The developmental and organ specific expression of sucrose cleaving enzymes in sugar beet suggests a transition between apoplasmic and symplasmic phloem unloading in the tap roots

Plant Physiology and Biochemistry 44 (2006) 656–665 www.elsevier.com/locate/plaphy

Research article

The developmental and organ specific expression of sucrose cleaving enzymes in sugar beet suggests a transition between apoplasmic and symplasmic phloem unloading in the tap roots D. Godt, T. Roitsch* Lehrstuhl für Pharmazeutische Biologie, Universität Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany Received 25 September 2006; accepted 26 September 2006 Available online 11 October 2006

Abstract Sucrose utilisation in sink tissues depend on its cleavage and is mediated by two different classes of enzymes, invertase and sucrose synthase, which determine the mechanism of phloem unloading. Cloning of two extracellular (BIN35 and BIN46) and one vacuolar invertase (BIN44) provided the basis for a detailed molecular analysis of the relative contribution of the sucrose cleaving enzymes to the sink metabolism of sugar beets (Beta vulgaris) during development. The determination of the steady state levels of mRNAs has been complemented by the analysis of the corresponding enzyme activities. The present study demonstrates an inverse regulation of extracellular invertase and sucrose synthase during tap root development indicating a transition between functional unloading pathways. Extracellular cleavage by invertase is the dominating mechanism to supply hexoses via an apoplasmic pathway at early stages of storage root development. Only at later stages sucrose synthase takes over the function of the key sink enzyme to contribute to the sink strength of the tap root via symplasmic phloem unloading. Whereas mRNAs for both extracellular invertase BIN35 and sucrose synthase were shown to be induced by mechanical wounding of mature leaves of adult plants, only sucrose synthase mRNA was metabolically induced by glucose in this source organ supporting the metabolic flexibility of this species. © 2006 Published by Elsevier Masson SAS. Keywords: Beta vulgaris; Sucrose; Invertase; Sucrose synthase; Assimilate partitioning; Sink metabolism; Gene regulation

1. Introduction In plants sucrose is the ubiquitous energy and carbohydrate source which is synthesised in photosynthetically active source tissues that are characterised by a net export of carbohydrates. Assimilates are transported to photosynthetically less active or inactive sink tissues such as roots, fruits or tubers, which are characterised by a net import of sugars and that compete for a common pool of carbohydrates. The supply of the main transport sugar sucrose is a limiting step for the growth and metabolism of sink tissues [19]. The long distance transport of assimilates occurs in the phloem and is driven by differences in solute concentrations and osmotic potentials [13,18]. Since removal of sucrose steepens the gradient and thus enhances the flow towards sinks, * Corresponding

author. Tel.: +49 931 888 6174; fax: +49 931 888 6182. E-mail address: [email protected] (T. Roitsch).

0981-9428/$ - see front matter © 2006 Published by Elsevier Masson SAS. doi:10.1016/j.plaphy.2006.09.019

enzymes involved in immediate sucrose metabolism are expected to be important both for phloem unloading and import of sucrose into sink organs. Thus they are critical links between photosynthate production in source leaves and growth capacity of sink organs thereby determining sink strength [5,32]. Sucrose utilisation is initiated by cleavage catalysed by invertase and sucrose synthase, with several isoforms differing in their subcellular localisation, which determine two different mechanism of phloem unloading. Invertases (ß-fructosidase, β-fructofuranosidase; EC 3.2.1.26) catalyse the irreversible cleavage of sucrose to glucose and fructose. Plants contain multiple forms of invertases which have been purified from a wide variety of plant species and tissues. Plant invertases are characterised by their subcellular localisation, by pH optima, and isoelectric points [51,64]. An intracellular invertase with an acidic pH-optimum and a low isoelectric point is localised in the vacuole and responsible to regulate the level of sucrose stored in this compartment [39, 41]. The function of the usual low enzymatic activity of solu-

