Journal of Experimental Botany, Vol. 55, No. 396, pp. 397-409, February 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
Structure and expression profile of the sucrose synthase multigene family in Arabidopsis
Received 4 August 2003; Accepted 28 October 2003
Unité de Biologie des Semences, UMR 204, INA-PG-INRA, RD 10, 78026 Versailles cedex, France
* Present address: Groupe de Physiologie Cellulaire et Moléculaire des Plantes, Université Pierre et Marie Curie/CNRS, UMR 7632, 75252 Paris cedex 05, France. To whom correspondence should be addressed. Fax: +33 1 30 83 31 11. E-mail: rochatc{at}versailles.inra.fr
Abbreviations: DAF, days after flowering; RT-PCR, reverse transcriptase-polymerase chain reaction.
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Abstract |
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The release of the complete genome sequence of Arabidopsis enabled the largest sucrose synthase family described to date, comprising six distinct members, for which expression profiles were not yet available, to be identified. Aimed at understanding the precise function of each AtSUS member among the family, a comparative study of protein structure was performed, together with an expression profiling of the whole gene family using the technique of real-time quantitative reverse transcriptase-polymerase chain reaction. Transcript levels were analysed in several plant organs, including both developing and germinating seeds. A series of treatments such as oxygen deprivation, dehydration, cold treatment, or various sugar feedings were then carried out to characterize the members of the family further. The AtSUS genes exhibit distinct but partially redundant expression profiles. Under anaerobic conditions, for instance, both AtSUS1 and AtSUS4 mRNA levels increase, but in a distinct manner. AtSUS2 is specifically and highly induced in seeds at 12 d after flowering and appears as a marker of seed maturation. AtSUS3 seems to be induced in various organs under dehydration conditions including leaves deprived of water or submitted to osmotic stress as well as late-maturing seeds. AtSUS5 and AtSUS6 are expressed in nearly all plant organs and do not exhibit any transcriptional response to stresses. These results add new insights on the expression of SUS genes and are discussed in relation to distinct functions for each member of the AtSUS family.
Key words: Arabidopsis, functional genomics, seed development, stress response, sucrose synthase, transcriptional profiling.
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Introduction |
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Sucrose synthase (SUS; UDP-glucose: D-fructose 2-glucosyltransferase, EC 2.4.1.13 [EC] ) is a key enzyme involved in sucrose metabolism. This enzyme catalyses the reversible conversion of sucrose and UDP to UDP-glucose and fructose. Its activity has been studied in various plants and has been shown to play a major role in energy metabolism, controlling the mobilization of sucrose into various pathways important for the metabolic, structural, and storage functions of the plant cell (Hesse and Willmitzer, 1996; Sturm and Tang, 1999, for a review). First of all, SUS activity associated with vascular tissues appears to play a key role in supplying energy in the companion cells for phloem loading by providing substrate for respiration (Fu and Park, 1995; Hänggi and Fleming, 2001). SUS is also proposed to supply UDP-glucose for cell wall biosynthesis in association with the cellulose synthase complex (Delmer and Amor, 1995; Haigler et al., 2001; Ruan et al., 2003). SUS cleavage activity correlates with the sink strength of storage organs, providing substrates for starch synthesis in potato tubers, maize kernels, or pea embryos (Sun et al., 1992; Zrenner et al., 1995; Déjardin et al., 1997). SUS expression is also associated with important processes such as nodule development and metabolism in legumes (Craig et al., 1999; Hohnjec et al., 1999) or apical meristem functioning in tomato (Pien et al., 2001). Several pieces of evidence indicate that SUS exists both free in the cytosol and in association with the plasmalemma (Amor et al., 1995; Carlson and Chourey, 1996). Moreover, a tonoplast-associated form of SUS has been described recently in red beet (Etxeberria and Gonzalez, 2003). In vitro experiments suggest that the phosphorylation status of the enzyme could determine the extent to which SUS is soluble or membrane-bound (Winter et al., 1997), but the mechanisms that act in vivo remain highly debated.
