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Sugars regulate sucrose transporter gene expression in citrus
BBRC Biochemical and Biophysical Research Communications 306 (2003) 402–407 www.elsevier.com/locate/ybbrc
Sugars regulate sucrose transporter gene expression in citrus Chun Yao Li, Jian Xin Shi, David Weiss, and Eliezer E. Goldschmidt* The Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel Received 13 May 2003
Abstract We report the isolation and characterization of two sucrose transporter cDNAs (CitSUT1 and CitSUT2) from citrus. CitSUT1 and CitSUT2 encode putative proteins (CitSUT1 and CitSUT2) of 528 and 607 amino acids, respectively. CitSUT1 and CitSUT2 share high similarities with sucrose transporters isolated from other plants. The expression of CitSUT1 in mature leaf discs is repressed by exogenous sucrose, glucose, mannose, and the glucose analog 2-deoxyglucose but not by another glucose analog 3-Omethylglucose, indicating a hexokinase (HXK)-mediated signaling pathway. CitSUT2 expression is not affected by exogenous sugars. Whereas CitSUT1 expresses strongly in source, sugar exporting organs, CitSUT2 expresses more strongly in sink, sugar importing organs, suggesting different physiological roles for these sucrose transporters. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Citrus; Glucose; Hexokinase; Source/sink; Sugar response; Sucrose; Sucrose transporter
Assimilate partitioning is an important and highly integrated process in higher plants which involves not only transport of sugars from source to sink organs but also the regulation of genes triggered by sugars. Longdistance transport of sucrose from source to sink tissues takes place in the sieve element/companion complex (phloem) and may require apoplastic loading and unloading processes facilitated by sucrose transporters. Evidence for apoplastic loading of sucrose has been obtained from several plant species such as tobacco [1], potato [2], and Arabidopsis [3,4]. With the isolation of first sucrose transporter gene from spinach [5], various molecular cloning strategies have been employed which led to the isolation and characterization of sucrose transporter genes from a number of plant species [6,7]. Functional analyses of sucrose transporters by antisense repression [8–10] and immunolocalization [11] have helped in elucidating the role of these proteins in carbohydrate partitioning in some plants such as potato and Arabidopsis. Physiological and molecular studies have shown that sugar transport is a highly regulated process. A variety * Corresponding author. Fax: +972-8-9363731. E-mail address: [email protected] (E.E. Goldschmidt).
of genes are now known to be transcriptionally regulated by changing cellular levels of sugars. However, little is currently known about the regulatory mechanisms underlying the sugar responses [12]. Both hexokinase (HXK)-dependent and independent signaling pathways have been proposed for sugar sensing and signaling [12,13]. In the HXK-dependent signaling pathway, HXK would supposedly activate a signaling cascade through HXK-interacting proteins or affect transcription directly after nuclear translocation [12]. In HXK-independent signaling pathways, gene expression would be regulated directly by the disaccharides, sucrose or thehalose [12]. The source–sink physiology of citrus as related to fruit development and productivity has attracted great attention in the last decades at the quantitative tree level [14,15]. Alternate bearing, a common phenomenon in some citrus cultivars, involves a change in carbohydrate distribution between the heavily fruiting ÔonÕ year and non-fruiting ÔoffÕ year [16,17]. Carbohydrates accumulate in all tree organs during the ÔoffÕ year and are subsequently depleted during the ÔonÕ year. With the transition of the trees from ÔonÕ to Ôoff,Õ the function of the roots as source and sink organs is also reversed. The roots of non-fruiting ÔoffÕ year function as a strong sink
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00978-1
C.Y. Li et al. / Biochemical and Biophysical Research Communications 306 (2003) 402–407
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organ [17,18]. During the ÔonÕ year, the roots no longer serve as a sink organ. The starch previously stored in roots is reconverted into sucrose that is mobilized upwards into the developing fruit and the roots now function, during the ÔonÕ year, as a source organ. As part of a study of the molecular aspects of citrusÕ carbohydrate allocation [17], we investigated the sucrose transporters of citrus. Two sucrose transporter cDNAs have been isolated and characterized. The expression of these transporters in tree organs has been examined. One of the transporter genes, CitSUT1, is strongly suppressed by sucrose and glucose, suggesting a HXK-mediated signaling mechanism.
50 -GCC(T)GGT(A)GTTCAA(G)TTC(T)GG-30 and 50 -A(G)CCCAT CCAA(G)TCA(T)GTA(G)TC-30 were used to amplify a DNA fragment of about 800 bp by PCR. Amplified products from different tissues were recovered after agarose–gel electrophoresis and cloned into T-easy vector (Promega). To get the full-length cDNAs, 32 P-labeled fragments of CitSUT1 and CitSUT2 were used to screen a cDNA library of citrus [20]. Full-length cDNA clones were identified after screening a library of 1.0 106 pfu. Library screening was done according to the instruction manual of Stratagene. Sucrose determinations. Root samples were dried in the oven (80 °C) for 48 h and then ground into fine powder. Dry tissue samples (100 mg) were extracted four times with 80% (v/v) ethanol at 55 °C. All the extracts were collected and quantified to a final volume of 50 ml. Three tissue samples were extracted for each treatment. Sucrose was determined using anthrone, according to the method of van Handel [21].
