JXB Advance Access originally published online on March 3, 2003
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Journal of Experimental Botany, Vol. 54, No. 385, pp. 1143-1151,
April 1, 2003
© 2003 Oxford University Press
Physiological, biochemical and molecular analysis of sugar-starvation responses in tomato roots
Received 2 August 2002; Accepted 25 November 2002
Unité Mixte de Recherche en Physiologie et Biotechnologie Végétales, Institut de Biologie Végétale Moléculaire et Institut National de la Recherche Agronomique, Centre de Recherche de Bordeaux, BP 81, F-33883 Villenave d’Ornon Cedex, France
1 To whom correspondence should be addressed. Fax: +33 5 57 12 25 41. e-mail: baldet{at}bordeaux.inra.fr
Abbreviations: RT-PCR, reverse transcriptase polymerase chain reaction; DW, dry weight.
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Abstract |
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Two-month-old tomato plants were submitted to day/night cycles and to prolonged darkness in order to investigate the physiological and biochemical response to sugar starvation in sink organs. Roots appeared particularly sensitive to the cessation of photosynthesis, as revealed by the reduction of the growth rate and the decline of the carbohydrate and protein content. Therefore, excised tomato roots were used as a model to deepen the characterization of sugar starvation symptoms. In excised roots, the endogenous sugars were rapidly exhausted and significant degradation of protein was observed. Glutamine and asparagine accounted for most of the nitrogen released by protein breakdown. Respiration declined and proliferation- and growth-associated genes were repressed soon after the beginning of the sugar depletion. Among the genes studied, only the gene encoding asparagine synthetase was strongly induced. All the starvation symptoms were reversible when the roots were resupplied with sugar. When the culture conditions deteriorated, the metabolic and molecular changes led to the triggering of apoptosis of the root cells.
Key words: Apoptosis, carbohydrate limitation, cell cycle, Lycopersicon esculentum, root, sink strength.
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Introduction |
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Plant growth depends largely upon the partitioning of assimilated carbon between photosynthetic sources such as mature leaves, and photosynthetically less active or inactive sink tissues such as roots, flowers and fruits (Farrar and Williams, 1991). The controls that actually regulate the partitioning of assimilates between sink organs are still poorly understood. Although this is still a matter of debate, it is now thought that the partitioning of assimilates between sink organs is not a property of the sink alone, but a combination of co-ordinated mechanisms involving the whole plant (Farrar, 1996; Marcelis, 1996; Minchin and Thorpe, 1996). In this respect, sink strength can be defined as the competitive ability of an organ to import photoassimilates. Among sink organs, roots are defined as low priority sinks under the concept of competition for assimilates between alternative sinks (Wardlaw, 1990).
During the life of a plant, any exogenous factors such as abiotic or biotic stress that lowers the total sugar production or increases the total sugar demand may influence carbohydrate partitioning and lead to carbohydrate limitation. This may be the case for instance when competition occurs between sink tissues such as roots, flowers, or fruits (Ho, 1996), as well as when the rate of photosynthesis decreases. In tomato, as the assimilate supply is limited, the carbohydrates exchanged in the plant are essentially accumulated by fruit that act as high priority organs (Wardlaw, 1990; Baldet et al., 2002), and this probably diminishes strongly the availability of sugars for other sink organs, such as roots.
The metabolic consequences of carbohydrate starvation have been studied in a number of plant models. In maize root tips the response of cells can be divided into three phases: an acclimation phase, a survival phase, and a cell-disorganization phase leading to cell death (Brouquisse et al., 1992). The acclimation phase consists of a decrease in respiratory and growth capacities and a remobilization of carbon resources. During the very first hours of carbohydrate starvation, maize roots cease to grow, which indicates the impairment of cell divisions. When the conditions deteriorate, plant cells must accommodate the depletion and survive by substituting with alternative carbon sources from cellular constituents such as lipids, proteins and other cellular materials. In the co-ordinated response of the cell to carbohydrate limitation leading to cell disorganization, the fragmentation of DNA appears as a trait of apoptosis, a genetically defined process associated with common morphological and biochemical changes (Kerr et al., 1995). It can be initiated by a variety of stimuli, including developmental and environmental signals. Apoptosis is well documented in animals, and similarly in plants chromatin condensation and the internucleosomal fragmentation of the nuclear DNA are seen. Few data dealing with the changes in physiological and biochemical parameters of sink organs submitted to carbon limitation are available at the whole plant level. In the main sink organs of maize plants, the consequences of carbon depletion induced by an extended darkness were identical to those established for the excised root tip, especially the decrease in sugar content and protein degradation (Brouquisse et al., 1998).
