J Plant Res (2007) 120:219–228 DOI 10.1007/s10265-006-0040-5
REGULAR PAPER
Physiological implications of metabolite biosynthesis for net assimilation and heat-stress tolerance of sugarcane (Saccharum officinarum) sprouts Abdul Wahid
Received: 30 May 2006 / Accepted: 16 August 2006 / Published online: 6 October 2006 The Botanical Society of Japan and Springer-Verlag 2006
Abstract Global increase in ambient temperature is a critical factor for plant growth. In order to study the changes in growth over short intervals, various primary and secondary metabolites, and their relationships with thermotolerance, 1-month-old sugarcane (Saccharum officinarum) sprouts were grown under control conditions (28C) or under heat-stress conditions (40C), and measurements were made at six 12-h intervals. Heat stress greatly reduced dry matter and leaf area of sprouts initially but only nominally later on. Changes in the rates of relative growth and net assimilation were greater than relative leaf expansion, indicating an adverse effect of heat on assimilation of nutrients and CO2 in producing dry matter. Although reduction in leaf water potential was an immediate response to heat, this effect was offset by early synthesis of free proline, glycinebetaine and soluble sugars (primary metabolites). Among secondary metabolites, anthocyanin synthesis was similar to primary metabolites; carotenoids and soluble phenolics accumulated later while chlorophyll remained unaffected. Relationships of growth attributes and metabolite levels, not seen in the controls, were evident under heat stress. In summary, observed changes in metabolite levels were spread over time and space and were crucial in improving net assimilation and heat tolerance of sugarcane. Keywords Heat stress Metabolites Net assimilation Osmotic balance Oxidative stress
A. Wahid (&) Department of Botany, University of Agriculture, Faisalabad 38040, Pakistan e-mail:
[email protected]
Introduction Heat stress, defined as a rise in temperature beyond threshold levels sufficient to cause irreversible damage, greatly limits growth and yield of crop plants when it occurs transiently or continually. Rising temperatures lead to altered geographical distribution and altered growing season of crops by changing threshold temperatures for growth, and causing plants to reach maturity sooner (Porter 2005). According to the current prognosis, global mean temperature will rise by 0.3C per decade and about 1C above the present value by 2025 (Houghton et al. 1990; Jones et al. 1999). With a rise in temperature comes diversion of and increase in metabolic activities leading to increased energy demand for growth (Rawson 1988; Karim et al. 2000). Leaf photosynthesis is directly impinged upon by heat stress, and any decline in it limits the supply of photoassimilates to keep pace with normal growth (Ebrahim et al. 1998; Karim et al. 2000; Camejo et al. 2005). Elevated temperature affects the metabolic pathways mainly through oxidative damage to cells, thereby affecting the levels of both primary and secondary metabolites, which are of great biological significance. Primary metabolites, originating from initial carbon reactions, directly participate in the osmotic adjustment and build-up of cellular structures (Taiz and Zeiger 2002). Of these, accumulation of free proline, glycinebetaine and soluble sugars is of great significance in regulating osmotic activities and protecting cellular structures from water, salt and other stresses that produce osmotic strain on the cells (Matysik et al. 2002; Wang et al. 2003; Bohnert et al. 2006). Characteristic roles of these metabolites under temperature extremes certainly merit further investigation.
