entire genome, were analyzed after hybridization with cRNA from 14-day-old plants subjected to four different light and/or carbon treatment conditions as shown in Figure (-C-L, -C+L, +C-L and +C+L) (see Materials and methods for experimental details). Comparative analyses of these RNA samples by microarrays were used to identify genes responding to light only or carbon only, and genes that are regulated by both light and carbon, as described below.
Figure 1 Light and carbon treatments and their effects on gene expression. (a) Diagrammatic representation of various carbon and light treatments given to plants before microarray analyses. -C-L, no carbon and no light; -C+L, no carbon and 70 μE/m2/sec (more ...)
Models for predicting light and carbon signaling interactions
Light and carbon signaling may interact in the regulation of a hypothetical gene (for example, X, Y or Z) by one of three models shown in Figure . In model 1, carbon and light are shown to act independently of one another to affect expression of gene X, in either a positive or negative manner in various combinations. In this example, both carbon and light are shown to act in a positive manner. However, for another gene, light may induce transcription, while carbon may independently repress (depress) transcription. Alternatively, for another gene, carbon and light may independently act to repress transcription. Model 2 predicts that carbon and light are two signals that are dependent, in that they are both required to affect expression of gene Y, where either signal alone has no effect on gene expression (Figure ). This could happen in a positive or negative manner. Model 3 predicts that carbon and light signals act both independently of and dependent on one another to influence the expression of gene Z. In the example shown in Figure for model 3, gene Z is induced to a certain level in the presence of carbon alone and is unresponsive to light alone (in the absence of carbon). However, in the presence of both carbon and light, the expression level of that particular gene exceeds that observed in the presence of carbon alone. Thus, the carbon and light regulation of gene Z is responsive to carbon alone, yet requires carbon for induction by light. This is but one of many examples of interactions between carbon and light in model 3. We validate the existence of all three models for carbon and light interactions through the analysis of genome-wide responses to carbon and/or light, as detailed below.
Using InterAct Class to classify genes regulated by carbon and/or light on the basis of their expression profiles
RNA samples obtained from plants subjected to four different light and/or carbon conditions (see Figure and Materials and methods) were hybridized to Affymetrix AG gene chips. Gene-expression analysis was carried out using Affymetrix's Microarray Suite 5.0 software. Microarray data obtained from plants treated with no carbon and no light (-C-L) were used as the control background (Table , -C/-L) to which data from all other treatments (that is, +C/-L, +C/+L and -C/+L) were compared (see Table ). Using this comparative approach, genes responding to carbon or light, as well as genes responding to both carbon and light, were identified at the genomic level as I, D or NC (I, induced; D, depressed; NC, no change). To analyze further the relative response of a gene under each of the three treatments, a classification system termed InterAct Class [27
] was used. InterAct Class allowed us to classify genes on the basis of their relative regulation by carbon and/or light. The InterAct Class program takes Affymetrix difference calls of treatment to control (NC, I, D), and further classifies a gene on the basis of its relative expression in each of the three treatments: carbon alone; carbon and light combined; or light alone. For example, in Table , row 1, gene X is induced by carbon alone (fivefold), and by light alone (fivefold), and is induced 10-fold in the presence of both carbon and light. Gene X thus falls into InterAct class 121 (1 C/ 2 C+L/ 1 L; Table , row 1). In another example, shown in Table , row 2, gene Y shows no change in expression in the presence of carbon or light alone, yet is induced threefold in the presence of both carbon and light. As such, gene Y is placed in the 010 InterAct class (0 C/ 1 C+L/ 0 L). In another example, gene Z shows a similar level of induction by carbon alone, or light alone, or by carbon plus light (Table ). Gene Z falls into InterAct class 111 (1 C/ 1 C+L/ 1 L).
Example of how genes are assigned an InterAct class
For this study, InterAct Class ranked gene-expression values from -3 to 3, with -3 being the most depressed value and 3 being the highest expression value. Therefore, for each comparison any one gene was assigned one of seven possible values: -3, -2, -1, 0 (no change), 1, 2 or 3. Taking into consideration all possible combinations, genes were theoretically categorized into 73 or 343 permutations of gene regulation by carbon and/or light. However, the actual number of possible InterAct classes was lower, because InterAct Class ranks the change in expression levels of the conditions relative to one another for each gene. Thus, in order to have a value of 3, there must be a 2 in the InterAct class, and in order to have a value of 2, there must also be a 1 in the InterAct class. Using this criteria, 72 potential InterAct classes were generated rather than the theoretical 73 permutations. Microarray expression data was used to place A. thaliana genes into one of 72 potential InterAct classes. Thus, the in vivo significance of various models proposed for gene responses to light and carbon (Figure ) were validated by identifying genes whose expression profiles fit into one of these 72 InterAct classes.