Materials and methods
Results
Plant materials. Field-grown Murcott (a Citrus reticulata hybrid of unknown origin) sour orange (Citrus aurantium) trees, about 15 years old, revealing an absolute biennial fruit-bearing habit [17], were used. Trees were divided into two groups, trees in the ÔonÕ year (with a heavy fruit load) and trees in the ÔoffÕ year, without fruit. Mature leaf, bark (peeled from one-year-old branches) and root (5 mm in diameter) samples were harvested from ÔoffÕ and ÔonÕ trees in late autumn for sucrose content and gene expression analysis. Sugar response experiments. Five-year-old greenhouse, containergrown defruited Murcott trees were used. For CitSUT1 sugar response analysis, trees were held in the dark (28 °C) for 3 days before the experiment. Mature leaves were surface cleaned with soft paper towels and leaf discs (0.8 mm in diameter) were removed with a disc-borer. Leaf discs were immediately incubated in sugar and sugar analog solutions in Erlenmeyer flasks (ca. 140 discs per flask). Incubation was in the dark at 28 °C, with constant shaking (150 rpm). For CitSUT2 sugar response experiment, leaves were collected from trees at noon without darkening pretreatment and the incubation was under 90–120 lmol photons m2 s1 continuous white fluorescent light. After the incubation, discs were rinsed three times with water, briefly dried on paper towels, and frozen in liquid nitrogen; then stored at )80 °C. DNA extraction and Southern blot analysis. Genomic DNA was extracted from young leaves of citrus according to the method of Gawel and Jarret [19]. Twenty micrograms per lane of genomic DNA was digested with restriction enzymes and then separated by electrophoresis in 0.9% agarose gels. Membrane transfer was conducted according to Amersham Pharmacia manual with alkaline solution (0.5 M NaOH) as a transfer buffer. The DNA blot was hybridized with specific cDNA probes described below. RNA extraction and Northern blot analysis. Total RNAs were extracted with a standard SDS–phenol method. Total RNA sample (20 lg per lane) was separated by electrophoresis in 1% agarose gels containing formaldehyde and blotted to Hybond-NX nylon membrane (Amersham Pharmacia Biotech) by capillary transfer. The RNA blots were hybridized with specific cDNA probes (CitSUT1: AY098891, nt 257–942; CitSUT2: AY098894, nt 555–1410) labeled with random priming method (Boehringer–Mannheim). Hybridization was carried out at 42 °C in a solution with 10% Dextran sulfate (Na salt, MW 500,000), 1% SDS, 2 SSC, 50% formamide, and 5 DenhardtÕs solution for 16 h followed by high stringent wash (45 °C, 2 SSC, 20 min, two times; 55 °C, 0.5 SSC, 20 min, two times followed by 65 °C, 0.1 SSC, 20 min, two times). The hybridized membranes were exposed and analyzed by Phosphorimager (FUJI BAS 1000). After exposure, the membranes were stripped and re-hybridized with citrus 18S rRNA. Isolation of cDNA clones for sucrose transporter. First-strand cDNA was obtained by reverse transcription of 5 lg total RNA isolated as above from roots of ÔonÕ and ÔoffÕ trees. Degenerated primers
Isolation of sucrose transporter cDNA clones from citrus To isolate sucrose transporter cDNAs from citrus, conserved regions of known cDNA sequences from several plant species (Arabidopsis, tomato, potato, grape berry, and spinach) were used to design degenerated primers for RT-PCR. Total RNAs from citrus leaves, bark, and roots were reverse transcribed and amplified by PCR. Expected length of amplified fragment from each reaction was cloned and sequenced. Two distinct sequences, isolated from roots of ÔonÕ trees and leaves of ÔoffÕ trees, respectively, were identified. Sequence analysis revealed that both clones are homologs of sucrose transporters from other species. These two clones, designated as CitSUT1 and CitSUT2, were used to screen a citrus cDNA library generated from fruit peel [20]. Fulllength CitSUT1 and CitSUT2 were found to be 1756 and 2386 bp in length encoding putative proteins of 528 and 607 amino acids, with calculated molecular masses of 55.9 and 65 kDa, respectively. Multiple alignments with deduced protein sequences of other known sucrose transporters (Fig. 1) showed that both proteins contain 12 predicted transmembrane domains as identified in other known sucrose transporters. Both CitSUT1 and CitSUT2 shared high similarities in structure with known sucrose transporters isolated from other plant species (Fig. 1). However, the similarity between CitSUT1 and CitSUT2 is low (57.8%). A phylogenetic tree based on multiple alignments showed that CitSUT1 and CitSUT2 belong to different subfamilies of sucrose transporters (Fig. 2). Southern blot analysis was carried out to estimate the copy number of genes coding for CitSUT1 and CitSUT2 in the citrus genome (Fig. 3). Digested genomic DNAs were hybridized separately with CitSUT1 and CitSUT2 probes. Results suggested that CitSUT1 and CitSUT2 exist as single copies in the citrus genome. Fig. 3 also showed that the CitSUT1 and CitSUT2 probes used for Southern blot hybridization are specific and do not cross-hybridize with each other
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Fig. 1. Alignment of the deduced amino acid sequences of the two putative citrus sucrose transporter cDNAs (CitSUT1: AY098891 and CitSUT2: AY098894) with two other putative sucrose transporter proteins (AtSUC1, X75365 and LeSUT2, AF166498). The boxes with roman numerals indicate the location of 12 proposed membrane-spanning regions. The two highly conserved sequence motifs (CCB1 and CCB2), which were suggested to play a role in sucrose sensing, are also highlighted. The sequence alignment was performed using PILEUP from GCG package (Wisconsin Package Version 10.1, Genetics Computer Group, Madison, WI).
under the hybridization and washing conditions used in the present study. Effects of sugars on CitSUT1 and CitSUT2 expression in a leaf disc system Leaf discs were incubated with 100 mM sucrose and 100 mM glucose solutions and analyzed for CitSUT1 expression. Both sugars were found to repress CitSUT1 expression (Fig. 4A). Further experiments with different
sugar concentrations showed that 25 mM of sucrose or glucose was sufficient for maximum CitSUT1 repression (Fig. 4B). Short time (30 min) incubation with 100 mM sucrose or glucose did not result in CitSUT1 repression; 4 h or longer were required to obtain the repression effect (Fig. 4C). A repression of CitSUT1 by fructose was also observed (data not shown). To examine whether the CitSUT1 repression by sucrose and glucose is mediated by the HXK signaling pathway, we applied additional sugars and glucose analogs (Fig. 4D). While mannose
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Fig. 2. Phylogenetic relation of sucrose transporter protein sequences. The sequences used are the deduced amino acid sequences from the nucleotide sequence. AtSUC1, AtSUT2, AtSUT4, and AtSUC5 (X75365, AC004138, AF175321, and AJ252133) from Arabidopsis; CitSUT1, CitSUT2 (AY098891, AY098894) from citrus; vvsuc11, vvsuc12, and vvsuc27 (AF021808, AF021809, and AF021810) from Vitis vinifera; LeSUT1, LeSUT2, and LeSUT4 (X82275, AF166498, and AF176950) from tomato; PsSUT1 (AF109922) from Pisum sativum; ZmSUT1 (AB008464) from Zea mays; OsSUT1 (D87819) from rice; SoSUT1 (X67125) from spinach; and NtSUT1 (X82276) from tobacco. The phylogenetic tree was made with ClustalW and read by TreeView (http://taxonomy.zoology.gla.ac.uk/rod/rod.html, Version 1.6.6). The scale bar indicates a distance value of 0.1.
