¹Department of Botany, St. John’s College, Anchal, Kollam

²Plant Biochemistry and Molecular Biology Laboratory, Department of Botany, University College, Trivandrum, 695 034, Kerala, India

Article Publishing History

Introduction

Desiccation is the most alarming stress faced by plants. The loss of water in the tissues leads to the denaturation of essential biomolecules and subsequently, degeneration of cell organelles (Alpert, 2006). Animals actively avoid desiccation by movements, while plants are static and therefore subjected to water loss and recover back slowly during rehydration (Alpert, 2006). Resurrection plants such as Myrothamnus flabellifolius, Xerophyta viscose, and Sporobolus stapfianus were proven drought tolerant species (even up to 90% water loss) (Alpert, 2006). Adaptation to desiccation is based on the ability of the organism to equilibrate its internal water potential with the desiccating environment, and also their drastic ability to regain normal activities after rehydration (Alpert, 2000).

Compared to vascular plants (Vicre et al., 2004), the mechanisms involved in desiccation among lower plants like ferns is poorly understood. Most of the drought stress initiates the activation of antioxidant enzymes such as catalase, superoxide dismutase, ascorbate peroxidase and glutathione reductase (GR) etc. (Burritt et al., 2002) to counter balance desiccation-mediated oxidative stress. Similarly, many macro algae resist desiccation tolerance through the photosynthetic system including chloroplast (Zou and Gao, 2002a,b). Fucus vesiculosus was evaluated by molecular approaches to unravel the responses to desiccation via the genes encoding photosynthetic and ribosomal proteins (Pearson et al., 2010 Maghsoudi et al., 2015).

The photosystem II reaction center was critical in photosynthetic pathway against drought stress (Wen et al., 2005) i.e., desiccation stress reduces the photosynthetic electron transport activity and also the fluorescence of photosystem II (PSII). Inactivation of PSII leads to derailment of the water-splitting complex, disturbance of pigment protein complexes in thylakoids, which further influences regulation of energy transfer and finally, the photochemical reaction center of PSII was deactivated (Wise et al., 2004). Impact of plants exposed to desiccation stress was drastic but, its recovery was gradual or ceased due to the injury to PSII components (Sinsawat et al., 2004 and Kifah and Jaroslav, 2015).

The use of fluorescence parameters permit to analyze the reduction in electron transport disorder via the emission of heat in the form of IR radiation or by fluorescence. This technique is based on the light kinetics absorbed by antenna pigments and the excitation energy transferred to the reaction centers of photo­system I and II (Zhani et al., 2012).

Contreras-Porcia et al., (2011) analyzed the interrelationship between Fm and F0 in Porphyra columbina collected from the different intertidal regions. Generally, in the optimal conditions the proportion of radiant energy emitted as fluorescence is decreased. Meanwhile, during stressed conditions, the chlorophyll fluorescence will be altered (Kadir and Von Weihe 2007). So, in vivo fluorescence of chlorophyll pro­vides an early sign of photosynthetic malfunc­tion and can be used as marker to localize the possible sites of damage induced by stress within the cells. In this juncture, the present study is aimed to analyze the photosynthetic pigments and their efficiency in the fern against different duration of desiccation and rehydration.

Material And Methods

Quantification Of Photosynthetic Pigments

Photosynthetic pigments were estimated in 80% acetone extract. 1 g tissue was homogenized with 1.5 ml of 80% chilled acetone. The homogenate was centrifuged at 3000 rpm for 5 min. The aliquots were made up to 3 ml by using 80% acetone and the absorbance was read at 470, 648 and 664 nm spectrophotometrically against 80% acetone as blank. Total chlorophyll as well as chlorophyll a and b concentrations and carotenoids were calculated according to the protocol of Arnon (1949).

Chlorophyll fluorescence emission from the upper and lower surface of the leaves of the fern was measured by a modulated fluorometer (OS 500;Opti Sciences;Inc;Tyngsboro;Mass). Maximum fluorescence yield (F_m) was determined during saturating flash (3000 μmol m^-2s-²)^. The actual fluorescence level (F) was monitored to ensure that it was stable. To obtain the maximal fluorescence yield under illumination (F_m), the leaf was exposed to a saturating flash during exposure to actinic light (210 μmol m^-2s^-1). To determine the minimal level of fluorescence during illumination (F₀), the leaf was continuously illuminated with far-red light (730 nm) to rapidly reoxidize the PSII centers. All measurements were conducted at 25ºC (Demmig-Adams et al., 1996).

The minimal fluorescence level (F₀) with all PSII reaction centers open and the maximal fluorescence level (F_m) with all PSII reaction centers closed were determined on dark-adapted leaves. Then the leaves were continuously illuminated with a white actinic light at an irradiance of 180 µmol m^-2 s^-1 to measure the steady-state value of fluorescence (F_s), which occurred at about 6 min after the initiation of white actinic light. The maximal fluorescence level in the light-adapted state (F_m’) was recorded after subjecting the leaf to a second saturating pulse at 8000 µmol m^-2 s^-1.

