Plant Cell, Vol. 11, 1253-1266, July 1999, Copyright © 1999, American Society of Plant Physiologists
Overexpression of Arabidopsis Hexokinase in Tomato Plants Inhibits Growth, Reduces Photosynthesis, and Induces Rapid Senescence
Nir Daia,
Arthur Schaffera,
Marina Petreikova,
Yosepha Shahakb,
Yuri Gillerb,
Kira Ratnerb,
Alex Levinec, and
David Granota
a Institute of Field and Garden Crops, Agricultural Research Organization, Volcani Center, Bet Dagan 50250, Israel
b Institute of Horticulture, Agricultural Research Organization, Volcani Center, Bet Dagan 50250, Israel
c Department of Plant Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel
Correspondence to:
David Granot, granot{at}agri.huji.ac.il (E-mail), 972-3-9669642 (fax)
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ABSTRACT |
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Sugars are key regulatory molecules that affect diverse processes in higher plants. Hexokinase is the first enzyme in hexose metabolism and may be a sugar sensor that mediates sugar regulation. We present evidence that hexokinase is involved in sensing endogenous levels of sugars in photosynthetic tissues and that it participates in the regulation of senescence, photosynthesis, and growth in seedlings as well as in mature plants. Transgenic tomato plants overexpressing the Arabidopsis hexokinase-encoding gene AtHXK1 were produced. Independent transgenic plants carrying single copies of AtHXK1 were characterized by growth inhibition, the degree of which was found to correlate directly to the expression and activity of AtHXK1. Reciprocal grafting experiments suggested that the inhibitory effect occurred when AtHXK1 was expressed in photosynthetic tissues. Accordingly, plants with increased AtHXK1 activity had reduced chlorophyll content in their leaves, reduced photosynthesis rates, and reduced photochemical quantum efficiency of photosystem II reaction centers compared with plants without increased AtHXK1 activity. In addition, the transgenic plants underwent rapid senescence, suggesting that hexokinase is also involved in senescence regulation. Fruit weight, starch content in young fruits, and total soluble solids in mature fruits were also reduced in the transgenic plants. The results indicate that endogenous hexokinase activity is not rate limiting for growth; rather, they support the role of hexokinase as a regulatory enzyme in photosynthetic tissues, in which it regulates photosynthesis, growth, and senescence.
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INTRODUCTION |
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Sugars are central compounds in nature that serve as essential metabolic nutrients and structural components for most organisms. They are also major regulatory molecules that control gene expression, metabolism, physiology, cell cycle, and development in prokaryotes and eukaryotes (Newgard and McGarry 1995 
; Ronne 1995 ; Saier et al. 1995 ). In plants, it has been shown that sugars regulate the expression of a broad spectrum of genes involved in many essential processes (e.g., Sheen 1990 ; Thomas and Rodriguez 1994 ; Stitt and Sonnewald 1995 ; Graham 1996 ; Koch 1996 ; Smeekens 1998 ). Furthermore, sugars affect developmental and metabolic processes throughout the life cycle of the plant. These processes include germination, growth, flowering, senescence, sugar metabolism, and photosynthesis (von Schaewen et al. 1990 ; Dickinson et al. 1991 ; Stitt et al. 1991 ; Goldschmidt and Huber 1992 ; Huber and Hanson 1992 ; Bernier et al. 1993 ; Ding et al. 1993 ; Yang et al. 1993 ; Thomas and Rodriguez 1994 ; Dangl et al. 1995 ; Kovtun and Daie 1995 ; Jang and Sheen 1997 ; Perata et al. 1997 ; Prata et al. 1997 ; Smeekens and Rook 1997 ; Wingler et al. 1998 ).
Photosynthesis is regulated by sugar levels, and this regulation overrides that of light, tissue type, and developmental stage (Sheen 1990 ; von Schaewen et al. 1990 ; Krapp et al. 1993 ). Increased concentrations of externally supplemented sugar or internal modification of sugar content inhibits photosynthesis and causes stunted growth of various plants (von Schaewen et al. 1990 ; Dickinson et al. 1991 ; Sonnewald et al. 1991 , Sonnewald et al. 1993 ; Goldschmidt and Huber 1992 ; Jones et al. 1996 ; Jang et al. 1997 ). In addition, many species exhibit a progressive decline in the rate of photosynthesis after full leaf expansion, and it has been suggested that photosynthesis is feedback regulated by the accumulation of carbohydrates in source leaves (Hensel et al. 1993 ; Jiang et al. 1993 ; Wingler et al. 1998 ). Senescence is also thought to be regulated by sugars. It has been hypothesized that leaf senescence is initiated when the photosynthetic rate drops below a certain threshold. At this threshold, the leaf no longer contributes fixed carbon to the rest of the plant (Ding et al. 1993 ; Hensel et al. 1993 ; King et al. 1995 ; Gan and Amasino 1997 ; Wingler et al. 1998 ).
The mechanisms by which higher plants recognize and respond to sugars are largely unknown. Recently, it was demonstrated that hexokinase, which catalyzes sugar phosphorylation, the first step of hexose metabolism, is involved in glucose sensing (Jang and Sheen 1994 ; Jang et al. 1997 ; Perata et al. 1997 ; Prata et al. 1997 ). Sugar-repressible, photosynthetic, and metabolic genes have been used to show that phosphorylation of sugars by hexokinase is critical for sugar signaling. For example, only sugars that can be phosphorylated by hexokinase repressed expression of photosynthetic genes, such as chlorophyll a/b binding protein (CAB1) (Jang and Sheen 1994 ). Furthermore, steps in sugar metabolism beyond the phosphorylation of glucose are not required to elicit sugar-mediated regulation of such photosynthetic genes (Jang and Sheen 1994 ).
