| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online October 21, 2005; 10.1104/pp.105.069468 Plant Physiology 139:1163-1174 (2005) © 2005 American Society of Plant Biologists
Differential Expression of Sucrose-Phosphate Synthase Isoenzymes in Tobacco Reflects Their Functional Specialization during Dark-Governed Starch Mobilization in Source LeavesInstitut für Pflanzengenetik und Kulturpflanzenforschung, 06466 Gatersleben, Germany
Sucrose (Suc)-phosphate synthase (SPS) plays a crucial role in the synthesis of Suc in photosynthetic and nonphotosynthetic tissues. Several isoforms of SPS exist in dicotyledonous plants that can be grouped into the different families A, B, and C. To explore whether functional differences between the SPS gene families might exist, we characterized a representative for each family from tobacco (Nicotiana tabacum). RNA-blot analysis revealed a distinct expression pattern for each of the three SPS genes. While the A-family member (NtSPSA) was found to be expressed in all tissues examined, expression of the B isoform (NtSPSB) was mainly confined to the reproductive organs and NtSPSC mRNA was exclusively detected in mature source leaves. We used RNA interference to assess the in planta function of NtSPSA and C. While silencing of NtSPSA had no detectable influence on leaf carbohydrate metabolism, reduction of NtSPSC led to an increase in leaf starch content by a factor of 3 to 8. Further analysis revealed that starch accumulation in NtSPSC-silenced plants was not due to an increased partitioning of carbon into starch, but rather showed that starch mobilization was impaired. The transgenic plants were unable to efficiently mobilize their transitory leaf starch during a prolonged period of darkness and accumulated maltose as a major intermediate of starch breakdown. NtSPSC mRNA level increased appreciably during the dark period while transcript levels of the other isoforms showed no diurnal changes. Together, these results suggest that NtSPSC is specifically involved in the synthesis of Suc during starch mobilization in the dark. The roles of the other SPS isoforms are discussed.
During the light period, starch and Suc are synthesized together as the products of photosynthetic carbon assimilation in source leaves. Suc is exported to nonphotosynthetic parts of the plants to support their growth and development, and starch is retained in the leaf. At night, starch is degraded to provide the substrates for the continued synthesis and export of Suc (Geiger and Servaites, 1994  ). Beyond its role in energy metabolism, Suc is often accumulated in response to environmental stresses such as cold, salinity, and drought (Yang et al., 2001; Strand et al., 2003). Suc-P synthase (SPS; EC 2.4.1.14) is assumed to be the key enzyme controlling flux of carbon into Suc (Huber and Huber, 1996). SPS catalyzes the formation of Suc-6-P from Fru-6-P and UDP-Glc. Subsequently, Suc-6-P is hydrolyzed by a specific Suc-P phosphatase (SPP; EC 3.1.3.24) to release Suc. The reaction catalyzed by SPP is essentially irreversible and displaces the reversible SPS reaction from equilibrium into the direction of net Suc synthesis (Stitt et al., 1987). While SPP activity is abundant in source leaves and does not exert significant control of Suc synthesis under normal growth conditions (Chen et al., 2005), SPS is known to make a significant contribution to control of flux into Suc. The enzyme is regulated by a hierarchy of mechanisms involving posttranslational modification via phosphorylation, direct control by metabolic effectors, such as Glc-6-P and inorganic phosphate (Pi; Huber and Huber, 1996; Winter and Huber, 2000), and transcriptional regulation during sink-source transition (Harn et al., 1993; Klein et al., 1993; Chávez-Bárcenas et al., 2000).
The regulation of SPS from spinach (Spinacia oleracea) leaves has been well characterized. The enzyme contains three phosphorylation sites, Ser-158, Ser-229, and Ser-424, which are involved in light/dark regulation (Huber and Huber, 1996
There is growing evidence that higher plants contain more than one gene encoding SPS. The inspection of the Arabidopsis (Arabidopsis thaliana) genome revealed the presence of four genes putatively encoding SPS enzymes, all of which are transcribed (Langenkämper et al., 2002 These experiments targeted only one of the SPS gene families, and the extent to which other SPS genes were targeted or may have been up-regulated to compensate has not been investigated. Thus, the challenge remains to determine if there is any functional specification between the different SPS gene families. Toward this end, we carried out a systematic analysis of the SPS gene families in tobacco (Nicotiana tabacum) combining molecular, biochemical, and reversed genetics approaches. Based on the results, a specific functional role for individual families is discussed.
