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Keywords:

  • starch synthase;
  • starch granule;
  • starch granule initiation;
  • amylopectin;
  • Arabidopsis;
  • knock-out mutant

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

All plants and green algae synthesize starch through the action of the same five classes of elongation enzymes: the starch synthases. Arabidopsis mutants defective for the synthesis of the soluble starch synthase IV (SSIV) type of elongation enzyme have now been characterized. The mutant plants displayed a severe growth defect but nonetheless accumulated near to normal levels of polysaccharide storage. Detailed structural analysis has failed to yield any change in starch granule structure. However, the number of granules per plastid has dramatically decreased leading to a large increase in their size. These results, which distinguish the SSIV mutants from all other mutants reported to date, suggest a specific function of this enzyme class in the control of granule numbers. We speculate therefore that SSIV could be selectively involved in the priming of starch granule formation.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Although most eukaryotes bacteria and archea accumulate α-(1[RIGHTWARDS ARROW]4) linked and α-(1[RIGHTWARDS ARROW]6) branched storage polysaccharides in the form of glycogen, green algae and land plants have resorted to the synthesis of starch, a far more complex semi-crystalline structure that accumulates as large insoluble granules within their plastids.

Amylopectin defines the major branched polysaccharide fraction of starch. Its synthesis requires the concerted action of different enzymatic activities: ADP-glucose pyrophosphorylases, starch synthases, branching enzymes and starch-debranching enzymes (Ball and Morell, 2003; Myers et al., 2000). Starch synthases (SSs) catalyze the transfer of the glucosyl moiety of ADP-glucose (the activated glucosyl donor) to a pre-existing α-(1 [RIGHTWARDS ARROW] 4) glucan primer. In a recent study it has been demonstrated that, despite its tiny genome, Ostreococcus tauri, a prasinophyte alga thought to have diverged at the earliest stage within the green linage, displays the same number and family types of SSs as those documented in either the rice or the Arabidopsis genomes (Ral et al., 2004). This high degree of conservation of the pathway suggests that these enzymes play a conserved and specific function in the building of starch. Five distinct SS families have been reported in plants. Four of these (SSI–SSIV) are predominantly found as soluble enzymes, whereas the fifth enzyme is granule-bound (GBSSI). Mutants for all the SSs have been obtained and analyzed in distinct plant and algal systems with the noticeable exception of SSIV. Predicted SSIV proteins show a C-terminal region highly similar to other SSs, which include the catalytic and starch-binding domains (Cao et al., 1999). On the contrary, the N-terminal half of SSIV protein differs significantly from other SS isoforms. Recently, Hirose and Terao (2004) have shown that the two SSIV genes found in rice (SSIV-1 and SSIV-2) are expressed throughout the plant and at relatively constant levels during the grain filling. However, genetic evidence for a role of this enzyme in starch biosynthesis is still lacking.

The picture emerging from the studies performed with all other SSs (SSI–III and GBSSI) in Chlamydomonas, pea, potato, Arabidopsis and cereals is that each enzyme is responsible for the synthesis of specific size classes of glucans within the amylopectin structure (Craig et al., 1998; Delvalléet al., 2005; Fontaine et al., 1993; James et al., 2003; Maddelein et al., 1993; Morell et al., 2003; Zhang et al., 2005). In addition, GBSSI seems to be the only elongation enzyme involved in the biosynthesis of amylose, the second unbranched and dispensable polysaccharide fraction found within starch (Delrue et al., 1992; Klösgen et al., 1986). Despite these specialized functions, some SSs display some degree of functional overlap whereas others do not. We now report on the characterization of a novel class of SS mutant in Arabidopsis. Mutants of SSIV are shown to display a modest but significant decrease in starch levels. At variance with all other SS mutants, the SSIV-lacking plants display a severe growth defect phenotype with either little or no modification in either starch granule composition or chain length (CL) distributions. The mutants, however, have dramatically decreased the number of starch granules synthesized within the plastids. Consequently they have significantly increased the size of the latter. We believe these results demonstrate an important and selective function of SSIV in the control of starch granule numbers. We speculate that SSIV is involved in the priming of starch granule formation.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Levels of SSIV mRNA in different organs

As a first step in the characterization of the function of the SSIV isoform, the spatial pattern of expression of the AtSS4 gene (locus At4g18240) was established. Using quantitative real-time RT-PCR, this pattern was compared with that of the other classes of SSs (SSI, SSII and SSIII encoded by AtSS1, AtSS2 and AtSS3 loci respectively). The four genes were expressed in all organs studied (leaves, roots, flowers and immature fruits). In all cases the steady state level of AtSS1 mRNA was one order of magnitude higher than that of the other AtSS genes (Figure 1a). On the other hand, AtSS4 mRNA accumulated at similar levels in all organs analyzed with values equivalent to those obtained for the AtSS3 gene (Figure 1b).

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Figure 1.  Expression profile of Arabidopsis starch synthase genes. The absolute mRNA levels of all the Arabidopsis soluble starch synthase encoding genes (AtSS1, At5g24300; AtSS2, At3g01180; AtSS3, At1g11720; AtSS4, At4g18240) were determined by real-time quantitative RT-PCR as described in Experimental procedures. Data shown in Panel B are the same as for Panel A, but the AtSS1 gene expression was omitted to make a clearer comparison between the other AtSS genes. Values are the average of three determinations of at least two cDNA preparations from different experiments. Samples were harvested at midday from 21-day-old plants.