Further evidence for the involvement of hexokinase in the repression of photosynthetic genes was recently presented by Jang et al. 1997 . Using transgenic Arabidopsis seedlings expressing sense and antisense Arabidopsis hexokinase (AtHXK1), they analyzed the role of hexokinase in whole plants. Transgenic seedlings overexpressing AtHXK1 were reported to be hypersensitive to increasing concentrations of exogenous glucose (2 to 6%), with reduced hypocotyl elongation, low expression of photosynthetic genes, and repressed greening of the cotyledons. Transgenic seedlings in which the expression of hexokinase was reduced by using an antisense approach showed a markedly reduced ability to sense the presence of exogenous glucose, and they displayed accelerated hypocotyl elongation and enhanced expression of photosynthetic genes. In a separate experiment, in which a heterologous hexokinase from yeast was introduced and overexpressed in Arabidopsis plants, no enhanced sensitivity to glucose was observed. Therefore, it was suggested that Arabidopsis hexokinase is a dual-function enzyme that plays a role in sugar sensing and signaling in addition to its role in phosphorylating hexoses (Jang et al. 1997 ).
In this study, we analyzed under natural growth conditions the role of hexokinase in transgenic tomato plants that exhibited varying levels of hexokinase activity. We were thereby able to assess the role of hexokinase in the response to endogenous sugar levels. The effects of increased hexokinase activity were observed for seedlings as well as for mature plants that were grown using standard horticultural procedures. We demonstrate that hexokinase regulates photosynthesis, growth, and senescence in plants overexpressing AtHXK1 in photosynthetic tissues.
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RESULTS |
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Analysis of AtHXK1
To analyze the role of hexokinase in plant development, we previously isolated an Arabidopsis hexokinase cDNA (Dai et al. 1995 ) by complementation of a yeast triple mutant (hxk1 hxk2 glk1) that lacks glucose and fructose phosphorylating ability and therefore is unable to grow either on glucose or on fructose (Fraenkel 1982 ). The Arabidopsis hexokinase cDNA complemented the inability of the mutant yeast cells to grow on glucose and fructose. The hexokinase cDNA that we isolated is identical to the AtHXK1 clone isolated by Jang et al. 1997 . They determined that AtHXK1 encodes hexokinase on the basis of analogous results from complementation assays and on its similarity to other known hexokinases.
We analyzed the enzymatic properties of AtHXK1 in protein extracts prepared from the mutant yeast cells expressing AtHXK1. The enzyme showed Km values of 44 µM for glucose and 17 mM for fructose (Figure 1A and Figure 1B), which are values similar to those of hexokinases from plant sources (Renz and Stitt 1993 ); therefore, we characterized the enzyme as hexokinase (EC 2.7.1.1) that has a preferential affinity for glucose.
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Figure 1.
Kinetic Plots of AtHXK1 When Expressed in Yeast.
(A) Hexose phosphorylation activity of AtHXK1 with fructose.
(B) Hexose phosphorylation activity of AtHXK1 with glucose.
Activity was measured using extracts prepared from DFY632 yeast cells transformed with pFL61–AtHXK1. Each data point is the mean of three independent experiments. DFY632 cells transformed with pFL61 without AtHXK1 showed no phosphorylation activity. The insets show a double-reciprocal plot of the data. Km values were calculated from the intercept on the horizontal axis of the linear portion of the double-reciprocal plot. V, nanomoles of fructose 6-phosphate or glucose 6-phosphate produced per milligram of protein extract per minute.
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Generation of Transgenic Tomato Lines with Differing Levels of AtHXK1 Expression and Activity
To analyze the effects of modified hexokinase activity on fruits and vegetative parts of the plant, we introduced AtHXK1 under the control of the cauliflower mosaic virus 35S promoter into tomato by means of Agrobacterium-mediated transformation. We obtained 64 independent R0 regenerants and analyzed them by using polymerase chain reaction and DNA and RNA gel blot analyses to determine the presence, genomic integration, copy number, and expression level of AtHXK1. Three independent transgenic plants, HK4, HK37, and HK38, each with a single copy of AtHXK1 and with differing expression levels of the gene, were chosen. Plants homozygous for AtHXK1 were obtained by self-pollinating each transgenic plant, and the expression of AtHXK1 in the homozygous progeny was analyzed.
HK4 and HK38 plants had high expression of AtHXK1, whereas HK37 plants had a moderate expression level (Figure 2A). Expression in leaf, stem, root, and fruit tissues was similar within each line. To test whether the differing expression levels of AtHXK1 among the three lines could be correlated with activities of hexokinase, we measured phosphorylation activity with glucose and fructose in protein extracts prepared from leaves of the transgenic plants and compared it with phosphorylation activity of control plants. As shown in Figure 2B and Figure 2C, hexokinase activities with glucose and fructose directly correlated with the AtHXK1 expression level.
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Figure 2.
AtHXK1 mRNA levels and Hexokinase Activity in Transgenic Tomato Plants.