Tobacco Expresses a Representative for Each SPS Gene Family
The isolation of a cDNA encoding SPS from a tobacco leaf cDNA library using the SPS A isoform from spinach as a probe has previously been reported (Börnke, 2005
As mentioned above, SPS A from spinach has three known regulatory phosphorylation sites involving Ser-158, Ser-229, and Ser-424. A comparison between the spinach protein and those from tobacco revealed that the light-regulated motif surrounding Ser-158 is well conserved in all sequences (Table I). However, NtSPSC lacked a basic residue at position –6 relative to the phosphoserine. The presence of a basic residue at this position has been shown to be an important positive recognition element for the phosphorylation of the motif by different protein kinases (Huang and Huber, 2001 ). The NtSPSB protein lacks both Ser-229 and Ser-424 involved in 14-3-3 binding and osmotic regulation, respectively. The phosphoserine contained in the 14-3-3 binding site has been altered to Thr in NtSPSC, while the protein kinase recognition element remains conserved (Table I).
Expression of SPS Genes Varies Greatly in Different Tissues Northern-blot analysis was carried out on total RNA from different tobacco tissues using each NtSPS cDNA as a probe. All tissues examined expressed the NtSPSA mRNA, albeit to different levels (Fig. 2). Highest expression was found in petals and stems, while source leaves and ovaries showed intermediate expression. Significant levels of NtSPSA transcript were also found in heterotrophic tissues such as roots, sink leaves, and anthers. NtSPSB expression was largely confined to reproductive organs such as anthers and ovaries, although a very weak signal was also present in source leaves (Fig. 2). Interestingly, NtSPSC transcript seemed to be exclusively expressed in mature source leaves as there was no detectable expression for this isoform in any of the other tissues investigated (Fig. 2).
From these data it can be concluded that SPS families in tobacco are differentially expressed in different tissues. Furthermore, NtSPSA and C seem to represent the major leaf isoforms in tobacco.
To investigate specific roles of NtSPSA and C in leaf carbohydrate metabolism, their expression was decreased, either separately or in combination, using an RNA interference (RNAi) approach. To differentially decrease selected SPS isoforms, RNAi was targeted against a region within the 3' end of the respective cDNAs sharing only 27% identity at the nucleotide level between the two isoforms. The RNAi cassettes, containing the SPS fragment in antisense orientation in front and in sense orientation behind a spliceable intron sequence, were transferred into a binary vector behind the cauliflower mosaic virus 35S promoter yielding the constructs NtSPSAi and NtSPSCi. To down-regulate NtSPSA and C simultaneously, the two fragments used to create the isoforms-specific RNAi cassettes were fused by PCR and the resulting chimeric DNA fragment was assembled into a 35S-driven RNAi construct as described above, giving rise to the construct chiSPSi. All three constructs were used to generate transgenic tobacco plants via Agrobacterium-mediated gene transfer. Approximately 80 regenerated plants for each construct were transferred into the greenhouse. To screen the transformants for reduced levels of SPS expression, western-blot analysis was carried out using polyclonal antiserum raised against an SPS A-family member from potato (Reimholz et al., 1997
Three lines from each transgenic genotype were chosen and the T1 generation was subjected to a detailed analysis. The selected transgenic lines along with a transgenic control line harboring a
The mRNA steady-state level of the three SPS isoforms was determined in all three genotypes of transgenic plants using either northern-blot analysis or semiquantitative RT-PCR. As expected, the steady-state level of NtSPSA was drastically reduced in NtSPSAi plants (Fig. 3A). Interestingly, the expression of NtSPSB was modestly induced in NtSPSAi transgenics while NtSPSC transcript levels remained unchanged as compared to the control specimen (Fig. 3A). NtSPSCi plants displayed a strong reduction of NtSPSC mRNA, however, in this case expression of NtSPSA was markedly up-regulated while NtSPSB transcript levels appeared to be unaffected (Fig. 3B). In chiSPSi plants, RNAi caused a strong reduction of both SPS isoforms targeted, while the expression of NtSPSB was induced in lines 8 and 27 (Fig. 3C).
