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Samara is the reproductive organ (seed) for many tree species in the arid land of northwestern China. It is ecologically important in population development due to its dispersal function. However, information on its photosynthesis and effect of environmental stresses on its photosynthesis is still very limited. In the present study, comparative responses of photosystem II (PSII) activity in samara and leaf of Siberian maple to short-term chilling/freezing and subsequent recovery potential were comparatively investigated by using polyphasic fluorescence test. The samara had more efficient photosynthesis (Fv/Fm and PIABS) and more efficient electron transport (fEo) but lower energy dissipation (DIo/RC) than leaf. Generally, the PSII performance and the electron transport for both samara and leaf were inhibited under low temperature stress, accompanied by an increase of energy dissipation in PSII reaction centers (RCs). PSII of both samara and leaf was not markedly affected by chilling and can acclimate to chilling stress. Short-term freezing could completely inhibit PSII activity in both samara and leaf, indicated by the drop of values of Fv/Fm, PIABS, fEo to zero. PSII functional parameters of short-term dark frozen samara could be largely recovered whereas those of frozen leaf could not be recovered. The higher tolerance of samara to short-term low temperature stress than leaf is of great ecological significance for seed development, population establishment of Siberian maple.
Keywords: samara; photosystem II; JIP-test analysis; chlorophyll fluorescence
Samara has an evolutionary advantage because the seed can be dispersed to a long distance through wings by wind (Osada et al. 2001). Samara wing was reported to have photosynthetic activity to support its own development (Ashton 1989). Evidences from anatomy and physiology show that green samaras photosynthesize and contribute to the carbon balance and growth of the fruit (Bazzaz and others 1979; Peck and Lersten 1991). Since the seed is embedded in the wings, the quality of the seed should be partly dependent on the photosynthetic performance of samara. However, information on photosynthetic process in samara is still very limited.
Siberian maple (Acer ginnala) is widely present in the arid land of northwestern China. Its fruit is a v-shape samara composed of a seed embedded in two membranous wings. The Siberian maple samara appears in late March and early April. At the same time (from March to April), the air temperature in northwestern China often drastically fell to chilling temperature or freezing temperature. Therefore the samara often encounters chilling or freezing stress during its development. The effect of low temperature (chilling and freezing) on the photosynthesis of samara is still unknown.
The fast rise Chl a fluorescence (O-J-I-P) rise transient was believed to provide important information on the photochemical activity of photosystme II (PSII) and the associated filling of the plastoquinone pool (Krause and Weis, 1991). Strasser and Strasser (1995) established a procedure for quantitatively calculating several phenomenological and biophysical parameters on the basis of O-J-I-P curve, known as the JIP-test. The fast rise Chl a fluorescence and the JIP-test has been proved to be a useful tool for investigation of PSII function under various environmental stresses (Strasser and Strasser, 1995; Eullaffroy et al., 2007; Pan et al., 2008; Pan et al., 2009).Â
In the present study, comparative effect of short-term chilling and freezing stresses on PSII activity in samara and leaf of Siberian maple and subsequent recovery were investigated by using polyphasic rise Chl a fluorescence and JIP-test analysis. The objectives of this study were to: (1) compare the responses of PSII activity between samara and leaf to low temperature stresses; (2) elucidate the mechanisms involved in the effect of low temperature on PSII in samara and leaf; and (3) assess the recovery potential of samara and leaf from the inhibition induced by low temperature stress.
2 Materials and methods
2.1 Plant material
On April 20, 2009, Siberian maple (Acer ginnala) shoot cuttings with about four-week old samaras and leaves were obtained from Xinjiang Branch of Chinese Academy of Sciences, Urumqi, China. The cuttings were grown in glassy containers containing tap water in a growth chamber with 12 h photoperiod and photosynthetic photon flux density (PPFD) of 200 Âµmol m-2 S-1 at 22-25?. The chlorophyll fluorescence of samara and leaf were monitored periodically. The JIP-test parameters for samara and leaf of cuttings grown in tap water are stable for at least two days. Therefore, tap water was used as the supporting medium for cuttings.