(A) AtHXK1 mRNA expression in HK4 and HK38 plants is higher than that in HK37 plants. Control wild-type plants, which did not express AtHXK1, did not cross-hybridize with the AtHXK1 probe under stringent conditions and showed no signal.
(B) and (C) Hexokinase activity measured in crude protein extracts prepared from mature leaves. Hexokinase activity of control wild-type plants (1.8 and 7 nmol/mg protein/min for glucose and fructose, respectively), which is a result of endogenous native hexose phosphorylating enzymes, was determined as 100%.
Error bars represent standard deviations. HK/HK, plants homozygous for AtHXK1; -/-, control wild-type plants.
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AtHXK1 Expression and Activity within Heterozygous and Homozygous Plants
To analyze the effect of AtHXK1 copy number on hexokinase expression and activity, we developed segregating populations of HK38 and HK4 plants. These plants carried one (heterozygous), two (homozygous), or no copies of AtHXK1. Hexokinase expression and activity levels in HK38 heterozygous plants were approximately half those in homozygous plants (Figure 3A and Figure 3B). Similar results were obtained with segregating HK4 plants. We concluded that hexokinase activity in the transgenic plants is also determined by gene dosage.
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Figure 3.
Expression of AtHXK1 and Hexokinase Activity in Segregating HK38 Plants.
(A) AtHXK1 mRNA expression in HK38 plants heterozygous and homozygous for AtHXK1. AtHXK1 mRNA expression is relative to HK37. (See the legend to Figure 2A.)
(B) Total relative hexose phosphorylation activity is the mean of the relative phosphorylation activities with glucose and fructose. (See the legend to Figure 2B.)
Error bars represent standard deviations. HK/HK, plants homozygous for AtHXK1; HK/-, heterozygous plants; -/-, control wild-type plants without AtHXK1.
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Glucose 6-Phosphate/Glucose and Fructose 6-Phosphate/Fructose Ratios in Transgenic Tomato Plants
Increased activity of hexokinase is expected to increase the level of phosphorylated sugars in the plant and accordingly may change the ratios of glucose 6-phosphate and fructose 6-phosphate to the respective nonphosphorylated sugars. We measured the levels of fructose, glucose, fructose 6-phosphate, and glucose 6-phosphate in young and mature leaves of heterozygous and homozygous HK4 plants. There was an approximately twofold increase in the amount of phosphorylated sugars in mature leaves of heterozygous plants and an approximately fourfold increase in those of homozygous plants when compared with plants without AtHXK1. The increase in the phosphorylated sugars was accompanied by a net reduction in the total amount of glucose and fructose. Hence, the fructose 6-phosphate/fructose and glucose 6-phosphate/glucose ratios increased approximately three- to fourfold in heterozygous plants and eightfold in homozygous plants (Figure 4A and Figure 4B). Similar results were obtained with young leaves (data not shown). We concluded that the increased activity of hexokinase affected the amounts and proportions of sugars and phosphorylated sugars in the plant.
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Figure 4.
Increased Level of Phosphorylated Sugars in Segregating Plants.
(A) Ratio of glucose 6-phosphate to glucose.
(B) Ratio of fructose 6-phosphate to fructose.
The ratio of phosphorylated sugars to nonphosphorylated sugars was measured in mature leaves of HK4 plants. Error bars represent standard deviations. HK/HK, plants homozygous for AtHXK1; HK/-, heterozygous plants; -/-, control wild-type plants without AtHXK1; P, phosphate.
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Overexpression of AtHXK1 Inhibits Growth
Transgenic plants overexpressing AtHXK1 exhibited several unique phenotypes. One of these was growth inhibition, the degree of which could be directly correlated with AtHXK1 expression and activity (Figure 5A). HK37 plants were stunted compared with wild-type plants, and HK4 and HK38 plants were more stunted, showed reduction of the internodal distances, and had smaller leaves. Accordingly, the growth of homozygous HK4 plants, with two copies of AtHXK1, was more inhibited than was growth of heterozygous plants (Figure 5B). Similar results were obtained with heterozygous and homozygous HK38 plants, indicating that growth inhibition was directly correlated with AtHXK1 expression and activity. To verify that growth inhibition was related to the increased activity of hexokinase, we germinated seeds of HK4 plants on half-strength Murashige and Skoog medium (Murashige and Skoog 1962 ) in the presence of mannoheptulose, which is a competitive inhibitor of hexokinase. Growth inhibition was partially attenuated in the presence of 10 mM mannoheptulose and was further attenuated in the presence of 50 mM mannoheptulose (Figure 6), supporting the hypothesis that the increased hexokinase activity was responsible for the growth inhibition.
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Figure 5.
Growth Inhibition of AtHXK1 Transgenic Plants.
(A) The wild-type (w.t.) plant and HK37, HK4, and HK38 plants homozygous for AtHXK1 grown in regular soil under normal growth conditions. The arrows indicate senescing cotyledons. Yellowing of old leaves is also evident for HK37, HK4, and HK38.
(B) Growth inhibition in segregating HK4 plants. HK/HK, plants homozygous for AtHXK1; HK/-, heterozygous plants; -/-, control plants without AtHXK1.
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Figure 6.
Mannoheptulose, a Hexokinase Inhibitor, Releases Growth Inhibition.
HK4 (homozygous for AtHXK1) seeds were germinated on half-strength Murashige and Skoog medium with or without 10 or 50 mM mannoheptulose. Mannitol was used to adjust the osmotic pressure. As a control, seeds of wild-type (w.t.) plants germinated in the same medium are shown.