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Isolation of mutant lines defective in SSIV

The AtSS4 (At4g18240) gene is located in chromosome 4 and is composed of 16 exons and 15 introns. It encodes a 1040 amino acid protein with a predicted mass of 117 747 Da. This protein shows a high level of similarity with the previously annotated SSIV proteins found in other species such as Vigna unguiculata (71% identity, accession number AJ006752), wheat (58.2% identity, accession number AY044844) or rice (56.8% identity with SSIVa, accession number AY373257; 58.3% identity with SSIVb, accession number AY373258). Bioinformatic analysis predicted the presence of a chloroplast-targeting signal comprising the first 42 amino acids, rendering a mature protein of 112 997 Da (ChloroP at http://www.cbs.dtu.dk/services/ChloroP/; Emanuelsson et al., 1999). Two independent mutant alleles, Atss4-1 [Columbia-0 (Col-0) ecotype] and Atss4-2 [Wassilewskija (WS) ecotype], designed to correspond to T-DNA insertions in the AtSS4 gene were found in the GABI-KAT (http://www.gabi-kat.de/; Rosso et al., 2003) and the Génoplante (http://www.evry.inra.fr/public/projects/bioinfo/flagdb.html; Balzergue et al., 2001) mutant collections, respectively. The T-DNA insertions are located in intron 11 and 2 (position +3763 and +227 bp with respect to the start codon for Atss4-1 and Atss4-2, respectively; Figure 2).

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Figure 2.  Analysis of mutant lines in locus AtSS4. (a) Genomic structure of the AtSS4 locus. Exons and introns are indicated as thick and thin black bars, respectively. Insertion sites of T-DNA in mutant lines Atss4-1 and Atss4-2 at introns 11 and 2 respectively are indicated by triangles. (b) Western blot analysis of crude leaf extracts of Atss4-1 and Atss4-2 mutant alleles and their respective wild-type ecotypes. Proteins (25 μg) were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose filters and immunolabelled with rabbit antiserum raised against a 178 amino acids fragment of the N-terminal region of soluble starch synthase IV (SSIV) protein (see Experimental procedures). The molecular markers (kDa) positions are indicated. Bands of approximately 112 kDa matching the predicted SSIV mass in Columbia-0 (Col-0) and Wassilewskija (WS) wild-type ecotypes are indicated by an arrow.

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Homozygous mutant plants were selected and expression of the AtSS4 mutant alleles was analyzed by RT-PCR using specific oligonucleotides for different regions of the gene. This analysis indicated that a modified messenger corresponding to AtSS4 was still present in both alleles (data not shown). Western blot analysis was then performed to check for the absence of SSIV protein in both mutant lines. The rabbit antiserum used was raised against a 178 amino acids polypeptide fragment of SSIV protein corresponding to a region that displays no similarity with all other SS isoforms (from Glu236 to Glu414) (see Experimental procedures). Western blots showed the presence of two close bands with a mass of approximately 112 kDa in both wild-type (WT) ecotypes (Figure 2b). These bands match the size of the expected mature SSIV protein and are both absent in the two mutant alleles. Truncated versions of the SSIV protein were not detected in the mutant lines either (smaller, unspecific bands were found in both mutant and WT lines; Figure 2b). The presence of these two bands in both WT ecotypes is not yet understood but could be a result of a post-translational modification of the protein.

Phenotypic characterization of Atss4 mutant alleles

The absence of the SSIV protein has a deleterious effect on plant growth. Both mutant lines showed lower growth rates under a 16-h day/8-h night photoregime when compared with their respective WT ecotypes (Figure 3, panels b and d). Rosette leaves of mutant alleles were smaller than those of WT (Figure 3, panels a and c). In addition we recorded a delay in flowering time: 31 ± 3 days for Atss4-1 versus 25 days for Col-O, and 21 days for Atss4-2 versus 18 ± 1 days for the WS ecotype. However the number of rosette leaves at bolting was the same in mutant and WT plants, indicating that the delay in flowering time comes as a consequence of the reduced growth rate in the mutant lines. Fruit size, number of seeds per silique and germination ratios were not altered in the mutant lines.

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Figure 3.  Growth of Atss4 mutant alleles and their respective wild-type ecotypes. Seeds of mutant and wild-type plants were incubated in water at 4°C for 3 days before being sowed in soil. Plants were cultured in a growth cabinet under a photoregime of 16-h light/8-h dark. Pictures were taken 21 days after sowing. Panel A: Atss4-1 mutant (left) and Columbia-0 (Col-0) plant (right). Panel C: Atss4-2 mutant (left) and Wassilewskija (WS) plant (right). Panels B and D: the above-ground organs fresh weight (FW) in mg per plant (y-axis) during the time course experiment plotted against the number of days after the germination of seeds (x-axis). •, wild type plants (Col-0 and WS in panel B and D respectively). bsl00066, mutant plants (Atss4-1 and Atss4-2 in panel B and D, respectively).

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The quantity of leaf starch was also determined in the mutant lines. The starch content at the end of the illuminated period was reduced in both cases: a 35% decrease for the Atss4-1 and a 40% decrease for the Atss4-2 line with respect to their WT genetic backgrounds. A more detailed analysis of starch accumulation over the day/night cycle was carried out on both mutant lines. As shown in Figure 4, starch turnover along a diurnal cycle was lower in mutant plants than in WT plants, with a clear reduction of both synthesis and degradation rates. Starch levels at the beginning of the light period were higher in the Atss4 alleles than in their respective WT plants. However, the reduced rate of starch synthesis in the mutant led to a lower starch level at the end of the illuminated period.

image

Figure 4.  Starch accumulation in leaves of wild-type and Atss4 mutant plants during a day/night cycle. Plants were cultured under a 16-h light/8-h dark photoperiod over 21 days and then one leaf from three plants for each line was collected at the indicated time. The starch content in leaves was determined by enzymatic assay as described in Experimental procedures. Values are the average of three independent experiments (error bars, ± SE). Panel A: Columbia-0 (bsl00043) and Atss4-1 (•). Panel B: Wassilewskija (bsl00043) and Atss4-2 (•). The bar at the top indicates day (gray bar) and night (black bar).

