Journal of Experimental Botany, Vol. 52, No. 365, pp. 2381-2385,
December 1, 2001
© 2001 Oxford University Press
Short Communication |
In wounded sugar beet (Beta vulgaris L.) tap-root, hexose accumulation correlates with the induction of a vacuolar invertase isoform
Heidelberger Institut für Pflanzenwissenschaften (HIP), Ruprecht-Karls-Universität, D-69120 Heidelberg, Germany
Received 28 February 2001; Accepted 5 July 2001
Abstract
Wounding of sugar beet tap-root causes an induction of invertase activity, which contributes to post-harvest sucrose losses. In this first comprehensive monitoring of wound-induced invertase mRNAs, proteins, enzyme activities, and tissue hexose concentrations, the VI isoform responsible for wound-induced hexose accummulation in mature tap-root could be identified.
Key words: Cell wall invertase, vacuolar invertase, sugar beet, tap-root, wounding.
Introduction
During the life span of higher plants, wounding is a common event. The open wound surface causes uncontrolled water loss and offers an entry point for pathogens. Therefore, the response to mechanical wounding involves molecular mechanisms to counter osmotic stress and pathogen attack (Reymond and Farmer, 1998
; Reymond et al., 2000). To prevent both, many plants react with the formation of a wound periderm which builds an efficient barrier against invaders and reduces evaporation (Barkhausen, 1978; Kahl, 1978). As the newly formed wound meristem forms a strong local sink, energy resources have to be mobilized either locally, or have to be provided by mobilization from neighbouring tissue. The induction of acid invertases, including cell wall (CWI) and/or vacuolar (VI) isoforms, in response to mechanical wounding has been previously reported (Sturm and Chrispeels, 1990; Zhang et al., 1996; Ehness et al., 1997; Godt and Roitsch, 1997; Ohyama et al., 1998), however, none of these systems has been comprehensively monitored for invertase transcripts, proteins, activities and in vivo hexose formation.
The role of acid invertase isoforms was studied in sugar beet tap-root, a specialized tissue which stores up to 22% sucrose. During harvest, mechanical wounding (‘decapitation’) of tap-roots causes a sustained metabolic response, leading to significant post-harvest sucrose losses (Berghall et al., 1997, and references cited therein). Upon wounding acid invertase activity increased several-fold (Leigh et al., 1979; Milling et al., 1993; Berghall et al., 1997). To identify the invertase isoform(s) responsible for the observed hexose accumulation, the expression was monitored of CWI and VI mRNAs, CWI and VI proteins, and invertase activities in the cell wall and the soluble (vacuolar) fraction, respectively, of wounded tap-root tissue. The results demonstrate that the wound-induction of the CWI-1 isoform (=Bin35; X81795) clearly preceded the induction of the VI-1 isoform (AJ277457), however, the wound-induced increase of hexoses coincided with the delayed VI-1 induction. The expression of two other invertase isoforms (CWI-2 [=Bin46; X81797] and VI-2 [=Bin44; X81796]) remained unaffected. The identification of VI-1 as the relevant isoform for hexose accumulation will allow strategies to be developed for its genetic manipulation by antisense expression and/or in vivo silencing via expression of an invertase inhibitor (Pfister et al., unpublished results).
Materials and methods
Plant material
Beta vulgaris L. (diploid inbred line VV-I/ZR 10676 from KWS Saat AG, Einbeck, Germany) plants were grown under field conditions. For wounding experiments, tap-roots from 24-week-old plants were harvested and stored at 6 °C for 3 months. After equilibration to room temperature, tissue cylinders of 2 cm diameter were taken from the cortex with a cork borer (4 cylinders per tap-root) at an equal distance from the tap-root centre. Tissue cylinders were immediately sliced by a set of fixed razor blades. Two mm slices were incubated in moist atmosphere at room temperature for different time intervals.