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Fig. 4. Response of CitSUT1 and CitSUT2 to exogenous sugar and sugar analogs in a leaf disc system. Total RNAs of 20 lg extracted from leaf discs were fractionated and analyzed. RNA gel stained with ethidium bromide was used to normalize the loading differences. (A) Leaf discs incubated in 100 mM of sucrose and glucose solutions (20 h). (B) Leaf discs incubated with different concentrations of sucrose and glucose (20 h). (C) Leaf discs incubated with 100 mM sucrose and 100 mM glucose for different times. (D,E) Leaf discs incubated with different sugar and sugar analog solutions (20 h).
The expression of CitSUT2 in response to exogenous sugar treatments was also examined. As indicated in Fig. 4E, sucrose and glucose did not repress CitSUT2 expression. Expression of CitSUT1 and CitSUT2 in citrus tree organs
Fig. 3. Southern blot analysis of CitSUT1 and CitSUT2 genes in genomic DNA. Citrus genomic DNA (20 lg per lane) was digested to completion with restriction enzymes as indicated and hybridized separately with 32 P-labeled CitSUT1 or CitSUT2 cDNA probe.
inhibited CitSUT1 expression, sorbitol and mannitol had no effect. 2-Deoxyglucose, a glucose analog that can be phosphorylated by HXK [22], strongly repressed CitSUT1 expression, whereas another glucose analog, 3O-methylglucose, which can be transported into the cell but is a poor substrate for HXK [23], did not cause any repression (Fig. 4D).
The expression of CitSUT1 and CitSUT2 was examined in leaves, bark, and roots of ÔoffÕ and ÔonÕ trees by Northern blot analysis. CitSUT1 transcript level in ÔonÕ roots was higher than in ÔoffÕ roots (Fig. 5). No difference between ÔoffÕ and ÔonÕ was found in the expression of CitSUT1 in leaves and bark. In view of the difference in CitSUT1 expression between the ÔoffÕ and ÔonÕ roots, we determined the levels of sucrose in leaves, bark, and roots of ÔoffÕ and ÔonÕ trees. Sucrose concentrations in ÔoffÕ roots were about three times higher than in ÔonÕ roots (Fig. 6). Sucrose concentrations in leaves and bark of ÔonÕ trees were only slightly lower than in ÔoffÕ trees. The CitSUT2 gene, on the other hand, revealed the same level of expression in ÔoffÕ and ÔonÕ roots (Fig. 5).
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Fig. 5. Expression of CitSUT1 and CitSUT2 in alternate bearing ÔoffÕ and ÔonÕ tree organs. An equal amount of total RNA (20 lg) isolated from leaf, bark, and root tissues were separated and analyzed by Northern blot hybridization. RNA gel stained with ethidium bromide was used to normalize the loading differences.
Fig. 6. Sucrose levels SD in root, bark, and leaf tissues of alternate bearing ÔonÕ and ÔoffÕ trees. Results are means of three independent samples.
Fig. 7. Expression of CitSUT1 and CitSUT2 during four stages of leaf development. An equal amount of total RNA (20 lg) isolated from four leaf development stages (Y1, less than half size of the mature leaf; Y2, a full size young leaf, not yet in full photosynthetic capacity; M, mature leaf; and S, senescing leaf) were separated and analyzed by Northern blot hybridization. 18S rRNA was used to normalize the loading differences.
When the expression of CitSUT1 and CitSUT2 was analyzed during four stages of leaf development (Fig. 7), expression of CitSUT1 was found to increase with leaf development, reaching its highest level in the mature (M) stage, and then decrease as the leaves approached senescence (S). The expression of CitSUT2, on the other hand, was highest in young leaves and declined as leaves turned into mature organs.
Discussion Murcott, the citrus cultivar utilized in our present study, has an extreme alternate bearing habit that turns
the ÔoffÕ and ÔonÕ cycles bi-annually [14,24]. During the non-fruiting ÔoffÕ year, roots function as a strong sink whereas during the ÔonÕ year roots function as a source organ [17,18]. In the absence of supply of sucrose from the leaves to the roots the level of sucrose in roots declines markedly (t test, P < 0:01) (Fig. 6). The differences in root function as sink–source organs and levels of sucrose in root tissues might regulate CitSUT1 expression, as demonstrated by the in vitro experiments (Fig. 4). The differences in CitSUT1 expression, showing high expression in ÔonÕ roots and low expression in ÔoffÕ roots (Fig. 5), suggest a possible role of CitSUT1 in loading sucrose into phloem for export to the canopy. A relationship between sink–source activity and CitSUT1 expression was also found in leaves. Young developing citrus leaves are supposedly a strong sink [25], while the mature, fully photosynthetic leaves, which produce and export sucrose, are a source organ. Its high expression in mature leaves suggests once more a role for CitSUT1 in phloem loading. In contrast, the high expression of CitSUT2 in young leaves and low expression in mature leaves suggest that this gene is related to sink activity and is probably not involved in phloem loading. It has been found that sugars play an important role in plant metabolic and developmental processes [12,13,26,27]. Many genes have been identified to be sugar-regulated [26]. Genes were found to use different mechanisms for sugar sensing and signaling [12,13]. Sucrose transporter was reported to be negatively regulated by sucrose but not by glucose in sugar beet [28,29]. To the best of our knowledge, glucose has not been reported heretofore as a repressor of the expression of sucrose transporters. In the present study, we found that the expression of CitSUT1 in citrus leaf discs is repressed by both sucrose and glucose (Fig. 4) while the expression of CitSUT2 is insensitive to sugar treatments (Fig. 4E). Results from glucose analog applications and transgenic plants with HXK over-expression or suppression [22,30,31] suggest that HXK is a glucose sensor in plants. Substrates of HXK, including 2-deoxyglucose and mannose, which are phosphorylated by HXK but are not or poorly further metabolized, caused repression of photosynthetic gene expression at low concentrations (1–10 mM) in maize mesophyll protoplasts [22], while 3O-methylglucose, which can enter the cell but is not or only a poor substrate for HXK, could not trigger the same kind of repression [22,23]. We proposed, therefore, that the observed glucose repression of CitSUT1 expression operates probably via a HXK-mediated signaling pathway. Sucrose may repress the expression of CitSUT1 indirectly, through the hexoses formed during sucrose breakdown, or directly via a HXK-independent signaling pathway [28]. An indirect mechanism apparently operates in sucrose repression of photosynthetic genes,
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which can be mimicked by a lower concentration of hexoses [31,32]. In our leaf disc system, the non-metabolizable sucrose analogs, palatinose and turanose [33], did not repress the expression of CitSUT1 (data not shown). This may be taken as an indication that the observed repression of CitSUT1 by sucrose in our system operates indirectly, via hexoses. Acknowledgments Thanks are due to Dr. D. Granot (The Agricultural Research Organization, Bet Dagan, Israel) for critical reading of the manuscript. The financial support by the Israel Citrus Marketing Board is gratefully acknowledged.
