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First published online October 1, 2004; 10.1104/pp.104.042606 Plant Physiology 136:3148-3158 (2004) © 2004 American Society of Plant Biologists Galactinol synthase1. A Novel Heat Shock Factor Target Gene Responsible for Heat-Induced Synthesis of Raffinose Family Oligosaccharides in Arabidopsis1,[w]Zentrum für Molekularbiologie der Pflanzen, Allgemeine Genetik (T.J.P., G.E.-S., M.W., F.S.) and Central Services (H.S.), Universität Tübingen, D–72076 Tubingen, Germany
Heat shock factors (HSFs) are transcriptional regulators of the heat shock response. The major target of HSFs are the genes encoding heat shock proteins (HSPs), which are known to have a protective function that counteracts cytotoxic effects. To identify other HSF target genes, which may be important determinants for the generation of stress tolerance in Arabidopsis, we screened a library enriched for genes that are up-regulated in HSF3 (AtHsfA1b)-overexpressing transgenic plants (TPs). Galactinol synthase1 (GolS1) is one of the genes that is heat-inducible in wild type, but shows constitutive mRNA levels in HSF3 TPs. The generation and analysis of TPs containing GolS1-promoter::  -glucuronidase-reporter gene constructs showed that, upon heat stress, the expression is transcriptionally controlled and occurs in all vegetative tissues. Functional consequences of GolS1 expression were investigated by the quantification of raffinose, stachyose, and galactinol contents in wild type, HSF3 TPs, and two different GolS1 knockout mutants (gols1-1 and gols1-2). This analysis demonstrates that (1) raffinose content in leaves increases upon heat stress in wild-type but not in the GolS1 mutant plants; and (2) the level of raffinose is enhanced and stachyose is present at normal temperature in HSF3 TPs. These data provide evidence that GolS1 is a novel HSF target gene, which is responsible for heat stress-dependent synthesis of raffinose, a member of the raffinose family oligosaccharides. The biological function of this osmoprotective substance and the role of HSF-dependent genes in this biochemical pathway are discussed.
Plants are sessile and hence cannot escape unfavorable environmental conditions such as heat, cold, drought, and salt stress. However, they have the potential to acclimate to these stresses by triggering a cascade of events that lead to changes in gene expression and, subsequently, to biochemical and physiological changes required for survival and growth during or after stress.
Plant acclimation to cold and drought induces to some extent common reactions, including signaling pathways, target gene expression, and biochemical/metabolic changes. One pathway that leads to the activation of a number of target genes is controlled by the transcriptional activator CBF/DREB (Stockinger et al., 1997
In Arabidopsis, there are seven members belonging to the GolS gene family, of which GolS1 and GolS2 mRNAs were detected in mature seeds (Taji et al., 2002
In contrast to drought and cold, the acquisition of tolerance to heat stress (HS) is correlated with the induction of heat shock protein (HSP) expression. HSPs are molecular chaperones whose functions are thought to counteract proteotoxic effects of stress-dependent protein denaturation and thus help to maintain the metabolic and structural integrity of the cell. The expression of HSPs is regulated at the level of transcription by HS-dependent activation of heat shock transcription factors (HSFs), which recognize conserved heat shock element (HSE)-binding sequences in the promoter upstream regions of HS genes. In Arabidopsis, 21 different HSF genes have been identified (Nover et al., 2001 The objective of this study was to identify other novel target genes of HSF regulation that may serve important functions in environmental stress responses of Arabidopsis. Taking advantage of AtHSF3 TPs, which show HSP synthesis at normal temperature, we were able to identify GolS1 mRNA in leaf tissue. Furthermore, by promoter::reporter gene expression and functional analysis of T-DNA insertion mutants, we clearly identify GolS1 as a novel HSF-dependent HS gene of Arabidopsis. The correlation between expression of GolS1 and the synthesis of RFOs in leaf tissue suggests an important role of this gene in stress-induced osmolyte synthesis in vegetative tissue and a common role of this pathway in environmental stress responses.