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High Expression of AtHXK1 Does Not Affect the Shoot/Root Ratio
AtHXK1 in the transgenic plants was under the control of the cauliflower mosaic virus 35S promoter, which is expressed in most tissues, including roots (Narvaez Vasquez et al. 1993 ). Therefore, the inhibited growth of these plants could result from the effects of AtHXK1 overexpression on root development. To analyze the effect of AtHXK1 on the roots, we grew transgenic tomato plants in an aerohydroponic system to allow continuous examination of root development without root disruption. Throughout 2 months of observation, the roots of HK4 and HK38 plants were found to be shorter than those of control plants. However, there was no visual evidence for inappropriate development of the roots (data not shown). In addition, whereas the total dry weight of 7-week-old transgenic plants was inversely correlated with hexokinase activity (Figure 7A), there was no significant difference between the shoot/root ratio in the transgenic plants and that in the control plants (Figure 7B), leaving open the issue of which part of the plant is primarily affected by AtHXK1 overexpression.
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Figure 7.
Total Dry Weight and Shoot/Root Ratio in Different Transgenic Plants.
(A) Total dry weight of 7-week-old transgenic and control (wild-type) plants.
(B) Shoot/root ratio of the same plants given in (A).
The dry weights of shoots and roots of five individual plants of each line were measured. Error bars represent standard deviations. HK/HK, plants homozygous for AtHXK1; -/-, control wild-type plants.
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Growth Inhibition Occurs When AtHXK1 Is Expressed in Photosynthetic Tissues
To analyze further in which part of the plant AtHXK1 overexpression mediates growth inhibition, we performed reciprocal grafting experiments. Shoots from HK4 plants were grafted onto wild-type roots, and wild-type shoots were grafted onto roots of HK4 plants. Whereas wild-type shoots grew normally on transgenic roots, indicating that the expression of AtHXK1 in the roots had no inhibitory effect, transgenic shoots on wild-type roots exhibited stunted growth (Figure 8), suggesting that the repressing effects of AtHXK1 are exhibited when AtHXK1 is expressed in the photosynthetic tissues. Moreover, wild-type shoots that emerged from wild-type roots on which HK4 shoots were grafted grew normally (Figure 8). In addition, HK4 roots on which wild-type shoots were grafted did not appear different from roots of wild-type plants. These results indicate that the suppressive effect was restricted to photosynthetic tissues expressing AtHXK1 and that no transmissible compounds were involved in the growth inhibition.
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Figure 8.
Reciprocal Grafting between HK4 and Wild-Type Plants.
Pots at right and left contain plants with HK4 shoots grafted onto wild-type (w.t.) roots; the pot at center contains a plant with a wild-type shoot grafted onto HK4 roots. The arrow indicates a wild-type shoot that emerged from the wild-type roots on which HK4 shoots were grafted.
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Overexpression of Hexokinase Reduces Photosynthesis
It had been reported previously that overexpression of Arabidopsis hexokinase in transgenic Arabidopsis plants inhibited expression of the sugar-repressible photosynthetic gene CAB1 (Jang et al. 1997 ). Because the growth inhibition in the transgenic tomato plants is associated with the expression of AtHXK1 in photosynthetic tissues, we analyzed photosynthetic activities in the transgenic plants. CO2 uptake rates of mature leaves were found to be inversely related to the expression and AtHXK1 activity. As shown in Figure 9A, HK37 plants had significantly lower rates of photosynthesis than did wild-type plants, and HK4 and HK38 plants had even lower rates. The expression of AtHXK1 in tomato plants also resulted in reduced chlorophyll content. Figure 9B shows that the chlorophyll a and b content (per unit leaf area) were also inversely related to AtHXK1 expression and activity. However, the reduced rate of CO2 uptake could not be solely attributed to the reduction in chlorophyll content. In HK37 plants, the reduction in chlorophyll content was negligible compared with the substantial (~32%) reduction in the rate of photosynthesis. In HK4 and HK38 plants, chlorophyll a and b content was reduced by ~45%, whereas CO2 uptake rate was reduced by ~70% (Figure 9A and Figure 9B).
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Figure 9.
Photosynthesis Rate and Leaf Chlorophyll Content in Different Transgenic Plants.
(A) CO2 uptake rates.
(B) Chlorophyll content.
Uptake rates and chlorophyll content were measured in mature leaves, as described in Methods. Eight leaves from four control and six transgenic plants were assayed. Error bars represent standard deviations.