In agreement with the strong reduction of NtSPSA transcript levels in NtSPSAi transgenic plants, NtSPSA protein amount was strongly reduced in the selected lines as compared to the control plants (Fig. 4). To investigate the influence of reduced SPS expression on enzyme activity, SPS Vmax and Vsel (activity under limiting substrate conditions and in the presence of Pi) activity was determined in all three transgenic genotypes. As shown in Table II, only minor differences were found between NtSPSAi and NtSPSCi and the control plants, respectively, for both SPS activities. In contrast, a marked reduction of both SPS Vmax activity (37%–41%) and Vsel activity (35%–38%) was observed in chiSPSi plants.
Carbohydrate Levels in SPS-Silenced Transgenic Tobacco Plants To determine how silencing of NtSPSA and C, separately or in combination, affects carbon metabolism, the levels of nonstructural carbohydrates were measured in mature leaves from all three transgenic genotypes 5 h after illumination. In NtSPSAi transgenic plants, neither soluble sugars nor starch levels were altered when compared to the control plants (Table III). While the levels of soluble sugars in NtSPSCi plants also remained unchanged, starch content was increased by a factor of 3 to 8 in these plants (Table III).
Based on northern-blot analysis it can be assumed that from the SPS genes investigated in this study, NtSPSA and C constitute the major leaf isoforms of SPS. Nevertheless, in accordance with the rather slight effect on SPS enzyme activity, Suc content was largely unaffected in both NtSPSAi and NtSPSCi plants. One possible explanation for this phenomenon could be that loss of one isoform is compensated by one or more other isoforms. If this was to be true, we expected an effect on Suc synthesis in chiSPSi plants. As shown in Table III, Suc content in chiSPSi plants was indeed reduced by 12%, 36%, and 45% in lines chiSPSi27, chiSPSi30, and chiSPS8, respectively, indicating that simultaneous silencing of the two major leaf isoforms of SPS in transgenic tobacco limits photosynthetic Suc biosynthesis. In addition to the reduction in Suc contents, chiSPSi plants also showed a drastic increase in starch contents (4- to 8-fold) as already observed in NtSPSCi plants (Table III). Despite the changes in carbohydrate metabolism, silencing of SPS had no effect on the photosynthetic rate or on phenotype in any of the transgenic genotypes as compared to control plants (data not shown).
Steady-state levels of leaf carbohydrates reflect the balance between synthesis and degradation. Therefore, the starch accumulation in NtSPSCi plants could either be brought about by an increased partitioning of newly fixed carbon into starch synthesis, or by a reduced rate of starch mobilization during the night, or both. To address the question of whether the alterations in carbon partition during photosynthesis cause the accumulation of starch, we investigated the fate of recently assimilated CO2 in NtSPSCi plants as compared to NtSPSAi plants and control plants, respectively. To this end, leaf discs from dark-adapted plants were incubated in a leaf-disk electrode under saturating light and 14CO2 for 20 min. Following ethanol extraction, radiolabel was determined in the insoluble and soluble fraction. As shown in Table IV, incorporation of 14C in the insoluble fraction (mainly representing starch) was unchanged in both transgenic genotypes as compared to the control, indicating that silencing of neither of the two SPS isoforms had an influence on photosynthetic carbon partitioning. The soluble fraction was furthermore divided into neutrals (mainly Suc), anions (phosphorylated intermediates and organic acids), and cations (amino acids). As before, no differences were observed concerning the incorporation of radiolabel into these fractions between transgenic plants and the wild type (Table IV).
To determine whether altered starch degradation rates might be responsible for the elevated starch accumulation, NtSPSCi plants along with control plants were subjected to a prolonged dark period and, subsequently, leaves were stained for remaining starch with iodine. In leaves of control plants, starch was totally degraded after 24 h of dark treatment while leaves from NtSPSCi plants still contained sufficient starch to stain blue in the presence of iodine (Fig. 5), indicating they were unable to efficiently mobilize their transitory leaf starch during the dark.
The diurnal changes in starch content were investigated in NtSPSCi as well as in NtSPSAi plants (Fig. 6). NtSPSAi tobacco plants exhibited a similar diurnal pattern of starch content as the wild type (Fig. 6A). The diurnal changes in starch content still operated in NtSPSCi transgenic plants, indicating that the starch-excess phenotype is not accompanied by a total absence of starch turnover. However, the starch level was substantially elevated throughout the entire diurnal period (Fig. 6B). This most likely is the result of a cumulative effect over a longer growth period, because the transgenics accumulate more starch in their leaves in comparison to the control specimen.