References [1] U. Sonnewald, M. Brauer, A. von Schaewen, et al., Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast—a powerful tool for studying sucrose metabolism and sink source interactions, Plant J. 1 (1991) 95–106. [2] D. Heineke, U. Sonnewald, D. Bussis, et al., Apoplastic expression of yeast-derived invertase in potato—effects on photosynthesis, leaf solute composition, water relations, and tuber composition, Plant Physiol. 100 (1992) 301–308. [3] J.R. Gottwald, P.J. Krysan, J.C. Young, et al., Genetic evidence for the in planta role of phloem-specific plasma membrane sucrose transporters, Proc. Natl. Acad. Sci. USA 97 (2000) 13979–13984. [4] A. von Schaewen, M. Stitt, R. Schmidt, et al., Expression of a yeast-derived invertase in the cell-wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants, EMBO J. 9 (1990) 3033–3044. [5] J.W. Riesmeier, L. Willmitzer, W.B. Frommer, Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast, EMBO J. 11 (1992) 4705–4713. [6] S. Lalonde, E. Boles, H. Hellmann, et al., The dual function of sugar carriers: transport and sugar sensing, Plant Cell 11 (1999) 707–726. [7] R. Lemoine, Sucrose transporters in plants: update on function and structure, BBA-Biomembranes 1465 (2000) 246–262. [8] C. Kuhn, W.P. Quick, A. Schulz, et al., Companion cell-specific inhibition of the potato sucrose transporter SUT1, Plant Cell Environ. 19 (1996) 1115–1123. [9] R. Lemoine, C. Kuhn, N. Thiele, et al., Antisense inhibition of the sucrose transporter: effects on amount of carrier and sucrose transport activity, Plant Cell Environ. 19 (1996) 1124–1131. [10] J.W. Riesmeier, L. Willmitzer, W.B. Frommer, Antisense repression of the sucrose transporter affects assimilate partitioning in transgenic potato plants, EMBO J. 13 (1994) 1–7. [11] C. Kuhn, V. Franceschi, A. Schulz, et al., Macromolecular trafficking indicated by localization and turnover of sucrose transporters in enucleate sieve elements, Science 275 (1997) 1298–1300. [12] F. Rolland, B. Moore, J. Sheen, Sugar sensing and signaling in plants, Plant Cell 14 (2002) s185–s205.
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[13] S. Smeekens, Sugar-induced signal transduction in plants, Annu. Rev. Plant Mol. Biol. Plant Physiol. 51 (2000) 49–81. [14] E.E. Goldschmidt, Carbohydrate supply as a critical factor for citrus fruit development and productivity, Hortscience 34 (1999) 1020–1024. [15] E.E. Goldschmidt, K.E. Koch, Citrus, in: E. Zamski, A.A. Schaffer (Eds.), Photoassimilate Distribution in Plants and Crops, Marcel Dekker, New York, 1996, pp. 797–823. [16] S.P. Monselis, E.E. Goldschmidt, Alternate bearing in fruit trees, Hortic. Rev. 4 (1982) 128–173. [17] C.Y. Li, D.Weiss, E.E. Goldschmidt, Effects of carbohydrate starvation on gene expression in citrus roots, Planta 217 (2003) 11–20. Available from:. [18] E.E. Goldschmidt, A. Golomb, The carbohydrate balance of alternate-bearing citrus trees and the significance of reserves for flowering and fruiting, J. Am. Soc. Hortic. Sci. 107 (1982) 206–208. [19] N.J. Gawel, R.L. Jarret, A modified CTAB DNA extraction procedure for Musa and Ipomoea, Plant Mol. Biol. Rep. 9 (1991) 262–266. [20] D. Jacob-Wilk, D. Holland, E.E. Goldshcmidt, et al., Chlorophyll breakdown by chlorophyllase: isolation and functional expression of the Chlase1 gene from ethylene-treated Citrus fruit and its regulation during development, Plant J. 20 (1999) 653–661. [21] E. van Handel, Direct microdetermination of sucrose, Anal. Biochem. 22 (1968) 280–283. [22] J.C. Jang, J. Sheen, Sugar sensing in higher plants, Plant Cell 6 (1994) 1665–1679. [23] S. Cortes, M. Gromova, A. Evrard, et al., In plants, 3-Omethylglucose is phosphorylated by hexokinase but not perceived as a sugar, Plant Physiol. 131 (2003) 824–837. [24] P. Smith, Collapse of ÔMurcottÕ tangerine trees, J. Am. Soc. Hortic. Sci. 101 (1976) 23–25. [25] A.A. Schaffer, O. Sagee, E.E. Goldschmidt, et al., Invertase and sucrose synthase activity, carbohydrate status and endogenous IAA levels during Citrus leaf development, Physiol. Plant. 69 (1987) 151–155. [26] K.E. Koch, Carbohydrate modulated gene expression in pants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47 (1996) 509–540. [27] S.I. Gibson, Plant sugar-response pathways. Part of a complex regulatory web, Plant Physiol. 124 (2000) 1532–1539. [28] T.J. Chiou, D.R. Bush, Sucrose is a signal molecule in assimilate partitioning, Proc. Natl. Acad. Sci. USA 95 (1998) 4784–4788. [29] M.W. Vaughn, G.N. Harrington, D.R. Bush, Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem, Proc. Natl. Acad. Sci. USA 99 (2002) 10876–10880. [30] N. Dai, A.A. Schaffer, M. Petreikov, et al., Overexpression of Arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence, Plant Cell 11 (1999) 1253–1266. [31] J.C. Jang, P. Leon, L. Zhou, et al., Hexokinase as a sugar sensor in higher plants, Plant Cell 9 (1997) 5–19. [32] L. Zhou, J.C. Jang, T.L. Jones, et al., Glucose and ethylene signal transduction crosstalk revealed by an Arabidopsis glucoseinsensitive mutant, Proc. Natl. Acad. Sci. USA 95 (1998) 10294–10299. [33] A.K. Sinha, M.G. Holmann, U. Romer, et al., Metabolizable and non-metabolizable sugars activate different signal transduction pathways in tomato, Plant Physiol. 128 (2002) 1480–1489.