GolS1 Expression in Wild-Type and AtHSF3 TPs
In an attempt to identify novel HSF target genes, we screened a differential suppression subtractive hybridization library (Diatchenko et al., 1996
GolS1 Promoter:: -Glucuronidase Expression in Transgenic Arabidopsis
To determine whether GolS1 expression is in fact regulated at the transcriptional level, we generated chimeric promoter-reporter gene constructs for expression analysis in transgenic Arabidopsis plants. The inspection of the putative promoter region of GolS1 (http://intra.psb.ugent.be:8080/PlantCARE) revealed several perfect and imperfect HSE sequences with the consensus -GAA-/-TTC- (see Fig. 2A), the binding sites of HSF, upstream of a putative TATA box. The binding of AtHSF3 to two subfragments (1 and 2), which include the putative binding sites, was investigated by electrophoretic mobility shift analysis (EMSA) using recombinant protein expressed in Escherichia coli. The shifted bands indicate that HSF3 forms DNA-binding complexes with both fragments (Fig. 2B). The formation of the HSE-HSF3-binding complexes is completed by an excess of unlabeled probe, which proves the specificity of binding. It should be noted that recombinant Arabidopsis HSF proteins have the capacity to bind to HSEs in vitro without HS (Hübel and Schöffl, 1994
A fragment spanning 1,902 bp upstream from the start codon, including the HSEs containing regions 1 and 2, was inserted in front of the uidA reporter of the plant vector pGPTV-BAR-Asc (Überlacker and Werr, 1996  ). Following transformation of Arabidopsis, TPs were selected by means of BASTA resistance and subsequent generations (T2, T3) were analyzed for the expression of the reporter gene by assaying -glucuronidase (GUS) activity in leaf tissue. Figure 2C exemplifies the results in the T3 generation: At normal temperature, there is low basal activity and, after HS, there is a significant increase in GUS activity. TPs of the T3 generation were chosen for determining the expression pattern of the GolS1 promoter by histochemical staining of GUS activity in different stages of development (Fig. 3). Without HS, 1-d-old seedlings show GUS activity in the axes and cotyledons (Fig. 3A), whereas after HS, GUS staining is observed throughout the seedling, with enhanced levels in the roots and vascular tissue (Fig. 3B). After 13 d of germination, the nonstressed plants show GUS staining in the shoot meristematic region, the vascular tissue of shoots, roots, and petioles (Fig. 3C), and in the emerging lateral roots as well (Fig. 3D). After HS, the staining of GUS activity spreads throughout the plant tissue (Fig. 3E), with enhanced staining of major veins in leaves (Fig. 3, E and F). In 4-week-old plants, the GUS activity patterns are very similar to that of plants at day 13 (Fig. 3, H and J). In addition, a developmental expression of GUS was observed in the abscission zones (Fig. 3K) and, after HS, in the basal part of the sepals/petals, expression was not observed in pollen, young siliques, mature siliques, or seeds (Fig. 3L). These data indicate that, at normal temperature, GolS1 expression is developmentally regulated but restricted to meristematic and vascular tissues. After HS, GolS1 expression is induced in almost all cells and tissues of the plant.
Isolation of GolS1 T-DNA Insertion Mutants To determine the biological function of GolS1, the Wisconsin alpha population was screened for T-DNA insertions in GolS1. Two insertion mutants, gols1-1 and gols1-2, were obtained from the subpopulation CSH266 and CSH113, respectively. The T-DNA insertions were mapped by sequencing, in gols1-1 to the first exon (336 nucleotides downstream from the ATG start codon) and in gols1-2 to the third exon (1,000 nucleotides downstream from the ATG), as depicted in Figure 4A. The two homozygous mutants showed no obvious phenotypic variations compared to wild-type plants.
The effects of the T-DNA interruptions on GolS1 transcript levels were investigated by quantitative reverse transcription (RT)-PCR. The results show that the heat-induced expression of GolS1 mRNA is almost completely blocked (<1% of heat-induced wild type) in mutant lines (gols1-1 and gols1-2; Fig. 4B). Surprisingly, at normal temperature, low levels of GolS1 transcripts were detectable in the mutant lines but not in the wild type. It should be noted that the primers chosen for PCR quantification of mRNA levels map downstream of the T-DNA-insertions in the 3'-terminal part of the mRNA, spanning a region of 144 nucleotides, including the last exon and the 3'-untranslated sequences of GolS1. The absence of heat-inducible GolS1 mRNAs (in quantitative RT-PCR analysis) indicates that the T-DNA insertions cause gene knockout mutations. However, using an upstream probe (upstream to the T-DNA insertion) in northern-blot hybridizations (Fig. 4C), we were able to detect heat-induced but truncated transcripts of different sizes in gols1-1 (1.09 and 0.68 kb) and gols1-2 plants (1.22 and 0.89 kb). The low levels of constitutive GolS1 mRNA at normal temperature in gols1-1 and gols1-2 (77% and 42.6% relative to actin 2 standard; Fig. 4B) are probably the result of an initiation or read-through of transcription starting within the T-DNA. This background transcription, not observed in wild-type plants (only 0.9% relative to actin 2 mRNA), is heat sensitive as indicated by the strong reduction to 2.32% and 4.4%, respectively, relative to actin 2 mRNA.
