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Originally published In Press as doi:10.1074/jbc.M411713200 on December 14, 2004

J. Biol. Chem., Vol. 280, Issue 9, 8143-8149, March 4, 2005
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Differential Pattern of Expression and Sugar Regulation of Arabidopsis thaliana ADP-glucose Pyrophosphorylase-encoding Genes*

Pedro Crevillén{ddagger}, Tiziana Ventriglia{ddagger}, Francisco Pinto§, Alicia Orea{ddagger}, Ángel Mérida{ddagger}, and José M. Romero{ddagger}

From the {ddagger}Instituto de Bioquímica Vegetal y Fotosíntesis and §Instituto de Investigaciones Químicas, Universidad de Sevilla-Consejo Superior de Investigaciones Cientificas (CSIC), Américo Vespucio 49, 41092, Seville, Spain

Received for publication, October 14, 2004 , and in revised form, November 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
ADP-glucose pyrophoshorylase (ADP-Glc PPase) catalyzes the first and limiting step in starch biosynthesis. In plants, the enzyme is composed of two types of subunits (small and large) and is allosterically regulated by 3-phosphoglycerate and phosphate. The pattern of expression and sugar regulation of the six Arabidopsis thaliana ADP-Glc PPase-encoding genes (two small subunits, ApS1 and ApS2; and four large subunits, ApL1–ApL4) has been studied. Based on mRNA expression, ApS1 is the main small subunit or catalytic isoform responsible for ADP-Glc PPase activity in all tissues of the plant. Large subunits play a regulatory role, and the data presented define a clear functional distinction among them. ApL1 is the main large subunit in source tissues, whereas ApL3 and, to a lesser extent, ApL4 are the main isoforms present in sink tissues. Thus, in source tissues, ADP-Glc PPase would be finely regulated by the 3-phosphoglycerate/phosphate ratio, whereas in sink tissues, the enzyme would be dependent on the availability of substrates for starch synthesis. Sugar regulation of ADP-Glc PPase genes is restricted to ApL3 and ApL4 in leaves. Sugar induction of ApL3 and ApL4 transcription in leaves allows the establishment of heterotetramers less sensitive to the allosteric effectors, resembling the situation in sink tissues. The results presented on the expression pattern and sugar regulation allow us to propose a gene evolution model for the Arabidopsis ADP-Glc PPase gene family.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Starch is the major storage polysaccharide in plants and is accumulated as granules in many different organs such as leaves, roots, shoots, fruits, or grains, where it is used as a carbon and energy source. The regulatory and rate-limiting step of starch biosynthesis is the synthesis of the glucosyl donor, ADP-glucose, by ADP-glucose pyrophosphorylase (ADP-Glc PPase)1 (EC 2.7.7.27 [EC] ) (1). ADP-Glc PPase is a heterotetramer ({alpha}2{beta}2) composed of two different types of subunits (1, 2). The small subunit (SS) is considered the fully catalytic one, whereas the large subunit (LS) plays a modulatory function regulating the activity of the enzyme (1, 3, 4). Nevertheless, different studies on ADP-Glc PPase from maize and potato have suggested that both subunits may have a regulatory role (5, 6). The activity of most plant ADP-Glc PPases is finely regulated by an allosteric mechanism (7). The enzyme is activated by 3-phosphoglycerate (3-PGA) and inhibited by Pi. This allosteric modulation is considered to represent the main control of the starch biosynthesis rate in photosynthetic tissues and in many non-photosynthetic tissues (1), and it is exerted by the relative ratio between 3-PGA and Pi. However, in some plants, the enzyme from developing seeds does not respond to these regulators (8–10). Besides the allosteric regulation of ADP-Glc PPase activity, a post-translational redox control that involves the action of thioredoxins has been shown (11) and acts in response to sugars and light (12, 13).