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Measuring chlorophyll fluorescence at room temperature is a noninvasive approach that provides insight into the organization and functionality of photosystem II (PSII) reaction centers (Schreiber et al. 1986 ; Karukstis 1992 ; Lavergne and Briantais 1996 ). We used this approach to examine whether malfunctional organization of the chlorophyll molecules in the reaction centers could account for our results. The ratio of variable to maximal chlorophyll fluorescence (Fv/Fm) is indicative of the photochemical quantum efficiency of PSII reaction centers, and it reflects the photoreduction efficiency of the primary electron acceptor QA. The ratio is determined by illuminating dark-adapted leaves with a saturating light pulse. The ratio between the contributions of phase I (also named FPI in the literature) and Fv (i.e., the Fi/Fv ratio) is thought to represent the fraction of QB nonreducing or closed nonfunctional centers (Karukstis 1992 ; Lavergne and Briantais 1996 ); it is derived from fluorescence induction curves obtained by illumination of dark-adapted leaves with continuous actinic light. As shown in Figure 10A and Figure 10B, HK37 leaves were identical to those of the wild type for both fluorescence parameters. However, in HK38, which has the highest expression of AtHXK1, Fv/Fm values were found to be very low, and Fi/Fv values were very high. These results suggest that increased levels of AtHXK1, such as in HK37 and HK4, cause inhibition of photosynthesis at the level of secondary electron transport (beyond PSII reaction centers) and/or at the level of the enzymatic reactions of CO2 fixation and do not affect the functionality of the PSII reaction centers. Higher expression of AtHXK1 as in HK38 leads to disorganization of the reaction centers. Therefore, there might be a threshold level of hexokinase activity beyond which PSII reaction centers are disrupted.
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Figure 10.
Chlorophyll Fluorescence Parameters Fv/Fm and Fi/Fv in Transgenic Plants.
(A) The Fv/Fm ratio is indicative of the photochemical quantum efficiency of PSII reaction centers.
(B) The Fi/Fv ratio is indicative of closed reaction centers.
These two chlorophyll fluorescence parameters were measured in dark-adapted leaves by using a portable pulse–amplitude modulated fluorometer (see Methods for assay details). Eight leaves for each variant from four (control) or six (transgenic) plants were assayed. Error bars represent standard deviations.
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Overexpression of AtHXK1 Decreases Fruit Yield
The reduced rate of photosynthesis and growth inhibition in plants overexpressing hexokinase could have affected the fruit yield of the transgenic tomato plants. Therefore, we analyzed the effects of AtHXK1 overexpression on the fruits. Fruit weight, starch accumulation in young fruits, and total soluble solids (TSS) in mature fruits were found to decrease as a function of hexokinase activity (Figure 11A to 11C). HK37 plants, whose hexokinase activity was approximately two to three times greater than the endogenous activity in wild-type plants (Figure 2), exhibited a nonsignificant yield reduction, whereas HK4 and HK38 plants, which had hexokinase activities approximately six times that in wild-type plants, displayed significant yield reduction. Similar results were obtained with segregating HK4 plants. HK4 heterozygous plants showed only two to three times the increase in hexokinase activity compared with wild-type plants (data not presented), and accordingly, their fruit weight, starch content, and TSS were higher than those of homozygous HK4 plants and were similar to those of HK37 (Figure 11). Thus, yield was inversely correlated with hexokinase activity.
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Figure 11.
Fruit Yield of Transgenic Plants.
(A) and (B) Fruit weight and TSS are the means of data obtained from >25 mature red fruits from five plants of each line.
(C) Starch content was determined in young (green) fruits.
Control plants are wild-type plants without AtHXK1. Error bars represent standard deviations. gfw, grams fresh weight; HK/HK, plants homozygous for AtHXK1; HK/-, heterozygous plants.
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AtHXK1 Overexpression Induces Rapid Senescence
In addition to growth inhibition, transgenic plants displayed rapid senescence of mature leaves, indicated by leaf yellowing proceeding from the margins toward the centers of the leaves (Figure 12). Rapid senescence was observed only in mature leaves and was more evident in older leaves than in younger leaves, thereby exhibiting a normal developmental regulation of leaf senescence. Accelerated senescence was already evident in HK37 plants, and HK4 and HK38 plants, which have higher hexokinase activity, senesced even faster (Figure 12). The differences in rates of senescence were also clear in the cotyledons' senescence (Figure 5). Whereas cotyledons of wild-type plants were still green, those of HK37 plants were yellow and those of HK4 and HK38 plants were already dry. These results indicate that AtHXK1-induced senescence correlated with hexokinase activity, suggesting that hexokinase plays a role in the regulation of senescence. In addition, rapid senescence was observed in HK4 shoots grafted onto wild-type stems and not in wild-type shoots grafted onto HK4 stems. Also, the rapid senescence was not evident in wild-type shoots that emerged from wild-type stems onto which shoots from HK4 plants were grafted. These observations indicate that accelerated senescence is dependent on expression of AtHXK1 in photosynthetic tissues.
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Figure 12.
Rapid Senescence in Transgenic Plants.
Shown are leaves from transgenic and wild-type (w.t.) plants of the same age grown under normal conditions. The leaves are numbered by the order of their appearance from the bottom of the plant. The first leaves of HK4 and HK38 and the second leaf of HK38 are not shown because they were already dry.
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DISCUSSION |
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Our study presents evidence that hexokinase is involved in the regulation of photosynthesis, growth, and senescence, based on analysis of transgenic tomato plants overexpressing AtHXK1. These plants had reduced chlorophyll content in their leaves and low rates of photosynthesis, and their growth was inhibited compared with wild-type plants. All of these phenotypes were correlated with the expression and activity levels of AtHXK1. Recently, Jang et al. 1997 showed that overexpression of AtHXK1 in Arabidopsis seedlings led to "sugar-sensing" phenomena, such as reduced expression of photosynthetic genes, inhibited hypocotyl elongation, and reduced greening of cotyledons. The phenotypes that we observed in our transgenic tomato plants suggest that AtHXK1 retains its regulatory functions within the heterologous host. Furthermore, our results suggest that there are similar sugar signaling pathways in tomato and Arabidopsis and that regulatory functions of hexokinase are conserved in plants.