The purpose of the present work was to characterize the effects of carbohydrate deprivation on the metabolism of tomato roots. First, the response to sugar limitation in young tomato roots from plants cultured under prolonged darkness was characterized. In the second part of the study, the symptoms of carbohydrate deprivation were validated in excised roots maintained on medium depleted of sugar at: (i) the physiological level, (ii) the biochemical level, and (iii) the molecular level. Al together, these complementary approaches allowed a model of the events that characterized the carbohydrate starvation responses in young tomato roots to be described.
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Materials and methods |
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Plant material and growth conditions
Cherry tomato (Lycopersicon esculentum var. cerasiforme Mill., cv. West Virginia 106) seeds were provided by Dr J Philouze (INRA Montfavet, France). Seeds were germinated in vermiculite soaked in Algospeed (Algochimie, Château Renault, France) fertilizing solution containing N:P:K:Mg in the ratio 13:13:24:3 (1 g l–1). The plants were raised under greenhouse conditions with temperature control within the range of 20–26 °C and 18–23 °C during the day and night, respectively. About 10 d after sowing, seedlings at the two-leaf stage were transferred to a growth chamber, under a 15 h photoperiod with a photosynthetic photon flux density of 400 µmol photons m–2 s–1. The day/night temperature was 25/20 °C and the relative hygrometry was maintained close to 70%. For hydroponic culture of plants, the culture medium consisted of Algospeed solution (1 g l–1), under constant bubbling with air using aquarium pumps. The pH of the medium was checked and readjusted daily close to pH 6. For control experiments (the incubation of excised roots with different carbohydrate sources) as well as the measurement of respiration and cellular viability of the roots, plants grown under aseptic conditions were needed. Therefore, tomato seeds were soaked in 90% ethanol, washed twice in sterile water and immersed in 2% sodium hypochlorite containing 0.2% SDS for 15 min. Seeds were washed four times in sterile water, and sown between two layers of Whatman paper previously soaked in a mineral solution, and put into sterile culture boxes (10x10x10 cm) (Saglio and Pradet, 1980). The boxes were maintained at 25 °C in darkness for 3 d, then the upper Whatman layer was removed and the culture medium was replaced by the same solution supplemented with 0.1 M sucrose. The boxes were transferred to a growth chamber with the same culture conditions as described above. After 4 d, plantlets were harvested, roots excised under sterile conditions and used for the experiment described in the following section.
Prolonged darkness experiment
After 2 months of hydroponic culture, tomato plants were submitted to prolonged darkness or maintained under the day/night cycle as a control. For both treatments, young roots (the last 10 cm of the primary and secondary roots) were harvested at the same time, corresponding to the end of the dark phase in the control. Roots were washed with distilled water and dried on filter paper. Immediately after harvest, the different tissues were snap frozen in liquid nitrogen and treated as described above.
Carbohydrate limitation in excised tomato roots
During plant culture, young roots were harvested at the end of the light period on 4-week-old plants. Immediately after harvest, the roots were decontaminated by immersion for 1 min in a 2% calcium hypochlorite solution, rinsed in a solution containing 0.1 M HCl and 1% Triton X100 (v/v), and then rinsed in sterile water several times. These roots and those obtained aseptically were incubated at 25 °C in darkness in a mineral solution described by Saglio and Pradet (1980) in the presence or absence of sugar. To avoid roots experiencing hypoxia, a gas mixture of 50% O2 and 50% N2 was continuously bubbled through the incubation medium. The nutrient solution was supplemented with an antibiotic–antimycotic mixture from Sigma (L’Isle d’Abeau Chesnes, France; ref. A-7292, 10 µl ml–1), the roots were rinsed with sterile water and the incubation medium was renewed daily. Excised roots were removed at different times, washed, dried on filter paper, promptly frozen in liquid nitrogen, ground, lyophilized until completely desiccated and stored at –20 °C.