SUS genes have been isolated primarily from starch-storing and sucrose- or hexose-storing plants (Sturm and Tang, 1999). Enhanced expression level in sink tissues is a widespread observation emerging from diverse SUS expression studies. SUS gene induction under stress conditions is also often described in roots and/or shoots. For instance, increased SUS transcript levels are thought to be involved in meeting the increased glycolytic demand during O2 deficiency (Ricard et al., 1998; Déjardin et al., 1999). Cold treatment, dehydration, or osmotic stress are also reported to induce SUS expression (Hesse and Willmitzer, 1996; Déjardin et al., 1999; Kleines et al., 1999). In the same manner, SUS expression is regulated by externally provided sugars (Koch, 1996; Déjardin et al., 1999). The vast majority of SUS genes characterized to date belong to small multigene families, both in monocot and dicot species (Komatsu et al., 2002). Due to partial redundancy in expression patterns, elucidating the precise function of each isoform in a family, if any, is far from being obvious. Moreover, the multigene families studied to date might be incomplete (Carlson et al., 2002), thus hindering the overall understanding of the family.
In the model plant Arabidopsis thaliana, the complete sequencing of the genome reveals six putative members in the SUS gene family (Barratt et al., 2001). The complete AtSUS family, which is the largest SUS family described to date, appears as a good system to elucidate the function of each isoform, which is mostly unknown. AtSUS1 (At5g20830) was shown to be expressed in the phloem of leaves and in roots (Martin et al., 1993). AtSUS2 (At5g49190) was cloned and characterized by Chopra et al. (1992). The two genes were found to be strongly and differentially regulated in leaves exposed to environmental stresses (Déjardin et al., 1999). To date, no data relative to AtSUS3 (At4g02280), AtSUS4 (At3g43190), AtSUS5 (At5g37180), or AtSUS6 (At1g73370) are available. Aimed at analysing the six AtSUS genes, the real-time quantitative reverse transcriptase (RT)-PCR technique appears both as a highly sensitive and specific method (Bustin, 2000). Because it allows the specific detection of each member of the family, the real-time RT-PCR technique is indeed a good tool to carry out an analysis of the expression level of genes belonging to a very conserved gene family. This was illustrated in A. thaliana on the members of a cell wall enzyme gene family (Yokoyama and Nishitani, 2001), on the NRT2 nitrate transporter family (Orsel et al., 2002), and on the shaggy-like kinase multigene family (Charrier et al., 2002).
This article reports on the comprehensive expression profile of the six AtSUS genes in various organs of A. thaliana, including time-scale studies on developing and germinating seeds. A series of abiotic treatments such as O2 deprivation, dehydration, cold treatment, or various sugar feedings was then considered to characterize the members of this multigene family further. Finally, the role of the different AtSUS isoforms will be discussed in the light of their expression patterns, which appear to be original.
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Plant material and growth conditions
Seeds of A. thaliana ecotype Wassilewskija (WS) were surface-sterilized and germinated on Murashige and Skoog (MS) medium (M02 555, pH 5.6; Duchefa, Haarlem, The Netherlands) solidified with 0.7% (w/v) agar. After a cold treatment of 48 h, at 4 °C, in the dark, the plates were transferred to a growth chamber and incubated at 21/15 °C day/night, under a 16/8 h light/dark regime. After 15 d, the plantlets were transferred to sterilized compost in individual pots, grown under the same conditions as above and irrigated twice a week with a complete mineral nutrient solution (Lesaint and Coïc, 1983). Primary shoots only were used and secondary shoots were removed. To harvest siliques of defined developmental stages, individual flowers were tagged using coloured tape on the day of flowering. For time-course studies during the seed maturation process, all the developing seeds of one shoot were harvested 3–4 weeks after the onset of flowering: siliques ranging from 6–18 d after flowering (DAF) were opened and the corresponding seeds removed and subsampled. To obtain root material, seedlings were grown in vertical plates and maintained in growth chamber for 4–5 weeks. For time-course study during the germination process, sterilized seeds were germinated on 0.8% (w/v) agar in the same conditions as described above. For stress treatments, germinating seeds and plantlets were incubated at 21/18 °C day/night, under a 8/16 h light/dark regime to avoid flowering. After 6–7 weeks, some plants were either deprived of water, transferred to a cold chamber (cold-stressed plants; 5 °C, same day/light regime), or submerged into degassed water. For the feeding experiments (sugar treatments), leaves were detached from intact plants, pre-adapted to the dark for 5 h, and placed into containers for 16 h with water or sugars/osmotica in 10 mM MES/KOH, pH 6.25. All the plant material were immediately frozen in liquid nitrogen after harvesting and stored at –80 °C prior to RNA extraction.