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Growth rate and levels of starch accumulation analogous to those in WT plants were restored when AtSS4 protein was expressed in the Atss4-1 mutant allele (Figure 5), indicating that those phenotypic alterations are a consequence of the absence of the AtSS4 protein.

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Figure 5.  Complementation of Atss4 mutation. Atss4-1 mutant allele, Atss4-1 mutant expressing AtSS4 protein (KO::P35SAtSS4) and wild type Columbia-0 (Col-0) ecotype plants were cultured under a 16-h light/8-h dark photoperiod and pictures were taken 15 days after sowing. A leaf from each plant was collected at the end of the light period, was depigmented by treatment with ethanol 80% and finally the starch was stained using Lugol solution.

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Finally, the levels of water soluble polysaccharides (WSP) and low molecular weight sugars closely related to starch metabolism, such as maltose, sucrose, fructose and glucose, were determined in both mutant strains (Figure 6). No difference in the WSP and maltose levels were observed between mutants and WT plants; however, the intracellular levels of sucrose, fructose and glucose were higher in SSIV mutant plants, and is likely to be a consequence of the lower rate of starch synthesis in those plants, which would divert the fixed carbon to soluble sugars.

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Figure 6.  Sugar content in the leaves of Atss4 mutants and wild-type plants. Leaves from 3-week-old plants cultured under a 16-h light/8-h dark photoperiod were collected at midday and the contents of sucrose, glucose, fructose, maltose and water soluble polysaccharides (WSP) were determined as described in Experimental procedures. Black columns: wild-type plants; gray columns, Atss4 mutant plants. Values represent the average of four independent experiments. Error bars,± SE.

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Enzymological characterization of Atss4 mutant lines

The levels of enzyme activities involved in the synthesis and the degradation of starch were determined in mutant lines using both zymograms and in vitro assays (Experimental procedures). No decrease in total soluble SS activity was observed in vitro in both mutants using either rabbit liver glycogen or amylopectin as primers (Table 2). Other starch metabolizing enzymes were unaffected (Table 1; Figure S2), except for the total activity of starch phosphorylase (PHS), which was increased by 1.4–2-fold, depending on the substrate used for the assay (Table 1). Zymograms analysis showed that this induction was caused by an increase in the activity of both the cytosolic (PHS2) and plastidial (PHS1) isoforms of starch PHS (Figure 7a). In order to test whether this increase could be attributable either to an interaction between SSIV and starch PHS proteins, which was missed in the mutant lines, or to another indirect effect, the mRNA level of both PHS genes in Atss4-1 was determined by real-time RT-PCR. Figure 7c shows that the expression of both genes was increased in the mutant line with respect to the WT ecotype. The extent of PHS mRNAs induction was comparable with that found for PHS activity (Figure 7b,c), suggesting that the absence of SSIV induced starch PHS activity through a metabolic alteration that triggers the induction of both plastidial and cytosolic PHS gene expression.

Table 1. In vitro assays of several starch metabolizing enzymes performed with crude extracts of leaves from AtSS4 mutant alleles (Atss4-1 and Atss4-2) and their wild-type ecotypes [Columbia-0 (Col-0) and Wassilewskija (WS), respectively]. Activities are expressed in nmol min−1 mg−1 of proteins ± SE (in each case n = 3). 1Starch-phosphorylase activity was assayed in the sense of glucan degradation. Samples were harvested at midday from 3-week-old plants
ActivityCol-0Atss4-1WSAtss4-2
Soluble starch synthase
 With rabbit liver glycogen7.86 ± 0.388.50 ± 0.476.35 ± 0.156.36 ± 0.10
 With amylopectin6.31 ± 0.256.42 ± 0.135.54 ± 0.235.39 ± 0.18
AGPase (biosynthetic assay)21.07 ± 3.521.77 ± 4.828.30 ± 2.127.48 ± 3.0
Starch branching enzyme617 ± 32582 ± 26378 ± 47447 ± 38
α-1,4-Glucanotransferase0.028 ± 0.0020.029 ± 0.0010.12 ± 0.0020.11 ± 0.003
Starch phosphorylase1
 With amylopectin16.0 ± 221.2 ± 1.714.7 ± 1.122 ± 1.2
 With DP732.2 ± 2.162.5 ± 3.136.8 ± 3.164.2 ± 2.8
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Figure 7.  (a) Zymogram of starch phosphorylase activities. Approximately 100 μg of proteins from leaf crude extract were loaded on starch-containing polyacrylamide gel. After separation and incubation overnight at room temperature in the presence of glucose-1-P at 20 mm, starch-phosphorylase activities (in the sense of glucan synthesis) were revealed by iodine staining. PHS2, cytosolic form of starch phosphorylase (At3g46970); PHS1, plastidial form of starch phosphorylase (At3g29320). (b) In vitro starch phosphorylase activities. Enzymatic assays were performed in the crude extracts of leaves using amylopectin as a substrate (as described in Experimental procedures). Activities in mutant lines are expressed as the percentage of values obtained for their respective wild-type ecotypes, which are considered to be 100%. Values are the average of three different experiments (Vertical bars = SE). Panel C) Expression of AtPHS1 and AtPHS2 genes in leaves of Atss4-1 and Col-0 plants. Levels of PHS1 and PHS2 mRNAs were determined using real-time quantitative RT-PCR as described in Experimental procedures. Data are normalized to values obtained for Col-0 plants, which are considered to be 100. Black columns: mRNA levels in Atss4-1 plants. White columns: mRNA levels in Col-0 plants (error bars, ± SE). In all cases, 3-weeks-old plants were used and samples were harvested at midday.