Amplification of partial cDNAs of sugar beet CWI and VI isoforms
The sequences of partial cDNAs for CWI-1, CWI-2 and VI-2 have been previously submitted to the EMBL Database (Roitsch et al., 1995; for accession numbers see Introduction). These cDNA fragments were amplified by RT-PCR, using MMLV-reverse transcriptase for first strand cDNA synthesis from total RNA of 2-week-old seedlings: CWI-2-sense and -antisense primers: 5'-GTTCTATCAGTACAACCCTTATTC-3' and 5'-GGAAGCGTAAAATTTACCTCCATA-3', respectively; CWI-1-sense and -antisense primers: 5'-TGGATIAACGATCCAAATGGACCIATG-3' and 5'-TCAAAGAAIGTCTT(G/T)GAIGC(A/G)TA-3', respectively; VI-2-sense and -antisense primers: 5'-CTTTTACCAGTACAACCCTGCAGG-3' and 5'-AGAAGCGTAAAATCTACCATAATC-3', respectively. For the cloning of a partial VI-1 cDNA (AJ277457), total RNA from wounded (3 d) tap-root tissue was reverse transcribed, followed by PCR amplification with the following primers: sense 5'-ATGGTI(G/C)C(G/T)GA(C/T)C(A/G)(A/T)TGGTA(C/T)GA-3', antisense 5'-TC(A/G)(G/C)T(A/G)TC(A/T)G(A/T)(C/T)TC(A/C/T)CCAA(C/T)CCA-3' (I, inositol). PCR products were cloned into the Bluescript SK+ vector (Stratagene, Amsterdam, The Netherlands) and sequenced in both directions. The partial cDNAs were used as templates for the synthesis of gene-specific biotinylated probes (see below) (Löw and Rausch, 1996).
RNA blot and Southern blot analysis
Total RNA was extracted as described previously (Logemann et al., 1987). Genomic DNA was isolated from sugar beet leaves (Murray and Thompson, 1980). RNA blot and Southern blot analysis with biotinylated probes were performed according to an established protocol (Löw and Rausch, 1996).
Generation of antisera and immunoblot analysis
For detection of CWI-1 protein, an antiserum directed against the N-terminal domain of tobacco CWI (X81834; Greiner et al., 1995) was generated. The corresponding partial cDNA sequence was amplified by PCR, using primers according to the peptide sequences NVHRTG and GEYTKHV (5'-GCCGAATCGAGCTCAATGTTCACAGAACTGG-3' and 5'-GCCGTTAGAAGCTTAACATGCTTAGTGTATTCG-3', respectively). These primers contained restriction sites (SacI and HindIII, respectively) for directional cloning into the pQE30 vector (Qiagen, Hilden, Germany). The His-tagged N-terminal CWI domain was expressed in E. coli M15[pREP4] and purified by Ni-NTA affinity chromatography. The purified protein was used to generate a polyclonal antiserum in rabbit. Following the same protocol, the partial VI-1 cDNA (see above) was amplified with the primers 5'-GCCGAATCGAGCTCATAAATGGTGTTTGGACAGG-3' and 5'-GCCGTTAGAAGCTTAGCCGAGGTATCCAACC-3', corresponding to the peptide sequences INGVWTG and GLDTSA, respectively. Immunoblot analysis was performed according to Weil and Rausch (Weil and Rausch, 1994), except that, for blocking, 5% dry milk powder was used instead of BSA, and blots were developed using the SuperSignal® West Dura Detection Kit (Pierce, Rockford, USA). In control blots, it was confirmed that the antisera were specific for CWI and VI, respectively.
Determination of vacuolar and cell wall invertase activities
VI activity:
tissue was extracted in 30 mM MOPS, pH 6, 250 mM sorbitol, 10 mM MgCl2, 10 mM KCl, and 1 mM PMSF. After centrifugation (10 min, 6500 g, 4 °C), the protein from the supernatant was precipitated with 80% acetone (-15 °C). The protein pellet was resuspended in assay buffer without sucrose. Aliquots were assayed for VI activity in the following buffer: 30 mM sucrose, 20 mM triethanolamine, 7 mM citric acid, 1 mM PMSF, pH 4.6. Released glucose was determined as described earlier (Weil and Rausch, 1994).
CWI actvity:
the pellet collected after the 6500 g centrifugation step was resuspended in extraction buffer including 1% (v/v) Triton X-100. The suspension was shaken vigorously for 10 min at 4 °C. After another centrifugation, the pellet was washed in extraction buffer without Triton X-100, and finally resuspended in assay buffer (see above).
Determination of tissue sugar concentrations
Concentrations of hexoses (glucose and fructose) and sucrose in tap-root tissue were determined enzymatically (Stitt et al., 1989).
Results and discussion
Southern blot analysis revealed that each of the four invertase cDNAs hybridized to a single invertase gene, and corresponding genomic clones have been isolated for all four isoforms (data not shown). Gene families of similar complexity have been previously reported for other higher plant species (Sturm, 1999, and references cited therein). The absence of cross-hybridization allowed selective monitoring of the expression of all four invertase isoforms during the wound response in tap-root tissue.