Whether an analogous native sugar-phosphorylating enzyme in tomato mediates the regulatory functions needs to be determined. Although a glucokinase (Martinez-Barajas and Randall 1998 ) and fructokinases (Kanayama et al. 1998 ) have been isolated from tomato, no tomato hexokinase has been reported to date. However, the isolated glucokinase reported by Martinez-Barajas and Randall 1998 did show "extremely low" fructose phosphorylation activity and could perhaps be defined as a hexokinase. Recently, we (N. Dai and D. Granot, unpublished data) and others (B. Ricard and C. Rothan, personal communication) have independently isolated tomato hexokinase cDNA clones. The proteins corresponding to these cDNA clones are closely related to Arabidopsis hexokinase; they are therefore the primary candidates in efforts to identify the proteins that mediate sugar regulatory functions in tomato.
Although some of the phenotypes of transgenic tomato plants overexpressing AtHXK1 are similar to those characterized for Arabidopsis plants, there are several major differences between the two experimental systems used to obtain these results. These differences enable us to extend the conclusions drawn from studies in Arabidopsis. First, whereas the phenotypes of Arabidopsis plants were observed only in seedlings, transgenic tomato plants exhibited their phenotypes both as seedlings and as mature plants. Thus, hexokinase appears to regulate photosynthesis and growth throughout the life cycle of the plant. Second, in addition to exhibiting inhibition of photosynthesis and growth, transgenic tomato plants underwent rapid senescence, suggesting that hexokinase is also involved in the regulation of senescence. Third, hexokinase inhibited growth and induced rapid senescence only when expressed in photosynthetic tissues. Fourth, unlike transgenic Arabidopsis plants, which expressed their phenotype only when grown on high concentrations of extracellular sugar (i.e., 2 to 6%; Jang et al. 1997 ), transgenic tomato plants displayed their phenotypes under normal growth conditions and without any extracellular sugar, indicating that the regulatory roles of hexokinase are expressed in response to endogenous sugar levels in tomato.
This final difference between transgenic Arabidopsis and tomato plants could be attributed to differing activity levels of AtHXK1 in Arabidopsis and tomato. Transgenic tomato plants could have higher hexokinase activities than do transgenic Arabidopsis plants. Higher hexokinase activities might promote the phenotypes in the absence of extracellular sugar. Indeed, most of the 64 independent transgenic tomato plants had relatively low AtHXK1 expression and activity levels and exhibited either no phenotype or only minor phenotypes, such as those described above for HK37 plants. On the other hand, a comparison between the hexokinase activities in HK38 plants and the corresponding results published for Arabidopsis plants (Jang et al. 1997 ) shows that both were increased by approximately five- to sixfold over the regular endogenous hexokinase activity. Therefore, it is also possible that the sensitivity to hexokinase activity of the sugar signaling pathways in Arabidopsis and tomato differ.
Similar sensitivity differences between Arabidopsis and tobacco have been observed in transgenic plants expressing a yeast-derived apoplastic invertase, which is a sucrose-hydrolyzing enzyme (von Schaewen et al. 1990 ). Whereas expression of the invertase in Arabidopsis plants did not lead to drastic changes, its expression in transgenic tobacco plants elicited dramatic changes with respect to development and phenotype. Therefore, in the use of transgenic plants to study the role of hexokinase, tomato plants might be more effective tools than Arabidopsis plants.
The appearance of the phenotypes in transgenic tomato plants in the absence of extracellular sugar indicates that hexokinase is responding to normal endogenous levels of sugar. It has been reported that sugar accumulation in mature leaves is accompanied by a decrease in the rate of photosynthesis (Goldschmidt and Huber 1992 ; Hensel et al. 1993 ; Jiang et al. 1993 ; Wingler et al. 1998 ), and perhaps an increased flux of sugar through hexokinase plays a role in photosynthesis inhibition in mature leaves. In this study, inhibition of photosynthesis in transgenic tomato plants occurred without an increase in glucose and fructose content. In fact, sugar content in the transgenic plants was reduced in mature and young leaves compared with that in wild-type plants. These results demonstrate that increased hexokinase activity is sufficient to stimulate the signaling pathway, leading to inhibition of photosynthesis and growth, without a need for an actual increase in sugar levels. In addition, mannoheptulose, a competitive inhibitor of hexokinase, suppressed the growth inhibition of HK4 plants (Figure 6), indicating that increased hexokinase activity is necessary to express the phenotypes. Hence, high activity of hexokinase is necessary and sufficient to inhibit photosynthesis and growth, suggesting that sugar regulation of photosynthesis and growth is entirely mediated via hexokinase. The high hexokinase activity in the transgenic plants is probably perceived as a sign of elevated sugar levels, which naturally inhibit photosynthesis and growth.
In other organisms, such as animals and yeast, it has been implied that hexose-phosphorylating enzymes are involved in sugar signaling. In mammalian systems, glucokinase is required in pancreatic ß cells for glucose-stimulated insulin release and in liver to convert excess glucose into glycogen (Matschinsky et al. 1993 ; Grupe et al. 1995 ). In yeast, hexokinase is required for the glucose repression phenomenon (Entian 1980 ). Although it is not clear how hexokinase and glucokinase transmit the signal, downstream genes involved in glucose repression in yeast are well characterized. In particular, the SNF1 gene, which encodes a protein serine/threonine kinase, is essential for glucose repression (Celenza and Carlson 1986 ). It forms a high molecular mass protein kinase complex that, upon removal of glucose, phosphorylates and thereby inactivates a DNA binding protein (Mig1) that represses transcription of glucose-repressed genes (Ostling and Ronne 1998 ). Plant homologs of SNF1, called SNF1-related protein kinases 1s (SnRK1), which complement snf1 yeast mutants, have been identified (Alderson et al. 1991 ; Muranaka et al. 1994 ; Halford and Hardie 1998 ). It would be of interest to determine whether these genes are affected by hexokinase.