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Effect of eliminating SSIV on the structure and composition of starch

Interruption of the AtSS4 locus did not affect the total soluble SS activity in vitro (Table 1). However starch synthesis was clearly reduced in the mutants. In order to determine if Atss4 mutations were altering the structure and composition of starch, the amylose/amylopectin ratio and CL distribution of amylopectin were analyzed in both mutant alleles.

Amylose and amylopectin polymers were separated using size exclusion chromatography performed on sepharose CL-2B columns, and subsequently quantified by the amyloglucosidase assay. This analysis showed that the amylose/amylopectin ratio was not affected in Atss4 mutants (data not shown).

Purified amylopectin was then subjected to complete enzymatic debranching and the CL distribution was determined by fluorophore-assisted capillary electrophoresis (FACE) after coupling the resulting linear glucans with 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). Comparison of CL distribution profiles of Atss4 mutant alleles and their respective WT ecotypes indicated that the Atss4 mutation had minor effects on the structure of amylopectin (Figure 8). Indeed, only a slight reduction in the number of chains of degree of polymerization (DP) = 7–10 could be observed (note the scale of the difference of plots in Figure 8).

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Figure 8.  Amylopectin chain length (CL) distribution profiles for mutant alleles (Atss4-1 and Atss4-2) and their respective wild-type ecotypes [Columbia-0 (Col-0) and Wassilewskija (WS)]. Amylopectin was purified using a CL-2B column and was subsequently debranched with a mix of isoamylase and pullulanase. The resulting linear glucans were analyzed by fluorophore-assisted capillary electrophoresis (FACE) after coupling with a fluorescent molecule (8-amino-1,3,6-pyrenetrisulfonic acid, APTS) to their non-reducing ends. The relative proportion for each glucan in the total population is expressed as a percentage of the total number of chains. x-Axes represent the degree of polymerization (DP) of the chains; y-axes represent molar%. In the two bottom plots, the normalized value for each wild type was subtracted from that of their respective mutant allele, and in these cases the y-axes represent the molar% difference. Values are the average of three different experiments. The standard deviation was <±15% of the average values.

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Morphology and size of the starch granules were analyzed using both scanning (SEM) and transmission (TEM) electron microscopy. Starch granules were isolated from Atss4 mutant and WT leaves collected at midday and processed as described in Experimental procedures. Two major alterations were observed: first, a dramatic enlargement of granule size was detected in both mutant alleles (Figure 9b,d); second, starch granules in mutants showed a less electrodense zone in the granule core (Figure 9d), which could indicate the presence of a cavity in the hilum (the center of the starch granules) isolated from mutant plants. A more detailed study was carried out by TEM (Figure 10) and light microscopy (Figure 11) analysis on sections of leaves collected at 4 and 12 h during the light phase. Two relevant results arose from those analyses: the greatest difference in size between Col-0 and Atss4-1 starch granules was observed at the beginning of the day (after 4 h of light, panels A and B of Figures 10 and 11). This result is in line with the lower rate of starch degradation observed in mutant plants (Figure 4), which is expected to yield larger starch granules after the dark period. Secondly, most of the chloroplasts in Atss4-1 mutant plants contain a single starch granule with only a few exceptions (two granules per chloroplast) (See panels C and D in Figures 10 and 11). This observation comes in stark contrast with that obtained in WT chloroplasts, where 4–5 starch granules per chloroplast could be observed (Figure 11c). Different sections from different leaf samples were analyzed yielding the same results in all cases (data not shown). We therefore conclude that a loss-of-function mutation at the AtSS4 locus affects the number and size of starch granules synthesized in the chloroplast.

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Figure 9.  Scanning (a and b) and transmission (c and d) electron microscopy analysis of starch from 21-day-old mutant and wild-type plants. Starch granules were isolated by Percoll gradient as described in Experimental procedures from leaves collected after 8 h of illumination (midday). (a) Columbia-0 (Col-0). (b) Atss4-1. (c) Wassilewskija (WS). (d) Atss4-2.

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Figure 10.  Transmission electron microscopy analysis of sections leaves from Atss4-1 mutant and wild-type plants. Leaves of plants cultured under a 16-h light/8-h dark photoperiod were collected at 4 and 12 h of the light phase and subsequently fixed, embedded and sectioned as described in the Experimental procedures. (a) Columbia-0 (Col-0) chloroplast at 4 h. (b) Atss4-1 chloroplast at 4 h. (c) Col-0 chloroplasts at 12 h. (d) Atss4-1 chloroplasts at 12 h. CW, cell wall; S, starch; V, vacuole.

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Figure 11.  Light microscopy analysis of sections of leaves from Atss4-1 and wild-type plants. Sections of the same samples described in Figure 8 were stained with toluidine-blue. (a) Columbia-0 (Col-0) cells at 4 h. (b) Atss4-1 cells at 4 h. (c) Col-0 cells at 12 h. (d) Atss4-1 cells at 12 h. Scale bars in A and B = 25 μm; scale bars in C and D = 15 μm.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study we have analyzed the phenotypic alterations produced by the specific loss of SSIV. The analysis of two independent T-DNA insertion mutants obtained in two distinct genetic backgrounds, such as Col-0 and WS ecotypes, indicates that the shared phenotypic alterations found in the two mutants are specifically caused by the loss of the SSIV protein. The T-DNA insertions were located in intron 2 and 11 of the AtSS4 genomic locus (At4g18240) in the two mutant alleles studied in this work (Figure 2). These insertions determined in both cases the synthesis of a modified SSIV mRNA. However, western blot analysis indicated that these messengers failed to produce a WT SSIV protein (Figure 2). The antibody used in this analysis was raised against a 178 amino acids fragment of the amino-terminal region of the protein, upstream of the T-DNA insertion site, so that the presence of truncated versions of SSIV protein in the mutant alleles should be also ruled out, as no small mutant-specific polypeptide was detected by western blot (Figure 2).

