Journal of Experimental Botany, Vol. 52, No. 358, pp. 1063-1070,
May 1, 2001
© 2001 Oxford University Press
Original Papers |
Alterations in carbon and nitrogen metabolism induced by water deficit in the stems and leaves of Lupinus albus L.
1 Plant Biochemistry, Instituto de Tecnologia Química e Biológica, Apartado 127, 2781-901 Oeiras, Portugal
2 Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017 Lisboa, Portugal
Received 20 October 2000; Accepted 20 December 2000
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Abstract |
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Water deficit (WD) in Lupinus albus L. brings about tissue-specific responses that are dependent on stress intensity. Carbohydrate metabolism is very sensitive to changes in plant water status. Six days from withholding water (DAW), sucrose, glucose and fructose levels of the leaf blade had already increased over 5-fold, and the activities of SS and INVA had increased c. 1.5–2 times. From 9 DAW on, when stress intensity was more pronounced, these effects were reversed with fructose and glucose concentrations as well as INVA activity dropping in parallel. The stem (specifically the stele) responded to the stress intensification with striking increases in the concentration of sugars, N and S, and in the induction of thaumatin-like-protein and an increase in chitinase and peroxidase. At 13 DAW, the plants lost most of the leaves but on rewatering they fully recovered. Thus, the observed changes appear to contribute to a general mechanism of survival under drought, the stem playing a key role in that process.
Key words: L. albus, drought, sugars, sucrose-metabolizing enzymes, pathogenesis-related proteins.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Lupins (Lupinus spp.) are important grain legume crops for animal and human nutrition because of their high seed protein content and adaptation to dry climates (Hill, 1986
; Lopez-Bellido and Fuentes, 1986). Those plants are frequently subjected to periods of water constraint and therefore their metabolism may exhibit some adaptation to water deficit (WD).
The specific plant responses to WD are dependent on the amount and rate of water loss, the duration of the stress and the stage of plant development (Bray, 1997). Physiological alterations induced by WD in L. albus L. (mainly considered a drought avoider) have been described (Henson and Turner, 1991; Ramalho and Chaves, 1992; Rodrigues et al., 1995), but the modifications in plant metabolism remain relatively unexplored. It was proposed that during WD the lupin stem may contribute to plant survival by acting as a temporary storage organ (Rodrigues et al., 1995). If so, leaf nutrients must be transferred to the stem, which implies changes in the leaf carbon and nitrogen pools. The expression pattern of proteins may be altered by WD, as was observed for both osmotic and salt stresses (Bray, 1993; Chen et al., 1994; Zhu et al., 1995; Riccardi et al., 1998; Xiong et al., 1999). Some of the responsive proteins have a known function, but the role of others (e.g. osmotin, lipid transfer proteins, and some cell wall structural proteins) is unknown (Bray, 1993, 1997; Creelman and Mullet, 1995; Colmenero-Flores et al., 1997).
The changes induced by water deficit in the proteins and sugars of the leaf and stem that may be implicated in a general response strategy to cope with stress have been studied.
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Materials and methods |
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Plant material
Lupinus albus L. plants (cv. Rio Maior) were cultivated on sterilized 1:1:1 sand, soil, peat mixture under controlled conditions of light (240–250 µmol m-2 s-1 PAR), photoperiod (12 h), temperature (17/25 °C, night/day) and relative humidity (60–70%). Twenty-three days after sowing, WD was induced by withholding water, the plants being collected 6, 9 and 13 d later. Watering was then resumed in order to test the plant capacity for recovery. The leaf water potential was measured with a Scholander pressure chamber (model 3005 from Soil Moisture Equipment Crop, Santa Barbara, CA, USA) at predawn (
pd) and the relative water content (RWC) was determined using the formula (Rodrigues et al., 1995)
 | (001) |
Three independent experiments were performed. For each experiment 15–16 plants were used per treatment (well-watered, WW and water-stressed, WD). For enzymatic activities and sugar determinations, samples were collected at the middle of the photoperiod.
Biomass and element analysis
The samples were dried at 80 °C and weighted. For element analysis (C, N, H, and S), the dried samples were homogenized and analysed by combustion on a LECO CNHS-932 Micro Elemental Analyser from LECO Corporation, St Joseph, MI, USA.