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Secondary metabolites are synthesized from the intermediates of primary carbon metabolism. Of those, chlorophylls synthesized via the Beale pathway (Schoefs and Bertrand 2005), carotenoids formed via the terpenoid or isoprenoid pathway, and phenolics produced via the shikimate pathway (Buchanan et al. 2000) are the most studied in the plant kingdom. Enhanced synthesis of these metabolites under stressful conditions is believed to protect the cellular structures from oxidative damage (Chalker-Scott and Fuchigami 1989; Close and McArthor 2002; Winkel-Shirley 2002; Wahid and Ghazanfar 2006), while others promote defensive action against herbivores and pathogens (Harborne and Williams 2000; Taiz and Zeiger 2002). Decrease in chlorophyll content is associated with enhanced expression of chlorophyllase activity under stress (Majumdar et al. 1991). Carotenoids (carotenes and xanthophylls), in addition to acting as accessory light-harvesting pigments, also show antioxidant action (Havaux 1998; de Pascale et al. 2001). Moreover, they protect the photosystems by (a) reacting with lipid peroxidation products to terminate chain reactions, (b) scavenging singlet oxygens and dissipating the energy as heat, (c) reacting with triplet-excited chlorophyll molecules to prevent formation of singlet oxygen or (d) dissipating excess excitation energy through the xanthophyll cycle (Rmiki et al. 1999). A steady-state level of carotenoids was correlated with salt tolerance and forms important salinity tolerance strategies of sugarcane (Kanhaiya 1996; Ahmad et al. 2005; Wahid and Ghazanfar 2006), but comparable information is scanty for cases of heat stress. The phenolics are powerful antioxidants in plant tissues under stress (Dixon and Paiva 1995; Sgherri et al. 2004). They are chemically heterogeneous compounds and include flavonoids, lignins and tannins. They play a variety of roles, e.g., they defend against herbivores and pathogens, lend mechanical support, attract pollinators, absorb high energy radiations and reduce the growth of nearby competing plants (Harborne and Williams 2000; Taiz and Zeiger 2002). Recently, the role of phenolics has been revisited because of evidence about their greater involvement in oxidative stress tolerance than as a defense against herbivory (Close and McArthor 2002; Wahid and Ghazanfar 2006). Anthocyanins are highly water soluble and produced under a variety of stresses including UV-B (Mendez et al. 1999), drought (Balakumar et al. 1993), low temperatures (Krol et al. 1995), nutrient deficiency (Rajendran et al. 1992), ozone (Foot et al. 1996), and salinity (Wahid and Ghazanfar 2006). Their accumulation under heat stress merits further research.
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Growth analysis is a functional approach to study plant development (Poorter 1989) and can be employed for clear understanding of plant stress responses. Determination of metabolic changes and their implications for better crop growth under stressful conditions is a priority area of research (Bohnert et al. 2006). No previous report describes the time-course changes in plant growth and their relationships to metabolite accumulation. It is predicted that changes in cell metabolites are important in the modulating heat-stress response of plants. This paper describes the relationships of time-course changes in growth attributes and primary and secondary metabolite levels and their significance in heat tolerance of sugarcane, a multipurpose crop of great economic value and a prime source of global sucrose production.
Materials and methods Experimental details and growth conditions Single-node sets of sugarcane (Saccharum officinarum L. var. NCO-310) were sown in pots containing wellmanured loam and kept in a greenhouse (28–29/21– 24C day/night). Experimental design was completely randomized with three replications. The pots were watered every other day to keep the soil moisture up to required levels. Thirty days after sprouting, one half of the pots were transferred to a programmable illuminated growth chamber (Sherer-Gillett, Marshal, MI) set at day/night temperature of 28/23±1C (control), while the other half were moved to another similar chamber, in which temperature was raised from 28 to 40C in about 4 h. For the rest of the experimental period, the day/night temperature was set at 40/35±1C (heat stress). Growth conditions other than temperature in both chambers included soil moisture to field capacity; day length 13 h; PAR 500–550 lmol m–2 s–1 provided with fluorescent, incandescent and mercury lamps; and relative humidity 60/65% (day/night). Sprouts were harvested 12, 24, 36, 48, 60 and 72 h after exposure to heat stress. Leaf water potential and growth determinations Water potential of penultimate leaf was determined using a pressure chamber (Soil Moisture Equipment, Santa Barbara, CA) during the light period before harvest. Leaf area of intact plants was determined as leaf length · leaf width · 0.68 (correction factor calibrated for all leaves). To determine dry weight, shoots were put in paper envelopes and dried in an oven at 70C for