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ble invertases with a neutral/alkaline pH-optimum in the cytoplasm is not known and assumed to be of procaryotic origin. An extracellular invertase, characterised by a highly acidic pHoptimum and a usually high isoelectric point, is the key enzyme for supplying carbohydrates to sink tissues via an apoplasmic pathway [48]. Sucrose unloaded into the apoplast by sucrose transporters is cleaves by this cell wall bound invertase isoenzyme into the hexose monomers, which then are imported into the sink cells by high affinity hexose transporters [17,40, 46,74]. Whereas such an unloading pathways is essential to supply sucrose to symplastically isolated cells such as developing pollen [25], it has been shown that extracellular cleavage of sucrose is also important for assimilate partitioning to other sink tissues, source-sink regulation, developmental processes, and defence responses. The corresponding functional studies include the overexpression of a yeast invertase in the apoplast [70], the analysis of an invertase deficient maize mutant [43], antisense repression in carrots [62], modulation of invertase activity during leave senescence [5]. In addition, the temporal, tissue specific expression [22,50,72], and regulation by a variety of endogenous and exogenous stimuli (summarised in [48]) supports a key role in carbohydrate supply under a variety of conditions. Sucrose synthase (EC 2.4.1.13) is localised in the cytoplasm and catalyses the energy conserving hydrolysis of sucrose to yield UDP-glucose and fructose [36,60]. Although the reaction is reversible, under physiological conditions sucrose synthase is primarily involved in the breakdown of the disaccharide [37]. Sucrose synthase has been suggested to be involved in a variety of diverse pathways [3]. This enzyme is in particular assumed to be important for synthesising storage compounds [10,73] and for determining sink strength in association with symplasmic phloem unloading via plasmodesmata [36,61]. Experimental evidence from antisense suppression of sucrose synthase supports a crucial role for sink metabolism in specific species [11,63,76]. In sugar beet (Beta vulgaris) sucrose is not only the major transport form of assimilates but it is also accumulated to high concentrations. During the first year of their vegetative growth cycle, sugar beet plants develop a large succulent taproot, which is a dominating storage sink organ accumulating high levels of sucrose. Studies in sugar beet have so far concentrated on source-sink relations in mature plants with a developed tap root. A number of enzymatic studies indicated a pivotal role of sucrose synthase during sucrose accumulation. Despite the great economical importance of the partitioning of sugars in sugar beet, only little is known about the function and regulation of genes encoding enzymes involved in sucrose metabolism of this species. cDNA cloning of a sucrose synthase from sugar beet and molecular analysis of mRNA regulation supported the role in sink metabolism of mature roots [31]. In wounded tap roots hexose accumulation was shown to correlate with the induction of vacuolar invertase VI-1 [53] in adult plants and in petioles a circadian regulation has been demonstrated for vacuolar invertase BvInv-V3 [26]. To address the contribution of the different sucrose cleaving enzymes to the sink metabolism of sugar beets during develop-

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ment, cDNA sequences of two extracellular invertases and one vacuolar invertase have been cloned. The determination of the expression of the different invertase isoenzymes and of sucrose synthase was complemented by analysis of the corresponding enzyme activities. The present study revealed an important role of extracellular cleavage of sucrose by invertase during early stages of development whereas sucrose synthase was found to be the key sink enzyme in mature plants. In addition, the effect of mechanical wounding and metabolic regulation by glucose in leaves has been determined. 2. Results 2.1. Cloning of cDNAs for an invertase family of sugar beet Degenerate oligonucleotide primers based on conserved amino acid motifs in the N-terminal part of extracellular invertase of carrot [58] and intracellular invertases of mung bean [2] and tomato [34] were used to amplify cDNA sequences by reverse transcriptase PCR from sugar beet. Amplification by PCR resulted in the expected product of about 750 bp. The PCR products were subcloned into pUC18 and characterised by sequence analysis. This approach yielded three different cDNAs, BIN35 (X81795), BIN44 (X81796), and BIN46 (X81797) with high nucleotide sequence homology to invertase sequences. The deduced amino acid sequences are highly homologous to each other with identities ranging from 46% to 67% and to published invertase sequences. A number of invertases have been cloned in recent years including two additional vacuolar invertases from sugar beet, VI-1 (AJ277457 [53]) and BvInv-V3 (AJ422051 [26]). A dendrogram based on the deduced amino acid sequences shows clearly two separated groups, representing cell wall bound and vacuolar isoenzymes, respectively (Fig. 1). The identity of representatives of both clusters has been experimentally proven by purification of the corresponding proteins and sequence analysis of tryptic peptides [15]. The dendrogram of representative vacuolar and extracellular invertase shown in Fig. 1 demonstrates that both BIN35 and BIN46 have significantly higher evolutionary relationship to the cluster of extracellular invertases. The corresponding sequences show 65–77% identity to extracellular invertases versus 56–60% identity to intracellular invertases. In contrast, the BIN44 sequence shares a higher degree of sequence identity with the cluster of vacuolar invertases. The corresponding sequence shows 61–71% identity to vacuolar invertases versus 52–60% identity to extracellular invertases. The identity of BIN35 and BIN46 as extracellular, and BIN44 as vacuolar invertase is further supported by the presence of characteristic variants of the amino acids motif WEC-P/V-D that was shown to determine the different pHoptimum of extracellular and vacuolar invertases [24]. The calculated isoelectric points were 7.5, 6.9, and 4.7 for BIN35, 44, and 46, respectively. Since the nucleotide sequences of the three invertase sequences are 75–79% identical it has been analysed whether they cross hybridise. A DNA gel blot analysis with the three