Different isoforms of ADP-Glc PPase have been described, and many cDNAs and genomic DNAs have been isolated from both monocot and dicot plants. In most cases, a single SS gene and several LS genes are present in a given species (1, 4). It has been postulated that specific tissues should present different ADP-Glc PPases with distinct regulatory properties that would be dependent on the specific LS present in a given tissue (3, 14). However, there is no comprehensive study of the expression of all the different ADP-Glc PPase isoforms in a single plant system. Six genes encode for proteins with homology to ADP-Glc PPase in the Arabidopsis genome. Two of these genes encode for SS, ApS1 (At5g48300) and ApS2 (At1g05610); and four of these genes encode for LS, ApL1 to ApL4 (At15g19220, At1g27680, At4g39210, and At2g21590). We have shown recently that the Arabidopsis large subunits confer different regulatory properties to ADP-Glc PPase in vitro and would modulate the activity of the ADP-Glc PPase enzyme according to the biosynthetic starch necessities in a tissue-specific manner. Thus, according to their kinetic and regulatory properties, the large subunits were classified as sink and source isoforms (4). However, the distinction between sink LS and source LS needs further confirmation because in A. thaliana only partial information concerning some genes has been reported (15–18). To address this question, a complete study of the expression pattern of all ADP-Glc PPase-encoding genes is needed.

Sugars play a basic role as substrates in carbon and energy metabolism as well as precursors of starch biosynthesis. They are also important signaling molecules controlling the growth and development of plants (19). With regard to the starch synthesis pathway, it has been shown that sugars mediate the induction of several genes of the pathway (20–23), including some genes encoding for ADP-Glc PPase (24–26), and also trigger the redox regulation of ADP-Glc PPase activity (12, 13). Induction of ApL3 mRNA levels in response to sucrose (15, 27) and trehalose (28) has been described in Arabidopsis leaves and seedlings. However, information concerning the effect of sugars on the expression of the other ADP-Glc PPase genes from Arabidopsis is scarce.

The aim of this work is to understand the role of the different ADP-Glc PPase isoforms in Arabidopsis thaliana, a model system to study starch biosynthesis (29). We present novel data about the expression pattern of the six genes encoding for ADP-Glc PPase isoforms in different organ and tissues, and we characterize the effect of sugar supply on the expression of those genes under physiological conditions. The data presented provide solid evidence that confirms a working model proposed previously by us assigning specific roles to the different ADP-Glc PPase subunits (4).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material and Growth Conditions—A. thaliana (Columbia ecotype) was grown in growth cabinets under a 16-h light/8-h dark photo-regime at 23 °C (day)/20 °C (night), 70% humidity, and a light intensity at the plant level of 100 microeinsteins m-2 s-1. Plants were sown in soil for mRNA expression studies assays or in perlite for sugar inductions assays. Sugar induction treatments consisted of irrigation of the mature plants with MS medium (30) supplemented with 100 mM sorbitol, 100 mM glucose, 100 mM sucrose, or 25 mM trehalose. We chose sugar concentrations according to previous reports (15, 17). During sugar treatments, plants were maintained under the same growing conditions, and samples were harvested at the middle point of the photoperiod (8 h light) during 4 consecutive days, frozen in liquid nitrogen, and stored at -80 °C until use.

RNA Extraction and Reverse Transcription—Total RNA was isolated as described by Prescott and Martin (31). Before cDNA synthesis, in order to remove contaminating genomic DNA, the RNA preparations were incubated with 10 units of DNase I FPLC Pure for 10 min at 37 °C, extracted with phenol and chloroform, precipitated, and dissolved in nuclease-free MilliQ-water. First-strand cDNA was synthesized from 10 µg of total RNA using Moloney murine leukemia virus reverse transcriptase and oligo(dT)12–18 primer, according to the manufacturer's instructions (Amersham Biosciences). The reaction was incubated at 37 °C for 2 h and stopped by adding 1 ml of nuclease-free MilliQwater. All reagents were from Amersham Biosciences.

