Journal of Experimental Botany, Vol. 53, No. 366, pp. 61-71,
January 1, 2002
© 2002 Oxford University Press
Original Papers |
Analysis of sucrose synthase genes in citrus suggests different roles and phylogenetic relationships
1 Faculty of Agriculture, Meiji University, Kawasaki, Kanagawa 214-0033, Japan
2 Department of Citriculture, Okitsu, National Institute of Fruit Tree Science, Shimizu, Shizuoka 424-0292, Japan
Received 2 April 2001; Accepted 24 August 2001
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Abstract |
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The purpose of this work was 2-fold; first, a molecular/evolutionary characterization of three sucrose synthase genes from citrus, and second, an analysis of their differential expression related to potential physiological function. Three non-allelic genes (CitSUS1, CitSUSA and CitSUS2) encoding sucrose synthase were isolated from citrus fruit (Citrus unshiu Marc.). Phylogenetic analysis from the deduced amino acid sequences showed that CitSUS1 and CitSUS2 could be classified into a dicot group. However, CitSUSA, together with Arabidopsis SSA, sugar beet SS and pea SusA defined another dicot group designated SUSA. Unlike other dicot sucrose synthases, these show a distinctive, monocot-like arrangement of introns and exons. The CitSUS1 and CitSUSA were also differentially expressed in leaf, flower and fruit tissues. Contrasting expression patterns were observed for CitSUS1 and CitSUSA in edible tissue (juice sacs/segment epidermis) and peel tissue (albedo/flavedo) of fruit: CitSUS1 mRNA levels decreased throughout fruit development, whereas those of CitSUSA increased. Various sugars also influenced the transcript levels of the CitSUS1 and CitSUSA. These results indicate that the CitSUS1 and CitSUSA genes for sucrose synthase in citrus differ markedly in their molecular structure and potential physiological roles. Sucrose synthase activity in edible tissue was high in the early stages and decreased until mid-develoment, then rapidly increased during maturation. The increase in activity during maturation paralleled that of sucrose accumulation. This result suggests that sucrose synthase has important roles on sugar metabolism when sucrose is accumulated in fruit.
Key words: Citrus unshiu Marc., gene expression, phylogenetic relationships, sucrose synthase, sugar accumulation.
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Introduction |
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Sucrose synthase (UDP-glucose: D-fructose 2-glucosyl-transferase, EC 2.4.1.13) catalyses the reversible reaction: sucrose+UDP
UDP-glucose+fructose. It has a major role in energy metabolism and is involved in the movement of sucrose into diverse pathways important for metabolic structure and storage functions of the plant cell. Sucrose synthase activity correlates with sugar import (Sung et al., 1989
), cell wall synthesis (Chourey et al., 1991) and sink strength (Sun et al., 1992).
Sucrose synthase genes have been isolated primarily from starch-storing plants, such as maize (Werr et al., 1985), rice (Yu et al., 1992), barley (Sánchez de la Hoz et al., 1992), potato (Salanoubat and Belliard 1987), mung bean (Arai et al., 1992), and pea (Buchner et al., 1998). Sucrose synthase genes have also been isolated from sucrose- or hexose-storing plants such as tomato (Wang et al., 1993) and sugar beet (Heim et al., 1993).
Two or three genes encode sucrose synthase isozymes in monocot species such as maize (Shaw et al., 1994), barley (Martinez de Ilarduya et al., 1993), wheat (Maraña et al., 1990), and rice (Wang et al., 1992; Huang et al., 1996). These isoforms can be classified into two groups based on contrasting sequence features and/or expression patterns. One of the two genes in maize, the Sh1 gene encodes SH1 protein, which constitutes a critical link in starch biosynthesis in developing endosperm. The Sh1 gene is abundantly expressed in endosperm, but not in the embryo of the immature kernel. Another sucrose synthase, SUS1, is encoded by the Sus1 gene. The Sus1 gene is expressed in many tissues, including seedling roots and shoots, endosperm, and embryo (McCarty et al., 1986; Chourey et al., 1986, 1998; Chen and Chourey, 1989; Heinlein and Stalinger, 1989; Chourey and Taliercio, 1994). Furthermore, Sh1 and Sus1 genes show markedly different responses to change in the carbohydrate status of maize roots (Koch et al., 1992). They observed that the Sh1 mRNA is maximally expressed under conditions of minimal glucose supply, whereas Sus1 mRNA level is maximally expressed with abundant glucose (
100 mM). Rice sucrose synthase expression is also induced by high levels (300 mM) of glucose, fructose and sucrose (Karrer and Rodriguez, 1992).