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1 week. One set of plants was harvested just before exposure to heat stress in order to get the baseline information for deriving relative growth rate, relative leaf expansion rate and net assimilation rate using the formulae of Hunt (1982). Determination of metabolite levels Freshly excised leaves were immediately frozen in liquid N, ground to a fine powder, transferred to falcon tubes and stored at –80C until analysis. Free proline was determined from the frozen fresh leaf powder that was extracted with sulphosalicylic acid and the extracts reacted with acid-ninhydrin, as described by Bates et al. (1973). Glycinebetaine was determined using the method of Grieve and Grattan (1983). Tissue extracts prepared by vigorous shaking in 2 N H2SO4 were cooled and mixed with an equal volume of periodide, vortexed and kept at 0–4C for 16 h. The mixture was centrifuged at 10,000g at 4C for 15 min and the supernatant was aspirated while cool. The periodide crystals were dissolved in 1, 2-dichloroethane to measure the absorbance at 365 nm using a spectrophotometer (DU 650, Beckman-Coulter, CA). For the determination of soluble sugars, the frozen powder was extracted in water at 80C by continuous shaking for 4 h and vacuum filtered. An aliquot of the filtrate was reacted with anthrone reagent by heating in a water bath at 100C for 20 min and the absorbance of the colored complex was measured at 620 nm (Yoshida et al. 1976). For the extraction of chlorophylls and carotenoids, the above powder was extracted in 80% acetone in black bottles and vacuum filtered; absorbance of the extract was determined immediately using a spectrophotometer and quantities determined as described by Gitelson et al. (2001). Specific absorption coefficients of chlorophylls and carotenoids were used as reported by Lichtenthaler (1987). Tannic acid-equivalent soluble phenolics were determined spectrophotometrically from 80% acetone extracts of leaves as described by Julkenen-Titto (1985). Anthocyanins were determined after extraction of leaves in acidified methanol (1% HCl v/v), vacuum filtered and quantified using spectrophotometer at 535 nm according to the method described by Stark and Wray (1989). Statistical analysis The experiment was conducted twice and all the determinations were made in triplicate. Data from both the experiments were pooled to perform statisti-
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cal analysis using COSTAT software. LSD values were determined, and Tukey’s test (Steel et al. 1996) was used to ascertain the significance of temperature treatments and time points. Correlations were drawn between various growth attributes and levels of primary (free proline, glycinebetaine and soluble sugars) and secondary (soluble phenolics, anthocyanins, chlorophylls and carotenoids) metabolites of sugarcane sprouts both under control and heat-stress conditions.
Results Growth characteristics Data revealed significant differences (P£0.001) under control and heat stress conditions as well as across different sampling times for dry weight and leaf area of sugarcane shoots (Table 1). Increase in dry weight with time was greater in the control than heat-stressed shoots. Leaf area differed significantly (P£0.001) between the treatments. After an initial decrease, a steady increase in leaf area was noted at later hours of heat stress (Table 1). Changes in shoot dry weight and leaf area substantially changed the derived growth attributes. Heat stress substantially reduced the relative growth rate (an index of dry matter production over time) of sprouts compared with the control, resulting in significant differences in treatments (P£0.05) and time points (P£0.001). Although relative growth rate decreased in the initial hours of heat stress, it showed a steady gain later on (Table 1). Relative leaf expansion rate (an estimate of changes in photosynthetic area) of control sprouts did not differ much at different time points. However, under heat stress, relative leaf expansion rate of sprouts indicated no remarkable increase during initial hours of stress but a gain later on. These changes resulted in a significant (P£0.001) difference between the treatments but not among the time points (Table 1). Net assimilation rate (an estimate of plant’s efficiency in using CO2 and available nutrients in dry matter accumulation) revealed significant differences between control and heat stress, as well as across time points (P£0.01). Although net assimilation rate increased both under control and heat-stress conditions, a greater increase (59%) was discernible in control than heat-stressed (50%) sprouts. Net assimilation rate increased steadily in control plants at all time points, but under heat stress it stopped initially and increased exponentially at later hours (Table 1).