Real-time Quantitative RT-PCR Analysis—Real-time quantitative RT-PCR assays were achieved using an iCycler instrument (Bio-Rad). The PCR mixture contained (in a total volume of 25 µl) 5 µl of cDNA, 0.2 mM deoxynucleotide triphosphates, 2.5 mM MgCl2, a 1:100,000 dilution of SYBR® Green I nucleic gel stain (Molecular Probes)/fluorescein calibration dye (Bio-Rad), 0.3 unit of Taq polymerase, 2.5 µl of 10x Taq polymerase buffer, and 0.2 µM of each primer. Specific oligonucleotides used were as follows: SA253 (5'-GAAAAACCAAAAGGGGAGCA-3') and SA254 (5'-CCTCAGTCCAAGGGAAGTGG-3') for ApS1; SA255 (5'-CTCGGTGTTTGCCTCCAAG-3') and SA256 (5'-TCTCCTTCCCTTTTCTTCTCACA-3') for ApS2; SA257 (5'-TCCCCACAGCAAACGACTT-3') and SA258 (5'-GGTGGCAGGTTTCTCCTTGA-3') for ApL1; SA259 (5'-TCGGAAAAACCAAAGGGAGA-3') and SA260 (5'-GGCCAACGGGATAATTTCAG-3') for ApL2; SA261 (5'-AGATCGGGAAAAACGTGGTG-3') and SA262 (5'-CTCTTTTTAACTTCCGGCCAAAC-3') for ApL3; SA263 (5'-AGCAGATAGGCCAGAGGAAGG-3') and SA264 (5'-GCGGGAAAGAAAAGATCGAAG-3') for ApL4; and UBQF (5'-GATCTTTGCCGGAAAACAATTGGAGGATGGT-3') and UBQR (5'-CGACTTGTCATTAGAAAGAAAGAGATAACAG-3') for UBQ10. Thermal cycling consisted of 94 °C for 3 min, followed by 40 cycles of 10 s at 94 °C, 15 s at 61 °C, and 15 s at 72 °C. After that, a melting curve was generated to check the specificity of the amplified fragment. In the case of ApL4 (primers SA263 and SA264), the annealing temperature was 64 °C. The efficiency of all the primers at the conditions described above was between 75% and 110% in all the tested samples, and product identity was confirmed by sequence analysis. Arabidopsis Ubiquitin 10 (32) was used as a housekeeping gene in the expression analysis. Absolute Quantification (33) was performed by cloning the amplified products in pGEM®-T vector (Promega) and using them as external calibration standards.

In Situ Hybridization Analysis—For mRNA in situ hybridizations, sense and antisense RNA probes were obtained by transcription of the 3'-untranslated region of the Arabidopsis ADP-Glc PPase-encoding genes cloned into pBluescript SK(-) vector (Clontech) from the T7 promoter. The digoxigenin-labeled riboprobes were prepared following the manufacturer's instructions (Roche Applied Science). In situ hybridizations were performed as described by Mérida et al. (34), at 50 °C in a solution containing 2x SSC and 50% formamide; signal was detected, and the sections were mounted in Entellan mounting medium (Merck). The sections were observed through an Olympus BX60 microscope by Normarski microscopy and photographed using a JVC GC-X3E digital camera.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Arabidopsis ADP-Glc PPase Genes Show Differential mRNA Expression Levels—We have determined the absolute steady-state mRNA levels of the six Arabidopsis ADP-Glc PPase-encoding genes by real-time quantitative RT-PCR. Specific primers for the six ADP-Glc PPase genes and for the housekeeping gene, Ubiquitin 10 of Arabidopsis (32), were designed, and their efficiency and reliability were checked. The fragments amplified by these primers were cloned and sequenced, and the resulting plasmids were used as external calibration standards (33).

All six mRNAs were detected in leaves, inflorescences, fruits, and roots of mature plants, but at very different levels (Fig. 1). ApS1, ApL1, ApL3, and ApL4 showed elevated mRNA steadystate levels, whereas ApS2 and ApL2 mRNA were accumulated at very low levels. These data are in accord with previous observations in our laboratory, in which ApS2 and ApL2 could not be detected by Northern blot.2 ApS1 was the major SS isoform in all organs of the plant. A more complex situation was observed for ApL genes. ApL1 was the most abundant LS in leaves, with levels of mRNA up to three times higher than that of ApL3 and more than 1 order of magnitude higher levels of transcript than ApL4. In inflorescences and fruits, ApL3 was the most abundant ADP-Glc PPase large subunit transcript. In roots, the level of expression of all ADP-Glc PPase genes was very low compared with leaves, and ApL3 was the predominant LS with up to 3-fold and 1 order of magnitude higher levels of transcript than ApL4 and ApL1, respectively. These data show that there is a tissue-specific level of expression of the different ADP-Glc PPase subunits in A. thaliana.



