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Journal of Stress Physiology & Biochemistry, Vol. 9 No. 3 2013, pp. 262-271 ISSN 1997-0838 Original Text Copyright © 2013 by Jain, Mittal, Gadre
ORIGINAL ARTICLE
Effect of PEG-6000 Imposed Water Deficit on Chlorophyll Metabolism in Maize Leaves
Meeta Jain*, Mini Mittal and Rekha Gadre
School of Biochemistry, Devi Ahilya University, Takshashila Campus, Khandwa Road, Indore - 452 017, India.
*E-Mail: meetajainind@yahoo.com
Received April 2, 2013
Drought stress is one of the major abiotic constraint limiting plant growth and productivity world wide. The current study was undertaken with the aim to investigate the effect of water deficit imposed by PEG-6000, on chlorophyll metabolism in maize leaves to work out the mechanistic details. Leaf segments prepared from primary leaves of etiolated maize seedlings were treated with varying concentrations of polyethylene glycol-6000 (PEG-6000; w/v- 5%, 10%, 20%, 30%) in continuous light of intensity 40 Wm-2 at 26±2 °C for 24 h in light chamber. The results demonstrate a concentration dependent decline in chlorophyll content with increasing concentration of polyethylene glycol-6000 (PEG-6000). Reduction in chlorophyll 'a' level was to a greater extent than the chlorophyll 'b'. The RNA content decreased in a concentration dependent manner with PEG, however, proline content increased significantly. Relative water content decreased significantly with the supply of 30% PEG only. A substantial decrease in chlorophyll synthesis due to significant reduction in ALA content and ALAD activity, with no change in chlorophyllase activity with the supply of PEG suggests that water deficit affects chlorophyll formation rather than its degradation.
Key words: Chlorophyll metabolism, PEG-6000, Zea mays
ORIGINAL ARTICLE
Effect of PEG-6000 Imposed Water Deficit on Chlorophyll Metabolism in Maize Leaves
Meeta Jain*, Mini Mittal and Rekha Gadre
School of Biochemistry, Devi Ahilya University, Takshashila Campus, Khandwa Road, Indore - 452 017, India.
*E-Mail: meetajainind@yahoo.com
Received April 2, 2013
Drought stress is one of the major abiotic constraint limiting plant growth and productivity world wide. The current study was undertaken with the aim to investigate the effect of water deficit imposed by PEG-6000, on chlorophyll metabolism in maize leaves to work out the mechanistic details. Leaf segments prepared from primary leaves of etiolated maize seedlings were treated with varying concentrations of polyethylene glycol-6000 (PEG-6000; w/v- 5%, 10%, 20%, 30%) in continuous light of intensity 40 Wm'2 at 26±2 °C for 24 h in light chamber. The results demonstrate a concentration dependent decline in chlorophyll content with increasing concentration of polyethylene glycol-6000 (PEG-6000). Reduction in chlorophyll 'a' level was to a greater extent than the chlorophyll 'b'. The RNA content decreased in a concentration dependent manner with PEG, however, proline content increased significantly. Relative water content decreased significantly with the supply of 30% PEG only. A substantial decrease in chlorophyll synthesis due to significant reduction in ALA content and ALAD activity, with no change in chlorophyllase activity with the supply of PEG suggests that water deficit affects chlorophyll formation rather than its degradation.