Sugar analysis
The samples were extracted with 80% (v/v) hot ethanol (1 ml per 0.2 g sample fresh weight) for 20 min. Freeze-drying eliminated the remaining ethanol. Then the samples were extracted twice with water and the supernatants combined and analysed. Glucose, fructose, sucrose, galactose, and 
-galactosides were quantified enzymatically (Roche Molecular Biochemicals cat. no. 716260 and 428167), using the Hatterscheid and Willenbrink modification, which involved the oxidation of NADPH and reduction of INT (p-iodonitrotetrazolium violet) catalysed by diaphorase and measured at 490 nm (Hatterscheid and Willenbrink, 1991).
Enzymatic activities
The proteins were extracted twice (2.5 ml g-1 fresh weight) with Tris-HCl 50 mM, pH 7.5 containing 1 mM MgCl2, 5 mM EDTA (ethylenedinitrilotetraacetic acid), 10 mM DTT (dithiothreitol), 2 mM AEBSF [4-(2-aminoethyl)-benzenesulphonyl fluoride], 20 µM leupeptin, 20 µl ml-1 chymostatin, 1.5 µM pepstatin A, 10% (w/v) NaCl, and 5% (w/v) PVPP (polyvinylpolypyrrolidone). The extracts were centrifuged at 15 000 g at 4 °C for 15 min. The supernatant was used after desalting in PD-10 columns (Amersham, Pharmacia) to Tris-HCl 50 mM, pH 7.5. After protein and enzyme activity determinations, the samples were concentrated by centrifugation in Centricon 10 (Amicon) and equilibrated with Tris-HCl 50 mM, pH 7.5, containing 5 mM DTT, 1 mM AEBSF, 2.5 mM EDTA, 10 µM leupeptin, 10 µl ml-1 chymostatin, and 1 µM pepstatin A.
Peroxidase activity (EC 1.11.1.7) was measured and isoelectric focusing (IEF) was performed according to the methods described previously (Jackson and Ricardo, 1994, 1998). Sucrose synthase (SS, EC 2.4.1.13) was incubated at 30 °C according to Dancer et al. (Dancer et al., 1990) and the fructose determined by the Somogyi–Nelson method (Nelson, 1944). Invertase (INV, EC 3.2.1.26) was incubated at 30 °C in phosphate-citrate buffer 0.1 M and 0.1 M sucrose at pH 4.5 (acid invertase, INVA) or pH 7.0 (neutral invertase, INVN) and the reducing sugars determined by the Somogyi–Nelson method. It was observed that at pH 7.0 activity was detected for both invertases so, INVN activity was considered as activity at pH 7.0—30% activity at pH 4.5.
Western blot analysis
Protein was quantified by the Lowry method (Bensadoun and Weinstein, 1976) and the SDS-PAGE (15% T, 2.5% C) performed according to Laemmli (Laemmli, 1970). Western blots to detect TL (thaumatin-like protein), ChT (class III chitinase) and PR 10-like protein were performed as described previously (Pinto and Ricardo, 1995; Regalado and Ricardo, 1996), on PVDF (polyvinylidene difluoride) membrane with antibodies previously prepared in the laboratory. The three proteins were sequentially detected, first ChT, next TL and finally PR-10. Between each detection the membrane was soaked overnight in TBS containing 0.2% (w/v) Tween 20. No reaction was detected with the pre-immune sera.
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Results |
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When withholding water from the lupin plants a small decrease in the leaf RWC,
pd and gs was observed by day 6. By days 9 and 13 these parameters had markedly decreased, but the plants maintained the capacity to recover fully on rewatering. Indeed, 4 d after rewatering the RWC, pd and gs had been equalized in the stressed and control plants (Fig. 1). As expected, withholding water led to tissue desiccation that was intensified with time but in a tissue-specific way (Table 1 ). The leaf blade and the petiole had the highest water content but it was the blade that showed the highest water loss. The stem stele was quite peculiar in having the lowest water content and showing throughout the experiment the smallest water loss. It should be pointed out that by day 6 the leaf blade, the petiole and the stem cortex had lost a similar percentage of water.