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  		FIG. 1.
Expression profile of the Arabidopsis ADP-Glc PPase-encoding genes. The absolute mRNA levels of all the Arabidopsis ADP-Glc PPase-encoding genes were determined by real-time quantitative RT-PCR as described under "Experimental Procedures." In the figure, quantities are represented in a logarithmic plot in order to compare the data among the different genes. The values are the average of the quantification of three cDNA preparations from different experiments. The error bars in the plot represent the S.D.

 

Expression Pattern at the Cellular Level—The mRNA expression pattern of Arabidopsis ADP-Glc PPase genes was studied by in situ hybridization in leaves, inflorescences, and fruits. Fig. 2 shows the expression pattern of the SSs, ApS1 and ApS2. Both genes are detected in the main starch-producing tissues of leaves, the mesophyll and the vascular companions cells (Fig. 2, a–d). In flowers, a strong signal is detected in the stamens and pistil, as well as in the receptacle. This pattern was observed in both young (Fig. 2f for ApS1; ApS2 data not shown) and mature flowers (Fig. 2, e and g). ApS1 and ApS2 are also clearly expressed in the embryo (Fig. 2, h and i). A different situation was observed for the ADP-Glc PPase LSs. In leaves, ApL1, ApL2, and ApL3 transcripts are localized in the mesophyll and vascular companions cells (Fig. 3, a–e), but ApL4 appeared to be restricted only to the surrounding cells of the vascular veins (Fig. 3, f and g). Similar to the SS mRNAs, all the LS mRNAs were detected in all tissues of flowers at different developmental stages (Fig. 3, h–k). In fruits, ApL1 is expressed uniformly in the embryo (Fig. 3, l and m), whereas ApL2, ApL3, and ApL4 expression was restricted to the pro-vascular cells of the embryo (Fig. 3, n–r). An unspecific signal in the seed coat (testa) was observed in most of the hybridizations with fruit sections. No signal was detected with the control sense riboprobes for all the genes (Fig. 2, j–m for ApS1; ApS2 and LS control sense hybridizations not shown).



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  		FIG. 2.
Pattern of expression of the Arabidopsis ADP-Glc PPase small subunits. a–d, leaf sections hybridized with the antisense probe against ApS1 (a and b) and ApS2 (c and d); e–g, flowers at different developmental stages hybridized with the antisense probe against ApS1 (e and f) and ApS2 (g); h and i, longitudinal sections of fruits showing the seed with the embryo hybridized with the antisense probe against ApS1 (h) and ApS2 (i). j–m, control samples hybridized with the ApS1 sense probe; hybridizations with the ApS2 sense probe are not shown. The mRNA in situ hybridization experiments were performed as described under "Experimental Procedures" using specific digoxigenin-labeled riboprobes against each SS gene.

 



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  		FIG. 3.
Pattern of expression of the Arabidopsis ADP-Glc PPase large subunits. a–g, leaf sections hybridized with the antisense probe against ApL1 (a and b), ApL2 (c), ApL3 (d and e), and ApL4 (f and g); h–k, flowers at different developmental stages hybridized with the antisense probe against ApL1 (h), ApL2 (i), ApL3 (j), and ApL4 (k); l–r, magnifications of longitudinal sections of fruits showing the seed and the embryo hybridized with the antisense probe against ApL1 (l and m), ApL2 (n), ApL3 (o and p), and ApL4 (q and r). Control samples hybridized against the sense probes are not shown. The mRNA in situ hybridization experiments were performed as described under "Experimental Procedures" using specific digoxigenin-labeled riboprobes against each LS gene.

 
The results shown indicate that there is not a differential pattern of expression between the two SSs. On the contrary, a clear differential expression pattern of the LS genes was observed in leaves and fruits. This fact, together with the different mRNA expression levels (Fig. 1), suggests a tissue specificity that could be related to the role of each LS isoform in regulating the activity of the heterotetrameric ADP-Glc PPase enzyme.