RNA extraction and cDNA preparation
Frozen tissues were ground in liquid nitrogen and total RNA was extracted with the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. The extracts were treated with 30 units of Rnase-free Dnase I (Qiagen) to eliminate the residual genomic DNA present in the preparation and eluted with 40 µl of diethyl pyrocarbonate-treated water. For the expression study in plant organs, two independent extractions per organ were performed, while three independent extractions were carried out for each data point in stress studies. For reverse transcription (RT)-PCR studies, DNA-free RNA was converted into first strand cDNA using the SuperScript preamplification system for first-strand cDNA synthesis (Gibco BRL, Cergy Pontoise, France) with oligo(dT)22. In all cases, three independent RT were performed.
Real-time quantitative RT-PCR
The reaction was performed on the LightCycler Instrument (Roche, Meylan, France) with the LightCycler-FastStar DNA Master SYBR Green I kit for PCR (Roche) according to the manufacturer’s protocol. Each reaction was performed with 5 µl of 1:10 to 1:100 dilution of the first cDNA strands in a total volume of 20 µl. The reactions were incubated at 95 °C for 8 min to activate the hot start recombinant Taq DNA polymerase, followed by 45 cycles of 10 s at 95 °C, 6 s at 55 °C, and 20 s at 72 °C (Orsel et al., 2002; Baud et al., 2003). Specific primer sets were designed for each gene (Table 1). The specificity of the PCR amplification was checked with a heat dissociation protocol (from 65 °C to 95 °C) following the final cycle of the PCR; PCR products were then purified and sequenced. The efficiency of the primer sets was calculated by performing real time PCR on several dilutions of first strands. Efficiencies of the different primer sets used were checked to be almost similar. The specificity of each primer set was checked by sequencing PCR products. The results obtained for the different tissues analysed were standardized to the constitutive EF1A4
gene expression level (Liboz et al., 1990).
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Construction of the distance tree
For the construction of the rooted tree, only the glucosyl-transferase domains of each selected sucrose synthase protein were used. The MultAlin program (Corpet, 1988) was used to create an alignment of the sequences that was then submitted to a Neighbor–Joining analysis to generate a branching pattern thanks to the ClustalX package (Thompson et al., 1997). Bootstrap analysis with 1000 replicates was performed to assess the statistical reliability of the tree topology and nodes with bootstrap score of <50% were discarded. The consensus tree was drawn using the TreeView program (version 1.5.3, Roderic DM Page, University of Glasgow, UK).
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Structure of the AtSUS family
The A. thaliana genome sequence was searched with genomic and cDNA sequences of AtSUS1 (Martin et al., 1993) and AtSUS2 (Chopra et al., 1992) using the BLAST program: four new putative AtSUS genes were isolated (Barratt et al., 2001). A complete alignment of the deduced amino acid sequences, beginning and ending at the putative start and stop codons, respectively, was performed using the Needle program (Needlman and Wunsch, 1970; Table 2). Three distinct AtSUS couples arose from this analysis (Fig. 1). The highest percentage of amino acid identity/similarity was found between AtSUS1 and AtSUS4 (89% and 95%, respectively). AtSUS2 and AtSUS3 then appeared to be closely related as well (74% and 86%). AtSUS5 and AtSUS6 are two isoforms very different from the four other AtSUS. A schematic representation of the AtSUS gene structures is presented in Fig. 1. The predicted exon/intron structure of these genes was analysed between the start and stop codons, regardless of the presence (AtSUS2; Chopra et al., 1992) or absence (AtSUS1; Martin et al., 1993) of a non-coding leader exon 1. The six genes shared the two characteristic sucrose synthase and glucosyl-transferase domains. The AtSUS1/AtSUS4 couple was characterized by the presence of a 6th/5th 336 bp long and a 12th/11th 564/567 bp long unsplit exon. In AtSUS2 and AtSUS3, the 564 bp exon was split by an intron. AtSUS5 and AtSUS6 were characterized by the presence of an 11th 567 bp unsplit exon, while the 336 bp exon was split by an intron. Moreover, AtSUS5 and AtSUS6 genes exhibited a 3' extension that had never been observed so far among the plant SUS genes. As a consequence, the predicted molecular masses of the corresponding proteins (96 and 107 kDa; Table 2) were higher than those calculated for other AtSUS peptides (92/93 kDa in average).