Recent evidence shows that maltose serves as a major metabolic intermediate in the conversion of starch to Suc during the night (Chia et al., 2004 ; Lu and Sharkey, 2004; Niittylä et al., 2004; Weise et al., 2004, 2005). To determine whether the impaired ability of NtSPSCi plants to mobilize leaf starch during the night would lead to an accumulation of primary products of starch breakdown, maltose levels were determined in NtSPSCi plants as well as in NtSPSAi plants toward the end of the dark period. It was found that maltose levels in NtSPSCi plants were elevated by a factor of 2 to 3 as compared to the control (Fig. 7B), further indicating the starch breakdown per se is operating in these plants but further metabolism of the primary products of this process is restricted. In contrast, maltose levels in NtSPSAi plants remained unchanged as compared to the control plants (Fig. 7A). This is in keeping with the unaltered starch content in these plants.
NtSPSC Transcript Levels Undergo Diurnal Changes To learn more about the reasons of the different metabolic phenotypes observed in NtSPSAi and NtSPSCi transgenic tobacco plants, the expression of the three NtSPS genes was investigated in wild-type tobacco plants throughout the diurnal cycle (Fig. 8). Northern-blot analysis revealed that NtSPSA and B transcript levels were largely independent of light/dark conditions. In contrast, the steady-state level of NtSPSC mRNA displayed a pronounced diurnal oscillation, exhibiting a doubling in signal intensity between the end-of-light period and the end-of-dark period. This result is consistent with the biochemical data from NtSPSCi plants and strongly supports the notion that NtSPSC is the major SPS isoform operating in the night path of Suc synthesis within a source leaf.
SPS Gene Families Are Differentially Expressed in Tobacco
We show here that tobacco expresses at least three different SPS genes, denoted NtSPSA, NtSPSB, and NtSPSC, each representing one member of the three SPS gene families present in dicot plants (Langenkämper et al., 2002
NtSPSB was only very weakly expressed in mature leaves, but high expression was found in anthers and ovaries. A similar expression pattern has been reported for a member of the rice (Oryza sativa) SPS B family that was highly expressed in immature inflorescences, and it has been proposed that in rice the SPS B isoform might be involved in the supply of carbon to developing pollen grains (Chávez-Bárcenas et al., 2000
NtSPSC expression was found to be confined to mature source leaves, suggesting it has a source-specific function. This is broadly consistent with the expression of the C isoform from wheat (Triticum aestivum) being most abundant in the flag leaf blade and sheath, but also in germinating seeds as another type of source tissue (Castleden et al., 2004
Specific knock down of NtSPSA or NtSPSC had only slight effects on overall SPS activity and left Suc steady-state levels largely unaltered. This indicates that during the day A and C function can be mutually substituted by each other or alternatively by NtSPSB. This is further corroborated by the substantial reduction in overall SPS activity as well as Suc levels observed upon simultaneous repression of both isoforms in chiSPSi plants. Furthermore, induction of the B isoform as seen in NtSPSA-silenced plants and of the A isoform as observed in NtSPSC-silenced plants might compensate for loss of SPS A and C, respectively. Despite its low expression level in source leaves, it seems possible that the B family accounts at least in part for the residual SPS activity in chiSPSi plants. Given the presence of more than one A-family member in some plant species such as Arabidopsis (see Fig. 1), kiwi (Fung et al., 2003
Recently, transgenic Arabidopsis plants with reduced expression of the A-family SPS have been produced (Strand et al., 2000
RNAi-mediated suppression of NtSPSC in transgenic tobacco plants lead to an increase in starch levels by a factor of 3 to 8, something that was not observed upon suppression of the tobacco A-family member. Several lines of evidence strongly indicate that starch accumulation in NtSPSCi plants was not due to changes in carbon allocation during photosynthesis but rather to an impaired mobilization of starch during the night. No changes in photosynthetic carbon partitioning under saturating light and CO2 conditions were observed in NtSPSAi and NtSPSCi plants, respectively, as compared to the wild type. This is consistent with the assumption that during the light period each isoform can compensate for the loss of the other and thus no restriction of Suc synthesis should occur. Interestingly, antisense inhibition of the SPS A family in Arabidopsis had also no effect on the ratio of Suc synthesis to starch synthesis in leaf-disc experiments (Strand et al., 2000 The changes in starch and maltose content observed upon silencing of NtSPSC were completely absent from NtSPSAi plants, showing that the A-family function is dispensable for maintaining Suc synthesis during the dark period. Taken together, our data strongly suggest a crucial function for SPS C in Suc synthesis during starch mobilization at night. In contrast to the light period, C function cannot be completely substituted by other SPS families during the dark.