The minimal fluorescence level in the light-adapted state (F₀) was determined by exposing the leaf to far-red light for 3s.Using both light and dark fluorescence data, the following parameters were calculated:

F_v (maximum variable chlorophyll fluorescence yield in a dark-adapted state) was calculated following Maxwell and Johnson (2000): F_v = F_m – F₀

F_v/F_m (the maximal efficiency of PSII photochemistry in the dark-adapted state) was calculated as: F_v/F_m = (F_m – F₀)/F_m

qP (the photochemical quenching coefficient): qP = (F_m´ – F_s) / ( F_m´ – F₀´)

qN (non-photochemical quenching coefficient): qN = 1 – (F_m´ – F₀´) / (Fm – F0).

ϕPSII (the actual quantum yield of PSII electron transport in the light-adapted state):

ϕPSII = (F_m´ – F_s)/ F_m´, which was equal to the product of qP and F_v´ /F_m´. Thus, ö PSII depends on the degree of closure of PSII reaction centers and the efficiency of excitation energy capture in PSII.

ETR (Apparent photosynthetic electron transport rate): Apparent electron transport rates (ETR) are derived from effective quantum yields of photosystem II (ΔF/F_m´ or Y(II)) according to ETR = Y(II) x PAR x 0.42. In this equation, the PAR corresponds to the quantum flux density of photosynthetically active radiation, and the 0.42 is the product of light absorptance by an average green leaf (0.84) times the fraction of absorbed quanta available for photosystem II (0.5).

Data were statistically analyzed using ANOVA followed by Tukey’s test (SPSS 14.0; SPSS Chicago, IL, USA). Significant differences were analyzed based on P < 0.05 and P < 0.01. Percentage data were subjected to arc sine transformation prior to statistical analysis.

Results And Discussion

Generally, desiccation tolerance was evaluated as the capacity of the plants to mitigate the excess ROS formed and thereby attenuating the oxidative stress within the plant. Photosynthetic components such as enzymes, chlorophylls, and carotenoids levels depend on the severity and duration of stress. In the present analysis, the total chlorophyll content was maintained by the fern during the different periods of desiccation stress (4 to 10^th day) i.e., showed an increase from 0.352 (in control) to 0.353 mg/g at 10^th day of desiccation treatment in the fern (Table-1). The optimal maintenance of chlorophyll content directly reflects the functional status of the fern against the desiccation stress management. Chlorophyll a/b ratio showed an initial increase (2^nd day) followed by an decrease with the degree of desiccation till the day 10 of treatment. Carotenoids, the major accessory pigments showed steady increase up to 6^th day and then decreased marginally (10^th day) (Table-1). The pigments are effective antioxidants and therefore, protect the cells from oxidative stress by mitigating ROSs formed during photo-oxidative stress.

Table 1: Pigment content in the fern treated with desiccation and rehydration stress (D – desiccated; R- rehydrated)
	Control	2D	2R	4D	4R	6D	6R	8D	8R	10D	10R
Chl a(mg/g)	0.226	0.197	0.261	0.240	0.285	0.237	0.309	0.284	0.325	0.21	0.226
Chl b(mg/g)	0.126	0.109	0.123	0.136	0.142	0.166	0.217	0.191	0.224	0.128	0.127
Chl a/chl b	1.79	1.8	2.12	1.76	2.00	1.42	1.42	1.48	1.45	1.64	1.78
Total chl	0.352	0.306	0.384	0.376	0.427	0.403	0.526	0.475	0.549	0.338	0.353
Carotenoids(mg/g)	0.051	0.057	0.066	0.062	0.059	0.083	0.113	0.070	0.088	0.075	0.089
Tot chl/tot car	6.9	5.37	5.81	6.06	7.23	4.85	4.65	6.78	6.23	4.50	3.97

Chlorophyll fluorescence emission is a versatile tool for quick and non-intrusive estimation of the photosynthetic activity and photoinhibition in the leaves against environmental stresses. Initially, after 2 d desiccation the F₀ level increased whereas, the F_m value decreased in the frond when compared to control. This leads to a decline in the maximum quantum yield of PSII (F_v/F_m) to 0.61. From 4^th to 8^th day of desiccation F₀, F_m, F_v and F_v/F_m values were maintained. The effective quantum yield of PSII (öPSII) and photochemical quenching (qP) also showed a similar trend. In contrast, the non-photochemical quenching (qN) was increased at 2 d desiccated fronds with improvement in apparent photosynthetic electron transport rate (ETR) also. F_o/F_m ratio, known as the basal quantum yield displayed a range from 0.198 to 0.38 (Table – 2). Rehydration application regained these parameters of desiccation stressed ferns compared to the stressed plants. A significant correlation was observed in 2 d desiccated ferns compared with in terms of marginal necrosis in the fronds.