Out of 72 potential InterAct classes, there were 43 classes whose existence was validated by the expression pattern of at least one gene. Of the 8,000 genes present on the partial A. thaliana genome array, 24.9% (1,997) unique genes were placed into InterAct classes on the basis of their expression profiles that met certain criteria. For a gene to be placed into an InterAct class, it must have been called present on both gene chip replicates, and have a similar expression profile in both biological replicates under all analyses (see Materials and methods for more details). For instance, if a gene was induced to similar levels by carbon or light alone, and was induced even further by carbon and light together in one replicate, but a similar expression profile was not observed in the second replicate, this gene would be discarded and not placed into an InterAct class. Because partial A. thaliana genome chips were used, the number of genes that were placed in an InterAct class is an underestimate. The number of genes that validate specific InterAct classes is shown in Table . Of the 43 InterAct classes that contained genes, 39 are shown in Table . The four InterAct classes that contained only one or two genes are not shown in Table .
Number of genes validating InterAct classes
InterAct classes that were validated by chip data were further used to categorize genes into specific models of carbon and light regulation (Table ). Table shows the number of genes regulated by carbon alone or light alone, and the number of genes whose regulation is affected by the interactions between carbon and light. Those genes that were categorized as being subject to regulation by carbon and light were further placed under one of the three models predicting carbon and light interactions presented in Figure . Within these models, the response of genes to carbon and/or light can be described as inductive or repressive (model 1 or model 2), antagonistic (model 1), suppressed, enhanced, equally affected by either signal, or dominated by either light or carbon (model 3). These responses of genes to carbon or light and the interaction between carbon and light become evident upon closer examination of the numbers of genes validating a particular InterAct class shown in Table . For instance, InterAct class 121 (40 genes), fits within model 1, because genes within this class are likely to be induced independently by both carbon and light (Table ). By contrast, InterAct class -1-2-1 (129 genes), is also under model 1, but the expression of genes within this class are likely to be repressed independently by both carbon and light (Table ).
Flow chart of the procedure used to identify common potential light- or carbon-responsive (LCR) cis elements in genes that share both a similar expression profile and a functional category.
Two hundred and seventy-eight genes are classified as being responsive to carbon alone or to light alone (Table ). In contrast, 1,247 genes comprise the InterAct classes that show regulation by carbon and light and fall under one of the three models presented in Figure (Table ). The responses to carbon and light and possible interactions between carbon and/or light within such InterAct classes are described in Table (carbon and light-response column), according to the various general models proposed in Figure .
Identifying primary functional categories of genes regulated by carbon and light
We next determined whether the signals - carbon and light - or interactions between carbon and light signaling regulate specific functional classes of genes involved in particular biological functions. For this analysis, the classification system of the Munich Information Center for Protein Sequences (MIPS) [28
] was used in conjunction with InterAct Class to determine whether gene function correlates with specific patterns of gene regulation by carbon and/or light. The MIPS system sorts genes according to functional categories called 'funcats'. MIPS classifications are separated into 29 major (primary) funcats that are further subdivided into minor (secondary, tertiary, and so on) functional categories [28
]. We tested whether the genes within a specific primary funcat were significantly (p
< 0.05) over- or under-represented in a particular InterAct class, as compared to the general population of genes on the chip that were assigned both a funcat and an InterAct class. This was determined by calculating the percentage of genes expected to be in a InterAct class on the basis of the general population, versus the percentage of genes in a specific funcat that were grouped into a single InterAct class. Using all chip data, there were 1,898 Affymetrix IDs that fell into a specific funcat and that were also assigned to a specific InterAct class (Table ). InterAct class 000 was used as an example for this analysis. The genes that fell into InterAct class 000 were not regulated by light and/or carbon. Therefore, if genes in a particular funcat were under-represented in the 000 class, this indicated that the particular funcat contained more genes that are regulated by carbon and/or light than expected from the general population of genes on the chip. In addition, a funcat that contains genes that were over-represented within the 000 class indicates that genes in that particular funcat are less regulated by carbon and/or light compared to the general population of genes on the chip. Thus, focusing on the 000 InterAct class and determining which funcats were under-represented directed us to the funcats that are significantly regulated by carbon and/or light.