Sugars regulate sucrose transporter gene expression in citrus Chun Yao Li, Jian Xin Shi, David Weiss, and Eliezer E. Goldschmidt* The Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel Received 13 May 2003
Abstract We report the isolation and characterization of two sucrose transporter cDNAs (CitSUT1 and CitSUT2) from citrus. CitSUT1 and CitSUT2 encode putative proteins (CitSUT1 and CitSUT2) of 528 and 607 amino acids, respectively. CitSUT1 and CitSUT2 share high similarities with sucrose transporters isolated from other plants. The expression of CitSUT1 in mature leaf discs is repressed by exogenous sucrose, glucose, mannose, and the glucose analog 2-deoxyglucose but not by another glucose analog 3-Omethylglucose, indicating a hexokinase (HXK)-mediated signaling pathway. CitSUT2 expression is not affected by exogenous sugars. Whereas CitSUT1 expresses strongly in source, sugar exporting organs, CitSUT2 expresses more strongly in sink, sugar importing organs, suggesting different physiological roles for these sucrose transporters. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Citrus; Glucose; Hexokinase; Source/sink; Sugar response; Sucrose; Sucrose transporter
Assimilate partitioning is an important and highly integrated process in higher plants which involves not only transport of sugars from source to sink organs but also the regulation of genes triggered by sugars. Longdistance transport of sucrose from source to sink tissues takes place in the sieve element/companion complex (phloem) and may require apoplastic loading and unloading processes facilitated by sucrose transporters. Evidence for apoplastic loading of sucrose has been obtained from several plant species such as tobacco [1], potato [2], and Arabidopsis [3,4]. With the isolation of first sucrose transporter gene from spinach [5], various molecular cloning strategies have been employed which led to the isolation and characterization of sucrose transporter genes from a number of plant species [6,7]. Functional analyses of sucrose transporters by antisense repression [8–10] and immunolocalization [11] have helped in elucidating the role of these proteins in carbohydrate partitioning in some plants such as potato and Arabidopsis. Physiological and molecular studies have shown that sugar transport is a highly regulated process. A variety * Corresponding author. Fax: +972-8-9363731. E-mail address: [email protected] (E.E. Goldschmidt).
of genes are now known to be transcriptionally regulated by changing cellular levels of sugars. However, little is currently known about the regulatory mechanisms underlying the sugar responses [12]. Both hexokinase (HXK)-dependent and independent signaling pathways have been proposed for sugar sensing and signaling [12,13]. In the HXK-dependent signaling pathway, HXK would supposedly activate a signaling cascade through HXK-interacting proteins or affect transcription directly after nuclear translocation [12]. In HXK-independent signaling pathways, gene expression would be regulated directly by the disaccharides, sucrose or thehalose [12]. The source–sink physiology of citrus as related to fruit development and productivity has attracted great attention in the last decades at the quantitative tree level [14,15]. Alternate bearing, a common phenomenon in some citrus cultivars, involves a change in carbohydrate distribution between the heavily fruiting ÔonÕ year and non-fruiting ÔoffÕ year [16,17]. Carbohydrates accumulate in all tree organs during the ÔoffÕ year and are subsequently depleted during the ÔonÕ year. With the transition of the trees from ÔonÕ to Ôoff,Õ the function of the roots as source and sink organs is also reversed. The roots of non-fruiting ÔoffÕ year function as a strong sink
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00978-1
C.Y. Li et al. / Biochemical and Biophysical Research Communications 306 (2003) 402–407
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organ [17,18]. During the ÔonÕ year, the roots no longer serve as a sink organ. The starch previously stored in roots is reconverted into sucrose that is mobilized upwards into the developing fruit and the roots now function, during the ÔonÕ year, as a source organ. As part of a study of the molecular aspects of citrusÕ carbohydrate allocation [17], we investigated the sucrose transporters of citrus. Two sucrose transporter cDNAs have been isolated and characterized. The expression of these transporters in tree organs has been examined. One of the transporter genes, CitSUT1, is strongly suppressed by sucrose and glucose, suggesting a HXK-mediated signaling mechanism.