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Expression analysis of AtSUS genes in A. thaliana organs
To date, among the six AtSUS genes present in the A. thaliana genome, expression analyses were reported for the AtSUS1 and AtSUS2 genes (Martin et al., 1993; Déjardin et al., 1999). A set of specific primers was designed to study the expression profile of each member of the family in various organs of the plant. The specificity of each primer was checked by sequencing of the PCR products. Figure 2 illustrates the relative transcript levels of the six AtSUS genes standardized to the constitutive EF1A4
(EF) gene expression level (Liboz et al., 1990). Due to high variations in the relative transcript levels observed between various plant organs for a single gene, a logarithmic scale was chosen to present these results. Each type of transcript was detected, demonstrating that all the members of this gene family were transcriptionnaly active. In the set of organs tested, the AtSUS1 gene exhibited transcript levels ranging from 0.1% EF in flowers to 1.7% EF in silique walls at 15 d after flowering (DAF). AtSUS2 transcripts were barely detectable and never exceeded 0.1% EF. The AtSUS3 expression pattern only differed from the one described for AtSUS2 in that increased transcript levels were observed in silique walls at 15 DAF (4.5% EF). The highest transcript levels for AtSUS4 were measured in roots (11% EF) and stems (3.4% EF), as well as in silique walls at 6 DAF (6.4% EF), while the lowest expression levels for this gene was observed in flowers and young siliques. Transcript levels measured for AtSUS5 and AtSUS6 were fairly constant in all organs studied, except for the oldest silique wall samples, where they could not be detected.
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Expression analysis of AtSUS genes in developing and germinating seeds
The relative expression profile of the six members of the AtSUS gene family was investigated in developing seeds. For each gene, a five-point kinetic was performed (Fig. 3A) that was representative of the three developmental stages of A. thaliana seed development (Baud et al., 2002): early embryo morphogenesis at 6 DAF, maturation at 9, 12, and 15 DAF, and late maturation at 18 DAF. The highest and most significant variations in relative AtSUS transcript levels observed during seed development were those encountered by AtSUS2 and AtSUS3. The AtSUS2 transcript level displayed a 2600-fold increase between 6 and 12 DAF, peaking at almost 80% EF, before falling sharply at 5% EF at 15 DAF. The AtSUS2 transcripts were not detectable anymore at 18 DAF. Conversely, the transcript level of AtSUS3 was lower than 1% EF until 12 DAF, then it increased steadily to reach almost 30% EF at 18 DAF. During all the seed development process, AtSUS1, AtSUS4, and AtSUS5 displayed a similar, fairly constant, and weak expression pattern, with no relative transcript level exceeding 0.5% EF. AtSUS6, though exhibiting a low expression profile too, peaked at 12 DAF, reaching 1.4% EF.
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The expression pattern of the six AtSUS genes was also analysed in germinating seeds. For each gene, a four-point kinetic was performed (Fig. 3B) from 0–72 h after the first exposure to light (fully germinated plantlets). Root protrusion was effective at 48 h. The AtSUS3-relative transcript level observed at 0 h (almost 20% EF) was comparable to the one measured in desiccating seeds at 18 DAF. It then decreased regularly until 24 h, reaching 1.6% EF, and then remained almost stable. During all the germination process, AtSUS1, AtSUS2, AtSUS4, AtSUS5, and AtSUS6 exhibited low and almost constant expression profiles, with no level exceeding 0.6% EF.