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), submitted to drought (•) for 6, 9 and 13 d and rewatered (shaded area). The water potential of the 5th or 6th leaf was measured before the beginning of illumination (
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The effects of WD in the lupin plants were studied in relation to several parameters. The ratios of the stressed plants to the controls were used for such studies. In order to judge the changes occurring in the controls with age, absolute values of different parameters for those plants are shown in Table 2, for the whole duration of the experiment.
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As a consequence of the water deficit, the leaf biomass decreased (up to 50%), but no change was detectable in the biomass of the stem (Fig. 2
). Likely, in what concerns soluble protein, its concentration decreased in the leaf but appeared to be unchanged in the stem (Fig. 3). However, when performing element analysis it was evident that the increase in stem N and S due to WD (Fig. 4). Despite this, the total amount of N and S in the blades of remaining leaves didn't decrease, while in the petiole an increase in the N content was observed. Presumably the increased stem N and S was a result of reserve translocation from senescing leaves before being discarded by the plant.
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Considering carbohydrates, changes were detected at a very early stage of the stress (by day 6 of withholding water; Fig. 5
). As shown in the figure, glucose, fructose and sucrose concentrations of the leaf blades increased 5–7 times relative to the well-watered plants. As the stress progressed, by day 9, the increments in these sugars were less evident and by day 13, while sucrose concentration still remained higher, that of reducing sugars had dropped. Concerning the petiole, the marked increase in sucrose with stress severity should be emphasized (c. 18-fold at day 13; Fig. 5) which suggests an increase in sucrose export out of the leaf. Detected levels of galactose and -galactosides were very low, and no marked changes were observed with stress. These sugars, normally associated with the seed desiccation process (Horbowicz and Obendorf, 1994; Tabaeizadeh, 1998), apparently are not involved in the WD responses of lupin vegetative tissues. The carbohydrate changes in the stem were not so impressive, but the 2-fold increase in the stele concentrations of glucose, fructose and sucrose, relative to the control should be noted (Fig. 5). It is striking that the changes in leaf blade glucose and fructose are paralleled by the changes in invertase activity, particularly of INVA (Fig. 6). SS activity shows a completely different pattern, continuously increasing in the blade with stress severity (Fig. 6) that is consistent with earlier findings (Wardlaw and Willenbrink, 1994; Tabaeizadeh, 1998), which described the increase in enzyme activity with WD and sugar accumulation.
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In addition to carbohydrate metabolism, other processes were affected by stress. When lupins were subjected to WD, the peroxidase level increased in the blade and, still more drastically, in the stem's stele (Fig. 7
). Despite this large peroxidase increase it was not possible to ascribe it to any particular isoform, a basic peroxidase (pI 8.8) being the major form present (results not shown). TL, ChT and PR-10 like protein were detected in the apoplastic fractions, but were only barely detectable in the tissues that were previously extracted for intercellular fluid (data not shown). The petiole Western blot for these proteins was identical to that of the blade and for the stele identical to that of the cortex (data not shown). The expression of these proteins was under developmental control in the leaves (both blade and petiole) and it seemed that WD intensified the expression of ChT and TL (Fig. 8A). In the stem, PR-10 and ChT, but not TL, were present in the unstressed plants and WD strongly induced TL and ChT and barely affected PR-10 (both in the cortex and stele; Fig. 8B).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Studies of water stress have been mainly directed to the physiology of the whole plant and to leaf blade metabolism, namely photosynthesis (Schulze, 1986
; Chaves, 1991; Tabaeizadeh, 1998; Teraza et al., 1999; Cornic, 2000), and less attention has been paid to other organs. In these studies with L. albus tissue-specific reactions dependent on stress intensity were detected, which appear as distinct strategies to cope with WD. Typical stress avoidance mechanisms occurred such as stomata closure and leaf senescence while the metabolic changes observed in the stem cortex and stele could be indicative of some tolerance to dehydration. Indeed, despite the massive loss of leaf biomass, plants were able to survive for up to 13 d and regrow on rewatering.
When withholding water, the first signs of stress in L. albus involved pronounced changes in sugar metabolism despite small variations in the water status parameters. Losses in water content of the same order of magnitude (10–15%) have been found to cause large changes in plant growth and metabolism (Cheng et al., 1993; Mullet and Whitstitt, 1996). The observed increase in the concentration of soluble sugars may be the result of growth being more inhibited by WD than photosynthesis, as well as an increased partitioning of fixed carbon to sucrose, as shown for various species (including lupins) under WD (Chaves, 1991; Quick et al., 1992). This accumulation of soluble sugars may be related to osmoregulation and desiccation tolerance (Morgan, 1984; Hare et al., 1998) contributing to plant survival.