Key words: Chlorophyll metabolism, PEG-6000, Zea mays
Plants are subjected to various abiotic stresses due to unfavourable environmental conditions that affect their growth, metabolism and productivity (Kaur and Gupta, 2005). Drought is one of the major abiotic stresses which is the most limiting factor for better plant performance and higher crop yield (Szilgyi, 2003; Hirt and Shinozaki, 2003). Several physiological processes are found to be affected by
water stress, both at whole plant and cellular levels (Morgan, 1984). Inhibition of leaf growth is a primary whole plant response to water stress, which has been reported in maize, barley and rice seedlings (Lu and Neumann, 1998). Decrease in the percentage and rate of germination and seedling growth by polyethylene glycol (PEG) stress is observed in Senna occidentalis (Delachiave and de
Pinho, 2003) and Zea mays (Khayatnezhad et al, 2010). Reduction in chlorophyll level by water stress has been shown in a few systems (Albert and Thornber, 1977; Tomati et al, 1978). Drought stress also inhibits the photochemical activities and decreases the activities of enzymes of the Calvin cycle (Monakhova and Chernyadev, 2002). Exposure of plants to different stresses induce the overproduction of reactive oxygen species. As a consequence, plants evolved cellular adaptive responses, like up-regulation of oxidative stress protectors and accumulation of protective solutes (Horling et al, 2003). For drought stress induction, one of the most popular approaches involves the use of high molecular weight substances, such as, PEG (Turkan et al, 2005; Landjiva et al, 2008). It is known that PEG does not enter the cell wall space (Rubinstein, 1982) and PEG molecules with a molecular weight greater than 3000 are apparently not absorbed (Tarkow et al, 1996). In the present study, PEG-6000 was used for drought stress induction in maize leaves.
Chlorophyll is the molecule that traps 'the most elusive of all powers' and thus acts as a photoreceptor. Entire pathway of chlorophyll biosynthesis is operated in plastids by a complex set of reactions involving many intermediates. 5-aminolevulinic acid (ALA) is the universal precursor of tetrapyrroles, which is synthesized from the intact carbon skeleton of glutamate and/or 2-oxoglutarate in plants. Condensation of two molecules of ALA to form porphobilinogen (PBG) is catalyzed by 5-ALA dehydratase (5-aminolevulinate-hydrolyase EC 4.2.1.24, ALAD). ALAD is a key enzyme of common biosynthetic pathway leading to formation of tetrapyrroles and plays a major role in the regulation of chlorophyll biosynthesis (Prasad et al, 1989; Padmaja et al, 1989; Prasad and Prasad,
1990). Chlorophyllase (chlorophyll- chlorophyllido-hydrolase, EC 3.1.1.14) degrades chlorophyll into chlorophyllide and phytol. It is one of first plant enzymes identified biochemically and its enzymatic activity is widespread in plant and algal species (Shioi and Sasa, 1986; Takamiya et al, 2000). Determination of chlorophyll content of plants is often accomplished to assess the impact of most environmental stresses, as the pigment content is linked to the visual symptoms and photosynthetic plant productivity. Hence, the present investigation was aimed to investigate the effect of water deficit imposed by PEG-6000, on chlorophyll metabolism in maize leaves to work out the mechanistic details.
MATERIALS AND METHODS
Seeds of Zea mays L. cv. Ganga Safed - 2 were surface sterilized with 0.1% HgCl2 for 1-2 minutes and then washed thoroughly with tap and distilled water. Seeds were sown in small plastic pots containing acid washed sand for 6-7 days in continuous dark at 26 ± 2 °C. They were watered with V strength Hoagland's solution without N. For various experiments the first and fully elongated leaves were used.
Water stress conditions were simulated to polyethylene glycol-6000 at one of four concentrations: 0, 5, 10, 20 and 30%. The osmotic potentials of the solutions was measured using a water potential meter (Psypro Wescor Corporation, US).
PEG-6000
0% 5% 10% 20% 30%
concentration
OP (MPa) 0.00 -0.02 -0.27 -1.27 -1.80
Leaves were cut into small segments (0.5 cm2) and were treated with varying concentrations of polyethylene glycol-6000 (PEG-6000; w/v); 5 %, 10 %, 20 % and 30 % in continuous light of intensity 30
Wm-2 at 26 ± 3 °C for 24 h in light chamber. Distilled water was used as control. At the end of treatment, leaf segments were thoroughly washed with distilled water prior to analysis.
The relative water content (RWC) was measured by the method of Barr and Weatherly (1962). After measuring the fresh weight and dry weight of treated leaf segments, RWC was calculated using the following equation:-
RWC (%) = (FW - DW) / (TW - DW) X 100 Where, FW is the fresh weight of the leaf segments; DW is the dry weight and, TW is the turgid weight of the leaf segments.
The Chl a, Chl b, and carotenoids were extracted in 80% acetone and their content was calculated in Mg ml-1 using equation of Linchtenthaler and Welburn (1983).
Chl a (Mg ml-1) = 12.21 (A663) - 2.81 ^)
Chl b (Mg ml-1) = 12.21 (A646) - 5.03 (A663) Carotenoids (Mg ml-1) = [1000 (A470) - 3.27 (Chl a) -104 (Chl b)] /227
Total RNA was extracted and estimated by the method of Webb and Levy (1958) using Orcinol reagent. The proline content was estimated as described by Bates et al (1973). Amino levulinic acid was extracted and estimated by the method of Tewari and Tripathy (1998).
5-Amino levulinic acid dehydratase (ALAD) activity was assayed by estimating colorimetrically the amount of porphobilinogen formed by using modified Ehrlich's reagent (Mauzerall and Granik, 1956) according to the method mentioned in Jain and Gadre (2004). The unit of enzyme activity is expressed as the number of nmoles PBG formed h-1.
The enzyme chlorophyllase was extracted and assayed according to the method of Nag et. al (1981). The chlorophyllase activity was expressed as % of chlorophyll degraded h-1.
The data presented in the text are the average values of at least four replicate experiments with ± standard errors. One way ANOVA was performed and F values i.e. Calculated variance ratio is given in Tables.
RESULTS
Supply of 30 % PEG to excised maize leaf segments from etiolated seedlings decreased the relative water content significantly, but it was reduced slightly by 20 % PEG and remained almost same by 5 and 10 % PEG (Table 1A).
When leaf segments were treated with 5 to 30 % PEG, a concentration dependent decrease in RNA, but increase in proline content was observed (Table 1B).
Supply of 5 to 30 % PEG to maize leaf segments decreased the total chlorophyll content and carotenoids in a concentration dependent manner; however, former was decreased to a greater extent than the latter (Table 2A).
Treatment of maize leaf segments with 5 to 30 % PEG to impose water deficit caused significant decline in chlorophyll 'a' level, while, chlorophyll 'b' content decreased to a lesser extent (Table 2B).
Incubation of maize leaf segments with different concentrations of PEG decreased the ALA content as well as ALAD activity significantly, while chlorophyllase activity remained unaffected, (Table 3). Decrease in ALA content was more severe at each concentration of PEG than the inhibition of ALAD activity.
Table 1A. Effect of water deficit on relative water content (RWC) in excised etiolated maize leaf segments
during greening
PEG concentration (%) Fresh weight (mg) Dry weight (mg) u 5 £ cc
00 206 ± 5.13 (100) 17 ± 1.20 (100) 103 ± 0.88 (100)
05 205 ± 5.23 (99) 17 ± 0.33 (100) 102 ± 1.20 (99)
10 202 ± 4.91 (98) 17 ± 0.57 (100) 105 ± 1.66 (102)
20 190 ± 2.02 (92) 17 ± 1.45 (100) 96 ± 0.88 (93)
30 127 ± 8.70 (61) 15 ± 1.60 (88) 83 ± 2.02 (80)
F value 1.96 (5.96) 1.26 (5.96) 0.33 (5.96)
Critical variance ratio from F-distribution table at 5 % level of significance is given in parentheses.
Leaf segments from maize seedlings grown in continuous dark were floated on distilled water and different concentrations of PEG 6000 for 24 h in continuous light of intensity 30 Wm'2 at 26 ± 3°C.
Values relative to control are given in parentheses.
Table 1B. Effect of water deficit on proline and RNA content in excised etiolated maize leaf segments during
greening
PEG concentration (%) Proline content (mg g-1 fr.wt.) Total RNA (mg g-1fr.wt.)
00 100 ± 11 (100) 7.58 ± 0.24 (100)
05 150 ± 12 (150) 6.92 ± 0.17 (91)
10 167 ± 13 (167) 6.31 ± 0.11 (83)
20 251 ± 17 (251) 4.67 ± 0.12 (61)
30 297 ± 25 (297) 3.57 ± 0.44 (47)
F value 3.05 (5.63) 43.86 (5.63)
Critical variance ratio from F-distribution table at 5 % level of significance is given in parentheses.
Leaf segments from maize seedlings grown in continuous dark were floated on distilled water and different concentrations of PEG 6000 for 24 h in continuous light of intensity 30 Wm-2 at 26 ± 3°C.
Values relative to control are given in parentheses.
Table 2A. Effect of water deficit on total chlorophylls and carotenoids in excised etiolated maize leaf segments
during greening
PEG concentration (%) Total chlorophylls (Mg ml-1) Carotenoids (Mg ml-1)
00 19.38 ± 1.71 (100) 5.89 ± 0.40 (100)
05 12.26 ± 0.56 (63) 4.16 ± 0.69 (70)
10 11.33 ± 1.05 (58) 3.86 ± 0.49 (65)
20 9.46 ± 0.77 (48) 3.23 ± 0.41 (55)
30 6.53 ± 1.10 (33) 3.06 ± 0.26 (52)
F value 17.75 (5.63) 6.40 (5.63)
Critical variance ratio from F-distribution table at 5 % level of significance is given in parentheses.
Leaf segments from maize seedlings grown in continuous dark were floated on distilled water and different concentrations of PEG 6000 for 24 h in continuous light of intensity 30 Wm-2 at 26 ± 3°C.
Values relative to control are given in parentheses.
Table 2B. Effect of water deficit on chlorophyll 'a', chlorophyll 'b' and chlorophyll a/b ratio in excised etiolated maize leaf segments during greening
PEG concentration (%) Chl a (W5 ml-1) Chl b (MS ml-1) Chl a /b ratio
00 15.75 ± 1.37 (100) 3.64 ±0.76 (100) 5.16 ± 0.99 (100)
05 9.71 ± 0.58 (62) 2.54 ± 0.19 (70) 3.89 ± 0.42 (75)
10 8.29 ± 0.64 (53) 3.03 ± 0.73 (83) 3.38 ± 0.50 (65)
20 6.92 ± 0.47 (44) 2.53 ± 0.60 (69) 3.61 ± 0.69 (70)
30 4.44 ± 0.40 (28) 2.08 ± 0.81 (57) 3.48 ± 0.63 (67)
F value 20.40 (5.63) 00.88 (5.63) 03.93 (5.63)
Critical variance ratio from F-distribution table at 5 % level of significance is given in parentheses.
Leaf segments from maize seedlings grown in continuous dark were floated on distilled water and different concentrations of PEG 6000 for 24 h in continuous light of intensity 30 Wm-2 at 26 ± 3°C.
Values relative to control are given in parentheses.
Table 3. Effect of water deficit on ALA content, ALAD and chlorophyllase activities in excised etiolated maize leaf segments during greening
PEG concentration (%) ALA content (nmole g-1 fr.wt.) ALAD (nmole PBG formed h-1 g-1 fr.wt.) Chlorophyllase (% Chlorophyll degraded h-1 )
00 437 ± 39 (100) 218 ± 34 (100) 95 ± 4 (100)
05 373 ± 25 (85) 205 ± 43 (94) 91 ± 5 (96)
10 315 ± 21 (72) 179 ± 32 (82) 94 ± 7 (99)
20 264 ± 21 (60) 166 ± 30 (76) 93 ± 5 (97)
30 192 ± 19 (44) 113 ± 22 (52) 95 ± 5 (100)
F value 13.47 (5.63) 18.34 (5.63) 00.59 (5.63)
Critical variance ratio from F-distribution table at 5 % level of significance is given in parentheses.
Leaf segments from maize seedlings grown in continuous dark were floated on distilled water and different concentrations of PEG 6000 for 24 h in continuous light of intensity 30 Wm-2 at 26 ± 3°C.
Values relative to control are given in parentheses.
DISCUSSION Environmental biotic and abiotic stress could evoke
compensatory metabolic changes through
The RWC is considered to be a measure of plant
modification and modulation of various biochemical
water status, reflecting the metabolic activity in
parameters. The present data indicate a
tissues and used as most meaningful index of
pronounced reduction in RNA content with
dehydration tolerance (Sinclair and Ludlow, 1986).
increasing water deficit (Table 1B). Reduced RNA
In the present study, incubation of leaf segments
synthesis with increased water stress has been
with high concentration of PEG decreased the RWC
reported by He et al (1999) in wheat leaves; they
(Table 1A). Decline in RWC due to PEG induced
also suggested upregulation of chloroplast RNAase
water stress has also been reported in barley leaves
as one of the possible reasons causing the
(Yuan et al, 2005) and tomato (Zgallai, 2005) and
degradation of RNA. Furthermore, many
pigeonpea (Kumar et. al, 2011) plants.
researchers showed that the ribosomes and the proportion of polyribosomes decreased remarkably during water stress (Mason et al, 1988; Scott et al, 1979). It is known that ribosomes cluster on mRNAs to protect them from degradation. So, disruption of ribosomes may be another reason for enhanced mRNA degradation. Proline accumulation under stress has been linked with its role as an osmolyte by contributing towards osmotic adjustment between cytoplasm and vacuoles (See Delauney and Verma, 1993). Moreover, because of its zwitter ionic and highly hydrophilic character it has a role as an osmoticum and acts as a compatible solute for plants subjected to low water potential and other environmental stresses (Sampras et al, 1995). Water deficit imposition resulted in an increased proline level and there is about three fold rise in proline content at highest concentration of PEG used (Table 1B). Increase in the free proline content during water stress condition due to PEG has also been shown in pigeonpea (Kumar et al, 2011).
Photosynthetic pigments determine the physiological status of the plants. The change in the chl a/b ratio provides further information about modification process taking place in the photosynthetic apparatus. In our study, the experiments indicate the concentration dependent reduction in total chlorophylls as well as carotenoids in maize leaf segments subjected to water deficit with more pronounced effect for chlorophylls (Table 2A). Decrease in the total chlorophyll content by PEG 6000 (-5 bar) has also been noticed by Pratap and Sharma (2010) in black gram and Guo et al (2013) in wheat seedlings. Unlike this, increase in chlorophyll content has been demonstrated in graminaceous chlorophyllic cell lines of grass Bouteloua gracilis exposed to different concentrations of PEG 8000 (Garcia
Valenzuela et al, 2005). The marked reduction of total chlorophylls in water deficit leaves was due to decrease in both the chl a and chl b contents but chl a was decreased to a greater extent than chl b (Table 2B). Reduction in chlorophyll content due to water stress is also reported by Zayed and Zeid (1997/98) in mung bean seedlings and by Jeyaramraja et al (2005) in tea leaves. Decrease in level of chlorophyll and increase in proline content by water deficit suggest that glutamate availability for chlorophyll biosynthesis could be checked under water stress, as both require it for biosynthesis.
PEG-6000 imposed water deficit has affected the activities of enzymes of chlorophyll metabolism also. Thus, ALA content and ALAD activity are severely inhibited while chlorophyllase activity remains almost same (Table 3). Salinity has been found to enhance the chlorophyllase activity in pigeonpea and Gingellay which results in lowering of chlorophyll content (Rao and Rao, 1981). ALA, the key compound in the chlorophyll biosynthetic pathway is synthesized via Beale's pathway from the intact carbon skeleton of 5 carbon compounds, glutamate and/or 2-oxoglutarate. Hence, it is possible that glutamate, being precursor of proline is not made available for ALA synthesis. Increased synthesis of glutamate for proline accumulation has been suggested in groundnut cotyledons during saline stress (Satakopan et al, 1989). Further, decreased ALA content may be responsible for reduced ALAD activity. Thus, a pronounced decrease in chlorophyll biosynthesis due to decline in ALA content and inhibition of ALAD activity suggests that water deficit imposed by PEG-6000 affects chlorophyll formation rather than its degradation.
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