Expression analysis of AtSUS genes in response to O2 deficiency
Induction of SUS genes by hypoxia or anoxia is a widely described phenomenon in dicot and monocot species, both in roots and shoots (Marana et al., 1990; Ricard et al., 1998; Zeng et al., 1998). To characterize the expression pattern of AtSUS genes in response to O2 deficiency, 6–7-week-old plants were submerged into degassed water for 2 d. AtSUS mRNA levels were analysed in the rosette leaves after 24 h and 48 h of treatment. The results showed that the AtSUS genes are differentially regulated by O2 deprivation (Fig. 4). While AtSUS2, AtSUS3, AtSUS5, and AtSUS6 mRNA levels were unaffected by the stress, a significant induction was observed for AtSUS1 and AtSUS4. After 1 d of treatment, their transcript levels exhibited a very significant 3-fold and 2-fold induction, respectively. This induction appeared to be transient for AtSUS1 and was not observed further after 2 d of stress. In the case of AtSUS4, this induction level rose steadily at 48 h, but this was not observed for all the leaves treated, thus explaining the relatively low level of significance of the variation observed.
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Expression analysis of AtSUS genes in response to dehydration, cold treatment, and sugar feedings
The relative transcript level of the six AtSUS genes was analysed in leaves of plants undergoing a gradual dehydration treatment. For each gene, a four-point kinetic was performed that began at the onset of water deprivation and ended with a quite drastic dehydration of the plant, 11 d later (Fig. 5). An increase in both AtSUS1 and AtSUS3 transcript levels was observed during the last days of treatment. While AtSUS3 mRNA level exhibited a 40-fold induction, the transcript level of AtSUS1 increased up to 8 times in response to water deprivation. In both cases, statistical analyses demonstrated that inductions observed in stressed leaves were significant. An increase in rab18 transcript level (Lang et al., 1994) was specifically observed 11 d after the onset of water deprivation (data not shown). Osmotic potential measurements confirmed that dehydration was not serious before day 11 (data not shown).
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The relative transcript level of the AtSUS genes was then analysed in leaves of plants undergoing a cold treatment (4 °C during 48 h). While AtSUS2, AtSUS4, AtSUS5, and AtSUS6 mRNA levels were unaffected by the stress, 5.5- and 24.5-fold inductions were measured for AtSUS3 and AtSUS1, respectively (Fig. 6). Statistical analyses showed that the increase observed in AtSUS1 mRNA level was very significant.
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Sugars are known to affect SUS gene expression levels in various species (Koch, 1996). Moreover, sugar contents are liable to change in leaves during stress exposure. As a consequence, the putative effect of exogenously provided sucrose and mannitol in the regulation of AtSUS gene expression was investigated. Before treatments, A. thaliana plants were kept in the dark for 5 h to lower the internal level of soluble sugars. The relative expression profile of the six members of the AtSUS family was analysed in darkened cut leaves fed for 14 h with sugar/osmotica (Fig. 7). The AtSUS1 mRNA level exhibited a slight (2.5-fold), but significant, increase in response to the two sucrose concentrations tested. In the same manner, the 4-fold (250 mM mannitol) and 7-fold (sucrose+mannitol) inductions observed in response to mannitol (penetrating osmotic agent) were statistically significant. An even stronger and more significant induction (up to 37-fold) due to mannitol was measured for the AtSUS3 transcript level, while no induction was observed with sucrose alone. Conversely, under the various feeding experiments tested, AtSUS2, AtSUS4, AtSUS5, and AtSUS6 displayed a fairly constant expression pattern.