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Fig. 1. Phylogenetic relation of BIN35, 44, and 46 to representative other cloned plant invertases. The dendrogram was generated by the PILEUP program of the University of Wisconsin Genetics Computer Group sequence analysis software package [11]. Sources: 1, [15]; 2, [53]; 3, [34]; 4, [66]; 5, [65]; 6, this work; 7, [2]; 8, [72]; 9, [56]; 10, [27]; 11, [22]; 12, [58]. Sequences where the identity has been proven by purification of the corresponding protein are marked by an asterix (*).

invertase fragments has been carried out at high stringency. Fig. 2 shows that each of the three invertase fragments specifically recognises only the corresponding sequence. This experiment proves that the cloned fragments are highly specific hybridisation probes for detection of the corresponding sequences by Southern and Northern blot hybridisation. 2.2. The invertase genes of sugar beet Sugar beet genomic DNA was digested with four restriction enzymes, XbaI, HindIII, BamHI, and EcoRI, respectively, and probed with the three different invertase clones. The comparison of the patterns from the Southern blot shown in Fig. 3 demonstrates that the invertase isoenzymes are encoded by

Fig. 3. Southern blot analysis of chromosomal sugar beet DNA. High molecular-weight DNA was digested with EcoRI (lane 1), BamHI (lane 2), HindIII (lane 3) and XbaI (lane 4) and used for DNA gel blots hybridised with probes for intracellular invertase BIN44 and extracellular invertases BIN35 and BIN46.

three distinct genes. The patterns obtained with all probes are rather simple and suggest that the different invertase genes are present only in one copy or very few copies per haploid genome. 2.3. Differential developmental regulation of invertase isoenzymes and sucrose synthase in roots The dominating sink organ of sugar beet plants is the root which differentiates into a tap root. To address the role of invertase isoenzymes during growth and development of sugar beets in sink metabolism, steady state levels of mRNAs for the cloned three invertases, the enzyme activity of the different invertase isoenzymes and the distribution of the invertase protein have been determined. Sucrose synthase, representing the second class of sucrose cleaving enzymes in plants, was included in this comparison. The sugar beet seeds were sown on different days and the plants were grown in the green house. Plants of different ages were harvested on the same day to rule out the exogenous effect of water and light conditions on mRNA regulation and enzyme activities. 2.3.1. Characterisation of the growth of sugar beet roots The increase in fresh weight of the roots of sugar beet plants was determined from 10 to 60 days after sowing. Fig. 4A shows that sugar beet roots grow exponentially between the whole period analysed.

Fig. 2. Southern blot analysis of fragments encoding invertase isoenzymes. cDNA fragments encoding BIN35 (1, 210 bp), 44 (2, 437 bp), and 46 (3, 589 bp) were amplified by PCR using sequence specific primers and used for DNA gel blots hybridised with probes for intracellular invertase BIN44 and extracellular invertases BIN35 and BIN46. 4, a mixture of fragments for BIN35, 44, and 46 was used.