50 -GCC(T)GGT(A)GTTCAA(G)TTC(T)GG-30 and 50 -A(G)CCCAT CCAA(G)TCA(T)GTA(G)TC-30 were used to amplify a DNA fragment of about 800 bp by PCR. Amplified products from different tissues were recovered after agarose–gel electrophoresis and cloned into T-easy vector (Promega). To get the full-length cDNAs, 32 P-labeled fragments of CitSUT1 and CitSUT2 were used to screen a cDNA library of citrus [20]. Full-length cDNA clones were identified after screening a library of 1.0 106 pfu. Library screening was done according to the instruction manual of Stratagene. Sucrose determinations. Root samples were dried in the oven (80 °C) for 48 h and then ground into fine powder. Dry tissue samples (100 mg) were extracted four times with 80% (v/v) ethanol at 55 °C. All the extracts were collected and quantified to a final volume of 50 ml. Three tissue samples were extracted for each treatment. Sucrose was determined using anthrone, according to the method of van Handel [21].
Materials and methods
Results
Plant materials. Field-grown Murcott (a Citrus reticulata hybrid of unknown origin) sour orange (Citrus aurantium) trees, about 15 years old, revealing an absolute biennial fruit-bearing habit [17], were used. Trees were divided into two groups, trees in the ÔonÕ year (with a heavy fruit load) and trees in the ÔoffÕ year, without fruit. Mature leaf, bark (peeled from one-year-old branches) and root (5 mm in diameter) samples were harvested from ÔoffÕ and ÔonÕ trees in late autumn for sucrose content and gene expression analysis. Sugar response experiments. Five-year-old greenhouse, containergrown defruited Murcott trees were used. For CitSUT1 sugar response analysis, trees were held in the dark (28 °C) for 3 days before the experiment. Mature leaves were surface cleaned with soft paper towels and leaf discs (0.8 mm in diameter) were removed with a disc-borer. Leaf discs were immediately incubated in sugar and sugar analog solutions in Erlenmeyer flasks (ca. 140 discs per flask). Incubation was in the dark at 28 °C, with constant shaking (150 rpm). For CitSUT2 sugar response experiment, leaves were collected from trees at noon without darkening pretreatment and the incubation was under 90–120 lmol photons m2 s1 continuous white fluorescent light. After the incubation, discs were rinsed three times with water, briefly dried on paper towels, and frozen in liquid nitrogen; then stored at )80 °C. DNA extraction and Southern blot analysis. Genomic DNA was extracted from young leaves of citrus according to the method of Gawel and Jarret [19]. Twenty micrograms per lane of genomic DNA was digested with restriction enzymes and then separated by electrophoresis in 0.9% agarose gels. Membrane transfer was conducted according to Amersham Pharmacia manual with alkaline solution (0.5 M NaOH) as a transfer buffer. The DNA blot was hybridized with specific cDNA probes described below. RNA extraction and Northern blot analysis. Total RNAs were extracted with a standard SDS–phenol method. Total RNA sample (20 lg per lane) was separated by electrophoresis in 1% agarose gels containing formaldehyde and blotted to Hybond-NX nylon membrane (Amersham Pharmacia Biotech) by capillary transfer. The RNA blots were hybridized with specific cDNA probes (CitSUT1: AY098891, nt 257–942; CitSUT2: AY098894, nt 555–1410) labeled with random priming method (Boehringer–Mannheim). Hybridization was carried out at 42 °C in a solution with 10% Dextran sulfate (Na salt, MW 500,000), 1% SDS, 2 SSC, 50% formamide, and 5 DenhardtÕs solution for 16 h followed by high stringent wash (45 °C, 2 SSC, 20 min, two times; 55 °C, 0.5 SSC, 20 min, two times followed by 65 °C, 0.1 SSC, 20 min, two times). The hybridized membranes were exposed and analyzed by Phosphorimager (FUJI BAS 1000). After exposure, the membranes were stripped and re-hybridized with citrus 18S rRNA. Isolation of cDNA clones for sucrose transporter. First-strand cDNA was obtained by reverse transcription of 5 lg total RNA isolated as above from roots of ÔonÕ and ÔoffÕ trees. Degenerated primers
Isolation of sucrose transporter cDNA clones from citrus To isolate sucrose transporter cDNAs from citrus, conserved regions of known cDNA sequences from several plant species (Arabidopsis, tomato, potato, grape berry, and spinach) were used to design degenerated primers for RT-PCR. Total RNAs from citrus leaves, bark, and roots were reverse transcribed and amplified by PCR. Expected length of amplified fragment from each reaction was cloned and sequenced. Two distinct sequences, isolated from roots of ÔonÕ trees and leaves of ÔoffÕ trees, respectively, were identified. Sequence analysis revealed that both clones are homologs of sucrose transporters from other species. These two clones, designated as CitSUT1 and CitSUT2, were used to screen a citrus cDNA library generated from fruit peel [20]. Fulllength CitSUT1 and CitSUT2 were found to be 1756 and 2386 bp in length encoding putative proteins of 528 and 607 amino acids, with calculated molecular masses of 55.9 and 65 kDa, respectively. Multiple alignments with deduced protein sequences of other known sucrose transporters (Fig. 1) showed that both proteins contain 12 predicted transmembrane domains as identified in other known sucrose transporters. Both CitSUT1 and CitSUT2 shared high similarities in structure with known sucrose transporters isolated from other plant species (Fig. 1). However, the similarity between CitSUT1 and CitSUT2 is low (57.8%). A phylogenetic tree based on multiple alignments showed that CitSUT1 and CitSUT2 belong to different subfamilies of sucrose transporters (Fig. 2). Southern blot analysis was carried out to estimate the copy number of genes coding for CitSUT1 and CitSUT2 in the citrus genome (Fig. 3). Digested genomic DNAs were hybridized separately with CitSUT1 and CitSUT2 probes. Results suggested that CitSUT1 and CitSUT2 exist as single copies in the citrus genome. Fig. 3 also showed that the CitSUT1 and CitSUT2 probes used for Southern blot hybridization are specific and do not cross-hybridize with each other
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Fig. 1. Alignment of the deduced amino acid sequences of the two putative citrus sucrose transporter cDNAs (CitSUT1: AY098891 and CitSUT2: AY098894) with two other putative sucrose transporter proteins (AtSUC1, X75365 and LeSUT2, AF166498). The boxes with roman numerals indicate the location of 12 proposed membrane-spanning regions. The two highly conserved sequence motifs (CCB1 and CCB2), which were suggested to play a role in sucrose sensing, are also highlighted. The sequence alignment was performed using PILEUP from GCG package (Wisconsin Package Version 10.1, Genetics Computer Group, Madison, WI).