2.2 Treatment with low temperature
The cuttings were incubated in the dark at 4? for chilling experiment and -4? for freezing experiment, respectively. After 12-hour chilling/freezing treatment, the cuttings were returned to normal conditions (photosynthetic photon flux density (PPFD) of 200 Âµmol m-2 S-1 and temperature of 22-25?). In order to assess their recovery potential from the damage, induced by low temperature stress, the cuttings without low temperature treatment were kept under normal conditions all the time and were used as the control. Â
2.3 Polyphasic fast fluorescence induction and JIP test
When illuminated with high intensity actinic light, dark-adapted oxygenic photosynthetic organisms shows the polyphasic rise with the basic steps from the 'origin' (O) through two 'inflections' (J and I) to a 'peak' fluorescence level (P) (Strasser and Strasser 1995). The polyphasic fast-phase fluorescence induction curve provides valuable information on the magnitude of stress effects on photosystem II (PSII) function. Strasser and Strasser (1995) has developed the JIP-test for quantifying PSII function by the biophysical parameters calculated from the O-J-I-P fluorescence transient data. The JIP-test has been proved to be a valid tool for investigating of the behavior of the photosynthetic apparatus under various environmental stresses (e.g., Nussbaum et al., 2001). In the present study, samples (samara and leaf) were adapted in the dark for 5 min before measurement of chlorophyll fluorescence. The chlorophyll fluorescence transient was recorded up to 1 s on a logarithmic time scale, with a data acquisition every 10 Âµs for the first 2 ms and every 1ms thereafter, by a handheld fluorometer (Fluopen-100, Brno, Czech). Each measured O-J-I-P induction curve was analyzed according to the JIP-test (Strasser and Strasser, 1995). The following data were directly obtained from the fast rise kinetic curves: Fo, the initial fluorescence, was measured at 50 Âµs, at this time all reaction centers (RCs) are open; FJ and FI are the fluorescence intensity at J step (at 2 ms) and I step at 30ms); FM, the maximal fluorescence, was the peak fluorescence at P step when all RCs were closed after illumination; F300Âµs was the fluorescence at 300 Âµs. Selected JIP-test parameters quantifying PSII behavior were calculated from the above original data as the formulae in Table 1 (Strasser et al., 2000).
Each experiment was at least triplicated and the results were presented as mean.
Table 1 Formulae and terms used in the JIP-test (Strasser et al., 2000)
Formulae and terms Illustrations VJ = (F2ms - Fo)/(Fm - Fo)Â Relative variable fluorescence intensity at the J-step Mo = 4(F300 Âµs - Fo)/(Fm - Fo) Approximated initial slope of the fluorescence transient fPo = TRo/ABS = [1-(Fo/Fm)] = FV/Fm Maximum quantum yield for primary photochemistry (at t=0) fEo = ETo/ABS = [1-(Fo/Fm)] â€¢ ?o Quantum yield for electron transport (at t=0) ?o =Â ETo/TRo = (1-VJ) Probability that a trapped exciton moves an electron into the electron transport chain beyond QA (at t=0) ABS/RC =Mo â€¢ (1/VJ) â€¢ (1/ fPo) Absorption flux per reaction centerÂ Â Â Â Â Â TRo/RC =Mo â€¢ (1/VJ) Trapped energy flux per reaction center (at t=0) DIo/RC = (ABS/RC) - (TRo/RC) Dissipated energy flux per reaction center (at t=0) PIABS = (RC/ABS)â€¢[fPo /(1-fPo)]â€¢ Performance index on absorption basis [?o /(1-?o)]
Table 1 selected JIP-test parameters for samara and leaf. Each data represents the mean value of at least three pieces of samaras or leaves.
Fv/Fm PIABS ?o fEo ABS/RC TRo/RC DIo/RC leaf 0.798 1.433 0.45 0.359 2.263 1.807 0.456 samara 0.826 3.987 0.627 0.518 2.006 1.658 0.349
3.1 PSII activities in samara and leaf
Typical OJIP fluorescence transient curves for samara and leaf were shown in Fig. 1. Selected JIP-test parameters for samara and leaf were presented in table 1. Samara had higher electron transport flux (fEo and ?o) and higher photosynthetic efficiency (Fv/Fm and PIABS) than leaf. In the case of energy flux, samara had lower value of ABS/RC and lower value of TRo/RC than those for leaf, resulting in lower dissipated energy flux (DIo/RC) in samara than in leaf. The difference of JIP-test parameters between leaf and samara suggesting that samara had more efficient photosynthetic process with more efficient electron transport and energy regulation.
Fig. 2 JIP-test parameters for samara and leaf during chilling treatment and their recovery: the samaras and leaves were dark chilled at 4? for 12 h; After 12-hour chilling treatment, the samaras and leaves were returned to the normal conditions (at 22-25? with 200 Âµmol m-2 S-1 PPFD) to recover from chilling stress; Chlorophyll fluorescence of the samaras and leaves were measured periodically during their chilling and recovery period. Each data point represents the mean value of at least three pieces of samaras or leaves.