Table 2: Chlorophyll fluorescence parameters
	control	2 D	4D	6D	8D	10D
F0	121	158	133	127	125	123
Fm	612	410	584	610	629	512
Fv	491	252	451	483	504	389
Fv/Fm	0.80	0.61	0.77	0.79	0.80	0.76
ö	0.67	0.50	0.62	0.67	0.68	0.60
qP	0.82	0.71	0.8	0.83	0.84	0.78
NPQ	0.68	0.76	0.65	0.66	0.65	0.70
ERT	78	66	75	78	79	70
F0/Fm	0.198	0.38	0.22	0.208	0.199	0.24

Generally, the chlorophyll fluorescence parameters are ideal markers of PSII and photosynthetic activity in stressed plants (Kifah and Jaroslav, 2015). Maximum quantum efficacy of PSII (F_v/F_m) refers the photosynthetic efficiency of the leaf (Maghsoudi et al., 2015). Thus, F_v/F_m is widely used to evaluate stress-induced impairment in the chloroplast. The present results revealed that desiccation stress resulted an initial reduced F_v/F_m (Table – 2) which may be due to the decreased efficiency of energy transfer from the antennae to the reaction centers and / or inhibition of the activity circumscribed around PSII reaction centers (Abdeshahian et al., 2010). The decline in F_v/F_m suggests the possible damage occurred to PSII (Yamane et al., 2008). Amirjani (2010) reported that the decline in F_v/F_m might retard the rate of photosynthesis, thereby inhibiting plant growth and development.

Lepedu et al., (2012) reported a reduction in photochemistry among drought-stressed cowpea plants but, its overall photosynthetic efficiency remained unaffected. The desiccation induced suppression noticed initially (2d after desiccation) with the apparent photosynthetic electron transport rate (ETR) revealing the initial imbalance of the fern against drought stress with an increase of qN. This may be to counter balance the excessive light energy with reduction in photosynthetic rate. Thus, the photochemical down-regulation related with the stress payway to reduction in ETR (Arabzadeh, 2013).

In this study, 2 day desiccation increased the non-photochemical quenching (qN) but later qN was maintained at optimal level. Yamane et al., (2008) reported chloroplast damage in rice due to photo-inhibition that was induced by high salinity condition. Photo-inhibition may also retard and reverse the reduction in photosynthetic efficacy that partially impairs transformation of radiation energy into net assimilatory products. Hazem et al., (2011) reported the effect of salt stress on photosystem II efficiency and CO₂ assimilation of two Syrian barley landraces. Further, the excessive light energy could be dissipated as heat through qN. Cha-um (2013) suggested that adequate supply of CO₂ for carbon reactions may prevent photoinhibition, which has been reflected as significantly higher F_v/F_m value in the cowpea plants and others against salinity stress. In the present study, desiccation initially reduced the F_v/F_m values, but was subsequently maintained significantly in the fern during 4, 6, 8^th days of desiccation.There were different interpretations regarding the variation in the level of F₀ such as an estimation of the relative size of the antenna pigment complexes of the PSII (Kadir and Von Weihe, 2007).

Contreras-Porcia et al., (2011) also suggested that in Porphyra columbina an increase in F₀ leads to symptom of damage to the PSII reaction center, resulting in a reduction in absorbed light and a subsequent increase in unused emitted light. The results of present study showed that desiccation stress reduced F_m initially, but maintained the photosynthetic quenching. Maghsoudi et al., (2015) also showed a reduction in F_m but an increase in F_o/F_m in wheat seedlings under water deficit stress. Additionally, De Lucena et al., (2012) reported in mango that a reduction in F_v/F_m ratio, under stress conditions, is often an indicator of photoinhibition or injury to PSII complex. Therefore, the increase in non-photochemical quenching can be expected under desiccation stress as a result of decrease in the utilization of light energy due to a drought-induced reduction in PSII activity (F_v/F_m). This might explain the increase in the value of F₀/F_m in silicon treated wheat under water-deficit conditions (Maghsoudi et al., 2015).

Several studies have reported stress-induced increases in the values of F₀/F_m and qN and decreases in F_v/F_m, qP, F₀, and cpPSII (Pellegrini et al., 2011; De Lucena et al., 2012; Contreras-Porcia et al., 2011). In the fern, D. linearis, under desiccation stress at 4, 6, 8 days significantly increased the value of F_v/F_m as well as that of qP (Table – 2). Reductions in the values of F₀/F_m and qN and increases in F_v/F_m, qP, F0, and ö PSII have been reported in many plants under abiotic stress conditions (Pellegrini et al., 2011; De Lucena et al., 2012; Contreras-Porcia et al., 2011). Similarly, Al-aghabary et al., (2004) have reported in tomato that addition of silicon to the root growing medium of salt-stressed plants enhanced F_v/F_m as well as improved the photochemical efficiency of PSII through antioxidant machineries.

Conclusion

The present results revealed that the ferns can withstand desiccation via maximum quantum yield of PSII and photochemical quenching. The chlorophyll fluorescence and photosynthetic pigments suggest enhanced drought tolerance of the ferns. The fern alleviate the adverse effects of desiccation through the pigments and also effective ROSs scavenging mechanism. Further studies are warranted at molecular level to analyze the antioxidant enzymes and stress protein up regulation in the fern.

Acknowledgement

The authors thank the University Grant Commission regional office, Bangalore for providing FDP status to the teacher fellow for completing the Ph.D. work (Order No.F.No.FIP/12th plan/KLKE021 TF 06).

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