ApL3 and ApL4 Are Induced by Sugars in Leaves—Most studies on sugar-regulated transcription of genes involved in the synthesis of starch have been performed with seedlings or detached leaves (15, 24, 27). This type of assay, although very informative, may not reflect the real physiological response of the plant, could magnify the differences in the mRNA levels, and is restricted to specific tissues. To overcome this problem, we have designed a sugar induction assay in which all the different organs of the plant could be harvested under the same experimental conditions (see "Experimental Procedures"). Arabidopsis plants irrigated with MS medium were grown to maturity. Then, plants were irrigated with MS medium supplemented with different sugars for several days. Samples were harvested at the middle point of the photoperiod, and the mRNA levels were determined by real-time quantitative RT-PCR. This experimental design reflects the carbon status of mature plants in a better way than in detached leaves or seedlings and at the same time allows global analysis of different organs.

Fig. 4 shows the steady-state ADP-Glc PPase mRNA levels in Arabidopsis leaves after irrigation with MS medium supplemented with 100 mM sucrose. A time-course analysis showed that only ApL3 and ApL4 mRNAs were sucrose-induced and that, after 72 h of treatment, a significant induction of both genes could be observed (a 10-fold and an 18-fold increase in ApL3 and ApL4 mRNA levels, respectively), and induction of both genes was even higher at 96 h. No variation in the transcript levels was observed for ApS1, ApS2, ApL1, and ApL2 transcripts. A similar behavior was observed when Arabidopsis plants were supplemented with glucose (100 mM) or trehalose (25 mM) (data not shown). Results in Fig. 5 show the induction of ApL3 and ApL4 after 72 h of sugar treatment. The mRNA levels of ApL3 were 4-fold higher in the presence of glucose (100 mM), 10-fold higher in the presence of sucrose (100 mM), and 6-fold higher in the presence of trehalose (25 mM) compared with control plants in the absence of added sugars. Induction of ApL4 was even higher, reaching 20- and 30-fold the control levels in the presence of sucrose and trehalose, respectively. No significant effect was observed when plants were supplemented with sorbitol, thus suggesting that sugar induction of ApL3 and ApL4 is not due to osmotic stress (Fig. 5). We also studied the effect of sugar treatment in other organs of mature Arabidopsis plants, and no sugar induction of ADP-Glc PPase-encoding genes was observed in fruits, inflorescences, or roots (data not shown). In summary, our data clearly show that only two genes encoding for Arabidopsis ADP-Glc PPase, ApL3 and ApL4, are sugar-regulated in source tissues (leaves), whereas sugar regulation in sink tissues does not control the level of any ADP-Glc PPase-encoding gene.



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  		FIG. 4.
Effect of sucrose on the mRNA level of ADP-Glc PPase-encoding genes. The figure shows a time-course mRNA expression analysis of the six ADP-Glc PPase-encoding genes in Arabidopsis leaves of plants induced with 100 mM sucrose. The plants were treated with sucrose, and the mRNA levels were determined by real-time quantitative RT-PCR as described under "Experimental Procedures." The data in the plot are normalized to the value in the control sample without induction. The values are the average of three determinations of two cDNA preparations. The S.D. was less than ±10% for each data point.

 



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  		FIG. 5.
Sugar induction of ApL3 and ApL4 mRNA expression level. Arabidopsis plants were fed with 100 mM glucose, 100 mM sucrose, 25 mM trehalose, or 100 mM sorbitol, and the ADP-Glc PPase-encoding gene mRNA levels in leaves were determined by real-time quantitative RT-PCR as described under "Experimental Procedures." The figure shows the results for the ApL3 and ApL4 genes. The data are normalized to the value of the control without induction. The values are the average of three determinations of two cDNA preparations. The S.D. was less than ±10% for each data point.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have characterized the expression of the Arabidopsis ADP-Glc PPase-encoding genes in different organs and tissues, determining their absolute transcript levels by real-time quantitative RT-PCR and their expression pattern by in situ hybridizations. Although the six transcripts were detected in all the studied organs, our results clearly show that the different ADP-Glc PPase subunits are expressed at different levels and have specific expression profiles among the different organs of the plant. Both SS-encoding genes are expressed in all the starch-producing tissues of the plant. However, ApS1 expression level is up to 2 orders of magnitude higher than that of ApS2. It has been shown previously that ApS1 plays a critical role in starch biosynthesis because adg1-1 mutant, affected in APS1, shows very low levels of starch accumulation (35). Besides, in vitro studies with the wild type (4) and a mutated APS2 H137D protein3 have suggested that ApS2 is a non-active isoform, with ApS1 being the only catalytic ADP-Glc PPase subunit in Arabidopsis. Our results on the expression data reported in this work confirm the role of ApS1 as the main, if not the sole, SS isoform responsible for ADP-Glc PPase activity in A. thaliana.