The loss of function of all previously characterized classes of soluble SSs have systematically lead to a clear alteration of the amylopectin structure, thereby changing the CL distribution. Those changes of CL distribution have allowed the assignment of preferential contributions for each of the three soluble SS isoform classes in the synthesis of different chain subclasses within amylopectin (Craig et al., 1998; Delvalléet al., 2005; Fontaine et al., 1993; Gao et al., 1998; Morell et al., 2003; Zhang et al., 2004, 2005). At variance with this behaviour, Atss4 mutants showed amylopectin CL profiles similar to those found in the respective WT ecotypes. Only a weak, although reproducible, reduction of the number of short chains (DP 7–10) could be observed (Figure 8). The amylose/amylopectin ratio and levels of WSPs remained unchanged in both mutant alleles, with respect to values in their respective WT (data not shown and Figure 6). Zymogram analysis did not reflect any change in the activity levels of either SSI or SSIII isoforms (see Figure S2), thereby ruling out the existence of compensation mechanisms leading to the selective increase in the activity of other SSs making up for the loss of SSIV. These results taken together suggest that the major function of the SSIV isoform might be different from the elongation of amylopectin chains during the process of starch biogenesis.

The most visible effect of Atss4 mutations is the growth inhibition observed in Atss4 mutant lines cultured under a 16-h day/8-h night cycle (Figure 3). Previous studies of Arabidopsis mutants affected in starch degradation, such as sex1 (Caspar et al., 1991) and sex4 (Zeeman et al., 1998), have shown that an efficient mobilization of transitory starch in a diurnal cycle is required for normal growth. Thus, the growth alteration of Atss4 mutants may be caused by the decreased rate of starch degradation during the night phase (Figure 4). The finding that a normal growth phenotype is restored when mutant plants are cultured under continuous light corroborates this idea (Figure S1). The deficient starch mobilization found in Atss4 lines may also be responsible for the induction of expression of both plastidial (PHS1) and cytosolic (PHS2) starch PHS genes (Figure 7). It was suggested that these enzymes may play a role in the tolerance to abiotic stress (Zeeman et al., 2004). Thus, the nutritional starvation induced by a poor starch mobilization during the night period could be the metabolic signal that triggers the induction of the expression of both PHS isoforms. The same behavior was observed in the case of double mutants of Arabidopsis defective for both AtBE2 (locus At5g03650) and AtBE3 (locus At2g36390) in which starch synthesis is completely abolished (Dumez et al., 2006).

The main alterations in starch metabolism in the Atss4 mutants consisted in the reduction of the starch synthesis and degradation rates (Figure 4) that correlated with an enlargement of starch granule size and a decrease in granule numbers per chloroplast (Figures 9–11). The reduced rate of starch synthesis cannot be attributable to a concomitant degradation of starch during the light period as maltose levels were unaffected in plant mutants (Figure 6), but is rather attributable to a block in the synthesis of starch that deviates the photosynthetically fixed carbon to soluble sugars such as glucose and fructose (Figure 6). The decreased rate of starch turnover along the diurnal cycle cannot be explained by a reduction in activity of the starch metabolizing enzymes, as no significant changes in such activities could be detected by both in vitro and zymograms analysis (Table 1 and Figure S2). A possible explanation for this reduced rate of starch turnover could be the impairment in the ability of Atss4 mutants to synthesize more than one or two starch granules per chloroplast (Figure 11). In that case, the overall surface area of starch granules accessible to either the starch synthesis or the starch degradation machinery would be dramatically reduced. Indeed, if we consider that starch granules are true spheres, and if we consider that there is only one granule in the Atss4 mutant but four in the WT, then the available surface at the end of the day in the WT is almost 10 times larger than in the mutant. Hence the rates of both starch synthesis and degradation could be reduced if we assume that both operate at, and are limited by, the available mutant granule surface.

The reduced number of starch granules in Atss4 mutants would also be responsible for the size enlargement observed in these granules (Figures 9 and 10). In this case, all the ADP-glucose pool must be channeled to one or two granules, leading to considerably bigger starch granules in the mutants in comparison with WT plants.