Two or more genes encoding sucrose synthase isoforms have also been isolated from dicot plants, such as Arabidopsis (Chopra et al., 1992; Martin et al., 1993), potato (Fu and Park, 1995), tomato (Chengappa et al., 1998), and carrot (Sturm et al., 1999). These genes have clear similarities in sequence and overall structure that distinguish them from two monocot-types and suggest a markedly different evolutionary origin.
Sucrose synthase activity in juice sacs of grapefruit (C. paradisi Macf.) has a potentially important role during the early stages of fruit development, when cell division, cell wall synthesis, and respiration rates are maximal. In addition, sucrose synthase is active in transport tissues of fruit (Lowell et al., 1989), to companion cells in vascular bundles (Nolte and Koch, 1993). However, analysis of function and expression of sucrose synthase have been limited, especially in sucrose- or hexose-storing fruits such as Citrus. In this study, three sucrose synthase cDNAs were isolated (designated CitSUS1, CitSUS2 [partial clone] and CitSUSA) from the mRNA of citrus (Citrus unshiu Marc. cv. Miyagawa Wase) juice sacs. A comparison of their molecular structures, phylogenic origin, and sugar responsiveness is presented here, and there is an analysis of the relationship between sucrose accumulation and differential expression of sucrose synthase genes in citrus fruit.
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Materials and methods |
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Plant materials
Fruit of satsuma mandarin (Citrus unshiu Marc. cv. Miyagawa Wase) grown at the National Institute of Fruit Tree Science, Okitsu, Shimizu, Shizuoka were used to isolate and characterize the genes for sucrose synthase. For gene expression, sugar content and enzyme activity, satsuma mandarin (C. unshiu Marc. cv. Okitsu Wase) cultivated in the experimental field at Meiji University (Kawasaki, Kanagawa, Japan) was used. Fruit from the immature stage (July) to the mature stage (December) were harvested from three 15-year-old trees and were used to analyse the sugar content and enzyme activity, and for Northern blot analysis. The fruits were immediately divided into two portions, albedo/flavedo peel and juice sacs/segment epidermis, which were frozen in liquid nitrogen and stored at -80 °C until use. The leaves and fruits were used to isolate DNA and RNA.
Amplification of sucrose synthase partial cDNA by RT-PCR using the mRNA from fruit
Total RNA was isolated from edible tissue (juice sacs/segment epidermis) of citrus fruit using a modified single-step method (Chomczynski and Sacchi, 1987). Poly (A)+ RNA was purified from the total RNA using an Oligotex Kit (TaKaRa), and was used to synthesize first-strand cDNA using a First-Strand cDNA Synthesis Kit (Pharmacia). By using the first strand cDNA as the template, cDNA was synthesized using the following conditions. The reverse transcription-polymerase chain reaction (RT-PCR) was conducted with 1 min at 94 °C, 1 min at 50 °C and 2 min at 72 °C, using sense a primer (SUS1F: 5'-TGA AGT CTG GCC ATA CCT GGA-3') and an antisense primer (SUS3R: 5'-ATT GCG GAC ACG GTT CAT CTG-3'). These were designed from the conserved region of other sucrose synthase genes from the database. The amplified fragments were cloned into the pCRII vector using a TA Cloning System Kit (Invitrogen).
Screening of a cDNA library from fruit
A cDNA library from the poly (A)+ RNA of citrus fruits (juice sacs/segments epidermis) harvested 124 d after flowering (DAF) was constructed by using a cDNA Synthesis Kit (Pharmacia). The candidate CitSUS1 and CitSUS2 cDNA clones were screened from approximately 1.0x105 pfu of the fruit cDNA library with the probe of a partial cDNA of CitSUS1 and CitSUS2 using the ECL direct nucleic acid labelling and detection system (Amersham). The filters were washed twice for 20 min each in 6 M urea and 0.2xSSC and 0.1% SDS at 42 °C. The candidate CitSUSA cDNA clones were also screened with the probe of a partial cDNA of CitSUS1. The filters were washed twice for 20 min each in 2 M urea and 0.5xSSC and 0.1% SDS at 42 °C. The nucleotide sequences of cDNA clones were determined using a Taq Dye Terminator Cycle Sequencing Kit (Applied Biosystems Instrument) and were analysed using the GENETYX software version 10.0 (Software Development).