GolS is a key enzyme in the biosynthesis of RFOs: raffinose, stachyose, and verbascose. To determine the functional consequences of changes in GolS1 expression, we assessed the levels of raffinose and stachyose in leaves of HSF3 TPs and in wild-type plants. Leaf samples representing the same developmental stage were subjected to HS for 2 h or incubated at normal temperature as a control. Carbohydrates were extracted and samples were subjected to HPLC analyses. In wild-type (Arabidposis C-24) leaves, raffinose is present at control temperature conditions (33 nmol mg–1 carbohydrate); however, there is a 3.5-fold increase (to 117 nmol mg–1 carbohydrate) of the raffinose content after HS (Fig. 5A). HSF3 TPs show significantly higher raffinose content at control temperature (378 nmol mg–1 carbohydrate) and after HS (541 nmol mg–1 carbohydrate) compared to wild-type plants (Fig. 5A). Interestingly, stachyose was detected only in HSF3 TPs but not in wild type, and the level was significantly higher (approximately 3-fold) at control temperature compared to HS (Fig. 5B). After HS and at control temperature, the level of Suc did not show any significant change in wild-type and HSF3 TPs (data not shown). This indicates that the increase in RFO levels is a specific effect of HS.
In wild-type Arabidopsis, ecotype Wassilewskija, there is also a strong increase in raffinose levels after HS but not in the knockout mutant lines, gols1-1 and gols1-2, which show only the basal raffinose contents present in wild type under nonstress conditions (Fig. 6A). In these lines we also examined the levels of galactinol, the primary product of GolS activity. Galactinol was detected and quantified in derivatized metabolite fractions after gas chromatography-mass spectrometry (GC-MS) analysis (Fig. 6B). In wild type, there is a strong (approximately 20-fold) increase in galactinol levels in leaves after HS. In gols1-1 and gols1-2 mutant lines, there is no increase after HS; they show only low basal galactinol contents as present in unstressed tissue.
These results indicate that there is a correlation between the induction of GolS1 mRNA (Figs. 1 and 4B) and an increase in raffinose-galactinol content after HS in wild-type plants. In HSF3 TPs, the largely enhanced levels of raffinose and the presence of stachyose correlate with the constitutive expression of GolS1 mRNA (Fig. 1).
The GolS1 mutant lines, gols1-1 and gols1-2, show no obvious alterations in growth and morphology compared to wild-type plants. To test stress tolerance phenotypes, seedlings or grown plants were subjected to different HS regimes, which had been previously used for determination of thermotolerance of Arabidopsis (Lee et al., 1995
Even after using electrolyte leakage as a more quantitative assay for detecting subtle differences in stress tolerance (Lohmann et al., 2004
Heat-Induced GolS1 Expression
Our analysis showed that GolS1 is a novel heat-inducible, HSF-dependently expressed gene in Arabidopsis. At normal temperature, GolS1 mRNA is hardly detectable in the leaf tissue of wild-type plants, but its abundance is greatly enhanced by a factor of >3,000 after HS (Fig. 1). The kinetics of mRNA accumulation (data not shown) are very similar to that observed for most HSP genes, which are transiently expressed with a maximum after about 1 h HS (Lohmann et al., 2004
The HS-induced GUS activity also provides evidence that GolS1 expression is controlled at the transcriptional level (Fig. 2). Histochemical GUS staining is observed in almost all cells and tissues upon HS, with only little variation during plant development. The generalized HS induction of GolS1 expression in all cells is reminiscent of the global expression of HSPs, and it suggests that this gene/enzyme represents another important pathway that may be required for plant cell protection from deleterious effects of HS. In the absence of HS, the GUS-staining pattern indicates that GolS1 is expressed at a low level, but only in certain tissues, including meristematic and major vascular tissue of roots, shoots, and leaves. Neither basal nor heat-induced expression of GolS1::GUS showed a preference for minor veins in leaves, which would have been consistent with a role of this gene in phloem loading, as inferred from cucurbits (Turgeon, 1996