2.3.2. Analysis of mRNA levels for invertase isoenzymes and sucrose synthase Northern blot analysis was performed to address the expression of the different sucrose cleaving enzymes. Only transcripts for extracellular invertase BIN35 and sucrose synthase could be detected in total RNA preparations with the expected sizes

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Fig. 4. Regulation of sucrose cleaving enzymes of sugar beet roots during development. The results of a representative experiment is shown. (A) Increase of root fresh weight. (B) Steady state levels of mRNAs. Thirty micrograms of total RNA were separated on a formaldehyde gel, transferred to a nitrocellulose membrane, and hybridised with probes for extracellular invertase BIN35 and sucrose synthase (Susy). (C) Activities of extracellular invertase (6, Inv-CW), vacuolar invertase (■, Inv-V), neutral invertase (▲, Inv-N), and sucrose synthase (●, Susy). (D) Immunoblot analysis of steady state levels of extracellular invertase BIN35. Proteins were released from cell wall preparations from roots derived from plants harvested at day 17 (2) and after 4 months (3). Five micrograms of protein were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with an anti-BIN35 polyclonal antiserum. Two micrograms of purified extracellular invertase CIN1 were used as positive control (1). (E) Tissue print analysis of extracellular invertase BIN35. Extracellular proteins of longitudinal sections of seedlings were blotted on a nitrocellulose membrane, and immunoblotted with an anti-BIN35 polyclonal antiserum (aBIN35) or preimmuneserum (Pre). Co, cotelydons, Hy, hypocotyl, ra, radicula.

of 2.2 and 2.8 kb, respectively (Fig. 4B). In contrast, no mRNA could be detected for extracellular invertase Bin46 and vacuolar invertase BIN44 (data not shown). Fig. 4B demonstrates that extracellular invertase BIN35 and sucrose synthase show an inverse expression pattern. High levels of mRNA for BIN35 are present at early stages of development up to day 24, whereas the transcript could not be detected in later stages. In contrast, the mRNA for sucrose synthase is absent at day 10 and then gradually increases to high levels up to day 60. 2.3.3. Analysis of the enzyme activities of the invertase isoenzymes and sucrose synthase The activities of the two intracellular invertases, the neutral and the vacuolar invertase, and of the extracellular invertase have been determined (Fig. 4C). A similar trend was obtained both for the activity of extracellular and vacuolar invertase. A high level of enzyme activity was present at day 10 that declines during development. However, whereas the vacuolar invertase reached very low levels already after 53 days, about 27% of the initial extracellular invertase was still present after 78 days. Experiments with mixed extracts rule out that the decrease in the invertase activity during development is due to the presence an invertase inhibitor: addition of extracts from day 78 did not result in the inhibition of the high activity

in the extracts of day 10. The initial low levels of neutral invertase declined to undetectable levels during the time period analysed. The activity profile of sucrose synthase was inverse to the invertase activities. The enzyme activity could not be detected at day 10 and then steadily increased, with a most pronounced increase after day 53. 2.3.4. Determination of the protein of extracellular invertase BIN35 To address the question whether the decrease of the mRNA for BIN35 and the extracellular invertase activity is also reflected at the protein level isoenzyme specific antisera have been generated. C-terminal in frame fusions between the cDNAs of BIN35, 44, and 46 and the open reading frames of LacZ and the maltose binding protein, respectively, were engineered. The corresponding fusion proteins were overproduced in E. coli and purified by preparative SDS-gel electrophoresis (ß-galactosidase fusions) or affinity chromatography (maltose binding protein fusions). The β-galactosidase fusion proteins of BIN35, 44, and 46 were used to immunise New Zealand White rabbits. The specificity of the obtained antisera was tested by probing with the corresponding maltose binding protein fusions. Western blot analysis revealed that highly specific

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antisera have been obtained that essentially recognise only one specific sugar beet invertase isoenzyme (data not shown). The differential expression of extracellular invertase BIN35 and of the extracellular invertase activity is also reflected at the protein level. Fig. 4D shows that the BIN35 antiserum can detect a strongly crossreacting band only in samples from 17 days old seedling roots but not in samples from fully developed tap roots from 4-month-old plants. The high level of extracellular invertase at the seedlings stage is also supported by immunocytolocalisation in tissue prints. Ionically bound extracellular proteins were blotted on a nitrocellulose membrane and probed with the BIN35 antiserum. Fig. 4E shows that high levels of extracellular invertase BIN35 can be detected in the cotelydons and the hypocotyl of the roots of seedling but not in the radicula. No signals were obtained with the corresponding preimmune serum supporting the specificity of the signals obtained with the BIN35 serum.

could be determined only for BIN44 and BIN46. The highest mRNA levels were found in sink leaves for vacuolar invertase BIN44 and in the cortical part of mature roots for extracellular invertase BIN46 (Fig. 5). A differential, sink tissue specific distribution of the transcript for sucrose synthase could be determined in total RNA preparations (Fig. 5). The sucrose synthase mRNA could not be detected in leaves. In contrast, high levels of sucrose synthase mRNA could be detected in samples of roots with the highest steady state level in the cortical region. A high level of expression of both vacuolar invertase BIN44 and sucrose synthase could be detected in flowers from greenhouse grown plants in the second year (data not shown).