Extraction and assay of sugars, amino acids, proteins, and NH4+
Soluble metabolites (soluble sugars, amino acids) and starch were extracted from 10–15 mg dried powder using the alcoholic extraction method (Brouquisse et al., 1991; Moing et al., 1994). Assays for glucose, fructose and sucrose were achieved (Kunst et al., 1984) and adapted to a micro assay using an MR 5000 reader (Dynatech, St Cloud, France). Free amino acids were analysed by reverse-phase HPLC after derivation according to the AccQ-Tag method (Cohen and De Antonis, 1994). Amino acid quantification was achieved with the Millennium 2.15 software (Waters, St Quentin en Yvelines, France). Total soluble proteins were extracted in 500 mM Tris–HCl (pH 7.5) and assayed using BSA as a standard (Bradford, 1976). NH4+ was extracted with 0.1 M HCl and assayed according to the phenol–hypochlorite method (King et al., 1990).
Respiratory activity and cellular viability measurements in excised roots
The respiration of excised roots was measured at 25 °C in the incubation medium using a Clark oxygen electrode (Strathkelvin Instruments, Glasgow, UK) connected to an oxygen analyser. Roots were transferred to 1.45 ml of medium previously saturated with O2. The measurement of O2 consumption was realized while stirring. Once assayed, roots were dried and weighed. Respiratory activity was expressed as 
[O2] h–1 mg–1 dry weight. The cellular viability of excised roots was based on the reduction of MTS [3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulpho phenyl)-2H-tetrazolium] by dehydrogenase activities that characterize living cells (Cory et al., 1991; Berridge and Tan, 1993). the reduction of MTS was measured following the instructions of the ‘Cell titer 96 AQueous one solution cell proliferation assay’ kit (Promega, Charbonières, France). At the end of the reaction, roots were dried, weighed and the value of OD min–1 mg–1 DW was used as a survival index.
Nucleic acid manipulation
Total RNA was extracted from roots according to the method described by Chevalier et al. (1995). The relative transcript level of each cDNA was determined specifically by RT-PCR starting with 5 µg total RNA and using the combination of a 3' primer corresponding to the 3' UTR sequence and a 5' primer corresponding to the C-terminal sequence of the protein (Table 1) as described by Joubès et al. (2000). For each RT-PCR product to be tested, a positive control amplification was performed using the recombinant plasmid harbouring the cDNA of interest. The RT-PCR products were separated in a 1.2% agarose gel, blotted onto Hybond N+ membrane (Amersham) and hybridized at 65 °C with the appropriate cDNA probe labelled with [
-32P] dCTP by random priming according to standard methods (Sambrook et al., 1989). Total DNA was extracted from root tissues by the method of Dellaporta et al. (1983). To analyse the apoptosis DNA profile 1 µg of total DNA was separated by electrophoresis in a 1.2% agarose gel and blotted onto Hybond N+ membrane. The membrane was then hybridized at 65 °C with a genomic DNA probe prepared as follows. Total genomic DNA (10 µg) was digested with 30 U of Sau3AI enzyme (Promega) for 4 h at 37 °C. The DNA probe consisted of 20 ng of the restricted DNA labelled by the random priming procedure in the presence of 50 µCi [-32P] dCTP (3000 Ci mmol–1, Amersham).
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Results |
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Analysis of the effects of sugar limitation on whole tomato plants submitted to prolonged darkness
Two-month-old plants were submitted to prolonged darkness or maintained in a day/night cycle (control plants) in order to monitor the occurrence of sugar starvation in roots. After 9 d of the day/night cycle, the whole root system from four control plants increased from 35 to 110 g fresh weight (FW) (Fig. 1). In the case of prolonged darkness, the growth of the root system from four treated plants stopped after 3 d and the FW remained close to 60 g. Five days after the beginning of darkness, roots displayed symptoms of senescence: root extremities turned to a dark brown colour (not shown).
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A strong decline in the soluble sugar and starch content was observed in roots during the first 2 d of darkness (Fig. 1). The protein level decreased strongly after 2 d of darkness, and reached 10% of its initial value after 6 d (Fig. 1). In the roots of control plants, the sugar and protein levels remained stable or increased slightly. Compared with other organs including flowers, leaves and fruits (Baldet et al., 2002; C Devaux, unpublished data), the decrease in the protein and sugar contents of roots was more pronounced. Since roots appeared particularly sensitive to a prolonged cessation of photosynthesis in whole plants it was decided to deepen the study of sugar starvation in vitro using excised roots.