The data reported here indicate that SSIV is required to determine the correct number of starch granules per chloroplasts, and that the area of polysaccharide surface is an important determinant of the rates of starch synthesis and degradation. The control of granule numbers by SSIV immediately suggests that the latter may be involved in the priming of starch granule synthesis. This idea is considerably strengthened by the finding of abnormal hilum structures, exemplified by the presence of cavities in the centre of mutant starch granules (Figure 9d). We propose that SSIV is necessary to establish an initial structure that will nucleate the crystallization and the biogenesis of a new starch granule. Priming of glycogen synthesis in mammals and fungi (uridine 5’-diphosphate (UDP)-glucose dependent systems) is carried out by an autoglucosylating protein, known as glycogenin, which synthesizes an α-1,4-linked glucan that is subsequently extended by the unique glycogen-synthase (Cheng et al., 1995). Recently, it was proposed that glycogenin-like proteins could play a role in the starch initiation process similar to that described in the animal glycogen synthesis (Chatterjee et al., 2005). In addition, other elements such as isoamylases (Bustos et al., 2004) seem to be operating in the control of the starch granule initiation process. Those data, taken together with results shown in this work, indicate that the process of starch granule initiation could be more complex than that described for mammal or fungal glycogen. Other elements, such as the one described in this work (SSIV), are likely to be involved. Indeed it has been recently demonstrated that starch metabolism is a mosaic of two distinct storage polysaccharide metabolism pathways that are likely to have existed in the first plant cells consecutively to endosymbiosis (Coppin et al., 2005). These probably consisted of a ‘eukaryotic’ pathway active in the cytoplasm, somewhat similar to fungal glycogen synthesis, and of a cyanobacterial pathway similar to that documented for other bacteria such as Escherichia coli. The priming mechanism used by plants to initiate starch granules may reflect this complexity, and therefore may be composed of both bacterial-like and eukaryotic-like components. In line with this, a selective function of glycogen-synthase in the priming of bacterial glycogen synthesis has been recently suggested (Ugalde et al., 2003). It must be stressed however that the loss of SSIV does not eliminate the ability of chloroplasts to make starch granules, thereby suggesting a certain degree of redundancy in the priming function assumed by SSIV. Further analysis of multiple mutants defective for SSIV and other genes (either directly involved or not in starch metabolism) will be necessary to ascertain if other proteins can to some extent supply part of the specialized SSIV function. In this respect, it is worth exploring both the ability of other SSs to initiate polysaccharide synthesis and the possible interaction of SSs with plant glycogenin-like proteins.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Arabidopsis lines, growth conditions and media

Mutants lines of Arabidopsis thaliana were obtained from the T-DNA mutant collections generated at URGV, INRA, Versailles (Bechtold et al., 1993; Bouchez et al., 1993) and the GABI-KAT (Köln, Germany) mutant collection (Rosso et al., 2003). WT ecotypes (WS line WS-4, accession number N5390 in the Nottingham Arabidopsis Stock Center (NASC, Nottingham, UK); Col-0, accession number N1093 in NASC) and mutant lines were grown in growth cabinets under a 16-h light/8-h dark photoregime at 23°C (day)/20°C (night), 70% humidity and a light intensity at the plant levels of 120 μE m−2 sec−1 supplied by white fluorescent lamps. Seeds were sown in soil and irrigated with 0.5X MS medium (Murashige and Skoog, 1962).

RNA extraction and reverse transcription

Total RNA was isolated according to the method described by Prescott and Martin (1987). Prior to cDNA synthesis, and in order to remove contaminating genomic DNA, the RNA preparations were incubated with 10 U of DNAse I FPLC Pure for 10 min at 37°C, extracted with phenol and chloroform, precipitated and then dissolved in nuclease-free MilliQ-water. First-strand cDNA was synthesized from 10 μg of total RNA using Moloney Murine Leukemia Virus (MMLV)-RT and oligo(dT)12−−18 primer, according to the manufacturer's instructions. The reaction was incubated at 37°C for 2 h and stopped by adding 1 ml of nuclease-free MilliQ-water. All the reagents were from Amersham Biosciences (Uppsala, Sweden).

Production of polyclonal antibody against SSIV

Leaf total RNA was used to obtain cDNA as described above. Oligonucleotides SA215 (5′-CATATGGAGACTGATGAAAGGATT-3′) and SA216 (5′-CTCGAGTTCTTTATAAACGTTGGC-3′) were used to amplify a 521-bp fragment of SSIV cDNA encoding the section from Glu236 to Glu414 of the SSIV amino acids sequence. Those oligonucleotides introduced restriction sites for NdeI and XhoI at the 5′ and 3′ ends of the cDNA fragment, respectively, which were used to clone the cDNA fragment in the expression vector pGEX-4T (Amersham Biosciences) fused in frame to the 3′-end of the glutathione-S-transferase (GST) gene. Construct was confirmed by DNA sequencing and transformed into E. coli BL21 (DE3) strain. Protein expression, purification of the GST-SSIV fragment fusion protein with glutathione agarose and purification of the SSIV fragment by cleavage of the matrix-bound GST fusion protein with thrombin were performed following the procedure described by Ausubel et al. (1987). Rabbit polyclonal antiserum was raised against the purified SSIV fragment. Finally, the immunoglobulin G fraction of antiserum was purified by FPLC using a Protein A Sepharose column (Amersham Biosciences) following the manufacture's instructions.