2.4. Analysis of the expression of sucrose cleaving enzymes in mature plants

Activation of defence responses requires energy and thus induction of sink metabolism [14,49]. Therefore the effect of mechanical injury by wounding on mRNA levels of the different invertase isoenzymes in leaves has been determined and compared to the regulation of the mRNA level of sucrose synthase. Leaves of sugar beet plants were cut into strips and shaken in sugar free MS medium. Samples were taken at various times after wounding and total RNAs were isolated and used for Northern blot analysis (Fig. 6A). Only the transcripts for extracellular invertase BIN35 and sucrose synthase were wound inducible, although with a different time course. The mRNA for sucrose synthase starts to accumulate after 3 h for sucrose synthase and for BIN35 after 10 h, respectively.

To address the distribution of invertases in 4-month-old mature plants the level of mRNAs for the extracellular invertases BIN35 and BIN46, for the vacuolar invertase BIN44 and for sucrose synthase have been determined in source and sink leaves, the undifferentiated root, and the central and cortical region of the developed tap root. No transcripts of any of the three cloned invertases could be detected in total RNA preparations from any of the tissues analysed. Using polyA RNA a differential, low expression level

Fig. 5. Tissue specific distribution of mRNAs for sucrose cleaving enzymes in mature, 4-month-old plants. Five micrograms of polyA RNA was separated on a formaldehyde gel, transferred to a nitrocellulose membrane, and hybridised with probes for extracellular invertase BIN35 and BIN46 and vacuolar invertase BIN44. Thirty micrograms of total RNA was separated on a formaldehyde gel, transferred to a nitrocellulose membrane, and hybridised with a probe sucrose synthase (Susy). Samples were prepared from sink leaves (1), source leaves (2), non differentiated roots (3), and the central (4) and cortical region (5) of a tap root.

2.5. Induction of mRNAs for an extracellular invertase and sucrose synthase by wounding in leaves

Fig. 6. Regulation of mRNAs sucrose cleaving enzymes by wounding and glucose in mature leaves. Thirty micrograms of total RNA was separated on a formaldehyde gel, transferred to a nitrocellulose membrane, and hybridised with probes for extracellular invertase BIN35 and sucrose synthase (Susy). (A) Source leaves were mechanically wounded by cutting into strips and samples removed at the times indicated. (B) Samples were prepared from control leaves (1) and leaves supplied either with 100 mM mannitol (2) or glucose (3) via the petiole after 48 h of incubation.

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2.6. Differential regulation of invertases and sucrose synthase in response to glucose feeding in leaves Metabolic regulation by sugars has been shown for invertases of several species [50] and was proposed to be a crucial element of a feed-forward mechanism to amplify endogenous and exogenous signal that regulate invertases [46,49]. To address the effect of hexose sugars on the mRNA level of the sucrose cleaving enzymes 100 mM glucose was supplied to mature leaves by petiole feeding for 48 h. To address the possible osmotic effect of the sugar applied control experiments were carried out with 100 mM mannitol. Northern analysis revealed that transcripts for the invertase BIN35, 44, and 46 could not be detected in any of the samples. In contrast, glucose specifically and strongly induced the mRNA for sucrose synthase (Fig. 6B), which is supported by the lack of induction by mannitol. The increase in the mRNA level of sucrose synthase is paralleled by a 320% increase of the corresponding enzyme activity. 3. Discussion The cDNA cloning of three invertases from sugar beet, extracellular invertases BIN35 and BIN46 and of the vacuolar invertase BIN44, provided the basis to study the function of invertases at the molecular level in this agricultural important species. Several lines of evidence support the identity of BIN35 and BIN46 as extracellular invertases, and of BIN44 as vacuolar invertase, respectively. First, the dendrogram shown in Fig. 1, including 18 cloned invertases, clearly displays two groups, comprising the extracellular and vacuolar invertases, respectively. This grouping is supported by two extracellular [15, 58] and three vacuolar [2,34,66] invertases that have been unequivocally identified to belong to the corresponding class of isoenzyme by the analysis of peptide sequences of the corresponding purified proteins. The deduced amino acid sequences of the BIN35 and BIN46 clearly show a higher phylogenetic relationship to the cluster of extracellular invertases, whereas BIN44 is more closely related to the cluster of vacuolar invertases. Second, invertases are characterised by variants of the conserved catalytic domain motif WEC(V/P)D. BIN35 and BIN46 share a proline with all other cloned extracellular invertases at the variable position, whereas BIN44 posses a valine at this position that is characteristic of vacuolar invertases. Third, the antiserum raised against BIN35 specifically recognises the cell wall bound invertase protein. Most extracellular invertases are characterised by a high isoelectric point (above 8). In contrast both cloned extracellular invertases from sugar beet, BIN35 and BIN46, are characterised by intermediate isoelectric points of 5.7 and 4.7 which they share with extracellular invertase Incw4 from maize [33] and Cin3 from Chenopodium rubrum [16]. The possible evolutionary and functional relationship to a fructan-6-exohydrolase from sugar beet (CAD48404 [69]), that shows sequence homology to