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The AtSUS genes exhibit distinct but partially overlapping expression patterns
Recent investigations (Sturm et al., 1999; Komatsu et al., 2002) as well as the publication of the A. thaliana genome sequence support the idea that SUS genes can be classified into four distinct groups. The comparison of predicted amino acid sequences as well as the intron/exon structure of the SUS genes corroborate this classification. The AtSUS family comprises six different genes distributed in three of these groups (Figs 1, 8A). AtSUS1 and AtSUS4 can be classified among the vast dicot SUS1 group, characterized at the gene structure level by the presence of two specific unsplit exons (Fig. 1; Komatsu et al., 2002). AtSUS2 and AtSUS3 belong to the SUSA group. Their 564 bp long exon is split by an intron in the same manner as for genes of the SUSA and monocot SUS1 groups (Fig. 1; Komatsu et al., 2002). AtSUS5 and AtSUS6 constitute a separate group on their own. The six predicted amino acid sequences exhibit a typical sucrose synthase organization with, in particular, the sucrose synthase and the glucosyl-transferase domains identified in the Pfam database (Bateman et al., 2002).
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In an attempt to elucidate the role of each AtSUS member, the expression profile of each of the six AtSUS genes was specifically described in various plant organs, including developing and germinating seeds, thanks to the sensitivity and the reliability of the real-time RT-PCR technique. To characterize the members of this family further, the relative expression profile of each AtSUS gene was investigated in plants undergoing various abiotic treatments. It was possible to show that there is no pseudogene in the AtSUS family and that the six AtSUS genes exhibit distinct but partially overlapping expression patterns. Such a high level of gene redundancy in both structure and expression profiles in a plant with the smallest genome size is remarkable. Thus, these results add a significant new contribution to the current understanding of the genome complexity in plants.
AtSUS5 and AtSUS6 constitute an independent group although the functions remain unknown
This study shows that AtSUS5 and ATSUS6, two genes composing a separate and new group on their own, are ubiquitously expressed in the plant (Fig. 2). Comparison of predicted amino acid sequences anchors these two genes on an independent branch of the distance tree, located between other plant SUS and bacterial SUS (Fig. 8A). Alignment of amino acid sequences then shows that the RXXS consensus sequence recognized by Ser/Thr protein kinase and classically located in the N terminal region of SUS proteins was modified in T/KXXS in AtSUS5 and AtSUS6. According to recent results obtained on the rice enzyme (Asano et al., 2002), this may suggest that AtSUS5 and AtSUS6 are not phosphorylated at this site. AtSUS5 and AtSUS6 putatively encode for proteins of relatively high molecular weight, the kinetic properties of which are not yet determined. This aspect is particularly striking in the case of AtSUS6, which encodes for a 107 kDa protein with a predicted pI of 8.1, the 3' extension of this particular gene containing some low complexity regions (Bateman et al., 2002). The two genes do not exhibit any transcriptional response to the stresses tested. Specific tissue localization and/or monitoring response to other types of stresses could help to improve current understanding of the specific role of these two particular genes. Moreover, post-transcriptional and post-translational data relative to these two particular genes would be of particular interest.
AtSUS1 and AtSUS4 are closely related but exhibit distinct expression profiles
AtSUS1 and AtSUS4, members of the dicot SUS1 group, exhibit the highest percentage of amino acid identity/similarity among the AtSUS family (Table 2). Transcripts of the corresponding genes are detected in the whole plant, and expression of AtSUS1 is fairly constant in all organs analysed, while AtSUS4 appears to be more expressed in roots, stems, and silique walls (Fig. 2). The relative expression profile observed in response to oxygen deficiency induced by water flooding (Fig. 4) then demonstrates that both genes exhibit significant induction after one day of treatment. AtSUS1 and AtSUS4 transcripts indeed increased by 3- and 2-fold, respectively, after 24 h of stress, and these data are in agreement with previous results. In A. thaliana transgenic plants, the AtSUS1 promoter transcriptionally fused to the ß-glucuronidase gene, was shown to be inducible by the same anaerobic treatment (Martin et al., 1993). Likewise, a cDNA microarray study, characterizing the expression profile of A. thaliana root cultures in response to low-oxygen treatment, pointed out the induction of both AtSUS1 and AtSUS4 in the same order of amplitude (2- and 5-fold, respectively; Klok et al., 2002). It was not possible to measure any induction or repression of the other AtSUS genes in response to anoxia induced by water flooding, in contrast to Déjardin et al. (1999). Using a northern blot strategy, these authors describe a strong increase in AtSUS2 mRNA level in leaves in response to O2 deficiency while they do not measure any increase in AtSUS1 transcript level. In other plant species, gene induction in response to oxygen deficiency was demonstrated for Daucus carota SUS1, Triticum aestivum SUS1, Beta vulgaris SUS, and Zea mays SUS1 and SH1 (Fig. 8B). In maize, SUS1 and SH1 are up-regulated preferentially by hypoxia (3% O2) and anoxia (0% O2), respectively (Zeng et al., 1998). Thus, it appears that fine differences in oxygen might have a strong effect on the expression of oxygen-responsive genes.