Real-time RT- PCR analysis

Real-time quantitative RT-PCR assays were achieved using an iCycler instrument (Bio-Rad, Hercules, CA, USA). The PCR reaction mixture contained in a total volume of 25 μl, 5 μl of cDNA, 0.2 mm of deoxyribonucleotide triphosphate (dNTPs), 2.5 mm of MgCl2, a 1 : 100 000 dilution of SYBR® Green I nucleic gel stain (Molecular Probes, Eugene, OR, USA) : fluorescein calibration dye (Bio-Rad), 0.3 U of Taq polymerase, 2.5 μl 10 × Taq polymerase buffer, and 0.2 μm of each primer. The specific oligonucleotides used were: SA198 (5′-TGATGAGAAGAGGAATGACCCGAAA-3′) and SA199 (5′-CCATAGATTTTCGATAGCCGA-3′) for AtSS1; SA126 (5′-GGAACCATTCCGGTGGTCCATGCCG-3′) and SA127 (5′-CTCACCAATGATACTTAGCAGCAACAAG-3′) for AtSS2; SA200 (5′-GTGCAAGACGGTGATGGAGCAA-3′) and SA201 (5′-CACGTTTTTTATATTGCTTTGGGAA-3′) for AtSS3; SA419 (5′-CGTGACTTAAGGGCTTTGGA-3′) and SA420 (5′-GCAGCTCGGCTAAAATACGA-3′) for AtSS4; SA546 (5′-TGGAAGGAAACGAAGGCTTTG-3′) and SA547 (5′-TGTCTTTGGCGTATTCGTGGA-3′) for AtPHS1; SA548 (5′-ACAGGTTTTGGACGTGGTGATT-3′) and SA549 (5′-ACAGGACAAGCCTCAATGTTCCA-3′) for AtPHS2; 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 sec at 94°C, 15 sec at 61°C and 15 sec at 72°C. After that, a melting curve was generated to check the specificity of the amplified fragment. The efficiency of all the primers at the above conditions was between 75% and 110% in all the tested samples, and product identity was confirmed by sequence analysis. Arabidopsis Ubiquitin 10 (Sun and Callis, 1997) was used as a house-keeping gene in the expression analysis. Absolute quantification (Ginzinger, 2002) was performed by cloning the amplified products in pGEM-T vector (Promega, Madison, WI, USA), and then using them as external calibration standards.

Complementation of the Atss4 mutation

The full-length cDNA of AtSS4 (At4g18240) was amplified by PCR using primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCATGGCGACGAAGCTATCGAGCTT-3′ (forward) and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTACGTGCGATTAGGAACAGCTCTT-3′ (reverse) designed to contain the attB sites for cloning using the GATEWAYTM system (Invitrogen, Carlsbad, CA, USA). Amplified fragment was cloned into pGEM-T easy (Promega), mobilized to vector pDONR221 (Invitrogen) and finally cloned into the GATEWAYTM binary vector pCTAPi (Rohila et al., 2004), which fused a small polypeptide containing protein A and calmodulin-binding protein domains at the C-terminal of the protein, and allows the expression of AtSS4 under a 35S promoter. The construct was introduced into Agrobacterium tumefaciens strain C58, which was used to transform Arabidopsis Atss4-1 mutant plants by the floral-dip method described by Clough and Bent (1998) Previously, Atss4-1 mutant plants were tested to be sensitive to BASTA herbicide. Twenty T3 progeny resulting from homozygous self-crosses were used for phenotypic characterization.

Extraction and purification of starch

For the analysis of the structure and composition of starch, Arabidopsis leaves were harvested at the end of the light period. Approximately 10 g of fresh material was homogenized using a Tissue Tearor (Biospec Products Inc., Bartlesville, OK, USA) in 30 ml of the following buffer: 100 mm 3-(N-morpholino) propanesulphonic acid (MOPS), pH 7.2, 5 mm EDTA and 10% (v/v) ethanediol. The homogenate was filtered through two layers of Miracloth (Millipore, MA, USA) and centrifuged for 15 min at 4°C and 4000 g. The pellet was resuspended in 30 ml Percoll 90% (v/v) and centrifuged for 40 min at 4°C and 10 000 g. The starch pellet was washed six times with distilled sterile water (10 min at 4°C and 10 000 g between each wash). Starch was finally stored at 4°C in 20% ethanol. For the analysis of starch content in leaves along the diurnal cycle, the method was scaled down and three leaves (approximately 300 mg) from three different plants were used in each point. Material was frozen with liquid nitrogen, homogenized with a mortar and pestle and resuspended in 1 ml of buffer. Starch isolation was performed using Percoll gradient centrifugation as described above.

Extraction and determination of sugars

Leaf tissue (0.5 g aprox.) was harvested and frozen in liquid N2. The material was powdered and extracted in 1.5 ml 0.7 m perchloric acid, as described in Critchley et al. (2001). Fructose, glucose and sucrose content in the buffered extract was determined by enzymatic analysis as described by Stitt et al. (1989). Maltose levels were determined by enzymatic analysis as described by Shirokane et al. (2000).

Determination of starch and WSP contents and spectral properties of the iodine–starch complex

Starch content in leaves was quantified enzymatically as described previously by Lin et al. (1988). A full account of λmax (the maximal absorbance wavelength of the iodine–polysaccharide complex) measures can be found in Delrue et al. (1992). Water-soluble glucan contents in leaves were determined as described in Zeeman et al. (1998).

Separation of starch polysaccharides by size exclusion chromatography

Starch (1.5–2.0 mg) was dissolved in 500 μl of 10 mm NaOH and subsequently applied to a Sepharose CL-2B column (inner diamater, 0.5 cm; length, 65 cm), which was equilibrated and eluted with 10 mm NaOH. Fractions of 300 μl were collected at a rate of one fraction per 1.5 min. Glucans in the fractions were detected by their reaction with iodine and levels of amylopectin and amylose were determined by amyloglucosidase assays.

Chain length distribution of amylopectin

FACE of debranched amylopectin  After purification on a Sepharose CL-2B column, 500 mg of amylopectin was dialyzed against distilled water and subsequently lyophilized. The amylopectin pellet was resuspended in 1 ml of 55 mm sodium acetate, pH 3.5 buffer, and incubated overnight at 42°C with 20 U of isoamylase isolated from Pseudomonas amyloderamosa (Hayashibara Biochemical Laboratories, Okayama, Japan) and 1 U of pullulanase from Klebsiella pneumoniae (Sigma, St Louis, MO, USA). Salts were subsequently removed by passage through an extract-clean carbograph column (Alltech, Deerfield, IL, USA).