Analysis of the effects of sugar deprivation on excised tomato roots
Changes in root carbohydrate contents: In order to induce sugar starvation, excised tomato roots were incubated in liquid medium in the absence of any exogenous sugar. The soluble sugar content showed a strong and rapid decrease as it reached 10% of the initial value after 12 h incubation (Fig. 2). In comparison, the decrease in starch content was less pronounced and decreased up to 50% of its initial value after 1 d. The response to sugar depletion was not the result of an osmotic stress, as excised roots grown in the presence of a non-metabolizable sugar (200 mM mannitol) behaved like sugar-starved roots (data not shown).
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Changes in root nitrogen compound contents: The protein content decreased to 75% of its initial level within 3 d of starvation treatment and did not change thereafter (Fig. 2). Regarding the change of sugar and protein content during starvation and similarly with dark-treated plants, excised roots appeared to be particularly sensitive to the lack of sugars. Previous studies on excised plant organs (Genix et al., 1990; Brouquisse et al., 1992) demonstrated that protein degradation led to the release of nitrogen as amino acids, mainly asparagine, and ammonium. Thus the fate of these metabolites was investigated in the course of sugar starvation (Fig. 2). The total amino acid content increased within 1 d, and then rapidly declined. Among the amino acids, glutamine as well as asparagine, both of which accounted for 50% of the total content, increased to 2.5- and 5-fold of the initial value, respectively, and then sharply decreased within the next 2 d. In the course of starvation, the NH4+ content sharply increased from 12 h to 4 d in tomato roots. The decline of NH4+ after 4 d suggests that this ion is released from the cells to the incubation medium. Table 2 summarizes the changes in the distribution of the endogenous nitrogen between the main N-compounds of the root. At the start of the experiment, nitrogen was predominantly localized in proteins (90%). During the time-course of starvation nitrogen was progressively reallocated, first from proteins into asparagine and glutamine and second into NH4+. Finally, amino acids and NH4+ represented 80% of intracellular nitrogen in roots.
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N represents the sum of (proteins+amino acids+NH4+) nitrogen compounds present in roots. On average, the standard deviation of the mean values varied between 5–17%.
Changes in the respiratory activity and cellular viability: The analysis of respiratory activity was investigated in roots from 1-week-old plantlets grown in aseptic cultures (Fig. 3). Under sugar depletion, the respiration rate declined to reach 25% of the initial value after 6 d, whereas in the presence of 100 mM sucrose or 200 mM glucose it was maintained at the initial level. The respiration rate was higher with sucrose than glucose, which confirms that sucrose is a better carbon source for tomato roots.
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The ability of root cells to survive under sugar starvation conditions was then investigated. A relative survival index was defined, which corresponds to the cell viability of the roots incubated with or without carbohydrate supply (Fig. 3). Up to 3 d in the absence of sugar, the survival index of the roots stayed close to its initial value, then decreased by 3-fold to reach a minimum after 5 d. In the presence of 200 mM glucose or 100 mM sucrose, the survival index was maintained at the initial value throughout the incubation. It is noteworthy that the survival index was the highest with sucrose as with the respiration rate.
Changes in gene expression in sugar-starved roots: In order to study the early response to sugar starvation in roots, the expression of genes involved in growth and metabolic pathways were analysed in excised roots in the presence and the absence of sugar supply over 4 d by semi-quantitative RT-PCR (Fig. 4). The level of sucrose synthase transcripts (Lyces;SuSy2), used as a marker for sugar metabolism (Koch, 1996), increased when roots were incubated or re-fed with sucrose while it decreased during starvation, thus suggesting that this gene was directly linked to sucrose availability in the roots. To investigate the effects of sugar starvation on cell proliferation, the expression profiles of cell cycle-associated genes were followed, e.g. CDKA;1, CycB1;1, histone H4, and an -subunit of the 20S proteasome (PSR5) (Joubès et al., 2000, 2001; Genschik et al., 1994). All these transcripts displayed a similar pattern of expression, with repression beginning after 2 d of starvation. As a marker for amino acid metabolism, a cDNA encoding asparagine synthetase (AS1) was used (Lam et al., 1996). The level of AS1 transcripts increased strongly under sugar limitation and was repressed in the presence of sugar or when roots were re-fed with sucrose. Genes encoding for cysteine and serine proteases (CYP1 and SBT2, respectively) were used as gene markers for protein degradation. While CYP1 was unaffected by sugar starvation, long-term sugar starvation decreases the steady-state SBT2 transcript levels to some extend, suggesting that these two cDNAs do not represent appropriate markers for protein breakdown during sugar starvation. The same conclusion can be made for the catalase gene Lyces;CAT1, although it has been shown to be induced in sugar-depleted Arabidopsis roots (M Dieuaide-Noubhani, personal communication).