Cloning and sequencing of CitSUS1 and CitSUSA genomic DNAs
Genomic clones of CitSUS1 and CitSUSA were isolated by PCR using a shuttle method (Barnes, 1994). The PCR was performed on the genomic DNA and LA Taq polymerase (TaKaRa) using 30 cycles each of 1 min at 94 °C and 4 min at 68 °C. For PCR of the CitSUS1, the sense primer was SUS1-full-F 5'-GAG AAT CAA TGG CCG AAC GTG CTT TG-3', and the antisense primer was SUS1-full-R 5'-TGT CCA ATC GCA ATC CCA GAA GG-3'. For amplification of CitSUSA gene the sense primer was SUSA-full-F 5'-TTC ACG AGC AAC AAA ATC ATG GCA GCC-3', and the antisense primer was SUSA-full-R 5'-AGC ACA GGA AGA TTA AGT GTC GAG AG-3'. In both instances these were synthesized from the 5' and 3' regions of CitSUS1 or CitSUSA cDNA sequences by a DNA synthesizer-1000 (Beckman). The amplified fragments were cloned into the pCRII vector using a TA Cloning System Kit (Invitrogen). The nucleotide sequences were determined using a Taq Dye Terminator Cycle Sequencing FS Kit (Perkin-Elmer) and were sequence analysed using GENETYX software version 10.0 (Software Development). A phylogenetic dendrogram of the deduced amino acid sequences of sucrose synthase was made by the multi-alignment analysis using UPGMA method of GENETYX software version 10.0 (Software Development).
Genomic DNA blot analysis
Genomic DNA was isolated from mature leaves of citrus. Ten µg DNA was digested with DraI, EcoRI or HindIII, followed by fractionation on 0.7% agarose gels and blotting onto a nylon membrane (Hybond-N, Amersham). The partial clones of CitSUS1, CitSUS2 and CitSUSA were labelled with Dig-11-dUTP using a random primer DNA labelling kit (Boehringer Mannheim) to make the probe. Three partial cDNA clones showed homologies in their nucleotide sequences in the range of 69.2 to 83.2%. Specificity of the three probes was verified by Southern blot analysis in which the probes did not cross-hybridize. The blots were hybridized with each probe in a hybridization buffer (5xSSC, 2.0% blocking reagent, 0.1% lauroylsarcosine, and 0.02% SDS) at 65 °C, washed twice in 0.2xSSC and 0.1% SDS at 65 °C for 20 min, and were exposed to X-ray film (RX; Fuji).
Northern blot analysis
Total RNA was isolated from young leaves, mature leaves, flowers, immature fruits, and mature fruit for Northern blot analysis. RNA samples from immature to mature fruits harvested 89, 120, 148, 187, and 223 DAF were isolated from edible (juice sacs/segment epidermis) and peel (albedo/flavedo) tissues. Immature and mature leaves were harvested at 2.5 and 10 cm long. Ten µg of RNA was electrophoresed on a 1.0% agarose gel containing formaldehyde (Ausubel et al., 1987) and was transferred to a Hybond-N membrane (Amersham). RNA blots were hybridized with each probe in a hybridization buffer (50% formamide, 5xSSC, 1.0% blocking reagent, 0.1% lauroylsarcosine, and 0.02% SDS) at 50 °C. The washing stringency was as described for the genomic DNA blot analysis.
Semi-quantitative RT-PCR
Total RNA from young leaves, mature leaves, flowers, immature fruits, and mature fruit was used to synthesize first-strand cDNA using a First-Strand cDNA Synthesis Kit (Pharmacia). RNA samples from immature to mature fruits harvested 89, 148 and 223 DAF were isolated from edible and peel tissues. Immature and mature leaves were harvested at 2.5 and 10 cm long. The reverse transcription-polymerase chain reaction (RT-PCR) was conducted with 30 s at 94 °C, 45 min at 56 °C and 60 s at 72 °C, using sense specific primer for CitSUS2 (SUS2 1F: 5'-CAC TGA GGA TGT TGC AAC-3') and an antisense specific primer for CitSUS2 (SUS2 2R: 5'-TTC TTC ACT CTG TCC AGC-3').