Derivatization procedure.  Glucans were derivatized with APTS according to the manufacturer's recommendations (Beckman Coulter, Fullerton, CA, USA). Briefly, 2 ml APTS in 15% acetic acid solution and 2 ml of 1 m of NaBH3CN in tetrahydrofolate were mixed and the coupling reaction was allowed to proceed overnight at 37°C in the dark.

Capillary electrophoresis analysis.  Separation and quantification of APTS-coupled glucans was carried out on a P/ACE System 5000 (Beckman Coulter, Fullerton, CA, USA) equipped with a laser-induced fluorescence system using a 4-mW argon ion laser. The excitation wavelength was 488 nm and the fluorescence emitted at 520 nm was recorded on the Beckman p/ace station software system (version 1.0). Un-coated fused-silica capillaries of 57 cm in length and 75 μm inner diameter were used. Running buffers were from Beckman Coulter. Samples were loaded into the capillaries by electroinjection at 10 kV for 10 sec and a voltage of 30 kV was applied for 20 min at a constant temperature of 25°C.

Zymograms techniques

A complete description of these techniques can be found in Delvalléet al. (2005).

In vitro assays of starch synthesis enzymes

ADP-glucose pyrophosphorylase was assayed in the synthesis direction according to the procedure described by Crevillén et al. (2003). Starch synthase activity was assayed as described by Delvalléet al. (2005) using either amylopectin or glycogen as primers. Branching enzymes, starch PHS and α-1,4-glucanotransferase activities were performed according to procedures described by Zeeman et al. (1998).

Western blot analysis

Proteins were transferred from an SDS-polyacrylamide gel to nitrocellulose membrane by electroblotting in a Trans-Blot SD transfer cell (Bio-Rad) according to the manufacturer's instructions. Blots were probed with rabbit anti-SSIV followed by horseradish peroxidase-conjugated goat-anti-rabbit serum and detected using ECL Plus Advanced Western Blotting Reagent (Amersham Biosciences).

Microscopy analysis

Fully expanded leaves from plants cultured under a 16-h light/8-h dark regime were collected at the indicated times. Small pieces (2 mm2) of leaves were cut with a razor blade and immediately fixed in 1% paraformaldehyde and 0.5% glutaraldehyde in 0.05 m Na-cacodylate buffer, pH 7.4, containing 25 mg of sucrose per ml (3.5 h at 4°C, under vacuum). After fixing and rinsing with the same buffer, tissues were dehydrated in an ethanol series and progressively embedded in LR White resin (London Resin Co., Reading, UK). Resin was polymerized with UV light at −20°C (Fedorova et al., 1999). Alternatively, some samples were fixed in 3% glutaraldehyde in the above buffer and embedded in Araldite Durcupan ACM as described by Lucas et al., 1998. Semi-thin (1-μm) and ultra-thin (60-nm) sections were cut with a Leika Ultracut microtome (Leika, Vienna, Austria) fitted with a diamond knife. Semi-thin sections for light microscopy were stained with 1% (w/v) toluidine blue in aqueous 1% sodium borate for direct observation with a Zeiss Axiophot photomicroscope (Zeiss, Oberkochen, Germany). Ultrathin sections for transmission electron microscopy were contrasted with 2% aqueous uranyl acetate and lead citrate (Reynolds, 1963). Observations were performed with a STEM LEO 910 electron microscope (Oberkochen, Germany) at 80 kV, equipped with a Gatan Bioscan 792 digital camera (Gatan, Pleasanton, CA, USA). Different sections from at least three different leaves samples were analyzed.

For scanning electron microscopy analysis samples were sputter-coated with gold and viewed with a JEOL JSM-5400 microscope (JEOL, Tokyo, Japan). Transmission microscopy analysis was performed as described by Delvalléet al. (2005).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to Cesar Morcillo and Fernando Pinto for technical assistance in the microscopy analysis. This work was supported by the Spanish Ministerio de Educación y Ciencias (grant no. BIO2003-00431), the French Ministère déléguéà la Recherche (ACI jeunes-chercheurs n°5145), the Génoplante consortium (grant n°Af2001030), the Région Nord Pas de Calais, the European Union-FEDER (ARCir projet en émergence), the Centre National de la Recherche Scientifique, and EMBO (IR was a recipient of an EMBO short-term fellowship in the C. D'Hulst laboratory).

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  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Growth of Atss4-1 mutant allele and Col-O wild type plants. Seeds of mutant and wild type plants were incubated in water at 4°C for 3 days before sowing in soil. Plants were cultured in growth cabinet under a short-day photoregime of 8 h light/16 h dark (SD, plants at the top) or continuous light (LL, plants at the bottom). Pictures were taken 21 days after sowing. Plants at the left, Atss4-1 mutants. Plants at the right, WT plants. Figure S2. (a) Zymogram of soluble starch synthases from leaf extract performed under native conditions. After migration of 150 μg of proteins on a glycogen-containing gel and incubation overnight at room temperature with 1 mM ADP-glucose, SS activities were revealed by soaking the gel for 30 min in lugol solution (I2/KI) and washed several times with distilled water before the picture was taken. Lanes WS and Col-0: wild-type references corresponding to ecotypes Wassilewskija and Columbia respectively. Lanes Atss4-1 and Atss4-2: SS4 mutant samples. (b) Zymogram of starch-modifying activities performed on a starch-containing polyacrylamide gel. After migration and incubation overnight at room temperature, starch-modifying enzymes were revealed by iodine staining. In all cases, three weeks old plants were used and samples were harvested at midday.

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TPJ_2968_sm_FigureS1.zip662KSupporting info item
TPJ_2968_sm_FigureS2.zip2284KSupporting info item

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