3.2 Effect of chilling on PSII activity and recovery
Selected JIP-test parameters for samara and leaf during their 12-hour chilling and subsequent two-hour recovery are presented in Fig. 2. The maximum PSII photochemical quantum yield (Fv/Fm) for samara and leaf declined slightly during the first 2-hour chilling and then gradually regained to the normal level during the subsequent 4-hour chilling. The values of Fv/Fm for samara and leaf changed little during the next 6-hour chilling, indicating the acclimation of the samara and leaf to chilling stress. The values of Fv/Fm fell within the range from 0.816 to 0.830 for samara and 0.783-0.804 for leaf, respectively over the chilling period. The photosynthesis performance (PIABS) for samara decreased during the first one-hour chilling and regained slightly during the next hour and then remained constant. PIABS for leaf generally showed a decreasing trend during the first six-hour chilling and then changed little. Electron transport flux (fEo and ?o) changed slightly under chilling stress. For leaf, fEo and ?o declined with increasing chilling time during the first six hours and then was kept nearly a constant value. Absorption flux per RC (ABS/RC) and trapped energy flux per RC (TRo/RC) for leaf increased during the first 6-hour chilling and then remained constant. In the case of samara, ABS/RC and TRo/RC increased during the first 4 h, decreased a little during the next 2 hours and then changed little. The change patterns of ABS/RC and TRo/RC resulted in the similar change pattern of dissipated energy flux per RC (DIo/RC).
After the samara and leaf were exposed to dark chilling stress for 12 h, they were transferred to the normal conditions for recovery. Fv/Fm, PIABS, fEo and ?o for samara changed little during recovery whereas these parameters for leaf rapidly returned to normal after cessation of chilling treatment. The energy flux (ABS/RC, TRo/RC and DIo/RC) for both samara and leaf rapidly decreased to the normal values during the 2-hour recovery.Â
3.3 Effect of freezing on PSII activity and recovery
JIP-test parameters for samara and leaf after exposed to dark freezing at -4? for 12 h and their recovery are presented in Fig. 3. Photosynthetic performance (Fv/Fm and PIABS) and electron transport per RC (fEo) were completely inhibited after the samara and leaf were exposed to freezing stress for 1h. Electron transport beyond QA for samara and leaf was reduced by 70.3% and 15.3%, respectively. On the contrary, energy flux per RC (ABS/RC, TRo/RC and DIo/RC) in samara and leaf increased drastically. The JIP-test parameters changed little during further 11-hour exposure to freezing stress. However, recovery potential of samara differed greatly from that of leaf. Photosynthesis efficiency and electron transport in 12-h frozen leaf could not recover. The values of energy flux parameters for leaf still remained at high level. For 12-h frozen samara, photosynthesis and electron transport activity significantly recovered during 3-hour recovery. The energy flux decreased to the values close to those of the control after 3-hour recovery. After 12-hour recovery, Fv/Fm for samara fully recovered to normal whereas PIABS and fEo only recovered to 20.8% and 57.6% of the control, respectively.Â
Fig. 3 Recovery of the JIP-test parameters for samara and leaf after exposed to -4? in the dark for 12 h: the samaras and leaves were exposed to -4? in the dark for 12 h and then were returned to the normal conditions (at 22-25? with 200 Âµmol m-2 S-1 PPFD) in order to recover from freezing stress; Chlorophyll fluorescence of the samaras and leaves were measured periodically during their recovery period. Each data point represents the mean value of at least three pieces of samaras or leaves.
Our study revealed that there is marked difference of PSII activity between the samara and the leaf (Fig. 1). The samara has higher photosynthetic efficiency (Fv/Fm and PIABS) and higher electron transport flux (fEo) than leaf (table 2). The higher value of ?o means that electron transport beyond QA- in samara was more efficient than leaf (Strauss et al. 2006). The higher Fv in samara further indicates the higher PSII capacity to reduce plastoquinone in samara (Bukhov et al., 1987). The differences in ?o and Fv resulted in higher electron transport flux (fEo) on the whole. The lower values of ABS/RC, TRo/RC and DIo/RC in samara than leaf shows that samara has more efficient energy regulation mechanisms than leaf.
PSII has been demonstrated to be one of the sensitive target sites for low temperature stress (Demmig-Adams and Adams, 1992; Jung and Steffen, 1997; Strauss et al., 2006; 2007; Pagter et al., 2008). In our study, OJIP fluorescence transient and the JIP-test analysis clearly showed that PSII function in samara and leaf of Siberian maple was affected by short-term chilling or freezing temperature treatment.