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Table 1 Time-course changes (mean±SD) in dry weight, leaf area, relative growth rate (RGR), relative leaf expansion rate (RLER) and net assimilation rate (NAR) of sugarcane shoots under heat stress applied during the seedling stage Treatments
Sampling time (h)
Dry weight (g shoot–1)
Leaf area (cm2 shoot–1)
RGR (mg g–1 12 h–1)
RLER (mm2 cm–2 12 h–1)
NAR (mg cm–2 12 h–1)
Control
12 24 36 48 60 72 12 24 36 48 60 72
1.63±0.09 1.70±0.13 1.78±0.15 1.90±0.14 2.02±0.15 2.19±0.20 1.58±0.04 1.62±0.07 1.66±0.08 1.73±0.12 1.80±0.10 1.89±0.09
13.62±0.59 14.21±0.31 14.92±0.40 15.58±0.37 16.21±0.82 16.90±0.63 13.06±1.13 13.21±1.08 13.36±1.01 13.61±0.79 14.05±0.91 14.53±1.02
1.46±0.53 1.42±0.83 1.76±0.54 2.38±0.88 2.28±0.21 2.88±0.94 0.90±0.59 0.86±0.49 0.91±0.35 1.44±0.62 1.62±0.48 1.68±0.51
0.017±0.0054 0.016±0.0063 0.018±0.0029 0.016±0.0019 0.014±0.0034 0.015±0.0011 0.003±0.0003 0.004±0.0020 0.004±0.0006 0.007±0.0007 0.012±0.0049 0.012±0.0095
0.30±0.13 0.34±0.21 0.42±0.14 0.58±0.22 0.56±0.07 0.73±0.25 0.22±0.15 0.21±0.11 0.22±0.08 0.36±0.15 0.41±0.13 0.44±0.14
0.08*** 0.15*** 0.20ns
0.62*** 1.07*** 1.51ns
0.423* 0.732*** 3.28ns
0.0034*** 0.0060 ns 0.0266ns
0.107** 0.185** 0.261ns
Heat
LSD (P<0.05) Treatment (T) Harvest time (H) T·H ns Non-significant *P£0.05, **P£0.01, ***P£0.001
Leaf water potential and primary metabolites Sugarcane sprouts under control conditions showed no change in the leaf water potential, but a sharp reduction in this attribute was noted at the 12-h time point under heat stress, followed by a steady state at later hours, thereby showing a significant (P£0.001) interaction of treatments and time points (Fig. 1). Linear accumulation of soluble sugars, free proline and glycinebetaine was evident in sprouts due to heat stress, although none of these metabolites accumulated under control conditions. Of these, accumulation of free proline indicated a more sharp and prolonged accumulation (up to 60 h) followed by glycinebetaine and soluble sugars (48 h each). Changes in the levels of these metabolites indicated significant (P£0.001) interactions of treatments and time points (Fig. 1). Secondary metabolites There was no significant (P‡0.05) change in the content of chlorophyll under control or heat stress, although during the initial hours of heat stress this metabolite decreased slightly, but increased to a level approaching the control leaves at later hours (Fig. 2). In contrast to chlorophyll, the carotenoids of sprouts showed significant (P£0.001) differences both in the time points and treatments as well as significant (P£0.001) interaction between these factors. The level of carotenoids remained steady in the control. Under heat stress, carotenoids did not change initially, began to increase at 36 h and attained a steady level at 48 h. The changes
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in both these photosynthetic pigments led to a significant (P£0.01) increase in the carotenoids-to-chlorophylls ratio under heat stress (Fig. 2). Levels of soluble phenolics increased after an initial lapse of 24 h, resulting in significant (P£0.01) differences for time points, treatments and their interactions (Fig. 2). Levels of anthocyanins showed a preferential and sharp increase within 24 h of exposure to heat stress, resulting in significant (P£0.001) difference in the treatments, time points and their interactions. The changes in the levels of soluble phenolics and anthocyanins indicated no change in soluble phenolics-to-anthocyanins ratio under control but a significant (P£0.001) decrease under heat stress (Fig. 2), thereby indicating the physiological implications of the accumulation of these metabolites due to high temperature. Relationships of growth attributes and metabolite levels Correlation coefficients of absolute and derived growth attributes with the levels of primary and secondary metabolites are presented in Table 2. Shoot dry weight and leaf area of sprouts exhibited no relationships with any metabolite under control conditions, but exhibited significant relationships with all the metabolites as well as ratios of carotenoids to chlorophylls, but not chlorophyll content and soluble phenolics-to-anthocyanins ratio under heat stress. Relative leaf expansion rate, although positively correlated with glycinebetaine and negatively with soluble sugars and soluble phenolics
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a
a
a
1.2
70 a
1.0 b
Control Heat stress
0.8 0.6 0.4
c
c
c
c
c
c
0.2
f
f
f
a
a
d
d
b
50 c 40 30
d e
20 10 f
f
f
35
35 a
30
b
b 25 c
a
c
20 15 d
d
d
d
d
d
5 0 12 24 36 48 60 72 Sampling time (h)
under control did not change in parallel with any of the metabolites under heat stress.