What is the function of GolS1? Previously, it was shown that the expression of both GolS1 and GolS2 was induced by drought and high-salinity stress (Taji et al., 2002
In our analyses of wild-type, HSF3 TPs, and GolS1 knockout plants, there is a strong correlation between the expression levels of GolS1 mRNA and the levels of raffinose or galactinol in leaf tissue. Heat-induced raffinose levels are significantly enhanced in both wild-type and HSF3 plants. In HSF3 TPs, the effect is more pronounced under nonstress conditions (11-fold higher basal raffinose level compared to wild type) than after HS (only about 5-fold difference). It has to be taken into account that the HS-induced RFO content was assayed immediately after the 2-h treatment, representing only a snapshot of a short-term effect, whereas the constitutive overexpression of GolS1 at normal temperature in HSF3 TPs represents long-term steady-state levels. An interesting phenomenon was the presence of stachyose, however, solely in leaves of HSF3 TPs, not in wild type. In Arabidopsis, stachyose accumulation has been observed during seed maturation but could not be detected in rosette leaves, neither without nor after drought, cold, or salinity stress treatments, which resulted in the induction of raffinose only (Taji et al., 2002
Why is stachyose formed only in HSF3 TPs? There are two observations that may explain this phenomenon: (1) the relatively high constitutive levels of raffinose; and (2) the down-regulation of stachyose levels after HS (by a factor of about 3). Raffinose is the substrate that, along with galactinol, is required for stachyose synthesis. In wild type, the levels of raffinose are 5 to 10 times lower than in HSF3 TPs. These low substrate levels may not be sufficient to cause conversion to detectable levels of stachyose. The down-regulation of stachyose after HS indicates that this step is thermosensitive, either at the expression or at the activity level of putative stachyose synthases. This may explain why wild-type leaves, which show increased raffinose levels after HS, are nevertheless unable to synthesize stachyose. The threshold levels for reaction and detection are unknown. The isolation and analysis of stachyose synthases will be required to verify this model. It cannot be excluded entirely that HS activates In the GolS1 knockout mutants (gols1-1 and gols1-2) the inability to induce GolS1 mRNA by HS correlates with a total loss of heat-inducible levels of galactinol and raffinose. Hence, GolS1 makes the major contribution to the heat-induced formation of these compounds. The low basal levels of raffinose, which are approximately the same in wild-type and knockout lines, may result from the combined basal activities of different GolS enzymes expressed in leaves of Arabidopsis.
RFOs accumulate to relatively high levels during seed maturation in Arabidopsis (Taji et al., 2002
Plant Material, Growth Conditions, and Heat Treatment
For experimental purposes, different ecotypes of Arabidopsis L. Heynh were used. In this study, Arabidopsis C-24 was the genetic background of HSF3-overexpressing TPs (Prändl et al., 1998
Unless stated otherwise, plants were grown on soil with a 16-h-dark/8-h-light cycle at 20°C, with a light intensity of 3,900 lux for 4 to 5 weeks. Prior to analysis, wild-type Arabidopsis C-24 and HSF3-overexpressing plants were incubated for 3 d at 28°C (Panchuk et al., 2002 For HS treatment, pools of fully expanded leaves from 4- to 5-week-old plants were cut and incubated at 37°C for 2 h in prewarmed section incubation buffer (1 mM potassium phosphate, pH 6.0, and 1% [w/v] Suc) placed in a water bath with 40 oscillations/min. As a control, the leaves were incubated under the same experimental conditions but at room temperature (approximately 25°C). After treating the leaves, excess buffer was rinsed off with water, blotted dry with a filter paper, and then immediately frozen in liquid nitrogen and stored at –70°C until further use.