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extracellular invertases including BIN35 and BIN48 (57% and 46% identity), needs to be explored. Characteristic features of the three different invertase isoenzymes are contained within the cloned sequences and Southern blot analysis revealed the absence of cross-hybridisation between the cloned cDNA fragments of BIN35, 44, and 46. Thus they represent highly specific hybridisation probes to analyse the genes by Southern blotting and to monitor the regulation of the expression of the corresponding mRNAs by gel blot analysis. Therefore, we refrained from obtaining fulllength clones. Genomic Southern blots demonstrate that the intracellular invertase BIN44 and the extracellular invertases BIN35 and BIN46 are encoded by three different genes that are present only in one copy or very few copies per haploid genome. Two additional invertase cDNAs encoding vacuolar invertases have been cloned from sugar beet, VI-1 (AJ277457 [53]) BvInv-V3 (AJ422051 [26]). Thus the extracellular invertases and vacuolar invertases from sugar beet are encoded by gene families comprising two and three members, respectively. Gene families of similar complexity have been previously reported from other species [60], although in other species up to six extracellular isogenes have been identified [51]. The cDNA cloning of two extracellular and one vacuolar invertase from sugar beet allowed the first detailed analysis of the contribution of the different sucrose cleaving enzymes to the sink metabolism of sugar beets during development. Carbohydrate partitioning into the developing tap root is of particular importance because the great agricultural importance of this species depend on the sucrose stored in this sink organ. In addition, so far no enzymatic analysis of the contribution of extracellular cleavage of sucrose to the sink metabolism of sugar beet root has been carried out. The analysis of the regulation of the mRNAs and the activity revealed an inverse regulation of invertase and sucrose synthase. At early stages of development high extracellular and vacuolar invertase activities are present in the roots that both declined during further development. The extracellular invertase activities correlate with the distribution of extracellular invertase protein and the mRNA level for extracellular invertase BIN35. The extracellular invertase BIN46 seems to have no function for carbohydrate partitioning at these developmental stages. Since no mRNA for the vacuolar invertase BIN44 could be detected, the measured high initial vacuolar invertase activity is possibly related to the vacuolar invertase VI-1 [53] that was not cloned by our experimental approach. It is unlikely that the recently cloned vacuolar invertase BvInv-V3 [26] accounts for the activity since it was shown to be expressed in developing tap roots only at a very low level. In mature plants the expression levels of the invertases were extremely low and did not show a sink tissue specific distribution except for a strong expression of vacuolar invertase BIN44 in flowers. In contrast to the invertase isoenzymes, neither the mRNA nor the activity of sucrose synthase could be detected in seedlings. Sucrose synthase mRNA level and enzymatic activity started to increase only at day 17 to be the dominating sucrolytic activity in mature roots already at day 75. In mature plants sucrose synthase shows a sink tissue