Discussion Plant performance under stressful conditions is best measured in terms of changes in visual growth (Vollenweider and Gunthardt-Goerg 2005), and timedependent absolute or relative changes in growth attributes are important indicators of stress effects (Poorter 1989; Guilioni et al. 1997; Ismail and Hall 1999; Wollenweber et al. 2003). Sugarcane sprouts in this study displayed time-dependent reduction in absolute growth and alterations in the derived growth attributes under heat stress (Table 1). Data revealed that, although relative leaf expansion rate was more affected by heat stress in initial hours, both relative growth rate and net assimilation rate were more affected than relative leaf expansion rate (showing 68, 66 and 20% reduction respectively at 72 h). This implied that short-term heat stress hampers the efficiency of plants to absorb and assimilate available nutrients from soil and CO2 from the atmosphere in dry matter production during initial hours of stress. It was, however, interesting to note that at later hours of heat stress, the
Glycinebetaine (µg g-1 fresh weight)
Soluble sugars (µg g-1 fresh weight)
a
60
0
0.0
10
a
a Free proline (µg g-1 fresh weight)
1.4
Water potential (-M Pa)
Fig. 1 Time-course changes in leaf water potential and levels of some primary metabolites in sugarcane sprouts under heat stress. P£0.001 for all these variables. Points marked by the same letters involve an interaction (P£0.001) between treatments and time points
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30 25
a
b
20
c
15 10
d
5 0
d
d
d
d
12 24 36 48 60 72 Sampling time (h)
sprouts showed a remarkable increase in both absolute and relative growth attributes (Table 1). This appeared to be an adaptive response to heat stress due to metabolic changes taking place in the sprouts. Plants exposed to supra-optimal temperatures show substantial alterations in the levels of both primary and secondary metabolites as assessed from apparent growth and tissue analysis (Winkel-Shirley 2002; Wang et al. 2003; Sharkey 2005; Wahid and Close 2007). Accumulation of these metabolites is of great significance in producing tolerance to environmental stresses (Havaux 1998; Rivero et al. 2001; Sharkey 2005). A greater growth reduction during initial hours of heat stress appeared to be related to the hampered leaf water potential as an initial response (Fig. 1) because the excessive evapo-transpiration generated osmotic strain on sprouts. Data further revealed that free proline, glycinebetaine and soluble sugars (primary metabolites) accumulated within first 12 h of stress, notwithstanding free proline accumulated more readily and in a greater relative quantity (Fig. 1). The production of both free proline and glycinebetaine has been noted in various plant species under different stresses (De Ronde et al. 2000; Wang et al. 2003; Wahid and Close 2007); they are believed to confer tolerance by maintenance of cell water balance,
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224 120 Soluble phenolics (µg g-1 fresh weight)
Chlorophylls (mg g-1 fresh weight)
1.6
1.2
0.8
Control Heat stress
0.4
b 80 c 60 d
d
40 20
d
d
d
d
d
d
a
a
e
e
0
a
a
bc bc 0.6
bc bc bc c
bc c
0.2
Anthocyanins (µg g-1 fresh weight)
ab a
0.8
0.4
a
100 80
b 60 40
e 20 0
0.8
2.0
0.6
0.4
0.2
12 24 36 48 60 72 Sampling time (h)
membrane stability and buffering cellular redox potential (Matysik et al. 2002; Sharma and Dubey 2004; Wahid and Shabbir 2005; Wahid and Close 2007). The parallels drawn between the time-course changes in growth variables and levels of primary metabolites revealed close correlations between the two, except relative leaf expansion rate (Table 2). This accentuated that short-term exposure to heat stress had a greater effect initially but ultimately little effect on shoot development. Therefore, changes observed in cell metabolic activities are primarily related to net assimilation efficiency of sprouts. The pattern of primary metabolite accumulation, noted here, appeared to bolster the sprout’s net efficiency in assimilating available resources to produce dry matter under heat stress.