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In summary, these data indicated that most of the genes studied here are globally repressed by sugar starvation, particularly transcripts for actin, which have been shown previously to be affected by sugar limitation (Sheu et al., 1994; Joubès et al., 2001).
Induction of DNA fragmentation in sugar-starved roots: The analysis of the genomic DNA pattern from tomato roots showed that DNA fragmentation was induced after 4 d of starvation and intensified afterwards (Fig. 5). This process of DNA fragmentation was characterized by the appearance of a ladder of DNA fragments calculated to be multiples of 160 bp corresponding to nucleosomal DNA. As shown in Fig. 5 (lane C), the DNA pattern remained unchanged when the roots were cultured with sucrose for 8 d, confirming that the observed DNA fragmentation was not the result of DNA extraction artefacts, but truly a specific cell-mediated degradation event induced by sugar starvation.
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Discussion |
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Attacks on plant roots by pathogens or hydric stress can significantly affect plant yield and, consequently, fruit production. Furthermore, tomato breeders recurrently encounter a reduction of the root growth a few weeks after the plants have produced the maximum number of fruit (P Baldet, personal communication). This emphasizes the importance of the root system in enhancing crop yield in relation to the regulation of assimilate partitioning between sink organs, a process that still remains poorly understood (Farrar, 1996; Marcelis, 1996; Minchin and Thorpe, 1996).
In this study, the biochemical and molecular processes occurring in tomato roots subjected to sugar depletion were investigated. In whole plants, the cessation of photosynthesis led to a strong decline in the sugar content of roots, and to a net degradation of proteins (Fig. 1). Compared with other sink organs such as fruit, tomato roots appear very sensitive to carbohydrate starvation. Indeed, both in planta or in vitro, signs of starvation are visible in roots a few hours after sugar depletion, whereas in young fruit signs of starvation appear only after several days (Baldet et al., 2002). Two explanations may account for this behaviour. Firstly, the kinetics of the response depend on the specificity of the sink organ; the roots appear as low priority organs, which is in accordance with the ‘priority rank order’ supporting the concept of competition for assimilates between alternative sinks (Wardlaw, 1990) and thus the concept of sink strength (Marcelis, 1996). Secondly, the response to starvation can be related to the level of carbohydrate reserves, such as starch, contained in tomato sink organs. During the rapid growth phase, young tomato roots accumulate much less starch than developing fruit, in which the starch can account for up to 25% of the dry weight (Rolin et al., 2000). In whole plants, starvation symptoms are more restrained and delayed than in excised roots incubated in the absence sugar (Fig. 2), as they take advantage of the mobilization of sugars from the reserve tissues, such as mature leaves and stems (Ho, 1976; Ammerlaan et al., 1986).
In the experiments using excised roots, it was demonstrated that the response to sugar limitation is similar to that described in maize root tips (Brouquisse et al., 1992; Dieuaide et al., 1992; James et al., 1993) and proceeds according to three phases: (1) the acclimation phase from 0–24 h, (2) the survival phase from 24 h to 4 d and (3) the disorganization phase after 4 d.
During the acclimation phase, sugar starvation leads to the exhaustion of endogenous carbohydrates within 12 h. a decrease in the expression of the Lyces;SuSy2 gene was observed (Fig. 4). In tomato roots, the decrease in the quantity of Lyces;SuSy2 transcripts could trigger a reduction in the sugar supply and contribute to the slowing down of respiration and tissue growth as the amount of sucrose synthase and corresponding activity would be lessened (Koch, 1996). Indeed sucrose synthase is a key enzyme in sink metabolism that supplies respiration and biosynthesis with carbon substrates and furthers sugar import into the cell (D’Aoust et al., 1999). The exhaustion of endogenous carbohydrates was concomitant with the decline in respiration (Figs 2, 3). In sycamore suspension cell cultures submitted to sucrose depletion, it has been suggested that the decline in respiration is the result of a decrease in the number of mitochondria per cell and not of an arrest in the supply of respiratory substrates (Journet et al., 1986). This process is, in fact, the result of an arrest in biosyntheses which limits the respiration of the maize root tips, the reduction of the mitochondria per cell occurring afterwards (Brouquisse et al., 1991).