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specific distribution with the highest mRNA level in the cortical part of the root. These data demonstrate a transition between the mechanisms of the functional unloading pathways during development. Since vacuolar invertase is responsible to regulate sucrose storage [35,45,71], extracellular invertase is the only sucrose cleaving enzyme at the seedling stage that contributes to phloem unloading via an apoplasmic pathway. Thus the present study shows that the dominating pathway to supply carbohydrate to sustain sink metabolism in the developing root is mediated via extracellular cleavage of sucrose and uptake of hexoses. Only at later stages of development sucrose synthase takes over the function of the key sink enzyme to contribute to the sink strength via uptake of sucrose into the sink cell and reversible sucrose cleavage in the cytoplasm. We can not rule out the possibility of unloading of sucrose into the apoplast and direct uptake of sucrose without prior cleavage into hexose monomers. This third pathway for supplying of sucrose to sink cells, in addition to direct unloading via plasmodesmata and cleavage by extracellular invertase, is also typically included in schemes on phloem unloading pathways. However, no strong experimental evidence exists supporting an apoplasmic pathway involving sucrose uptake by sink cells. In particular, the expression pattern of sucrose transporters do not support such a pathway. Whereas the function of sucrose transporters in phloem loading in source tissues is well established, they are typically not expressed in sink tissues. Although a sucrose transporters has been cloned from sugar beet [9], which also was shown to be expressed in leaves, no expression data have been published so far on the expression in the tap root. So far studies in sugar beet have concentrated on the sink metabolism of the mature tap root that indicated a dominating role of sucrose synthase for sink metabolism. The importance of sucrose synthase for sucrose accumulation in sugar beet has been suggested by enzymatic studies carried out in particular by various investigators from Russia in the 1970s ([21] and references cited therein). The first and only molecular study on sucrose synthase function in sugar beet demonstrated a high expression level of sucrose synthase in mature tap roots [31]. These data are extended by the present study that neither mRNA nor enzyme activity of sucrose synthase can be detected at the seedling stage and that both parameters gradually increase during development. The available literature data on the specific expression of both sucrose synthase [31] and in addition sucrose phosphate synthase [30] in sugar beet tap roots strongly support the operation of a futile cycle of initial sucrose cleavage via sucrose synthase and subsequent resynthesis via sucrose phosphate synthase before transport into the vacuole. This is in agreement with the suggestions that cleavage of sucrose via sucrose synthase prevents reloading of sucrose and consequently enhances sink strength in tap roots, providing a rationale for the observed mechanism [20]. The decline in extracellular invertase activity precedes the onset of the storage phase that starts around day 30 [6,21], whereas the increase in sucrose synthase activity correlates with sucrose accumulation. This further supports the conclusions that sucrose synthase is responsible for supplying sucrose

for storage processes whereas extracellular invertase is involved in supplying sucrose for active growth to support cell division [75]. The extracellular invertase possibly has a dual function by both providing the substrate for heterotrophic growth and generating a hexose based metabolic signal to stimulate the cell cycle [51,75]. In addition, the type of sucrolytic activity will also affect sugar related signalling processes, extracellular inverts generates two hexose sugars, sucrose synthase only one. It is a general observation that during development a high invertase activity is associated with active growth processes, characterised by a high hexose/sucrose ratio, whereas a high sucrose synthase activity is associated with storage processes and differentiation. The available information on the function of sucrose cleaving enzymes in carrot [57,59,63], radish [67,68], and potato [1, 54,76] indicate a general transition between an apoplasmic and symplasmic unloading pathway during storage root formation. A gradual decline of a high level of mRNA for extracellular invertase isoforms during development has also been found in carrot [59] and potato [1]. Specific increases in the activity or mRNA levels for sucrose synthase during root development and high steady state levels in fully developed tap roots or tubers were also present in radish [67,68], carrot [59], and potato [1,54]. Sucrose cleaving enzymes do not only have a role in carbohydrate partitioning to sink organs but they are also important during the activation of stress responses possibly to satisfy the increased demand for carbohydrates as energy source [4,47, 49]. In sugar beet the effect of mechanical wounding on sucrose cleaving enzymes has been so far analysed only in roots. Wounding of tap roots resulted in induction of vacuolar invertase VI-1, and extracellular invertases BIN35 and BIN46 [53], whereas sucrose synthase was repressed [31]. The present study demonstrates that both the extracellular invertase BIN35 and sucrose synthase are also strongly induced in wounded source leaves. This finding shows that although extracellular invertase is not important for carbohydrate partitioning to sink organs of mature sugar beet plants, it is part of the defence response of adult plants of this species that has been observed also in other species [22,29,48,50,58], supporting the metabolic flexibility. Both wound induction and repression of mRNAs for sucrose synthase has been observed on other species [50], indicating that a less uniform regulation by stress related stimuli compared to invertases. Metabolic regulation by sucrose seems to be a general property of sink specific enzymes [49,52]. The present study demonstrates that the expression of sucrose synthase is strongly carbohydrate responsive in leaves whereas it has been reported before that the high level mRNA level in the tap root can not be modulated by sugars [31]. This differential metabolic regulation in the sink and source organ suggests the presence of distinctly different sensing pathways. Both repression and induction by sugars has been reported for sucrose synthases [23,28,36,42,54]. None of the three invertases were carbohydrate responsive although sugar responsive vacuolar and extracellular invertase isoenzymes have been identified in different species [48], which may be related to the fact that