Determination of sugar content
The quantity and types of sugars in ethanol extracts were determined as described previously (Moriguchi et al., 1990). Soluble sugars were extracted by grinding tissues in 80% ethanol, adjusting to pH 7.0 with 0.1 N NaOH, and heating for 15 min at 80 °C. The extraction was repeated three times. The combined extract corresponding to 0.5 g fresh weight was dried in a vacuum, redisolved in water, passed over an ion-exchange column (Dowex 50W-X8, Dowex 1-X8), and eluted with water. Aliquots of the elute were analysed using high-pressure liquid chromatography (Shimadzu LC-6A) and a refractive index detector (Shimadzu RID-6A) equipped with an SP1010 (Showa Denko K.K.) column at 70 °C with water (0.5 ml min-1) as the eluate. Fructose, glucose and sucrose were identified and quantified by comparing the retention time and integrated peak areas of external standards.
Extraction and assay of sucrose synthase
Sucrose synthase activity was assayed in the cleavage direction, as previously described (Moriguchi et al., 1990). All assays were performed in triplicate. Sucrose synthase was extracted by homogenizing frozen tissue (edible and peel tissues) in liquid nitrogen using a Wareing blender. Ground tissue was then homogenized in a chilled 100 mM MOPS-NaOH buffer (pH 7.5) containing 5 mM MgCl2, 0.5 mg ml-1 bovine serum albumin (BSA), 2.5 mM dithiothreitol (DTT), 0.05% TritonX-100, 10 mM K-ascorbate, 2% glycerol, 1 mM EDTA, and 2% polyvinylpolypyrrolidone (PVPP). The tissue-to-buffer ratio was 1:3. After centrifugation at 10000 g for 10 min, the supernatant was passed through a PD-10 column previously equilibrated with the same buffer according to the manufacturer's instructions (Pharmacia LKB Biotech., Uppsala). Unless otherwise described, all procedures were conducted at 4 °C. Protein was quantified using BSA as the standard (Bradford, 1976).
Analysis of sugar-responsiveness by sucrose synthase genes
Detached mature leaves were incubated in water for 12 h in darkness at room temperature, then subjected to test treatments consisting of 12 h in light with water alone or 10% (w/v) sucrose, glucose or fructose. These concentrations were used for analysis of ADP-glucose pyrophsphorylase in potato (Müller-Röber et al., 1990) and for sucrose phosphate synthase in sugar beet (Hesse and Willmitzer, 1996). Untreated leaves served as controls. All samples were immediately frozen in liquid nitrogen, and total RNA extracted for Northern blot analysis as described above.
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Results |
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Amplification and sequencing of cDNAs encoding sucrose synthase
Two partial fragments of sucrose synthase were isolated using RT-PCR. Both were as the expected 750 bp, but differed in EcoRV restriction site (data not shown). The two partial cDNA clones, CitSUS1 and CitSUS2, had 83.2% identity at the nucleotide level and 90.0% identity at the amino acid level. The deduced amino acid sequences of the CitSUS1 and CitSUS2 proteins were very similar to those of tomato sucrose synthase (85% and 83%; Wang et al., 1993
) and mung bean sucrose synthase (86% and 85%; Arai et al., 1992), suggesting that these two clones did indeed encode sucrose synthase.
Isolation and sequence analysis of two full-length sucrose synthase cDNAs
The two candidate clones for CitSUS1 and CitSUSA, having coding regions from the start codon to the stop codon for sucrose synthase, were obtained from the 1x105 pfu by screening with the partial cDNA probe for CitSUS1. Full-length cDNA clones for CitSUS2 could not be obtained using a similar procedure with a partial CitSUS2 cDNA probe. CitSUS1 and CitSUSA were 2672 and 2752 bp long, respectively. Open reading frames encode 806 and 812 amino acids, correspond to ATG start codons at positions 25 bp and 61 bp, and TAA stop codons at positions 2440 bp and 2494 bp, respectively. In addition to the open reading frame, the two cDNAs also contained 24 bp and 60 bp of 5' untranslated sequence, and 230 bp and 256 bp of 3' untranslated sequence. The predicted molecular masses of CitSUS1 and CitSUSA were 92.1 kDa and 92.6 kDa, respectively, which were approximately equivalent to the molecular mass of the sucrose synthase subunit determined for other plants. The level of sequence homology of the three isoforms for citrus sucrose synthase amino acids was high between CitSUS1 and CitSUS2 (90.0% identical), but less so between CitSUSA and CitSUS1 or CitSUS2 (70.5%, 72.4% identical, respectively). Their N-termini contain no signal peptide-like sequences, but the phosphorylation site Ser-15 and some of the neighbouring amino acids of the Sus1 polypeptide of maize are conserved (Huber et al., 1996).