Similar to a number of previous studies (Demmig-Adams and Adams, 1992; Li et al. 2004; Jung and Steffen, 1997; Strauss et al., 2006; 2007; Pagter et al., 2008), the maximum primary PSII photochemical quantum yield (Fv/Fm) for samara and leaf declined under low temperature stress. Fv/Fm for samara and leaf responds to chilling stress in a different way to freezing stress. Chilling at 5? only slightly reduces Fv/Fm firstly and then returned to normal (Fig. 2-a), suggesting that PSII of both samara and leaf is not significantly affected and can acclimate to sudden chilling stress. However, freezing treatment at -4? decreased Fv/Fm for both samara and leaf to zero (Fig. 3-a), indicating that photochemical activity was completely suppressed by freezing treatment. PIABS for seems to be a more sensitive indicator under chilling stress than Fv/Fm (Fig. 2-a, b). Marked decreases in PIABS for both samara and leaf induced by chilling were observed. Several earlier studies also reported that PIABS was a much more sensitive parameter for distinguishing differences in dark chilling response than Fv/Fm (Strauss et al., 2006; 2007; Pagter et al., 2008). However, Pagter et al. (2008) pointed out that PIABS may not be more suitable than Fv/Fm if the differences in chilling sensitivity were not significant. Under freezing stress, PIABS responds in the similar pattern as Fv/Fm (Fig. 3-a, b), that is, PIABS for both samara and leaf was rapidly reduced to zero after one-hour freezing treatment.
Under chilling stress, electron transport (fEo and ?o) for leaf was markedly decreased but slightly altered for samara (Fig. 2-c). This result suggested that electron transport, especially beyond QA in leaf, was more susceptible to chilling stress than in samara. Electron transport in both samara and leaf was completely blocked under freezing stress (Fig. 3-c). Since from O to J in the reflects reduction of QA to QA- and J to I to P reduction of the PQ pool (Strasser et al., 1995), decrease in ?o could be attributed to the decreasing PQ pool size in response to low temperature stress (Martin et al., 1978; Ã-quist and Ã-gren, 1985). Ã-quist and Ã-gren (1985) reported that the considerable decrease in the ratio of PQ pool size to the primary electron acceptor (Q) under winter stress corresponded to the inhibition of whole chain electron transport.
An increase in apparent antenna size (ABS/RC) was induced by low temperature stress (Fig. 2-d, e, f and Fig. 3-d, e, f), indicating that some of RCs were inactivated (Krüger et al., 1997). The trapped energy flux per RC (TRo/RC) also increased but of much smaller magnitude. The large increase in ABS/RC accompanied with small increase in TRo/RC resulted in marked dissipated energy flux per RC (DIo/RC). The increased dissipation of energy flux led to decrease in efficiency for conversion of excitation energy flux to electron flux (?o). Similar response of energy flux through PSII in soybean to dark chilling was observed (van Heerden et al., 2003). Taking the changes of these parameters together, it can be concluded that the inhibitory effect of chilling temperature on photosynthetic performance (PIABS) was mainly resulted from the slowing down of electron transport beyond QA and an increase in thermal energy dissipation.
All the PSII functional parameters of chilled samara and leaf can rapidly recover to normal (Fig. 2). However, the PSII functional parameters of short-term dark frozen samara can be largely recovered whereas those of frozen leaf cannot be recovered (Fig. 3). Since samara is the reproductive organ, the higher tolerance of samara to short-term freezing and its better recovery potential from freezing stress than leaf was of great ecological significance for population establishment of Siberian maple and its northern distribution limit in northern hemisphere.
(1) There is pronounced difference of PSII activity between the samara and the leaf of Siberian maple. The samara has more efficient electron transport flux (fEo) and photosynthetic efficiency (Fv/Fm and PIABS) but lower energy dissipation (DIo/RC) than leaf.
(2) Generally, the PSII performance and electron for both samara and leaf were reduced under low temperature stress accompanied by increasing of energy dissipation in PSII reaction centers (RCs).
(3) PSII of both samara and leaf is not significantly affected and can acclimate to sudden chilling stress. Short-term freezing treatment can completely inhibit PSII activity in both samara and leaf. PSII functional parameters of short-term dark frozen samara can be largely recovered whereas those of frozen leaf cannot be recovered.
(4) Samara is more tolerant to short-term low temperature stress and has better recovery potential than leaf.
This work was supported by Knowledge Innovation Program of Chinese Academy of Sciences (kzcx2-yw-335), the Program of 100 Distinguished Young Scientists of the Chinese Academy of Sciences, and the National Natural Science Foundation of China (40673070, 40872169).
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