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invertases have no important function in sink metabolism of mature sugar beet plants. Whereas a controversial debate has been carried out for many years whether phloem unloading occurs predominantly symplasmically or apoplasmically (see discussion in [46]) since Münch [44] recognised the symplast around 1930 and Kursanov [38] favoured the apoplasmic metabolite transport there is accumulating evidence that both pathways are equally important and temporal and tissue specific factors as well as endogenous and exogenous stimuli determine the predominating mechanism. The data obtained in the present study on the contribution of sucrose cleaving enzymes to the sink metabolism of sugar beets is a further example of the metabolic flexibility of higher plants. The mechanism of phloem unloading to sustain heterotrophic growth of sink tissues, to provide storage compounds, and to provide the energy for defence responses is not strictly determined and may be adjusted to the actual requirement. This supported by the finding that extracellular invertases and thus an apoplasmic pathway for carbohydrate supply is induced in source leaves in response to wounding although phloem unloading of mature sugar beet plants predominantly occurs symplastically. The insight into the underlying mechanism will contribute to understand growth and development of higher plants as well as to successfully manipulate carbohydrate partitioning by transgenic approaches. 4. Methods 4.1. Plant material Sugar beet plants, cultivar Corinna, were grown in the greenhouse; the natural light was supplemented with additional illumination for 12 hours/day. 4.2. Amplification and cloning of invertase cDNAs Amplification of invertase sequences from cDNA as substrate using degenerate oligonucleotides was carried out as described previously in [50]. PCR products were subcloned into pUC18 according to standard procedures [55] to generate the plasmids pMP35, pMP44, and pMP46 and sequenced using the Sequenase 2.0 kit (Amersham, Braunschweig, FRG). Sequence analysis was performed using the sequence analysis software package of the University of Wisconsin Genetics Computer Group [12].

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C. rubrum [23], that is highly homologous to the sucrose synthase from sugar beet [31] were used as hybridisation probe. Hybridisation was performed in 50% formamide, 5 × SSC, 0.5% SDS and 5 × Denhardt’s solution at 42 °C for 18 h. After hybridisation, membranes were washed in solutions with decreasing salt concentration and increasing temperature. A final washing step was carried out in 0.1 × SSC at 56 °C. 4.4. Generation of antisera, immunoblot and tissue print analysis Fusions of the cDNA sequences of invertases BIN35, 44, and 46 with lacZ, or the maltose binding protein were constructed as follows: the 750 bp BamHI-EcoRI fragments of pMP35 and pMP44 were subcloned into pBluescriptKS(+) to generate plasmids pBS35 and pBS44. The inserts were subcloned as BamHI/HindIII fragments into the lacZ expression vector pTRB1 to generate plasmids pAK35 and pAK44 and into the maltose binding protein expression vector pMAL2c to generate the plasmids pML35 and pML44. A 600 bp SalIHindIII fragment of pMP46 was subcloned into pTRB1 to generate plasmids pAK46 and into pMAL2c to generate the plasmid pML46. Induction of protein synthesis and purification of the fusion proteins was carried out as described before for the lacZ fusions [7] and according to the instructions of the supplier for the maltose binding protein fusions (New England Biololabs, Frankfurt, Germany). Polyclonal antibodies directed against the purified lacZ fusion proteins were generated in New Zealand White Rabbits as described before [15]. Immunoblot [15] and tissue print [8] analyses were carried out according to published procedures. 4.5. Determination of enzymatic activities The activities of neutral and acidic intracellular invertase and of cell wall bound invertase [50] and sucrose synthase [23] were determined as described previously. Acknowledgements We would like to thank Nikolaus Burkhardt (Burgweinting, Germany) for supplying sugar beet seeds and field grown sugar beet plants, Edelgart Herold for skilful technical assistance and S. Berger for critically reading of the manuscript. References

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