Conversely, AtSUS1 and AtSUS4 differ in their response to dehydration (Fig. 5), cold treatment (Fig. 6), or sugar/osmotica feeding (Fig. 7). The AtSUS4 transcript level remains steady in all the conditions tested. On the contrary, AtSUS1 is up-regulated by cold treatment (24.5-fold) as already mentioned in other studies (Déjardin et al., 1999; Marana et al., 1990). An induction by sucrose (2.5-fold) was then reported, but this effect does not seem to be specific to sucrose, since mannitol (a penetrating osmotic agent) led to an even more pronounced induction (4–7-fold). Dehydration also induces an increase in the AtSUS1 transcript level (8 times). This can be interpreted either as a specific effect or as a side-effect of sugar and/or osmotic increase during stress (data not shown). These results corroborate previous observations (Déjardin et al., 1999) showing that AtSUS1 is up-regulated by a decrease in leaf osmotic potential rather than by the increase in sucrose concentration per se. Nevertheless, recent studies strongly suggest that sugars (sucrose, glucose, and mannose) regulate AtSUS1 via HXK-modulated mechanisms (Cieresko and Kleczkowski, 2002). Sugars are known to be inducers/repressors of gene expression and they have key roles in signal transduction pathways (Koch, 1996; Smeekens, 2000). As a conclusion, the regulation of AtSUS1 possibly involves two or more transduction pathways depending on the nature and strength of the signal (Cieresko and Kleczkowski, 2002).
AtSUS2 is a marker of seed maturation
AtSUS2 transcripts are barely detectable in all the plant organs analysed, with the outstanding exception of seeds undergoing maturation. SUS was classically described as a marker of sink strength in storage tissues such as developing tubers or seeds in which the SUS pathway of sucrose cleavage was usually associated with starch biosynthesis (Zrenner et al., 1995; Carlson et al., 2002). However, the very strong induction in relative AtSUS2 transcript level is measured between 6 and 12 DAF, corresponding in the growth conditions to the first period of the seed storage phase (Baud et al., 2002). This developmental phase is characterized by a decrease in starch concentration in the seed and by the onset of storage product biosynthesis, including both triglycerides and proteins. This result is consistent with a microarray study that examined changes in gene expression during A. thaliana seed development (Ruuska et al., 2002). Expression of AtSUS clones was indeed reported to increase strongly between 8 and 12 DAF and then coincide with an increase in oil and protein synthesis rather than with the transient starch accumulation in young seeds. Moreover, King et al. (1997) reported a peak in SUS activity in developing Brassica seeds, emphasizing the role of SUS in providing carbon for oilseed metabolism. In A. thaliana seeds, preliminary measurements of SUS activity corroborate the observed expression pattern (data not shown). AtSUS2 could, consequently, appear as a marker for storage compound accumulation, providing carbon skeletons through the glycolytic pathway for triglyceride and protein synthesis, rather than starch synthesis. AtSUS2 also appears as specifically expressed in maturing seeds, and is not induced in leaves in response to the various abiotic stresses tested, including O2 deficiency, dehydration, or sugar feedings.