During the day, Suc synthesis proceeds from triose-P exported from the chloroplast into the cytosol. Control of the pathway is achieved by coordinate regulation of cytosolic FBPase and SPS. According to the current understanding of SPS regulation, the protein's phosphorylation state is the largest determinant of catalytic activity (Huber and Huber, 1996
Another level of regulation where the SPS families clearly differ is at the point of transcription. While there is currently little evidence for transcriptional regulation of NtSPSA and B, mRNA steady-state level of NtSPSC showed pronounced diurnal changes. NtSPSC transcript levels peaked during the night, coinciding with the phase of starch mobilization (compare Figs. 6 and 8). Interestingly, a similar diurnal expression pattern has been observed for the Arabidopsis SPS C-family member (Harmer et al., 2000
This provides further evidence for the notion that SPS C is the key SPS isoenzyme involved in Suc synthesis during the dark period. However, this expression pattern is apparently different to that of other enzymes involved in the degradation of transitory leaf starch. In Arabidopsis the transcripts of several genes putatively involved in starch breakdown showed a coordinated decline in the dark followed by a rapid accumulation in the light (Smith et al., 2004
Here we provide evidence that the three SPS gene families A, B, and C present in dicots are functionally distinct. In tobacco, at least one representative for each SPS family is expressed and the three genes have distinct expression patterns. Transgenic tobacco plants with selective down-regulation of the two major SPS isoforms expressed in leaves revealed a specific role for NtSPSC during starch mobilization at night. Future studies will have to elucidate differences in kinetic properties, posttranslational modifications, and protein-protein interactions of the different SPS isoforms to understand the biochemical basis for their functional specification.
Transgenic Plants, Growth, and Maintenance Tobacco plants (Nicotiana tabacum cv Samsun NN) were obtained from Vereinigte Saatzuchten and grown in tissue culture under a 16-h-light/8-h-dark regime (irradiance 150 µmol quanta m–2 s–1) at 50% humidity on Murashige and Skoog medium (Sigma) containing 2% (w/v) Suc. Plants in the greenhouse were kept in soil under a light/dark regime of 16 h light (25°C) and 8 h (20°C) dark.
Standard cloning procedures were carried out as described (Sambrook et al., 1989 Primers CS118 5'-GAAGATGGTGATGACACTGATCTCGGAG-3' and CS119 5'-CTCCGAGATCAGTGTCATCACCATCTTC-3', respectively, were used in the 5'-RACE and 3'-RACE reactions to clone the missing sequence portion of NtSPSB. Primers CS25 5'-TTGGCACTTTGGCTCCTGGCGTGATGC-3' and CS26 5'-GCATCACGCCAGGAGCCAAAGTGCCAA-3' were designed to amplify 5' and 3' ends of SPS C, respectively. The entire predicted coding regions of SPS B and C were amplified by PCR using two pairs of primers (CS135 5'-AAGGATCCAAATGGCTGGTAATGAATGGATAAA-3'/CS136 5'-ATGTCGACTCATCTAGACACTTGCCTCAATGC-3' and CS38 5'-ATGGCGGAGAACGAATGGTTAAACG-3'/CS39 5'-TCACATGACCTCTAACTTCTCCAAGGCTG-3', respectively), subcloned, and sequenced.