RNA blot analysis revealed that only CWI-1 and VI-1 were wound-induced, whereas transcript amounts for CWI-2 and VI-2 remained almost unaffected; the apparent minimum induction of CWI-2 (5-fold longer film exposure time) was not consistently observed. CWI-1 mRNA was strongly induced within 10 h, whereas VI-1 mRNA increased only 24 h after wounding, reaching a maximum after about 5 d. Later, CWI-1 transcripts decreased slightly, but mRNAs of both genes remained high throughout the entire wounding period. For comparison, the expression of the wound-inducible phenylalanine ammonium lyase (PAL) gene (Sturm and Chrispeels, 1990; Fukasawa-Akada et al., 1996; Ehness et al., 1997) was also monitored. PAL mRNA was induced 3 h after wounding, reaching maximum levels 7 h later. After 5 d, PAL mRNA declined, but remained elevated compared with unwounded tissue. Formation of CWI-1 and VI-1 proteins was monitored with specific antisera (see Materials and methods). Regarding the selective wound-induction of CWI-1 and VI-1 in tap-root (Fig. 1), immunoblots with these antisera reflect the protein amounts of these isoforms. Consistent with the rapid induction of CWI-1 mRNA, CWI protein was already strongly induced after 24 h, whereas the delayed increase of VI protein correlated with the delayed induction of VI-1 mRNA (Fig. 2). Five days after wounding, the VI-1-antiserum detected an additional polypeptide of about 33 kDa, most likely representing a splitting product of VI-1. The detection of VI splitting products has been reported for many other plant species, however, it is not known whether this apparent VI instability has a physiological relevance (Sturm, 1999). Acid invertase activities in the cell wall (CWI) and the soluble fractions (VI) of wounded tap-root tissue correlated with the observed immunosignals (Fig. 3). CWI activity increased 10-fold 24 h after wounding, with a further 80% increase during the following 4 d. Conversely, VI activity increased only by 50% during the first 24 h, whereas a further 7-fold increase was detectable 4 d later. To address the contribution of CWI-1 and VI-1 to the wound-induced sucrose hydrolysis, sucrose and hexose concentrations were monitored during the first 72 h after wounding (Fig. 4). Apart from an initial transient increase of glucose after 24 h, hexose concentrations showed a strong and persistant increase only 48 h after the onset of wounding. This time-course indicates, that VI-1 is responsible for the sustained increase in hexoses during the wound response, whereas the minor and transient increase in glucose 24 h after wounding correlated with the earlier induction of CWI-1. Note, that the formation of hexoses after 48 and 72 h, respectively, is not matched by a corresponding decline in sucrose, due to about a 10% water loss of the tissue slices over the 72 h period (data not shown).
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As far as is known, this is the first comprehensive report on the roles of specific invertase isoforms during the wound response of a plant storage tissue. For sugar beet tap-root, it could be demonstrated that (i) specific wound-inducible isoforms of CWI and VI are sequentially induced, and (ii) that the observed increase of hexoses correlates with the delayed induction of VI. Earlier studies have indicated the existence of an invertase inhibitor in sugar beet (Pressey, 1968
), and invertase inhibitors may regulate CWI and VI in vivo (Krausgrill et al., 1998; Greiner et al., 1998, 1999). However, the observed correlation of wound-induced CWI-1 and VI-1 mRNAs and proteins with increased enzyme activities and hexose accumulation has clearly shown that in sugar beet tap-root wound-induced invertase activation is not the result of inhibitor inactivation but due to de novo enzyme synthesis. The delayed induction of VI-1, coinciding with a sustained and substantial increase in glucose and fructose, identifies VI-1 as the relevant isoform for wound-induced sucrose loss. The dramatic release of free hexoses, corresponding to about 70 mM hexoses 72 h after wounding, may serve three purposes, (i) to provide substrate for the wound-activated cellular metabolism, (ii) to increase the osmotic potential at the wound surface in order to reduce water loss via evaporation, and (iii) to adjust the hexose/sucrose ratio to the demands for cell division. While CWI-1 may be important for providing hexoses during the initial phase, VI-1 induction is required for the mobilization of the large vacuolar sucrose pool. Therefore, from a biotechnological point of view, attempts to reduce post-harvest sucrose losses in sugar beet should target the VI-1 isoform.
Acknowledgments
This work was supported by the KWS Saat AG, Einbeck, the Südzucker AG, Mannheim/Ochsenfurt, and the Bundesministerium für Bildung und Forschung (BMBF). We particularly acknowledge helpful discussions with Thomas Roitsch (Regensburg) and Arnd Sturm (Basel).
Notes
1 These authors contributed equally to this publication. 
2 To whom correspondence should be addressed. Fax: +49 6221 545859. E-mail: trausch{at}mail.bot.uni\|[hyphen]\|heidelberg.de
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