Primary functional categories whose genes show over-representation or under-representation in InterAct class 000
For example, it was observed that in the general population, 440 out of 1,898 genes were placed into the InterAct class 000 (Table ). Thus, 23.1% (440/1,898) of all the genes on the chip that were assigned both a funcat and an InterAct class are in class 000. Genes in nine of these funcats showed statistically significant under- or over-representation (-S or +S, respectively) in InterAct class 000 compared to the general population (Table ). Among these was the primary funcat metabolism (45 genes) which was found to be statistically under-represented within the 000 InterAct class (45/274; 16.4%), compared to the general population (440/1,898; 23.2%). Additionally, a number of other primary funcats were also under-represented in InterAct class 000, indicating these particular funcats contain more genes that are regulated by carbon and/or light than expected in the general population of genes on the chip. These primary funcats which contain a significant number of genes regulated by carbon and/or light include protein fate, protein synthesis, energy, cell rescue, defense and virulence (see Table , -S). Conversely, other funcats were over-represented in the 000 InterAct class. This indicates that these processes are more immune to regulation by carbon and light compared to the general population of genes on the chip that are assigned both a funcat and an InterAct class. These funcats included unclassified proteins, transcription, cellular communication/signal transduction, cell cycle and DNA processing (Table ). Other primary funcats that showed significance within any other InterAct class (besides 000) are shown in Table . The actual number of additional InterAct classes in which a particular funcat was under- or over-represented is shown in the last column of Table .
Further analysis of the regulation of genes in the primary funcat metabolism was carried out because metabolism was one of the primary funcats that had the largest number of genes that was assigned an InterAct class and because 45 genes in this funcat were significantly under-represented in the 000 InterAct class. This suggests that metabolism is a process highly regulated by light and/or carbon. Metabolism was used as a working example of the type of further analysis that could also be performed for any of the funcats listed in Table that are also regulated by light and carbon.
Secondary funcats of metabolism and significance of all models of carbon and light interactions
To identify the relative dominance and interaction of carbon and light in regulating genes in the metabolism funcat, we first examined whether any secondary funcats of metabolism were under- or over-represented in a particular InterAct class. A breakdown of the secondary funcats of metabolism according to their distinct carbon and/or light regulation models (Figure ) is shown in Table . In each column the models noted above correspond to InterAct classes shown in Table . Each row shows the number of genes in a secondary funcat of metabolism and their significance (over- or under-representation, +S or -S, respectively) in distinct models of carbon and/or light regulation. Bold type in the table indicates the funcats with a statistically significant number of genes that fell into an InterAct class that corresponds to a particular mode of carbon and/or light regulation. Funcats in plain type are not statistically significant. Upon further dissection of the primary funcat metabolism into secondary funcats, it was observed that the secondary funcats C-compound (carbon-containing compound)/carbohydrate metabolism and amino-acid metabolism were each under-represented in 000 InterAct class (Table ). This analysis indicates that genes in these processes are significantly more regulated by carbon and/or light than the general population of genes on the chip that were assigned both a funcat and an InterAct class. Indeed, genes in these secondary funcats were over-represented in InterAct classes corresponding to model 3, carbon and light interactions (Table ).
Number of genes in the metabolism primary and secondary funcats in each InterAct class that are regulated by light only, carbon only and light and/or carbon
For further analysis, we focused on secondary funcats of metabolism whose genes were over-represented in the InterAct classes that suggest regulation by carbon and light (Table ). In the general population, 386/1,898 genes (20.3%) validated the existence of model 1 (carbon and light independent) (Table ) of which 91/1,898 genes (4.8%) fell under the 'inductive' classification of model 1 (Table ). The primary funcat metabolism (274 genes) contained 21 genes whose expression patterns fit this model. Thus, the primary funcat metabolism was over-represented within model 1, 21/274 (7.6%) genes compared to the general population (Table ). Out of those 21 metabolism genes, seven genes within the secondary funcat lipid, fatty-acid and isoprenoid metabolism were over-represented within the InterAct classes belonging to this model, and two genes within the secondary funcat nitrogen and sulfur metabolism also showed over-representation in InterAct classes belonging to model 1 (Table ). This suggests that genes involved in lipid, fatty-acid, and isoprenoid metabolism are highly regulated by light and carbon independently. Whereas five genes in metabolism fell into model 2 (carbon and light are dependent), no specific funcat was under- or over-represented in this model, compared to the general population (Table ).