In addition, it is known that sucrose and other sugars regulate the expression of many genes involved in photosynthesis, respiration, N and secondary metabolism as well as defence processes (Koch, 1996; Jang and Sheen, 1997; Hare et al., 1998; Halford et al., 1999). Thus, sugar-regulated genes are a means of integrating cellular responses (sugar transport, allocation and utilization) affecting plant development and stress response (Koch, 1996; Yu, 1999). The large alterations observed in L. albus sugar metabolism preceded the drastic decrease of soluble leaf protein, the accumulation of N and S in the stem and petiole and, also, the increase in stem ChT, TL and peroxidase. These proteins are typically related to stress responses, such as freezing, osmotic and salt stress and pathogen attack (Chen et al., 1994; Yun et al., 1996; Moons et al., 1997; Riccardi et al., 1998; Tabaeizadeh, 1998; Trudel et al., 1998). Thus the WD response of lupins seems to have characteristics in common to other adverse conditions in agreement with suggestions made for other species (Shinozaki and Shinozaki, 1996; Tabaeizadeh, 1998).
Some reports suggest that the pathway for PR-gene induction (chitinase and turgor-responsive PR-proteins) is related to soluble sugars (Herbers et al., 1996) or to alterations in INVA activity (Herbers and Sonnewald, 1998). In this context, the particular response of L. albus INVA activity that seems to be related to WD intensity should be noted, increasing under mild stress and dramatically decreasing with severe WD. The conflicting data in the literature concerning invertase behaviour under WD could therefore be explained by the degree of stress. INVA may have a role not only in the sucrose-sensing pathway but also in the integration of signals for defence responses and could be considered as a central modulator of these processes (Kingston-Smith et al., 1999; Roitsch, 1999).
The physiological significance of the increase in L. albus peroxidase activity and in TL and ChT under WD could be related to changes in the cell wall properties potentially important for the stem in order to cope with the stress. TL can participate in the cell wall metabolism, due to the ability of some of its forms to bind cell wall polymers (Trudel et al., 1998) and to hydrolyse water-soluble and -insoluble complex ß-1,3-glucans (Grenier et al., 1999). Peroxidase can also alter the cell wall properties by promoting the cross-linking between molecules like lignin, suberin, proteins, hemicelluloses, and ferulic acid (Espelie et al., 1986; Fry, 1986; Barceló, 1995; Krishnamurthy, 1999). The fact that in the stele N and S content and peroxidase activity markedly increased could suggest that the stele is a region of protein insolubilization. Such insolubilization of cell wall proteins had already been observed as a result of osmotic stress and was considered to contribute to the adjustment of cell wall elasticity despite the water loss (Marshall et al., 1999). Since WD causes the formation of active oxygen species, an additional function of the increased peroxidase activity could be the protection against oxidative damage (Tabaeizadeh, 1998).
The fact that lupins subjected to WD recovered after rewatering, despite a massive loss of leaves, suggests that WD triggered a defence/resistance/adaptation mechanism in these plants, particularly in the stems. Sugars explain plant desiccation tolerance only in part (Farrant et al., 1993) and water stress-responsive proteins (ChT, TL and peroxidase) may play an additional important role in tolerating WD (Ingram and Bartels, 1996; Pelah et al., 1995, 1997). Thus, the stem appears to play a central role in the strategy to overcome WD being a transient reservoir of nutrients, a survival structure able to withstand the stress and allowing plant reconstruction when water is available.
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Acknowledgments |
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This work was financed by PRAXIS XXI programme (2/2.2/BIA/227/94 and BD/16137/98). We would like to thank Ana Paula Regalado and Maria Paula Pinto for the antibodies, and Leonor Osório for her help on water parameters assays.
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Notes |
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3 To whom correspondence should be addressed. Fax: +351 213 635031. E-mail: mchaves{at}isa.utl.pt
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Abbreviations |
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ChT, chitinase; INV, invertase; PR-10, pathogenesis-related protein of class 10; RWC, relative water content; SS, sucrose synthase; TL, thaumatin-like protein; WD, water deficit.
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