Experiments with combined heat and dehydration (drought) stress were conducted in a growth chamber under standard conditions. Soil-grown plants were subjected to 8 h/d HS (37°C) for 7 d. Dehydration stress was applied to one set of heat-stressed plants by withdrawing water supply for 7 d; the other set was irrigated every third day during the experiment (receiving only HS). Control plants were grown at room temperature (20°C) and either subjected to dehydration or watered. Electrolyte leakage assays were conducted after collecting leaves from stressed plants in 15 mL of deionized water. Ion leakage was measured using a Mettler-Toledo MC126 conductometer following a 1- to 3-h incubation at 20°C in a shaker. Samples were then frozen at –80°C, thawed, shaken for 1 h at 20°C, and total ion leakage measurements were taken. With the two measurements, percentage of electrolyte leakage was determined for each sample. In each experiment, three individual samples were taken from each treatment and analyzed in parallel.
Total RNA was isolated from leaf tissue using the RNeasy kit (Qiagen, Valencia, CA), and poly(A+) RNA was isolated directly from 1 g of frozen leaf tissues using oligo(dT)-Dynabeads (Dynal Biotech, Oslo, Norway), following the manufacturer's instructions.
One microgram of poly(A+) RNA or 15 µg of total RNA was electrophoresed on a 1% agarose gel containing formaldehyde (Maniatis et al., 1982
The hybridization probes were labeled using the Rediprime DNA Labeling System (Amersham-Pharmacia Biotech) with [
For quantitative RT-PCR, GolS1 promoter::GUS studies, and isolation of T-DNA insertions in GolS1, gene-specific primers were designed using the Primer 3 program (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi). Primers used for quantitative RT-PCR amplification of GolS1 (AC002337) were: F10-52 ic L (5'-TTCATCACCGCTCTTACTGAAG-3') and F10-52 ic R (5'-AAGAGGTTGGGGATTATGTGTC-3'). The amplification product included 65 bp of the last exon and 79 bp of the 3'-UTR, respectively, to yield a gene-specific product of 144 bp. Actin 2 (U41998) primers used for quantitative RT-PCR were: left primer 5'-AAGCTGGGGTTTTATGAATGG-3', right primer 5'-GACTACGTGAACACACACTGTT-3', yielding a gene-specific product covering 118 bp of the 3'-terminal region of the mRNA. Gradient PCR was performed to determine suitable annealing and amplification conditions for generating a gene-specific product. The specificity of the performed reaction was indicated by a single band after agarose gel electrophoresis and was confirmed by DNA sequencing of the PCR product.
Total RNA was isolated from 100 to 200 mg of frozen leaf material using the RNeasy Plant kit (Qiagen), and mRNA was purified from total RNA using Oligotex kit (Qiagen); cDNA was synthesized from either 100 ng or one-fifth of mRNA using the Thermo Script RT-PCR system (Invitrogen, Carlsbad, CA) with oligo(dT)20 primers. Quantitative RT-PCR was performed in triplicate using undiluted and 1:10 diluted cDNA as templates. The 50-µL reactions contained 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 0.5 units platinum Taq polymerase (Invitrogen), 0.1x SYBR-Green (Molecular Probes, Eugene, OR), 1.5 mM MgCl2 (Invitrogen), 100 µM of each dNTPs (TaKaRa Shuzo, Shiga, Japan), and 0.5 µM GolS1 primers or Actin 2 primers. PCR was performed with the following program: initial polymerase activation at 95°C for 15 min, then 40 cycles at 95°C for 20 s, and 56°C for 1 min 10 s. The amplification was monitored via intercalation of SYBR-Green using the iCycler iQ system (Bio-Rad Laboratories, Hercules, CA). All experiments were repeated three times using independently prepared RNA/cDNA templates. Deviations of threshold values were less than 1 cycle for independent cDNA preparations and less than 0.5 cycles for replicates of the same cDNA. The values were normalized to an internal standard Act 2 mRNA that was set as 100%.
Double-stranded DNA fragments spanning the two HSEs containing regions 1 and 2 (58 bp each) of the GolS1 promoter (Fig. 2A) were generated from synthesized complementary single strands (Sigma-Aldrich, St. Louis) by slow annealing starting at 85°C with a decline of 0.5°C min–1. The double-stranded fragments were purified by polyacrylamide (10%) gel electrophoresis and eluted from the gel. These fragments were used to test binding of recombinant HSF3.