Model 3 was validated by the general population of genes in which 776/1,898 (40.8%) fell into model 3 (Table ). For the 'C-dominates' classification of model 3, 223 genes or 11.7% of the population (223/1,898) were found under this model (Table ). Fifty-one genes in the metabolism primary funcat were over-represented in InterAct classes that belonged to 'C-dominates', model 3 (51/274; 18.6%). Out of those 51 genes, the funcats C-compound/carbohydrate metabolism (17 genes) and amino-acid metabolism (12 genes) were each over-represented in the InterAct classes that correspond to 'C-dominates', model 3 (Table ). There were 124 genes in the general population (124/1,898; 6.5%) that fell into another variation of model 3, where light and carbon have equal effects on gene expression, (Table ). Genes in the primary funcat metabolism were not over-represented in the InterAct classes that belonged to this model. However, there were 10 genes in the secondary funcat metabolism C-compound/carbohydrate metabolism that were over-represented in the InterAct classes which belonged to 'equal effect', model 3 (Table ). Genes in InterAct classes that belonged to 'equal effect', model 3 were equally affected by light, carbon and carbon plus light (for example, 111 or -1-1-1). Thus, genes that fell under these two InterAct classes may contain a single cis element that responds to either light or carbon. Alternatively, carbon regulation of these genes may be due to an indirect effect of light. This would occur through an increase in carbon skeletons generated through photosynthesis. Below we show how a cis-analysis of genes in the 111 InterAct class (model 3) was used to identify putative cis elements responsive to either carbon or light. This analysis is an example of the type of data mining that can be carried out for other funcats that show genes over-represented in an InterAct class, implicating various models for carbon and light interactions.
Breakdown of model 3 shows over-representation of secondary funcats of metabolism in InterAct class 111
Model 3 (equal effect of carbon and/or light) comprises the two InterAct classes, 111 (85 genes) and -1-1-1 (39 genes) (Table ). Out of 274 genes that are categorized as being involved in metabolism and placed into any InterAct class, 22 fell into class 111. Thus, metabolism genes were over-represented within this InterAct class (22/274; 8%) compared to the general population (85/1,898; 4.5%) (Table ). Of these 22 metabolism genes, nine in the secondary funcat, C-compound/carbohydrate metabolism also showed over-representation in InterAct class 111 (9/84; 10.7%) compared to the general population (85/1,898; 4.5%) (Table ). This suggests that C-compound/carbohydrate metabolism has a significant number of genes that are equally regulated by light and/or carbon according to model 3. Although there were other genes from some of the metabolism secondary funcats that fell into InterAct classes 111 and -1-1-1, they were not statistically significant compared to the general population.
Significance of the number of genes in metabolism secondary funcats in InterAct classes 111 and -1-1-1 in model 3
Genes within C-compound/carbohydrate metabolism and InterAct Class 111
The genes categorized under the secondary funcat C-compound/carbohydrate metabolism were further subdivided according to the specific processes in which they are involved and by InterAct class. One subset of these genes, belonging to C-compound/carbohydrate metabolism and 111 InterAct class, all encode proteins involved in starch biosynthesis and include glucose-1-phosphate adenylytransferase, starch branching enzyme II and 1,4-alpha-glucan branching enzyme protein isoform SBE2 (Table ). Another subset of genes that belong to C-compound/carbohydrate metabolism and InterAct class 111 are involved in cell-wall metabolism/biosynthesis. These genes encode the cellulose synthetase catalytic subunit (AthA) and a putative pectate lyase A11 (Table ). Finally, five genes with unconfirmed C-compound/carbohydrate metabolism functions that fell into 111 InterAct class were grouped as 'other' (Table ). In addition to C-compound/carbohydrate metabolism, other secondary funcats were identified that were over-represented in the InterAct class 111 (see Additional data file 1). These genes corresponded to the secondary funcats for cell-wall biosynthesis genes (five genes), and genes involved in electron transport and membrane associated energy conservation (three genes) (Table ). By using InterAct class and MIPS to group genes according to similar expression profiles and biological function, we were able to find over-represented promoter motifs which may identify previously unknown shared cis elements involved in gene regulation by both carbon and light, as described below.