The ADP-Glc PPase large subunits are considered to play a regulatory role, modulating the activity in response to allosteric effectors (4). The mRNA levels of the four genes encoding for LS show significant differences. Whereas ApL1 is the most highly expressed ADP-Glc PPase LS in leaves (Fig. 1), ApL3 reaches the highest levels of expression in sink organs (inflorescences, fruits, and roots). Differentiation is also observed at the cellular level (Fig. 3). In seeds, ApL1 appeared to be expressed in all the embryo cells, whereas the others LSs are preferentially localized in the pro-vascular cells. In mature leaves, we clearly detect the ApL1, ApL2, and ApL3 mRNAs in the mesophyll cells as well as in the bundle sheet, whereas ApL4 was restricted to the companion cells of the vascular veins. We could not determine which subunit is expressed in the guard cells of the stomata, cells that accumulate significant amounts of starch. However, microarray analysis has shown that ApL4 is the most abundant LS in guard cells, reasserting the sink nature of this gene (36). Thus, a clear distinction among the LSs can be established. APL1 is the large subunit showing the highest sensitivity to 3-PGA and Pi (4) and, by its level of expression, would be the main isoform in source tissues, thus providing a fine modulation of ADP-Glc PPase by the 3-PGA/Pi ratio to adjust starch synthesis to carbon fixation. In sink tissues, APL3 and, to a lesser extent, APL4 would be the main LS isoforms participating in the establishment of the ADP-Glc PPase heterotetramer. These two LSs have been shown to confer less sensitivity to 3-PGA and Pi to the enzyme (4), and thus starch synthesis would be more dependent on the supply of substrates from source tissues than on the allosteric effectors. The results support our previous model in which a functional distinction among the different LSs was proposed on the basis of the kinetic and regulatory properties that they confer to the ADP-Glc PPase heterotetramer (4).

The specialized function of the different ADP-Glc PPase isoforms is also reflected in their response to sugars. Coordinated sugar transcriptional regulation of several starch biosynthetic genes has been suggested by numerous works (19). Induction of several ADP-Glc PPase-encoding genes has been reported in different species including Arabidopsis (16, 25–28). We show that, under physiological conditions, only two ADP-Glc PPase-encoding genes, ApL3 and ApL4, are controlled by sugars (Figs. 4 and 5). Sugar induction of ApL3 and ApL4 genes is leaf-specific and does not take place in sink tissue. Induction was stronger in response to disaccharides than to glucose (Fig. 5), suggesting that different signaling pathways may be involved in the transcriptional control of ApL3 and ApL4 by sugars. To date, trehalose-regulated expression of ApL3 has been shown (28), but no data regarding ApL4 regulation have been published. Trehalose feeding rescues the phenotype of the adg2-1 mutant lacking ApL1 (17), and it was proposed that under these conditions, APL3 would substitute for APL1. Nevertheless, our results demonstrate that trehalose would alter the subunit composition of ADP-Glc PPase by inducing both APL3 and APL4.

The low-starch mutant adg2-1 confirms our hypothesis that in normal situations, APL1 is the main LS isoform in the leaf mesophyll. In this tissue, there is also a significant amount of APL3 transcript (Figs. 1 and 3). However, it is very likely that in the metabolic environment of the leaf, most of the starch synthesis was driven by the APS1+APL1 heterotetramer. In plants fed with sugars, the intracellular level of hexoses increases (27, 37). In this situation, the change of the metabolic conditions in the mesophyll and the increased ApL3 and ApL4 expression would allow the establishment of heterotetramers less sensitive to the allosteric effectors (4), so that the enzyme would respond to or be controlled by the supply of substrates. This would lead to a higher rate of starch synthesis in response to the higher carbohydrate availability, resembling the situation in sink organs. In fact, sugar induction treatments increase starch levels in our plants (data not shown).