c d
0.0 Soluble phenolics:anthocyanins ratio
Carotenoids (mg g-1 fresh weight)
1.0
0.0
123
a
100
0.0
Chlorophyll:carotenoids ratio
Fig. 2 Time-course changes in the levels of some secondary metabolites in sugarcane shoots as affected by heat stress applied during the seedling stage. P‡0.05 for chlorophylls, P£0.01 for chlorophylls-to-carotenoids ratio and P£0.001 for carotenoids, anthocyanins and soluble phenolics-toanthocyanins ratio. The same letters indicate that an interaction (P£0.001) of treatments and time points exists
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e
e
e
e
1.6 1.2 0.8 0.4 0.0 12 24 36 48 60 72 Sampling time (h)
Among the secondary metabolites, heat stress produced the greatest increase in the levels of anthocyanins and soluble phenolics followed by carotenoids, whilst chlorophylls showed no significant changes (Fig. 2). Moreover, close relationships of soluble phenolics, anthocyanins and carotenoids with all growth attributes under heat stress, but not under control conditions, further substantiated the physiological implications of these metabolites in heat tolerance of sugarcane sprouts (Table 2). Modulations in the levels of carotenoids, anthocyanins and soluble phenolics are of great importance in the prevention of stress-induced oxidative damage and maintenance of osmotic balance (Chalker-Scott 1999; Gould et al. 2000; Rivero et al. 2001; Sgherri et al. 2004). However,
J Plant Res (2007) 120:219–228 Table 2 Correlations of changes in absolute and derived growth attributes with primary and secondary metabolite levels of sugarcane shoots under control and heat-stress conditions (n=6)
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Growth variables
Shoot dry weight
Leaf area
Relative growth rate
Relative leaf expansion rate
Net assimilation rate
ns Non-significant *P£0.05, **P£0.01, ***P£0.001
their mechanism of action may be spatially and temporally related. Heat stress causes the peroxidation of membrane lipids with the production of reactive oxygen species (ROS) and damaged chloroplastic membranes (Xu et al. 2006), whilst carotenoids have the ability to scavenge the ROS (Rmiki et al. 1999; de Pascale et al. 2001; Netto 2001). Considering the increased carotenoids-to-chlorophylls ratio (Fig. 2), it is plausible that carotenoids, in addition to harvesting light, provide a
Metabolites
Free proline Glycinebetaine Soluble sugars Soluble phenolics Anthocyanins Soluble phenolics-to-anthocyanins Chlorophylls Carotenoids Chlorophylls-to-carotenoids ratio Free proline Glycinebetaine Soluble sugars Soluble phenolics Anthocyanins Soluble phenolics-to-anthocyanins Chlorophylls Carotenoids Chlorophylls-to-carotenoids ratio Free proline Glycinebetaine Soluble sugars Soluble phenolics Anthocyanins Soluble phenolics-to-anthocyanins Chlorophylls Carotenoids Chlorophylls-to-carotenoids ratio Free proline Glycinebetaine Soluble sugars Soluble phenolics Anthocyanins Soluble phenolics-to-anthocyanins Chlorophylls Carotenoids Chlorophylls-to-carotenoids ratio Free proline Glycinebetaine Soluble sugars Soluble phenolics Anthocyanins Soluble phenolics-to-anthocyanins Chlorophylls Carotenoids Chlorophylls-to-carotenoids ratio
Correlation (r)
ratio
ratio
ratio
ratio
ratio
coefficient
Control
Heat stress
–0.533ns –0.737ns 0.563ns 0.795ns 0.124ns 0.179ns –0.388ns –0.730ns 0.007ns –0.458ns –0.770ns 0.535ns 0.807ns 0.174ns 0.145ns –0.406ns –0.723ns 0.029ns –0.333ns –0.647ns 0.451ns 0.765ns 0.003ns 0.254ns –0.245ns –0.808ns –0.182ns 0.707ns 0.836* –0.975*** –0.845* 0.279ns –0.580ns –0.171ns 0.263ns 0.314ns –0.394ns –0.706ns 0.482ns 0.754ns 0.094ns 0.181ns –0.302ns –0.802ns –0.099ns
0.966** 0.923** 0.974*** 0.964** 0.968** –0.709ns –0.324ns 0.944** 0.933** 0.939** 0.879* 0.952** 0.933** 0.942** –0.678ns –0.256ns 0.907* 0.896* 0.938** 0.917* 0.928* 0.960** 0.932** –0.564ns –0.279ns 0.953** 0.939** –0.718ns –0.642ns –0.660ns –0.693ns –0.710ns 0.504ns –0.227ns –0.634ns –0.597ns 0.938** 0.912* 0.931** 0.960** 0.932** –0.560ns –0.271ns 0.952** 0.938**
protective advantage against heat-induced oxidative damage. Similar accumulation and a similar role of carotenoids have been indicated in sugarcane and mungbean under salt stress (Ahmad et al. 2005; Wahid and Ghazanfar 2006). Since carotenoids are components of a light-harvesting complex and are biosynthesized in the chloroplast, it is believed that they protect the thylakoid lamellae from stress-induced lipid peroxidation due to heat, and therefore their role remains specific to the chloroplast. Although nominal,