AtSUS3 is a marker of dehydration
The expression profile of the AtSUS3 gene is very specific and highly inducible. It is mainly expressed in 15 DAF silique walls (as they began to turn yellowish), in late maturing seeds (the AtSUS3 transcript level increases while the seed water content steadily decreases; Baud et al., 2002), and in early germinating seeds (Fig. 3). Induction of this gene by drought and mannitol, an osmotic agent, is shown to be strong in stressed leaves (Fig. 5). Moreover, abundant AtSUS3 ESTs were described in dehydration-treated rosette plants (TAIR database). AtSUS3 also appears as a marker of dehydrating tissues and behaves as an osmoticum- but not sucrose-responsive gene. Likewise, recent studies already suggested, using the gin1/aba2 mutant, that glucose regulation of some genes was distinct from regulation by osmotic stress (Cheng et al., 2002). More experimentation will be required to dissect these regulatory pathways for AtSUS3.
Interesting parallels can be drawn between the AtSUS3 expression pattern and available data concerning different SUS isoforms closely related to AtSUS3 on a distance tree (Fig. 8). Like AtSUS3, citrus SUSA (Komatsu et al., 2002), pea SUS2 (Buchner et al., 1998; C Rochat, unpublished data), and maize SUS3 (Carlson et al., 2002) transcript levels increase greatly during late maturation in seeds and/or in fruits. The citrus SUSA mRNA level was shown to increase with maturation both in edible and peel tissues of the fruit (Komatsu et al., 2002). This induction was correlated with sucrose accumulation in both tissues, but sucrose-feeding experiments revealed that the abundance of SUSA mRNA was unaffected by sucrose itself, in leaves. The pea SUS2 transcript level was shown to rise both in the testa and the embryo during late maturation. Likewise, the steady-state level of maize SUS3 mRNA exhibits a gradual increase during the kernel maturation process (Carlson et al., 2002). In the resurrection plant Craterostigma plantagineum, two SUS genes were isolated, called CpSSI and CpSSII (Kleines et al., 1999). Corresponding mRNA levels were shown to be highly induced in leaves and roots in response to dehydration. This mRNA accumulation correlates with an increase in sucrose concentration in leaf tissues. As a conclusion, a group of closely related SUS genes emerges that includes citrus SUSA, pea SUS2, maize SUS3, AtSUS3, Cpss1, and Cpss2. They exhibit resembling expression patterns in addition to strong similarities in predicted amino acid sequences (Fig. 8). These expression data highlight the major contribution that sugar metabolism is thought to play in desiccation-tolerance processes. But the precise function of these SUS isoforms in such a phenomenon remains to be demonstrated. On one hand, an increased glycolytic demand during stress could first be considered (Kleines et al., 1999). On the other hand, a massive sugar conversion leading to sucrose accumulation could explain why some of these SUS expression patterns correlate with sucrose accumulation although no induction by sucrose itself was observed (Fig. 8). In A. thaliana, AtSUS3 could, consequently, be involved in the response to low moisture content through sustained sucrose cleavage activity in the late-maturing seed and increased glycolytic demand during dehydration. AtSUS3 could also participate in sucrose and oligosaccharides synthesis and storage in the late-maturing seed (Baud et al., 2002), thus allowing acquisition of desiccation tolerance. Finally, as proposed by Kleines et al. (1999), transcript accumulation may well be very important for desiccation tolerance in resuming full physiological activity during rehydration, in germinating seeds.
This study adds new significant insights to current understanding of the AtSUS family. These results being focused on correlated changes in transcript profiles and SUS genes being well demonstrated instances of both post-transcriptional and post-translational regulations (McElfresh and Chourey, 1988; Chourey and Taliercio, 1994; Zeng et al., 1998), consequently, other methods of analysis should now be added to face the complexity of the characterization of the AtSUS functions.
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We are grateful to Bertrand Dubreucq and Sandra Moreau for helpful discussions and precious technical advice for real-time PCR, and to Michel Caboche for motivating support. Annabelle Déjardin, Loïc Lepiniec, and Jean-Pierre Boutin are acknowledged for critical reading of the manuscript. This work was supported by a fellowship from the Ministère de l‘Enseignement supérieur et de la Recherche to SB.
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