To generate the NtSPSA RNAi construct, a fragment of NtSPPA comprising nucleotides 2,565 to 3,165 was amplified by PCR using primers FB196 (5'-GGATCCTTTTGTAGTTGACTTGTACTA-3'; a BamHI site is underlined) and FB69 (5'-GCGAATTCGTCGACTTATCCTTTGAGTAC-3'; a SalI site is underlined). The resulting DNA fragment was digested with BamHI and SalI before it was ligated in sense orientation into pUC-RNAi, a vector containing the first intron of the GA 20-oxidase gene from potato (Solanum tuberosum) flanked by a short polylinker sequence (Chen et al., 2003
Transformation of tobacco plants by Agrobacterium-mediated gene transfer using Agrobacterium tumefaciens strain C58C1:pGV2260 was carried out as described previously by Rosahl et al. (1987)
Protein extracts were prepared by homogenization of leaf material in a buffer containing 25 mM HEPES, pH 7.0, 12 mM MgCl2, 0.5 mM EDTA, 8 mM dithiothreitol, 10 µM phenylmethylsulfonyl fluoride, 0.1% Triton, and 10% glycerol. Protein content was determined according to Bradford (1976)
Total RNA was extracted from tobacco leaf material as described by Logemann et al. (1987)
For RT-PCR experiments, 2.5 µg of deoxyribonuclease-treated total RNA was reverse transcribed into cDNA with oligo(dT) (30-mer) using M-MLV[H–] RT (Promega). A fraction (about one-twentieth) of the first-strand cDNAs was used as a template for PCR with gene-specific primers in a volume of 100 µL with 1 unit of Taq-polymerase (Takara), 20 µM each dNTP, and 0.25 µM of each primer. An initial denaturation step for 5 min at 95°C was followed by 25 to 35 cycles of 5 s at 95°C, 45 s at 55°C, and 1 min at 72°C. PCR products were separated on 1% (w/v) agarose gels containing ethidium bromide and visualized by UV light. Amplification of actin using primers 5'-ATGGCAGACGGTGAGGATATTCA-3' and 5'-GCCTTTGCAATCCACATCTGTTG-3' served as an internal control.
SPS activity was assayed by quantifying the fructosyl moiety of Suc using the anthrone test exactly as described by Baxter et al. (2003)
The incorporation of 14CO2 into tobacco leaf discs was performed as described by Quick et al. (1989)
Soluble sugars and starch levels were determined in leaf samples extracted with 80% (v/v) enthanol/20 mM HEPES, pH 7.5, as described (Stitt et al., 1989
Following 12 h illumination plants were kept in complete darkness for 24 h. Subsequently, leaves were destained with 80% ethanol at 80°C and then stained with Lugol's solution to visualize the starch content.
The nucleotide sequences reported in this paper have been lodged with GenBank/DDBJ/EMBL under accession numbers DQ213015 (NtSPSB) and DQ213014 (NtSPSC), respectively.
We would like to thank Dr. Uwe Sonnewald for his continuous support of our work and for fruitful discussions. We are thankful to Andrea Knospe for plant transformation and tissue culture work, Christa Kallas and Elis Fraust for excellent technical assistance, and Martina Meier and colleagues for taking care of the greenhouse-grown plants. We thank Dr. Rita Zrenner for providing the SPS antibody. We thank two anonymous reviewers for their valuable suggestions. Received August 3, 2005; returned for revision September 20, 2005; accepted September 20, 2005.
1 Present address: Lehrstuhl für Biochemie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Frederik Börnke (fboernke{at}biologie.uni-erlangen.de). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.069468. * Corresponding author; e-mail fboernke{at}biologie.uni-erlangen.de; fax 49–9131–8528254.