For expression of N-terminal 6x-His-tagged full-length HSF3 (Lohmann et al., 2004 For binding reactions, 1.5 µg of purified protein were incubated for 20 min at room temperature in the presence of 1 µg of poly[d(I-C)] and 2 ng of radiolabeled promoter fragment in a total volume of 25 µL 0.5x Tris-borate/EDTA. In the competition experiment, 40 or 200 ng of unlabeled promoter fragment were included. After adding 5 µL of loading buffer (30% [v/v] glycerol, 0.02% [w/v] bromphenol blue), samples were resolved on a 5% polyacrylamide gel containing 3% (v/v) glycerol in 0.5x Tris-borate/EDTA. After electrophoresis at 350 V for 2.5 h at 4°C, the gel was dried on DE81 anion-exchange chromatography paper (Whatman Biometra, Clifton, NJ) and subjected to autoradiography.
Genomic DNA was isolated from Arabidopsis C-24 plants, according to Li and Chory (1998)
Arabidopsis (Col-0) was transformed, with the resulting promoter::GUS construct, according to Bechtold et al. (1993)
For GUS activity measurements, a single leaf located at an identical position within a rosette was collected from 7 to 10 individual T3-generation TPs (4–5 weeks old) and was subjected to HS or kept at control temperature. GUS activity was measured fluorometrically in total protein extracts from leaf tissue according to Jefferson (1987)
For histochemical localization of GUS, seeds of the T3 generation were germinated on solidified Murashige and Skoog medium. Seedlings and plants were heat stressed or kept at room temperature followed by vacuum infiltration (Jefferson et al., 1987
T-DNA insertions in GolS1 were screened using the alpha populations consisting of 60,480 T-DNA-tagged lines (ecotype Wassilewskija; Krysan et al., 1999
Leaves (150–200 mg) from identical positions within a rosette were pooled from different plants and subjected to incubation at control temperature or HS (37°C, 2 h) in buffer; subsequently leaves were washed with deionized water, briefly blotted on filter paper, and frozen in liquid nitrogen. Samples were crushed in liquid nitrogen with a prechilled mortar and pestle. Sugars were extracted by boiling in 80% methanol (800 µL, 2 min), followed by centrifugation (12,000g, 5 min) to sediment the insoluble fraction. The pellet was extracted again by boiling in 20% methanol (500 µL, 2 min), as mentioned above. The two supernatants were pooled and evaporated in a speedvac at room temperature overnight. Samples were resuspended in 0.5 mL of HPLC-grade water, centrifuged (12,000g, 3 min), and filtered (0.2 µm). HPLC separation was done using a high-pH anion-exchange column (Carbo Pac PA1; Dionex, Sunnyvale, CA) with 62 mM NaOH as eluent and pulsed amperometric detection (PED-300; Biometra, Göttingen, Germany). The equipment used was a modular HPLC system (pump 420, autosampler 460; Kontron, Eching, Germany) with KromaSystem 2000 software (Bio-Tek, Winooski, VT). Identity and amount of raffinose and stachyose were confirmed by cochromatography with corresponding authentic sugars (Sigma-Aldrich). The amounts of raffinose and stachyose were expressed in nanomoles (mg total carbohydrate)–1. Values obtained are mean ± SD (n = 3).
For the colorimetric determination of total carbohydrate, according to Dubois et al. (1956)
Extraction of Polar Arabidopsis Metabolites
Derivatization (Methoxymation and Trimethylsilylation)
GC-MS
Conditions Mass spectra of the putative galactinol peak from Arabidopsis leaves were matched with spectra obtained from pure galactinol provided by Fluka (code 79544; Buchs, Switzerland).
We thank Christine Leisgen of our laboratory for providing the SSH library, Marta Hoffman for excellent assistance in DNA sequencing and screening, Bettina Stadelhofer, Karl Wurster (Central Services, ZMBP-Tübingen) for technical assistance with HPLC and GC-MS, and Steffi Nagel (FG Seitz, ZMBP-Plant Physiology) for assistance with carbohydrate measurements. Received March 15, 2004; returned for revision July 2, 2004; accepted July 19, 2004.
1 This work was supported by Deutsche Forschungsgemeinschaft SFB 446, project A2. 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.042606. * Corresponding author; e-mail friedrich.schoeffl{at}zmbp.uni-tuebingen.de; fax 49–7071–29–5042.
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ABSTRACT
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-galactosidases that would specifically degrade stachyose but not raffinose. However, it has been shown that down-regulation of 