Several lines of evidence are in apparent contradiction with the results that we present on sugar regulation of ADP-Glc PPase isoforms. In previous studies, it was reported that the ApL3 promoter fused to the GUS gene was only expressed in the bundle sheet and that its expression was induced in the leaf mesophyll cells in response to sucrose (15). However, according to the data presented in Fig. 2, ApL3 can be detected in the mesophyll cells of plants grown in the absence of sugars. The simplest explanation for this discrepancy is that the ApL3 promoter used for the GUS construct lacked element(s) needed for its normal expression. Induction with sucrose would raise the mRNA levels in the mesophyll cells, making it possible to detect GUS. On the other hand, results have also been published reporting that in Arabidopsis detached leaves, ApS1, ApL3, and ApL2 genes were induced by sucrose and glucose, whereas ApL1 was repressed (ApS2 and ApL4 were not known at that time) (27). This discrepancy is explained by the probes and assays used in that work. Sokolov et al. (27) used probes based on the available sequences at that time, which corresponded to a conserved region of the LS genes (38). Thus, they detected ApL2, whereas our observations using specific probes indicate that this gene is undetectable by Northern blot. Besides, they used detached leaves maintained in the dark for the induction assay. In that case, the effect of sugar treatment could be more severe or unspecific due to higher water evaporation rates or dehydration stress (15). We have analyzed the effect of the sugars under normal physiological conditions, and the only ADP-Glc PPase-encoding genes regulated by sugars at the transcriptional level are ApL3 and ApL4.

The characterization of the expression pattern and sugar regulation of the ADP-Glc PPase gene family allows us to propose a model of gene evolution of this family in Arabidopsis. APS1 is essential for starch biosynthesis and is the fully catalytic subunit of the enzyme (4, 35), whereas APS2 has several amino acid substitutions and is not active in vitro (4, 39). Thus, our hypothesis is that the lone functional Arabidopsis SS is APS1. APS2, although it is still expressed at very low levels in the plant, is a nonfunctional ADP-Glc PPase subunit that could be accumulating mutations through a process of pseudogenization (40).

It is accepted that many genes appear by gene duplication (40, 41), and it has been proposed that the different ADP-Glc PPase LSs of a plant specie would result by divergence and specialization (7). It is also accepted that the Arabidopsis genome underwent several ancient rounds of duplication (42). Indeed, ApL3 and ApL4 genes are located in sister genomic regions that have been described in different genomic analysis (www.tigr.org/tdb/e2k1/ath1/Arabidopsis_genome_duplication.shtml). Our biochemical studies (4) showed that both LSs confer similar regulatory properties to the enzyme, and in the present report, we show that both ApL3 and ApL4 genes are similarly induced by sugars (Figs. 4 and 5). On the other hand, the expression pattern of the two genes is different (Figs. 1 and 3). These facts support the hypothesis that they are related genes under a process of functional divergence. Because it is not usual to find twin genes so well characterized, we think this is a great example of subfunctionalization that could be used in further genomic analysis (41).

In summary, we have determined the expression pattern and sugar regulation of the six Arabidopsis ADP-Glc PPase-encoding genes. A clear functional distinction can be made between the different large subunit isoforms that could be considered as sink (ApL2, ApL3, and ApL4)- or source (ApL1)-specific. Besides, a shift in the ADP-Glc PPase LS composition in response to sugar feeding allows the enzyme to be efficiently regulated according to the metabolic situation and the starch necessities of a given tissue. This hypothesis is in accordance with our previous data concerning the kinetic properties of the enzyme (4).


   FOOTNOTES
 
* This work was supported in part by Grants BMC2002-00984 and BIO2003-00431 from the Ministerio de Ciencia y Tecnología (Spain) and by Junta de Andalucía group CVI-281 (Spain). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 34-954-489526; Fax: 34-954-486500; E-mail: jmromero{at}ibvf.csic.es.

1 The abbreviations used are: ADP-Glc PPase, ADP-glucose pyrophosphorylase; SS, small subunit; LS, large subunit; 3-PGA, 3-phosphoglycerate; RT-PCR, reverse transcription-PCR. Back

2 P. Crevillén, unpublished observations. Back

3 T. Ventriglia, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We are grateful to Teresa Ruíz for critical reading of the manuscript. The Plant Culture facilities from the Centro de Investigaciones Científicas Isla de la Cartuja (CICIC) are also acknowledged.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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