We do not know how hexokinase activity stimulates sugar-signaling pathways in either yeast or plants. In previous studies, it has been suggested that yeast and plant hexokinases are bifunctional enzymes with catalytic and regulatory activities (Entian and Frohlich 1984 ; Jang and Sheen 1994 ; Jang et al. 1997 ). This conclusion was based on experiments demonstrating that hexokinase activity is necessary to stimulate the regulatory functions, and that neither downstream metabolism of the sugars nor accumulation of phosphorylated sugars is required for induction of the sugar-sensing pathway (Entian and Frohlich 1984 ; Jang and Sheen 1994 ; Jang et al. 1997 ). Our results neither confirm nor negate the dual function of AtHXK1, because we were unable to distinguish between the enzymatic activity and downstream effects of the products. The elevated levels of hexose phosphate in the transgenic plants may increase the flux toward glycolysis and respiration, and changes in intermediate metabolites could stimulate the regulatory pathway. For example, it is possible that altered ATP/ADP ratios or altered concentrations of cytosolic phosphate ions have a signaling function (Sadka et al. 1994 ; Halford et al. 1999 ). However, irrigation or spraying of HK4 and HK38 plants with phosphate did not reverse the phenotypes described above (N. Dai and D. Granot, unpublished results), suggesting that changes in the cytosolic phosphate might not be the signal.
Growth inhibition, reduced sugar levels in leaves, and decreased yield could be attributed to the inhibition of photosynthesis by AtHXK1. Accordingly, growth inhibition was manifested only when AtHXK1 was expressed in photosynthetic tissues. Accelerated senescence might also be a consequence of a decline in the rate of photosynthesis below a certain level at which senescence is initiated. However, we cannot rule out the possibility that hexokinase might have an additional role in the regulation of senescence: it is possible that a drop in the photosynthesis rate reduces the sugar flux, which might also be sensed by hexokinase. This hypothesis is supported by results obtained elsewhere with transgenic tobacco plants that express a yeast-derived invertase and that accumulate carbohydrates (Ding et al. 1993 ). The transgenic tobacco plants manifested the onset of early leaf senescence only when expressing an apoplastic invertase and not a cytosolic invertase. We speculate that hexokinase might sense changes in extracellular sugar flux and stimulate senescence.
Hexokinase may be involved in the regulation of additional pathways affected by sugars in addition to photosynthesis, growth, and senescence (Graham et al. 1994 ; Perata et al. 1997 ; Prata et al. 1997 ; Smeekens and Rook 1997 ). Photosynthesis, growth, and senescence are also regulated by light and by hormones, such as auxin, cytokinin, gibberellic acid, brassinosteroids, and ethylene. It has been suggested that sugars may interact with or regulate such internal signals (DeWald et al. 1994 ; Thomas and Rodriguez 1994 ; Sonnewald et al. 1995 ; Szekeres et al. 1996 ; Jang et al. 1997 ; Smeekens 1998 ; Wingler et al. 1998 ; Zhou et al. 1998 ). The availability of transgenic tomato plants with differing hexokinase activities provides a means to study the role of hexokinase in other pathways and in interactions with other signaling pathways.
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METHODS |
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Yeast Transformation and Hexokinase Complementation
The yeast (Saccharomyces cerevisiae) strain used was DFY632 (MATa ura3-52 hxk1::LEU2 hxk2::LEU2 glk1::LEU2 lys1-1 leu2-1). This strain is unable to phosphorylate glucose or fructose and therefore is unable to grow on these sugars as carbon sources. The yeast cells were transformed with Arabidopsis hexokinase (AtHXK1) previously isolated from an Arabidopsis thaliana cDNA expression library (Dai et al. 1995 ) in the pFL61 shuttle vector. Yeast transformation was performed according to Ito et al. 1983 . The transformants were grown on plates with galactose and without uracil and were tested on plates with glucose and without uracil and on plates with fructose and without uracil. Plates with sugar and without uracil contained 0.5% ammonium sulfate, 0.17% yeast nitrogen base without amino acids (Difco), 0.2% casamino acids (Difco), 0.004% adenine (Sigma), 0.008% tryptophan (Sigma), and 110 mM galactose, glucose, or fructose.
Protein Extraction from Yeast and Plants
DFY632 yeast cells, transformed with either pFL61 or pFL61–AtHXK1, were grown in 40 mL of liquid medium containing galactose and without uracil for 72 hr to ~5 x 107 cells per mL. Cells were spun down for 5 min at 6000 rpm, washed twice with water, and resuspended in 0.5 mL of water. A 250-µL sample of the cells was extracted twice with 500 mL of extraction buffer (50 mM Hepes, pH 7.6, 1 mM EDTA, 15 mM KCl, 1 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride) by vortexing with 250 µL of glass beads. After vortexing three times for 30 sec, the mixture was centrifuged for 5 min at 12,000g at 4°C, and the supernatant was brought to 80% ammonium sulfate saturation. After centrifugation at 12,000g at 4°C, the pellet was resuspended in 0.5 mL of washing buffer (50 mM Hepes, pH 7.5, 1 mM EDTA, and 1 mM DTT), desalted on a G-25 Sephadex column (55 x 11 mm), and used as a crude enzyme extract for subsequent enzymatic analysis.