under the hybridization and washing conditions used in the present study. Effects of sugars on CitSUT1 and CitSUT2 expression in a leaf disc system Leaf discs were incubated with 100 mM sucrose and 100 mM glucose solutions and analyzed for CitSUT1 expression. Both sugars were found to repress CitSUT1 expression (Fig. 4A). Further experiments with different
sugar concentrations showed that 25 mM of sucrose or glucose was sufficient for maximum CitSUT1 repression (Fig. 4B). Short time (30 min) incubation with 100 mM sucrose or glucose did not result in CitSUT1 repression; 4 h or longer were required to obtain the repression effect (Fig. 4C). A repression of CitSUT1 by fructose was also observed (data not shown). To examine whether the CitSUT1 repression by sucrose and glucose is mediated by the HXK signaling pathway, we applied additional sugars and glucose analogs (Fig. 4D). While mannose
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Fig. 2. Phylogenetic relation of sucrose transporter protein sequences. The sequences used are the deduced amino acid sequences from the nucleotide sequence. AtSUC1, AtSUT2, AtSUT4, and AtSUC5 (X75365, AC004138, AF175321, and AJ252133) from Arabidopsis; CitSUT1, CitSUT2 (AY098891, AY098894) from citrus; vvsuc11, vvsuc12, and vvsuc27 (AF021808, AF021809, and AF021810) from Vitis vinifera; LeSUT1, LeSUT2, and LeSUT4 (X82275, AF166498, and AF176950) from tomato; PsSUT1 (AF109922) from Pisum sativum; ZmSUT1 (AB008464) from Zea mays; OsSUT1 (D87819) from rice; SoSUT1 (X67125) from spinach; and NtSUT1 (X82276) from tobacco. The phylogenetic tree was made with ClustalW and read by TreeView (http://taxonomy.zoology.gla.ac.uk/rod/rod.html, Version 1.6.6). The scale bar indicates a distance value of 0.1.
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Fig. 4. Response of CitSUT1 and CitSUT2 to exogenous sugar and sugar analogs in a leaf disc system. Total RNAs of 20 lg extracted from leaf discs were fractionated and analyzed. RNA gel stained with ethidium bromide was used to normalize the loading differences. (A) Leaf discs incubated in 100 mM of sucrose and glucose solutions (20 h). (B) Leaf discs incubated with different concentrations of sucrose and glucose (20 h). (C) Leaf discs incubated with 100 mM sucrose and 100 mM glucose for different times. (D,E) Leaf discs incubated with different sugar and sugar analog solutions (20 h).
The expression of CitSUT2 in response to exogenous sugar treatments was also examined. As indicated in Fig. 4E, sucrose and glucose did not repress CitSUT2 expression. Expression of CitSUT1 and CitSUT2 in citrus tree organs
Fig. 3. Southern blot analysis of CitSUT1 and CitSUT2 genes in genomic DNA. Citrus genomic DNA (20 lg per lane) was digested to completion with restriction enzymes as indicated and hybridized separately with 32 P-labeled CitSUT1 or CitSUT2 cDNA probe.
inhibited CitSUT1 expression, sorbitol and mannitol had no effect. 2-Deoxyglucose, a glucose analog that can be phosphorylated by HXK [22], strongly repressed CitSUT1 expression, whereas another glucose analog, 3O-methylglucose, which can be transported into the cell but is a poor substrate for HXK [23], did not cause any repression (Fig. 4D).
The expression of CitSUT1 and CitSUT2 was examined in leaves, bark, and roots of ÔoffÕ and ÔonÕ trees by Northern blot analysis. CitSUT1 transcript level in ÔonÕ roots was higher than in ÔoffÕ roots (Fig. 5). No difference between ÔoffÕ and ÔonÕ was found in the expression of CitSUT1 in leaves and bark. In view of the difference in CitSUT1 expression between the ÔoffÕ and ÔonÕ roots, we determined the levels of sucrose in leaves, bark, and roots of ÔoffÕ and ÔonÕ trees. Sucrose concentrations in ÔoffÕ roots were about three times higher than in ÔonÕ roots (Fig. 6). Sucrose concentrations in leaves and bark of ÔonÕ trees were only slightly lower than in ÔoffÕ trees. The CitSUT2 gene, on the other hand, revealed the same level of expression in ÔoffÕ and ÔonÕ roots (Fig. 5).
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Fig. 5. Expression of CitSUT1 and CitSUT2 in alternate bearing ÔoffÕ and ÔonÕ tree organs. An equal amount of total RNA (20 lg) isolated from leaf, bark, and root tissues were separated and analyzed by Northern blot hybridization. RNA gel stained with ethidium bromide was used to normalize the loading differences.
Fig. 6. Sucrose levels SD in root, bark, and leaf tissues of alternate bearing ÔonÕ and ÔoffÕ trees. Results are means of three independent samples.
Fig. 7. Expression of CitSUT1 and CitSUT2 during four stages of leaf development. An equal amount of total RNA (20 lg) isolated from four leaf development stages (Y1, less than half size of the mature leaf; Y2, a full size young leaf, not yet in full photosynthetic capacity; M, mature leaf; and S, senescing leaf) were separated and analyzed by Northern blot hybridization. 18S rRNA was used to normalize the loading differences.
When the expression of CitSUT1 and CitSUT2 was analyzed during four stages of leaf development (Fig. 7), expression of CitSUT1 was found to increase with leaf development, reaching its highest level in the mature (M) stage, and then decrease as the leaves approached senescence (S). The expression of CitSUT2, on the other hand, was highest in young leaves and declined as leaves turned into mature organs.