Another important feature of the acclimation phase is the remobilization of alternative carbon sources (proteins and amino acids) together with nitrogen storage processes. Thus plant cells adapt, through protein and amino acid catabolism, to maintain respiratory and biosynthetic pathways using carbon skeletons (Brouquisse et al., 1992). Nitrogen that is released is then stored as NH4+ ions (Mazelis, 1980). Subsequently, NH4+ ions are reincorporated into amino acids via de novo synthesis (Givan, 1979; Sieciechowicz et al., 1988). It can be hypothesized that the increase in asparagine and glutamine observed during the first 24 h of starvation resulted from these recovery processes. Moreover, the accumulation of asparagine proceeds from the increase in asparagine synthetase gene transcription (Chevalier et al., 1996; Baldet et al., 2002) associated with a strong induction of the enzymatic activity (Brouquisse et al., 1992). As a result it cannot be excluded that this process tends partly to detoxify the released NH4+ ions observed in Fig. 2.
As a consequence of the remobilization processes targeting the carbon- and nitrogen-compounds, root growth and cell proliferation are likely to be impaired. the effects of sugar starvation on the expression of cell cycle genes (Lyces;CDKA;1, Lyces;CycB1;1 and Lyces;histone H4) were then analysed (Fig. 4). Indeed, the mitotic activity is related to the differential expression of CDK and cyclins in tomato roots (Joubès et al., 2001). As CDKA is characteristic of the competence of the cell to divide (Mironov et al., 1999), this suggests that under starvation conditions the ability of root cells to divide is reduced. The repression of the Lyces;PSR5 gene encoding an 20S proteasome -subunit has been observed under phosphate-starvation (Ziethe et al., 1998) and under carbon starvation (this work, Fig. 4). The 20S proteasome is a constituent of the 26S proteasome, which is an important regulator of the cell cycle progress through its specific proteolysis activity on cyclins (Genschik et al., 1994; Hilt and Wolf, 1996). It is thus tempting to associate the reduction in root growth with a strong reduction in mitotic activities inside root tissues.
Once in the survival phase, the roots exhibit changes in the process of remobilization. As shown in Fig. 2, the degradation of protein becomes very effective, the amino acid content does not increase any further and then declines strongly as the content in NH4+ ions increases concomitantly. Altogether these data suggest that the amino acids released by protein breakdown are not stored within the cells any more. Such changes in metabolism support a co-ordinated modification of the enzymatic machinery. The synthesis of enzymes involved in the adaptation to starvation was enhanced, like those for the catabolism of fatty acids (Dieuaide et al., 1992, 1993), amino acids (Brouquisse et al., 1991) and proteins (Tassi et al., 1992; James et al., 1993). Moreover, it is tempting to suggest that, in the survival phase, although the induction of the Lyces;AS1 transcript persists, the asparagine pool decreases as it is probably consumed to supply respiration (Fig. 3).
Finally, the disorganization phase is characterized by the decline in the NH4+ content, which is then released into the incubation medium (Fig. 2), and the drop in root viability and respiration (Fig. 3). Moreover, the most remarkable symptom of this phase is the occurrence of apoptosis characterized by the nuclear DNA fragmentation (Fig. 5). Hence after 4 d, carbohydrate depletion induces the arrest of the metabolism and triggers apoptosis.
This work shows that tomato roots are very sensitive to the shortage in supply of photoassimilates. Increasing knowledge of the mechanisms that regulate carbon partitioning in whole plant organs, as well as unravelling the reasons for the marked response of roots to sugar depletion, may be useful for tomato breeders in order to improve fruit production and, consequently, fruit quality.
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Acknowledgements |
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We thank A Roos and JP Desbiens for managing the phytotronic chambers and the cultures. We wish to thank Drs R Brouquisse for critically reading the manuscript and P Scott for language corrections. This work was partly funded by a grant from Région Aquitaine, and AIP Agraf-INRA (‘Elaboration de la qualité des fruit’).
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