Protein extraction from plants was performed in a similar way, with 1 to 2 g of plant tissue homogenized in 4 volumes of extraction buffer (50 mM Hepes, pH 7.6, 1 mM EDTA, 15 mM KCl, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 3 mM diethyldithiocabamic acid, and 0.2% PVP). The mixture was centrifuged for 25 min at 16,000g at 4°C, and the supernatant was treated as described above.
Analysis of Hexokinase Activity
Hexokinase activity was measured by enzyme-linked assay according to Schaffer and Petreikov 1997 . The assays contained a total volume of 1 mL of 30 mM Hepes-NaOH, pH 7.5, 2 mM MgCl2, 0.6 mM EDTA, 9 mM KCl, 1 mM NAD, 1 mM ATP, and 1 unit of NAD-dependent glucose-6-phosphate dehydrogenase (G6PDH from Leuconostoc mesenteroides; Sigma). To assay glucose phosphorylation, the reaction was initiated with 2 mM glucose. To assay fructose phosphorylation, 1 unit of phosphoglucoisomerase was added, and the reaction was initiated with fructose. Reactions were conducted at 37°C, and absorption at 340 nm was monitored continuously.
Tomato Transformation and Analysis of AtHXK1 mRNA Expression in Transgenic Plants
AtHXK1 (Dai et al. 1995 ) was introduced under the control of the cauliflower mosaic virus 35S promoter into binary vector pGA643 containing the neomycin phosphotransferase II gene as a selectable marker. Transformation was performed with MP-1, a tomato (Lycopersicon esculentum) line known for its high transformation efficiency (Barg et al. 1997 ). Essentially, the transformation procedure was as described by McCormick 1991 . T0 and T1 independent transgenic plants were analyzed by polymerase chain reaction and DNA gel blotting for the presence and copy number of the AtHXK1 transgene. Most plants had only one copy of AtHXK1. Heterozygous and homozygous plants were identified after segregation analysis of AtHXK1 within T1 seeds.
Expression of AtHXK1 was analyzed by RNA gel blotting using AtHXK1 as a probe. AtHXK1 mRNA was analyzed in the same amount of total RNA from each plant. The relative amount of AtHXK1 mRNA in each transgenic plant was calculated by using the National Institutes of Health Image software after data were obtained using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Aerohydroponic Growth and Shoot/Root Ratio Analyses
Four-week-old transgenic plants of the HK37, HK4, and HK38 lines were transferred and grown in aerohydroponic pots according to Bar-Tal et al. 1993 . The shoot/root ratio was measured on 7-week-old plants grown in soil. The soil was carefully washed off the roots, and the plants were dried at 80°C. Total dry weight was the sum of shoot and root weights.
Total Soluble Solids, Starch, and Sugar Determinations
Portions of a whole harvested fruit were used for determination of total soluble solids (TSS), using an N-1 hand-held refractometer (Atago, Tokyo, Japan). Portions (~500 mg fresh weight) were immediately placed in 80% ethanol for sugar and starch determination. Soluble sugars were extracted three times in hot ethanol, and starch content was measured in the insoluble fraction after treatment with amyloglucosidase, as described previously (Schaffer et al. 1987 ).
The content of soluble sugars in mature leaves was determined after extraction with ethanol and separation by HPLC, as described by Miron and Schaffer 1991 .
Glucose 6-phosphate and fructose 6-phosphate were extracted and assayed spectrophotometrically on 1 g fresh weight of mature leaves, according to Tobias et al. 1992 .
Photosynthetic Activity and Chlorophyll Content
The CO2 uptake rate was measured with a portable photosynthesis measuring system (model CI-301; CID, Vancouver, WA) in mature leaves under greenhouse natural saturating light (600 to 800 mmol m-2 sec-1 photosynthetically active photon flux density) in the morning (10:00 to 11:00 AM).
Chlorophyll fluorescence was measured with a portable pulse–amplitude modulated fluorometer (model PAM 2000; Heinz Walz GmbH, Effeltrich, Germany) in the same leaves. Dark adaptation was applied by dark-clips at 15 to 20 min before each measurement. The ratio of variable to maximal chlorophyll fluorescence (Fv/Fm) was measured directly by the "run 2" mode of the PAM 2000, whereas the ratio of phase I contributions to Fv (Fi/Fv) was determined from the induction curves obtained by "run 6" at the high modulation frequency (Schreiber et al. 1986 ).
Chlorophyll content was determined by acetone extraction according to Zhang and Kirkham 1996 .
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ACKNOWLEDGMENTS |
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This work was supported by the United States–Israel Binational Agricultural Research and Development fund (BARD; Grant No. IS-2894-97) and by the Binational Science Foundation (BSF; Grant No. 97-00250). This paper is Contribution No. 165/98 (1998 series) of the Agricultural Research Organization of the Volcani Center.
Received December 23, 1998; accepted April 13, 1999.
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ABSTRACT
INTRODUCTION