Discussion Murcott, the citrus cultivar utilized in our present study, has an extreme alternate bearing habit that turns
the ÔoffÕ and ÔonÕ cycles bi-annually [14,24]. During the non-fruiting ÔoffÕ year, roots function as a strong sink whereas during the ÔonÕ year roots function as a source organ [17,18]. In the absence of supply of sucrose from the leaves to the roots the level of sucrose in roots declines markedly (t test, P < 0:01) (Fig. 6). The differences in root function as sink–source organs and levels of sucrose in root tissues might regulate CitSUT1 expression, as demonstrated by the in vitro experiments (Fig. 4). The differences in CitSUT1 expression, showing high expression in ÔonÕ roots and low expression in ÔoffÕ roots (Fig. 5), suggest a possible role of CitSUT1 in loading sucrose into phloem for export to the canopy. A relationship between sink–source activity and CitSUT1 expression was also found in leaves. Young developing citrus leaves are supposedly a strong sink [25], while the mature, fully photosynthetic leaves, which produce and export sucrose, are a source organ. Its high expression in mature leaves suggests once more a role for CitSUT1 in phloem loading. In contrast, the high expression of CitSUT2 in young leaves and low expression in mature leaves suggest that this gene is related to sink activity and is probably not involved in phloem loading. It has been found that sugars play an important role in plant metabolic and developmental processes [12,13,26,27]. Many genes have been identified to be sugar-regulated [26]. Genes were found to use different mechanisms for sugar sensing and signaling [12,13]. Sucrose transporter was reported to be negatively regulated by sucrose but not by glucose in sugar beet [28,29]. To the best of our knowledge, glucose has not been reported heretofore as a repressor of the expression of sucrose transporters. In the present study, we found that the expression of CitSUT1 in citrus leaf discs is repressed by both sucrose and glucose (Fig. 4) while the expression of CitSUT2 is insensitive to sugar treatments (Fig. 4E). Results from glucose analog applications and transgenic plants with HXK over-expression or suppression [22,30,31] suggest that HXK is a glucose sensor in plants. Substrates of HXK, including 2-deoxyglucose and mannose, which are phosphorylated by HXK but are not or poorly further metabolized, caused repression of photosynthetic gene expression at low concentrations (1–10 mM) in maize mesophyll protoplasts [22], while 3O-methylglucose, which can enter the cell but is not or only a poor substrate for HXK, could not trigger the same kind of repression [22,23]. We proposed, therefore, that the observed glucose repression of CitSUT1 expression operates probably via a HXK-mediated signaling pathway. Sucrose may repress the expression of CitSUT1 indirectly, through the hexoses formed during sucrose breakdown, or directly via a HXK-independent signaling pathway [28]. An indirect mechanism apparently operates in sucrose repression of photosynthetic genes,
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which can be mimicked by a lower concentration of hexoses [31,32]. In our leaf disc system, the non-metabolizable sucrose analogs, palatinose and turanose [33], did not repress the expression of CitSUT1 (data not shown). This may be taken as an indication that the observed repression of CitSUT1 by sucrose in our system operates indirectly, via hexoses. Acknowledgments Thanks are due to Dr. D. Granot (The Agricultural Research Organization, Bet Dagan, Israel) for critical reading of the manuscript. The financial support by the Israel Citrus Marketing Board is gratefully acknowledged.
References [1] U. Sonnewald, M. Brauer, A. von Schaewen, et al., Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast—a powerful tool for studying sucrose metabolism and sink source interactions, Plant J. 1 (1991) 95–106. [2] D. Heineke, U. Sonnewald, D. Bussis, et al., Apoplastic expression of yeast-derived invertase in potato—effects on photosynthesis, leaf solute composition, water relations, and tuber composition, Plant Physiol. 100 (1992) 301–308. [3] J.R. Gottwald, P.J. Krysan, J.C. Young, et al., Genetic evidence for the in planta role of phloem-specific plasma membrane sucrose transporters, Proc. Natl. Acad. Sci. USA 97 (2000) 13979–13984. [4] A. von Schaewen, M. Stitt, R. Schmidt, et al., Expression of a yeast-derived invertase in the cell-wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants, EMBO J. 9 (1990) 3033–3044. [5] J.W. Riesmeier, L. Willmitzer, W.B. Frommer, Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast, EMBO J. 11 (1992) 4705–4713. [6] S. Lalonde, E. Boles, H. Hellmann, et al., The dual function of sugar carriers: transport and sugar sensing, Plant Cell 11 (1999) 707–726. [7] R. Lemoine, Sucrose transporters in plants: update on function and structure, BBA-Biomembranes 1465 (2000) 246–262. [8] C. Kuhn, W.P. Quick, A. Schulz, et al., Companion cell-specific inhibition of the potato sucrose transporter SUT1, Plant Cell Environ. 19 (1996) 1115–1123. [9] R. Lemoine, C. Kuhn, N. Thiele, et al., Antisense inhibition of the sucrose transporter: effects on amount of carrier and sucrose transport activity, Plant Cell Environ. 19 (1996) 1124–1131. [10] J.W. Riesmeier, L. Willmitzer, W.B. Frommer, Antisense repression of the sucrose transporter affects assimilate partitioning in transgenic potato plants, EMBO J. 13 (1994) 1–7. [11] C. Kuhn, V. Franceschi, A. Schulz, et al., Macromolecular trafficking indicated by localization and turnover of sucrose transporters in enucleate sieve elements, Science 275 (1997) 1298–1300. [12] F. Rolland, B. Moore, J. Sheen, Sugar sensing and signaling in plants, Plant Cell 14 (2002) s185–s205.
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[13] S. Smeekens, Sugar-induced signal transduction in plants, Annu. Rev. Plant Mol. Biol. Plant Physiol. 51 (2000) 49–81. [14] E.E. Goldschmidt, Carbohydrate supply as a critical factor for citrus fruit development and productivity, Hortscience 34 (1999) 1020–1024. [15] E.E. Goldschmidt, K.E. Koch, Citrus, in: E. Zamski, A.A. Schaffer (Eds.), Photoassimilate Distribution in Plants and Crops, Marcel Dekker, New York, 1996, pp. 797–823. [16] S.P. Monselis, E.E. Goldschmidt, Alternate bearing in fruit trees, Hortic. Rev. 4 (1982) 128–173. [17] C.Y. Li, D.Weiss, E.E. Goldschmidt, Effects of carbohydrate starvation on gene expression in citrus roots, Planta 217 (2003) 11–20. Available from: