ABSTRACT: In this investigation an attempt has been made to compare the action of different polyamines such as putrescine (Put), spermidine (Spd) and spermine (Spm) in delaying the dark incubation induced alterations in primary reactions of photosynthesis in maize plants. Among these polyamines spermine (25µM) is able to provide more protection in terms of loss of chlorophyll (Chl), protein content and regaining of various electron transport activities. In addition to this, these PAs stabilize the thylakoid membranes of 72 h dark incubated samples in valency dependent manner and restore the excitation energy distribution between two photosystems (PS II and PS I)

G Bhanumathi

Murthy S D S

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Received: 6th Aug-2014                           Revised: 29th  Aug-2014                          Accepted: 30th Aug-2014 
Research article 
ROLE OF POLYAMINES IN RETARDATION OF DARK INCUBATION INDUCED 
ALTERATIONS IN THE PRIMARY PROCESSES OF PHOTOSYNTHESIS IN MAIZE (ZEA 
MAYS) LEAVES. 
 
Bhanumathi,G. and Murthy S.D.S* 
Department of Biochemistry, SV University, Tirupati-517502, Andhra Pradesh, India.                                
Email: sdsmurthy@rediffmail.com 
 
ABSTRACT: In this investigation an attempt has been made to compare the action of different polyamines such as 
putrescine (Put), spermidine (Spd) and spermine (Spm) in delaying the dark incubation induced alterations in 
primary reactions of photosynthesis in maize plants. Among these polyamines spermine (25µM) is able to provide 
more protection in terms of loss of chlorophyll (Chl), protein content and regaining of various electron transport 
activities. In addition to this, these PAs stabilize the thylakoid membranes of 72 h dark incubated samples in valency 
dependent manner and restore the excitation energy distribution between two photosystems (PS II and PS I). 
Key words: Chl a fluorescence, energy distribution, electron transport, polyamines, maize plants 
 
INTRODUCTION 
Polyamines (PAs) are unique chemical agents which affect the plant growth and development [1, 2,]. Generally these 
PAs are able to stabilize the cell membranes and help in the retardation of senescence [3]. PAs have ability to 
increase the ion flux and restore the age induced alterations [4]. Several workers showed that PAs are able to delay 
the loss of photosynthetic pigments in higher plant systems [5, 6]. PAs at high concentrations (50-100µM) are able to 
cause the destruction of thylakoid membrane and inhibit the photosynthetic electron transport mediated by PS II as 
well as PS I [7]. But at low concentrations (less than 50 µM) these PAs are able to delay the aging process to protect 
the plants from senescence induced damage. Therefore in this investigation an attempt has been made to study the 
protective role of PAs in a comparative manner in dark incubated maize primary leaf segments. The parameters 
studied are contents of photosynthetic pigments, protein, electron transport activities and spectral properties.  
 
MATERIALS AND METHODS 
Primary leaf segments (4-5 cm long) were cut from 7 d old maize (Zea mays) seedlings grown under continuous 
“white” radiation of 30-35 µmol m-2 s-1. Four sets consisting of 20-25 primary leaf segments were maintained in 
double distilled water or 25 µM putrescine or 25 µM spermidine or 25 µM spermine solutions in separate test tubes. 
The tubes were kept at 25 ± 1ºC in dark for 72 h. Leaf segments were sampled from each test tube and used for 
experimental work.The Chl content was estimated according to Arnon [8] and total protein content according to 
Lowry et al [9] using bovine serum albumin as standard. Thylakoid membranes were isolated in a medium 
containing 50 mM Hepes-NaOH, pH 7.8, 400 mM sucrose, 2mM MgCl2, and 5 mM KCl, by a procedure similar to 
that of Saha et al [10] as described in Swamy et al [11]. Electon transport activities of thylakoid membranes were 
assayed according to Sabat et al [12] in 2 ml reaction buffer (50mM Hepes-NaOH, pH 7.5, 100mM sucrose, 2 mM 
MgCl2, and 5mM KCl) using a Hansatech (Kings Lynn, England) electrode. PS II activity was measured by adding 
freshly prepared p-benzoquinone (p-BQ) to 2 ml reaction buffer to a concentration of 0.5 mM.  
 
 
 
International Journal of Plant, Animal and Environmental Sciences               Page: 191                              
Available online at www.ijpaes.com 
 
Bhanumathi.G. and Murthy S.D.S                                 Copyrights@2014     IJPAES       ISSN 2231-4490 
Whole chain electron transport was assayed with 0.5 mM methyl viologen (MV). By using 0.1 mM 2,6-
dichlorophenol indophenol (DCPIP), 0.5 mM MV, 5mM ascorbate (Asc), and 10 mM 3-(3,5-dichlorphenyl)-1,1-
dimethyl urea (DCMU), PS 1 activity was measured. In all the assays, thylakoid membranes equivalent to 40 µg Chl 
were used.  Chl a fluorescence emission of control (0 h) and treated (72 h) leaf thylakoid membranes equivalent to 
20g (Chl) m-3 were investigated in presence or absence of 10 µM DCMU in a medium containing 50mM Hepes-
NaOH, PH 7.5, 100 mM sucrose, 2 mM MgCl2 and 5 mM KCl on a Jasco-FP 777 (Tokyo, Japan) 
spectrofluorometer at 25 ± 1 ºC with an excitation wavelength of 440nm. The excitation and emission slit width was 
5nm. Besides, 77 K fluorescence emission spectra were recorded on a Hitachi MPF4 (Tokyo, Japan) 
spectrofluorometer in samples prepared by mixing equal volume of 60 % glycerol and thylakoid membrane 
suspension. The samples were excited at 430 nm. The slit width of excitation and emission was 8 and 2 nm, 
respectively. 
RESULTS AND DISCUSSION 
Dark incubation for 72 h induced  loss of Chl. In control sample the Chl observed to be 2.4 mg/g.f.w of leaf tissue 
(Table 1). 72 h of dark incubation brought the Chl content 0.9 mg/g.f.w of leaf segment. This loss in Chl could be 
due to induction of certain enzymes like chlorophyllases during dark incubation. When leaf segments of maize are 
incubated for 72 h in the presence of different polyamines. Spermine seems to be more protective in minimizing the 
loss of total Chl content [13]. Similarly there is a loss of carotenoids and protein content in 72 h dark incubated 
samples and was regain to 80% of the original content due to the application of PAs during dark incubation. The 
protective effect of PAs on Chl and protein content could be due to stabilization of thylakoid membrane and 
inhibition of certain enzymes like chlorophyllases and proteases [14, 15]. After studying pigment, protein contents an 
attempt has been made to analyze the photochemical activities of the thylakoid membranes during dark incubation in 
terms of oxygen exchange measurements (Table 2). PAs are able to retain the whole chain electron activity even 72 h 
of incubation. In order to ascertain the target photosystems, both PS II and PS I catalyzed transport activities were 
measured after isolating thylakoid membranes from different samples. There is a decrease of PS II activity by 53% 
and PS I activity by 17% after 72 h of dark incubation. By inclusion of PAs during treatment maintain the loss of 
both electron transport activities depending on the valency of PAs. Chl a fluorescence is an indicator of PS II 
photochemistry in thylakoid membranes [16,17,18].  Hence Chl fluorescence was measured in the presence and 
absence of DCMU in different samples. The ratio in control sample seems to be 1.7 indicating the proper functioning 
of PS II. Dark incubation for 72 h brought the ratio from 1.7 to 1.2 indicating the impairment of PS II activity (Table 
3). The treatment of PAs brought the ratio from 1.2 to1.6 depending upon the valency of PAs. Prakash et al [19] 
showed the similar results in seedling of Cucumis plants. In order to show the influence of PAs on an energy 
distribution between the two photosystems, an attempt has been made to study the Chl a low temperature emission 
spectra by estimating the ratio of F690/F735 (Table 4). The ratio was increased from 0.70 to 0.78 in dark incubated 
samples during 72 h suggesting the inhibition in energy transfer from PS II to PS I [20]. However the treatment of 
PAs brought the ratio from 0.78 to 0.71 indicating the improvement of energy distribution between two 
photosystems. Spm is able to restore the excitation energy transfer distribution from PS II to PS I in more efficient 
manner than spd and put. Thus PAs are able to stabilize thylakoid membrane and delay the loss of photochemical 
activity during dark incubation by shielding the negative charges of thylakoid membranes [21]. 
 
Table 1: Effect of polyamines (putrescine, spermidine and spermine) on pigment and total protein in maize 
primary leaves during dark incubation. The concentration of each polyamine was 25 µM. 
Incubation 
time (h) 
mg of photosynthetic pigments 
or protein/g.f.w. 
Total Chl (a+b) Carotenoids Total roteins 
H2O  (0 h) 2.40±0.12 0.061±0.004 26.66±2.0 
H2O  (72 h) 0.91±0.08 0.032±0.001 14.28±0.4 
Putrescine  (72 h) 1.10±0.06 0.036±0.001 18.12±0.8 
Spermidine  (72h) 1.15±0.07 0.040±0.002 20.50±0.5 
Spermine  (72 h) 1.35±0.09 0.046±0.002 21.50±0.5 
 
 
International Journal of Plant, Animal and Environmental Sciences               Page: 192                               
Available online at www.ijpaes.com 
 
Bhanumathi.G. and Murthy S.D.S                                 Copyrights@2014     IJPAES       ISSN 2231-4490 
Table 2: Effect of 25 µM putrescine, spermidine and spermine on electron transport activities [Whole Chain 
(O2 consumed), Photosystem II (O2 evolved), Photosystem I (O2 consumed)] in maize primary leaves during 
dark incubation. 
Incubation 
time (h) 
Electron transport activities (µM of O2↑ 
(evolved) or↓ (consumed)/mg Chl/h). 
WCE PSII PSI 
H2O (0h) 105±8 180±16 491±46 
H2O (72h) 40±3 85±7 409±38 
Putrescine (72 h) 49±3 96±8 423±40 
Spermidine (72 h) 56±4 104±9 432±41 
Spermine  (72 h) 66±5 115±10 445±42 
 
Table 3: Retarding effect of polyamines in the presence and absence of DCMU on Chl a fluorescence in maize 
thylakoid membranes. 
Sample +/- DCMU ratio of Chl a  
fluorescence  
Control(0) 1.7 
Control(72) 1.2 
Putrescine(72) 1.4 
Spermidine(72) 1.5 
Spermine (72) 1.6 
 
Table 4: Retarding effect of polyamines on low temperature Chl a fluorescence relative intensity of PS II and 
PS I in maize thylakoid membranes. 
Sample F
690
/F
735
 
Control(0) 0.70  
Control(72) 0.78  
Putrescine (72) 0.67  
Spermidine (72) 0.68  
Spermine (72) 0.71  
 
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Bhanumathi.G. and Murthy S.D.S                                 Copyrights@2014     IJPAES       ISSN 2231-4490 
 
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ROLE OF POLYAMINES IN RETARDATION OF DARK INCUBATION INDUCED ALTERATIONS IN THE PRIMARY PROCESSES OF PHOTOSYNTHESIS IN MAIZE (ZEA MAYS) LEAVES

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ROLE OF POLYAMINES IN RETARDATION OF DARK INCUBATION INDUCED ALTERATIONS IN THE PRIMARY PROCESSES OF PHOTOSYNTHESIS IN MAIZE (ZEA MAYS) LEAVES

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