Genes over-represented in primary and secondary metabolism funcats in InterAct class 111, model 3 where equal effects of carbon and light are observed
Putative co-regulated genes were used to identify shared cis-regulatory motifs responding to light and carbon
The InterAct class 111 was selected for cis-analysis because this class of genes showed equal responses to carbon, light, and carbon plus light. This suggests there may be a single cis element that responds to either carbon or light (among other possibilities, see Discussion). Three secondary funcats showed over-representation of genes in the InterAct class 111. These corresponded to metabolism: C-compound/carbohydrate metabolism (10 genes); control of cellular organization: cell wall (five genes); and energy: electron transport (three genes) (Table ).
Of the 10 genes in InterAct class 111 that are found in C-metabolism, three genes were initially selected for cis-analysis because they are involved in a specific biochemical process, starch metabolism (At2g36390, At5g03650, and At5g48300) (see Table and Figure , step 1). These three starch-metabolism genes were used as the basis for the discovery of carbon- and light-responsive cis elements because they satisfied two criteria that suggested they may be 'co-regulated'. First, they are functionally related (metabolism: C-compound/carbohydrate metabolism: starch metabolism); and second, they have similar expression patterns under varying carbon and light treatments (InterAct class 111).
The program AlignAce [30
] was used to find elements that were over-represented in the proximal 1,000 base pairs (bp) (-1 to -1,000) of the promoters of these three genes (Figure , step 2). Using this method, 59 degenerate motifs were found to be over-represented among the promoters of these InterAct class 111 genes encoding starch-metabolism enzymes. The promoters of all genes in the A. thaliana
genome were retrieved using the program RSA Tools [32
] (Figure 3, step 3). Finally, for each motif, we determined whether the genes containing any given motif were significantly over-represented (p
< 0.05) in all of the genes of a particular InterAct class (that is, InterAct class 111) (Figure 3, step 4). As a negative control, these motifs were also analyzed to determine whether they were also over-represented in all genes in InterAct class 000 (these are genes that are not regulated by carbon, light, or carbon plus light). Any motifs that were over-represented in genes in InterAct class 111 but not in genes in InterAct class 000 were identified as 'significant' motifs (Figure 3, step 4).
Thus, in this analysis the promoters of genes that shared both a similar expression profile and were involved in similar biochemical processes were used to identify putative shared cis elements. Using genome-scale data, those motifs that also shared the carbon and light regulation on a genome-wide level with similarly regulated genes (same InterAct class) were selected for further study. Using this analysis, eight putative cis elements (of the 59 motifs originally found to be over-represented in the promoters of the three starch-metabolism genes) were identified because they were statistically over-represented in all genes belonging to InterAct class 111 when the whole chip data were analyzed (Table ).
LCR cis-motifs are proposed to be responsive to light or carbon
This analysis was also carried out using the five genes (At1g04680, At1g23820, At1g69530, At2g33590, At4g39350) in the funcat 'Control of cellular organization: cell wall', that was also found to be over-represented in InterAct class 111 as compared to the general population (Table ). Forty-five motifs were found to be over-represented in the promoters of these five cell-wall genes that showed similar expression profiles. Genes with 10 (out of 45) of these motifs were found to be statistically over-represented in all genes that belonged to InterAct class 111 (but not in genes that belonged to InterAct class 000) when whole chip data were analyzed (Table ), which suggests they are putative cis elements.
The expression profiles of the eight genes (Table ) that fell into InterAct class 111 and that were used for cis-analysis were checked using quantitative polymerase chain reaction (PCR) (Additional data file 2). The absolute expression levels were translated into fold changes and were classified according to the InterAct Class classification system. Using quantitative PCR data, two (At5g48300 and At2g36390) out of three genes involved in starch metabolism fell into InterAct class 111 (Additional data file 2). The third gene, At5g03650, did not fall into any InterAct class because a criterion necessary to place a gene in a class was not met, which in this case was overlapping standard deviations. Similarly, using quantitative PCR to measure gene regulation, three (At2g33590, At4g39350, At1g23820) out of five genes that fell into the cell-wall biosynthesis funcat were placed into InterAct class 111. The other two cell-wall biosynthesis genes, At1g69530 and At1g04680, fell into InterAct class 221, a class similar to InterAct class 111 (Additional data file 2). Overall, the expression profiles of five out of the eight genes used for cis-analysis were verified using quantitative PCR. Upon closer examination of the fold changes and standard deviations that were calculated using quantitative PCR versus microarray data, quantitative PCR was found to follow the same trends as the microarray data. Differences are likely to be due to the small standard deviations for the expression levels of these genes in -C+L as determined through quantitative PCR analysis (Additional data file 2).
Motifs identified in genes regulated by light or carbon correspond to known light-response elements
The eight putative cis
elements identified from the starch-metabolism genes that both share a similar expression profile and are found to be statistically over-represented in all InterAct class 111 genes (Table ) were compared with known cis
-regulatory elements found in the PlantCARE database [34
] (Figure 3, step 5). All but one of these putative cis
elements that were identified in genes equally regulated by carbon and light were found to be similar to validated light-response cis
elements in PlantCARE. The exception was motif45/LCR1, which did not match any known elements in the database (Table ). One putative cis
element, motif2/LCR4, was found to have an exact match to sequences in the DREP-module, an element shown to be involved in light responses as well as other processes [36
Ten putative cis
elements were identified from the cell-wall genes in InterAct class 111 that responded equally to light and carbon (Table ). Three of these putative cis
elements were found to be similar to validated light-response elements in PlantCARE (Table ). It is significant that some elements in the PlantCARE database (ACE and RbcS-CMA7c), validated as involved in light responses [38
], were found to match putative cis
elements that we identified using the promoters of both starch metabolism and cell-wall genes in the 111 InterAct class. This indicates that the motifs we identified using the protocol outlined in Figure 3 may correspond to light- or carbon-responsive cis
elements of a more global nature, which are not limited to the regulation of one specific process such as starch metabolism or cell-wall synthesis.
Candidate cis elements responsive to either light or carbon are over-represented in InterAct class 111 but not 110
We identified a total of 18 putative cis elements (eight identified from starch-metabolism genes and 10 from cell-wall genes) that were over-represented in genes in the InterAct class 111 (Table ). Genes in InterAct class 111 are induced equally by carbon, light, and carbon plus light. However, what is perceived as a direct response to light could actually be an indirect response through carbon. That is, genes in InterAct class 111 could be induced either by light or carbon, or by carbon alone. We next sought to further classify the putative cis elements we identified according to one of these two models, as either a carbon-only response element, or a carbon-or-light response element, that is, a single cis element responsive to either carbon or light.
To investigate this question, we reanalyzed the putative cis elements that were identified using the promoters of the starch-metabolism and cell-wall genes in the 111 InterAct class. We now asked whether any of these putative cis elements were also over-represented in InterAct class 110 (carbon-only, model 1), a class that responds to carbon and carbon-plus-light, but not to light alone. Genes in InterAct class 110 are proposed to be regulated by carbon only, and not by light. The rationale for this analysis was that putative cis elements over-represented in InterAct class 111, but not 110, are candidate light-or-carbon responsive (LCR) cis elements. Motifs over-represented in both InterAct classes 111 and 110 are candidates for putative cis elements that are responsive to carbon only.
Our analyses found that seven of the putative cis
elements identified using the starch-metabolism genes were over-represented in InterAct class 111 genes, but were not over-represented in InterAct class 110 genes (Table ). (One motif, motif5, was over-represented in genes in both InterAct class 111 and 110.) We further eliminated an additional motif (motif55) by testing for over-representation in genes in InterAct class 011 (Table ; see also Discussion). The remaining six putative cis
elements are candidate LCR cis
elements that confer responsiveness to either light or carbon. When these six putative LCR cis
elements were compared to known elements in the PlantCARE database, four were similar to elements that had already been identified as involved in light responsiveness. Motif2/LCR4, one of the LCR cis
elements identified from the starch-metabolism genes, corresponded exactly to sequences in the DREP module, a known element in the PlantCARE database (Table ) [34
]. This cis
element has been experimentally shown to be involved in light regulation and other processes [36
]. None of the 10 putative cis
elements that we identified using the cell wall genes was significantly over-represented in genes in either InterAct class 110 or 011, and all 10 motifs are classified as LCR elements (Table ). We identified 16 LCR elements from the analysis of starch-metabolism and cell-wall genes in InterAct class 111 and most corresponded to known light-response elements including GT-1-binding sites, G-box, H-box and RE1 (Table ).
Thus, candidates for LCR cis elements that are responsible for responses to either carbon or light are proposed by the use of a combination of InterAct class analysis, MIPS analysis and statistical methods as outlined in Figure 3. Many of these candidate LCR cis elements have already been shown to be involved in light regulation. Our analyses suggest that these LCR cis elements may also be able to confer carbon responsiveness. Experiments are under way in our lab to test these hypotheses.