Baxter CJ, Foyer CH, Turner J, Rolfe SA, Quick WP (2003) Elevated sucrose-phosphate synthase activity in transgenic tobacco sustains photosynthesis in older leaves and alters development. J Exp Bot 54: 1813–1820 Börnke F (2005) The variable C-terminus of 14-3-3 proteins mediates isoform-specific interaction with sucrose-phosphate synthase in the yeast two-hybrid system. J Plant Physiol 162: 161–168[CrossRef][ISI][Medline]
Börnke F, Hajirezaei M, Sonnewald U (2001) Cloning and characterization of the gene cluster for palatinose metabolism from the phytopathogenic bacterium Erwinia rhapontici. J Bacteriol 183: 2425–2430 Bradford MM (1976) A rapid and sensitive method for quantification of microgram quantities of proteins utilizing the principle of protein-dye coupling. Anal Biochem 72: 248–254[CrossRef][ISI][Medline]
Castleden CK, Aoki N, Gillespie VJ, MacRae EA, Quick WP, Buchner P, Foyer CH, Furbank RT, Lunn JE (2004) Evolution and function of the sucrose-phosphate synthase gene families in wheat and other grasses. Plant Physiol 135: 1753–1764
Chávez-Bárcenas AT, Valdez-Alarcón JJ, Martínez-Trujillo M, Chen L, Xoconostle-Cázares B, Lucas WJ, Herrera-Estrella L (2000) Tissue-specific and developmental pattern of expression of the rice sps1 gene. Plant Physiol 124: 641–653 Chen S, Hajirezaei M, Peisker M, Tschiersch H, Sonnewald U, Börnke F (2005) Decreased sucrose-6-phosphate phosphatase level in transgenic tobacco inhibits photosynthesis, alters carbohydrate partitioning, and reduces growth. Planta 221: 479–492[CrossRef][ISI][Medline] Chen S, Hofius D, Sonnewald U, Börnke F (2003) Temporal and spatial control of gene silencing in transgenic plants by inducible expression of double-stranded RNA. Plant J 36: 731–740[CrossRef][ISI][Medline] Chia T, Thorneycroft D, Chapple A, Messerli G, Chen J, Zeeman SC, Smith SM, Smith AM (2004) A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night. Plant J 37: 853–863[CrossRef][ISI][Medline]
Doehlert DC, Huber SC (1983) Regulation of spinach leaf sucrose-phosphate synthase by glucose-6-phosphate, inorganic phosphate, and pH. Plant Physiol 73: 989–994 Ebneth M (1996) Expressions analyse des promotors einer cytosolischen fructose-1,6-bisphosphatase aus kartoffel in transgenen tabak- und kartoffelpflanzen. PhD thesis. Freie Universität, Berlin Fung RWM, Langenkämper G, Gardner RC, MacRae E (2003) Differential expression within an SPS gene family. Plant Sci 164: 459–470[CrossRef] Geigenberger P, Reimholz R, Deiting U, Sonnewald U, Stitt M (1999) Decreased expression of sucrose phosphate synthase strongly inhibits the water stress-induced synthesis of sucrose in growing potato tubers. Plant J 19: 119–129[CrossRef][ISI][Medline] Geiger DR, Servaites JC (1994) Diurnal regulation of photosynthetic carbon metabolism in C-3 plants. Annu Rev Plant Physiol 45: 235–256[CrossRef][ISI]
Gibon Y, Blaesing OE, Hannemann J, Carillo P, Hohne M, Hendriks JHM, Palacios N, Cross J, Selbig J, Stitt M (2004) A robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness. Plant Cell 16: 3304–3325
Harmer SL, Hogenesch JB, Straume M, Chang H-S, Han B, Zhu T, Wang X, Kreps JA, Kay SA (2000) Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science 290: 2110–2113
Harn C, Khayat E, Daie J (1993) Expression dynamics of genes encoding key carbon metabolism enzymes during sink to source transition of developing leaves. Plant Cell Physiol 34: 1045–1053 Heineke D, Kruse A, Flugge UI, Frommer WB, Riesmeier JW, Willmitzer L, Heldt HW (1994) Effect of antisense repression of the chloroplast triose-phosphate translocator on photosynthetic metabolism in transgenic potato plants. Planta 193: 174–180 Herbers K, Prat S, Willmitzer L (1994) Functional analysis of a leucine aminopeptidase from Solanum tuberosum L. Planta 194: 230–240[CrossRef][Medline] Höfgen R, Willmitzer L (1990) Biochemical and genetic analysis of different patatin isoforms expressed in various organs of potato (Solanum tuberosum). Plant Sci 66: 221–230[CrossRef] Horton RM (1997) PCR cloning protocols: from molecular cloning to genetic engineering. Methods Mol Biol 676: 141–149
Huang JZ, Huber SC (2001) Phosphorylation of synthetic peptides by a CDPK and plant SNF1-related protein kinase: influence of proline and basic amino acid residues at selected positions. Plant Cell Physiol 42: 1079–1087
Huber SC, Huber JL (1992) Role of sucrose-phosphate synthase in sucrose metabolism in leaves. Plant Physiol 99: 1275–1278 Huber SC, Huber JL (1996) Role and regulation of sucrose-phosphate synthase in higher plants. Annu Rev Plant Physiol 47: 431–444 Ingram J, Chandler JW, Gallagher L, Salamini F, Bartels D (1997) Analysis of cDNA clones encoding sucrose-phosphate synthase in relation to sugar interconversions associated with dehydration in the resurrection plant Craterostigma plantagineum Hochst. Plant Physiol 115: 113–121[Abstract] Klein RR, Craftsbrandner SJ, Salvucci ME (1993) Cloning and developmental expression of the sucrose-phosphate-synthase gene from spinach. Planta 190: 498–510[Medline] Komatsu A, Takanokura Y, Omura M, Akihama T (1996) Cloning and molecular analysis of cDNAs encoding three sucrose phosphate synthase isoforms from a citrus fruit (Citrus unshiu Marc). Mol Gen Genet 252: 346–351[Medline] Krause KP, Hill L, Reimholz R, Nielsen TH, Sonnewald U, Stitt M (1998) Sucrose metabolism in cold-stored potato tubers with decreased expression of sucrose phosphate synthase. Plant Cell Environ 21: 285–299[CrossRef] Langenkämper G, Fung RWM, Newcomb RD, Atkinson RG, Gardner RC, MacRae EA (2002) Sucrose phosphate synthase genes in plants belong to three different families. J Mol Evol 54: 322–332[CrossRef][ISI][Medline]
Li CR, Zhang XB, Hew CS (2003) Cloning of a sucrose-phosphate synthase gene highly expressed in flowers from the tropical epiphytic orchid Oncidium Goldiana. J Exp Bot 54: 2189–2191 Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem 163: 16–20[CrossRef][ISI][Medline] Lu Y, Sharkey TD (2004) The role of amylomaltase in maltose metabolism in the cytosol of photosynthetic cells. Planta 218: 466–473[CrossRef][ISI][Medline] Lunn JE, MacRae E (2003) New complexities in the synthesis of sucrose. Curr Opin Plant Biol 6: 208–214[CrossRef][ISI][Medline]
Niittylä T, Messerli G, Trevisan M, Chen J, Smith AM, Zeeman SC (2004) A previously unknown maltose transporter essential for starch degradation in leaves. Science 303: 87–89 Quick P, Siegl G, Neuhaus HE, Feil R, Stitt M (1989) Short-term water stress leads to a stimulation of sucrose synthesis by activating sucrose-phosphate synthase. Planta 177: 535–554[CrossRef] Reimholz R, Geigenberger P, Stitt M (1994) Sucrose-phosphate synthase is regulated via metabolites and protein-phosphorylation in potato-tubers, in a manner analogous to the enzyme in leaves. Planta 192: 480–488 Reimholz R, Geiger M, Haake V, Deiting U, Krause KP, Sonnewald U, Stitt M (1997) Potato plants contain multiple forms of sucrose phosphate synthase, which differ in their tissue distributions, their levels during development, and their responses to low temperature. Plant Cell Environ 20: 291–305 Rosahl S, Schell J, Willmitzer L (1987) Expression of a tuber-specific storage protein in transgenic tobacco plants—demonstration of an esterase-activity. EMBO J 6: 1155–1159[ISI][Medline] Sambrook J, Fritsch E, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Smith AM, Zeeman SC, Smith SM (2005) Starch degradation. Annu Rev Plant Biol 56: 73–98[CrossRef][Medline] Smith SM, Fulton DC, Chia T, Thorneycroft D, Chapple A, Dunstan H, Hylton C, Zeeman SC, Smith AM (2004) Diurnal changes in the transcriptome encoding enzymes of starch metabolism provide evidence for both transcriptional and posttranscriptional regulation of s | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
RESULTS
-glucuronidase transgene under control of the cytosolic Fru-1,6-bisphosphatase (FBPase) promoter (Ebneth, 1996
-globulin as the standard. After heat denaturation, 50 µg total protein was subjected to electrophoresis on a 10% (w/v) SDS-polyacrylamide gel and subsequently transferred onto nitrocellulose membrane (Porablot, Macherey and Nagel). Immunodetection was carried out using the ECL kit (Amersham Pharmacia Biotech) according to the manufacturer, using the rabbit antiserum raised against SPS from potato (Reimholz et al., 1994
-32P]-dCTP. Hybridization was carried out as described previously (Herbers et al., 1994