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an initial reduction and later increase in chlorophylls under heat stress is plausible in view of the ability of the carotenoids to protect the chlorophyll biosynthetic machinery and ensure intactness of thylakoid membrane components. The role of phenolics has been recently reappraised as a protection from oxidative stress rather than protection from herbivory (Close and McArthur 2002; Moyer et al. 2002; Sgherri et al. 2004). Available data show that phenolics accumulate under a range of environmental stresses (Chalker-Scott and Fuchigami 1989) including temperature extremes (Rivero et al. 2001) and salinity (Wahid and Ghazanfar 2006). Close relationships of growth parameters (both absolute and derived) with soluble phenolics revealed that soluble phenolics have a crucial role in the heat tolerance of sugarcane sprouts (Table 2). Since the metabolism of phenolics takes place in the cytosol, it is believed that soluble phenolics themselves are the scavengers of ROS (Moyer et al. 2002). A delayed accumulation of soluble phenolics may be due to the fact that biosynthesis and accumulation of secondary metabolites involve complex reactions. Levels of anthocyanins, a subcategory of soluble phenolics, are greatly modulated in plant tissues by prevailing temperature. They have been shown to protect sensitive tissues by acting as a UV screen (Singh et al. 1999). High temperature increases anthocyanin concentration in certain plant species (Oren-Shamir and Nissim-Levi 1999; Sachray et al. 2002). Sugarcane sprouts manifested a sharp increase in anthocyanins even during initial hours of heat stress and a gradual decrease in soluble phenolics-to-anthocyanins ratio towards the end. This revealed that, among the water-soluble phenolics, the synthesis of anthocyanins is preferential and greater during heat stress (Fig. 2). Close relationships of anthocyanin biosynthesis with increased absolute and derived growth attributes (Table 2) was due to the properties of anthocyanins being highly water soluble and osmotically active (ChalkerScott 1999), as envisaged from their accumulation quite early during heat stress in this study (Fig. 2). An observed increase in anthocyanins is attributable to increased activity of two key enzymes, phenyl ammonia lyase (PAL) and chalcone synthease (CHS), leading to flavonoids and anthocyanin synthesis under high temperature (Oren-Shamir and Nissim-Levi 1999; Franc¸a et al. 2001; Sachray et al. 2002). Although accumulation of anthocyanins was determined for the whole shoot, the most probable location of their synthesis is the epidermis, where they presumably shielded the underlying mesophyll tissues from heat-stress effects.
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Earlier studies suggested a decrease under heat and excess of light in fruits (Alpert 2000), but an increase in anthocyanin synthesis in flower buds at initial developmental stages and a decrease later on, but no change in plant parts other than buds (Dela et al 2003). In this study the light intensity in the chamber was not excessive enough to cause photo-oxidative damage to sugarcane leaves, which is a plausible reason for enhanced anthocyanin biosynthesis under heat stress. Nonetheless, more directed studies on the anthocyanin content in extracted epidermal peels will elucidate its characteristic role in sugarcane heat tolerance. In summary, these findings strongly suggest that biosynthesis of both primary and secondary metabolites improves the heat-stress response of sugarcane sprouts by enhancing their net assimilation capacity. Further studies on metabolic pathways operative in plants under heat stress will increase our understanding about potential dangers of global warming. Acknowledgements I thank the Ministry of Science and Technology, Government of Pakistan, for the postdoctoral award, Terrance Donovan for providing sugarcane sets, and T.J. Close for laboratory facilities.
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