Dendrogram and molecular structure of citrus sucrose synthase genes
A phylogenetic dendrogram of plant sucrose synthase was made using the deduced amino acid sequences by the multi-alignment analysis using GENETYX software (Fig. 1). The dendrogram shows that sucrose synthase genes of plants can be divided into four groups (New group, a monocot group, a dicot SUS1 group, and a dicot SUSA group). The deduced amino acid sequence of CitSUS1 and CitSUS2 (partial sequence) showed a high degree of similarity to those of sucrose synthase from other species, and was thus classified among the large group of dicot SUS1 genes (Fig. 1). By contrast, the deduced amino acid sequence of CitSUSA showed a stronger sequence similarity to distinctive sucrose synthase from Arabidopsis (SSA, Chopra et al., 1992), sugar beet (Hesse and Willmitzer, 1996) and pea (Buchner et al., 1998). These form a natural, previously unrecognized, SUSA group among the dicots.
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Analysis of the genomic sequence for CitSUS1 showed 13 exons which is typical of the dicot SUS1 group (Fig. 2
). Exons 6 and 12 were also of similar length to those exons of other sucrose synthase genes in the dicot SUS1 group, such as potato Sus3, Sus4 (Fu and Park, 1995), Arabidopsis Asus1 (Martin et al., 1993), carrot Sus1 and Sus2 (Sturm et al., 1999), and tomato Sus2 (accession no. AJ011535). However, CitSUSA had 15 exons and these were split at distinct positions otherwise observed only for sucrose synthase genes isolated from the monocot group or from SSA of Arabidopsis (Chopra et al., 1992). Sucrose synthase gene structures from sugar beet and pea have never been determined yet.
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Southern blot analysis with three sucrose synthase
Southern blot analysis of genomic DNA from citrus leaves was conducted using three partial sequences of CitSUS1, CitSUS2 and CitSUSA as probes. Hybridization patterns of the three cDNA probes differed, indicating that they were gene-specific under stringent conditions. Few fragments hybridized to either CitSUS1, CitSUS2 or CitSUSA probes, regardless of digestion with DraI, EcoRI or HindIII (Fig. 3). These results are consistent with the suggestion that the three sucrose synthase genes probably represent one copy or a low-copy-number gene and comprise a small gene family in citrus. Furthermore, the three sucrose synthase clones appeared to be from non-allelic genes by linkage analysis using these clones and RFLP markers; three sucrose synthase clones were severally mapped on different linkage group (data not shown).
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Accumulation of sugars in fruit
The sugar contents in edible and peel tissues of fruit (Fig. 4A, C) were determined during the development. Sugar levels in edible tissues were initially low, but sucrose gradually accumulated throughout fruit development, and then eventually comprised about 65% of the total sugars in the mature fruit. Although fructose and glucose also accumulated, their levels were lower than content of sucrose, at approximately 20% and 14% of the total sugars, respectively. In peel tissues, content and accumulation patterns for sucrose, fructose and glucose were almost identical, and increased steadily toward maturation. These results were similar to those of Takagi et al. for another variety of Citrus (Takagi et al., 1994).
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), glucose (
) and fructose (
) in edible tissue were measured using high-pressure liquid chromatography. (B) Sucrose synthase activity in edible tissue was measured g-1 FW (
Enzyme activity of sucrose synthase
The activity of sucrose synthase was measured in vitro in extracts of edible and peel tissues sampled at different developmental stages. Sucrose synthase activity g-1 fresh weight (FW) in edible tissue was low during the early stages of fruit development (89 and 120 DAF), then markedly increased during fruit development (187 and 223 DAF; Fig. 4B
). Sucrose synthase activity in the peel tissue was low at 89 DAF, decreased until 120 DAF, but rapidly rose at 148 DAF, increasing through the developed stages (187 and 223 DAF; Fig. 4D). When data were expressed mg-1 protein, the patterns of sucrose synthase activity in the edible and peel tissues were similar to those expressed on a FW basis (Fig. 4B, D).
Relative levels of sucrose synthase mRNAs
Expression of CitSUS1 and CitSUSA was investigated by Northern blot analysis of total RNA prepared from young leaves, mature leaves and flowers of citrus. The CitSUS1 mRNA was approximately 2.4 kb, as expected (Fig. 5). In Northern blot analysis, CitSUS1 transcript levels were high in young sink leaves and flowers, and lower in mature source leaves. The CitSUSA transcripts were also approximately 2.4 kb, but mRNA levels were higher in the mature source leaves and flowers, with lower levels in young sink leaves (Fig. 5). These results indicate that CitSUS1 and CitSUSA are expressed at different stages of leaf development.
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Transcripts of CitSUS1 and CitSUSA were also detected in edible and peel tissues of fruit at all stages analysed (Figs 6
, 7). As observed for leaves, CitSUS1 mRNA levels were maximal for fruit at the immature stage (89 DAF), then decreased throughout development of edible tissue. In contrast, levels of CitSUSA mRNA were low in the immature stages, then increased with maturation (187 and 223 DAF; Fig. 6). Similar overall patterns were evident for the CitSUS1 and CitSUSA mRNA in peel tissues (Fig. 7).
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CitSUS2 mRNA levels were also investigated, but they were no longer detected in all organs and stages investigated (Figs 5
, 6, 7). However, the CitSUS2 gene is apparently transcribed, because a partial cDNA clone encoding CitSUS2 was amplified using the first-strand cDNA from edible and peel tissues of fruit and flowers by RT-PCR (Fig. 8). The expression patterns in edible and peel tissues during fruit development showed the same pattern with expression of CitSUS1 by Northern blot analysis, but amplification bands of CitSUS2 in young leaves and mature leaves were not detected by RT-PCR (Fig. 8).
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X174/Hae III digest marker (1st and 14th lanes).
Sugar-responses of citrus sucrose synthase genes
Several sugars affected CitSUS1 and CitSUSA mRNA levels in leaves in the light (Fig. 9). Sucrose increased CitSUS1 transcript abundance compared to modest, if any, effects of glucose or fructose. By contrast, transcript abundance of CitSUSA was unaffected by sucrose, but enhanced by hexoses. CitSUS2 mRNA levels were also investigated by Northern blot analysis and semi-quantitative RT-PCR, but they were not detected in all sugars in the same conditions. Sugar responses of CitSUS1 and CitSUSA in the dark were less pronounced (data not shown).
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A subgroup of dicot sucrose synthase
A phylogenetic analysis of plant sucrose synthase genes has been reported previously (Fu and Park, 1995
; Sturm et al., 1999). Their data clearly show that plant sucrose synthase genes can be classified into at least three major branches: one monocot group (including Sh1 and Sus1 types) and two dicot groups. However, the phylogenetic position of the dicot groups including Arabidopsis SSA was unclear in dendrograms by DNA or amino acid sequence so this gene was previously excluded from evolutionary groupings of sucrose synthase (Chopra et al., 1992). Now, sequence similarities at the nucleotide and amino acid levels for CitSUSA, Arabidopsis SSA, pea SusA, and sugar beet Sus show a stronger relationship to one another than to other sucrose synthase genes from the dicot SUS1 group. These four sucrose synthase genes thus comprise an independent subgroup of dicot sucrose synthase genes, which have been designated as SUSA in this paper (Fig. 1). Based on these results, it is expected that, in addition to sucrose synthase genes from the dicot SUS1 group, individual dicot plants are likely to have another sucrose synthase gene from the dicot SUSA group. In carrot, for example, Southern blot analysis using a general probe for sucrose synthase and specific probes for Dc1 and Dc2 belonging to the dicot SUS1 group identified additional hybridization signals that could not be assigned to the two isolated genes (Sturm et al., 1999).
In this study, sucrose synthase genes newly discovered by the publication of the Arabidopsis genome sequence were also added for a phylogenetic analysis (Fig. 1). Two genes (MJG14.24 and T9L24.42) formed the new group. The amino acid sequence of the MJG14.24 gene showed 53.8% of homology to those of CitSUS1, and showed 55.5% to those of CitSUSA gene. The amino acid sequence of the T9L24.42 gene showed 55.8% of homology to those of CitSUS1, and showed 57.0% to those of the CitSUSA gene. Although it is not yet known whether these genes all encode expressed proteins, from now on, it will be necessary to investigate this in various plants in detail.
Interestingly, the structure of dicot SUSA genes has greater similarity to the gene structure of monocot sucrose synthase genes than to those of the dicot SUS1 group. The coding regions corresponding to exons 6 and 12 of dicot SUS1 group are intact, but those of the dicot SUSA and monocot groups are split with the insertion of one intron in each exon (Fig. 2). These specific structures of exons 6 and 12 in the dicot SUS1 group provide evidence for a distinct dicot SUS1 group.
Expression of CitSUS1, CitSUS2 and CitSUSA
Previous studies of dicot sucrose synthase expression have not compared genes from different groups (dicot SUS1 and SUSA groups) within a given species. Although two sucrose synthase genes from different groups have been isolated from Arabidopsis (Martin et al., 1993; Chopra et al., 1992) and pea (Buchner et al., 1998), their gene expression has thus far included only Arabidopsis Asus1. In tetraploid potato, three sucrose synthase genes, potssyn, Sus3 and Sus4 have been isolated, but they all appear to be members of the dicot SUS1 group (Salanoubat and Belliard, 1987; Fu and Park, 1995). The two sucrose synthase genes or cDNAs from tomato and carrot also belong to the dicot SUS1 group (Wang et al., 1993; Chengappa et al., 1998; Sturm et al., 1999). In the present study of diploid citrus plants, three sucrose synthase genes (CitSUS1, CitSUSA and CitSUS2) were identified from two different groups in the phylogenetic dendrogram (dicot SUS1 and dicot SUSA groups), and two of these were characterized by concurrent analysis of sequence similarity, gene structure, and expression. The CitSUS1 and CitSUSA genes showed clearly different expressions patterns in Northern blot analysis of leaves (Fig. 5), fruit during development (Figs 6, 7), and response to sugars (Fig. 9). The latter may influence numerous aspects of expression because sugar contents can differ from tissue to tissue, change during fruit maturation and may not be fully compartmentalized in vacuoles. The differential patterns of expression for CitSUS1 and CitSUSA transcripts might thus be due to structural differences observed in both the 5' and 3' flanking sequences, the leader intron, and the open reading frame, since all three can affect transcriptional and post-translational regulation by sugars. In potato, for example, a series of deletion and substitution constructs indicated that the 5' flanking sequence, leader intron, and 3' sequence of the Sus3 and Sus4 genes, all have important roles in expression (Fu et al., 1995a, b). In citrus, CitSUS1 mRNA is induced by high levels of sucrose in the light, whereas CitSUSA mRNA is induced by hexose under similar conditions. In the analysis of sugar regulation of sucrose synthase transcription (Maas et al., 1990), transcription from the Sh promoter, as estimated by comparison of NPTII activities, was 20-fold higher in protoplasts cultivated in medium supplemented with monosaccharides rather than with sucrose. Contrasting responses were also observed in maize roots by different genes depending on sugar levels (Koch et al., 1992). At low sugar concentrations (0.2% glucose), Sh1 mRNA is maximally expressed, whereas Sus1 transcript levels peaked when sugars were abundant (2% glucose [111 mM]). These results show that sugar levels can modify the expression of Sh1 and Sus1 genes in a cell-specific manner. This observation is further supported by results showing that rice sucrose synthase expression can also be induced by elevated levels (300 mM) of glucose, fructose and sucrose (Karrer and Rodriguez, 1992). Therefore, it is likely that the expression pattern of sucrose synthase may be influenced by physiological factors that include sugar concentration, possibly operating through structural differences in either the 5' or 3' flanking sequences, or the leader intron.
The CitSUS2 gene is apparently transcribed, because a partial cDNA clone encoding CitSUS2 was amplified using the first-strand cDNA from edible and peel tissues of fruit and flowers by semi-quantitative RT-PCR (Fig. 8). But the amplified bands were very weak in spite of RT-PCR by 40 cycles. These results suggest that the CitSUS2 gene is not a pseudogene, and CitSUS2 protein may be small compared with CitSUS1 and CitSUSA in citrus.
Relationships between sucrose accumulation, sucrose synthase activity and transcripts of sucrose synthase genes
Transcripts of CitSUS1 were abundant in both edible and peel portions and were high at the immature stage and decreased towards the ripening stage. By contrast, the mRNA levels for CitSUSA increased towards fruit ripening. Sucrose synthase enzyme activity was detectable when assayed in the cleavage direction throughout fruit development, and the activities notably increased towards fruit maturation (although sucrose synthase on DAF 89 shows high activity in both tissues). Suzuki et al. reported the presence of two sucrose synthase isozymes in pear fruits, and that one isozyme predominates in immature fruits and the other in mature fruits (Suzuki et al., 1996). In citrus, the early versus late roles of CitSUS1 and CitSUSA, respectively, in sucrose accumulation by fruit may correspond to those of the two isozymes in pear.
In citrus fruit, sucrose is translocated from the source to the fruit and is either apoplastically hydrolysed to fructose and glucose by invertase, or is cleaved symplastically to fructose and UDP-glucose by sucrose synthase (Lowell et al., 1989). However, acid invertase activity in both grapefruit and satsuma mandarin is initially high, decreasing to very low levels at the mature stage (Kato and Kubota, 1978; Lowell et al., 1989). These results suggest that only sucrose synthase can cleave the sucrose translocated to fruit tissue at a mature stage. In the present work, both levels of sucrose synthase activity (Fig. 4B, D) and CitSUS1 transcript decreased at the immature stage, and a positive correlation between sugar accumulation (Fig. 4A, C) and sucrose synthase activity (Fig. 4B, D) was indicated during fruit development. The increase in sucrose synthase activity of developing stages also paralleled CitSUSA transcript accumulation (Figs 6, 7). However, the increase of the activity and the transcripts during fruit development could not compare directly with each other. Because the protein contents of sucrose synthase increased, and the increase of transcripts may push up the activity of sucrose synthase, it is necessary to use specific antibodies of CitSUS1 and CitSUSA in Western blot analysis from now on. This is consistent with the suggestion that the two steps of sucrose degradation by sucrose synthase and the resynthesis by sucrose-phosphate synthase (SPS) in fruit could generate a locally increased sucrose concentration gradient in the zone of phloem unloading, thus favouring sucrose import. The potential capacity for resynthesis of sucrose by the action of SPS in citrus fruit is also supported by the presence of SPS activity and transcripts of CitSPS1 and CitSPS2, which increase until the mature stage in edible tissue (Komatsu et al., 1999). A series of actions by sucrose synthase and SPS during maturation could be important to sink strength in citrus fruit at this stage. Thus, CitSUS1 may have a role in supplying materials for growth and cell wall construction, whereas CitSUSA may supply SPS with substrates (UDP-glucose and fructose) for the resynthesis of sucrose.
In conclusion, the diploid plant, citrus has at least three sucrose synthase genes that support two subgroupings of dicot sucrose synthase based on distinctive phylogenetic analysis, and genomic structure. In addition, the CitSUS1 gene, having the same structure as sucrose synthase genes of the dicot SUS1 group, and the CitSUSA gene, having a structure similar to monocot sucrose synthase genes (Sus2 type), are differentially responsive to sugars and show contrasting expression patterns consistent with different functional roles. Because the increase in activity during maturation paralleled that of sucrose accumulation, it suggests that sucrose synthase has important roles on sugar metabolism when sucrose is accumulated into the fruit.
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Acknowledgments |
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We thank Professor KE Koch, University of Florida, for helpful advice and support. We acknowledge the receipt of a JSPS Fellowship for Japanese Young Scientists. This work was also supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan (no. 06304013 and no. 5326).
The nucleotide sequence data of CitSUS1, CitSUS2 and CitSUSA reported will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers AB022092, AB021745 and AB022091.
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3 Present address and to whom correspondence should be sent: National Institute of Crop Science (NICS), 3-1-1 Tsukuba Science City, 305-8666, Japan. Fax: +81 298 38 8949. E-mail: akomatsu{at}naro.affrc.go.jp
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