Photosynthetic adaptation of Euphorbia fractiflexa (Euphorbiaceae) and survival in arid regions of the Arabian Peninsula

Abstract

Arid regions of the Arabian Peninsula are characterized by a short wet season with erratic rainfall, high temperature, and high evaporation. In these arid regions, the leafless stem succulent Euphorbia fractiflexa S. Carter & J.R.I.Wood (Euphorbiaceae) is an abundant perennial. Work presented in this paper aimed at investigating crassulacean acid metabolism (CAM) as a physiological adaptation crucial for survival of E. fractiflexa in arid regions of the Arabian Peninsula. Work involved investigations of stomatal diffusive conductance, chlorophyll fluorescence, and cell sap titratable acidity. Results represent the first report of obligate CAM in E. fractiflexa. Low values of stomatal conductance and dampening of CAM acidification–deacidification cycles during the long dry season also denoted tendency of this species to shift from obligate CAM to CAM-idling. Results also showed water stress-induced reduction in Photosystem II (PSII) activity occurring in concomitance with increased non-photochemical quenching of chlorophyll fluorescence denoting operation of non-photochemical energy dissipation mechanisms that are important for photoprotection of the photosynthetic machinery under stress conditions.

Keywords:

• crassulacean acid metabolism (CAM)
• CAM-idling
• chlorophyll fluorescence
• Euphorbia fractiflexa
• water stress

Introduction

Arid regions of the Arabian Peninsula are characterized by high temperature, high evaporation, scarce water, and erratic rainfall (Fisher & Membery 1998). In such arid regions, leaf succulents reduce their transpirational surface area by seasonal partial leaf shedding (Sayed 1996, Sayed 1998, Sayed 2001a), whereas leafless stem succulents rely on ample water storage capacity, morphoanatomical traits, and physiological adaptations (Sayed 2001a; Masrahi et al. 2012a, 2012b). These physiological adaptations involve exhibiting crassulacean acid metabolism (CAM) photosynthesis for its unique nocturnal carbon acquisition which improves the plant water economy (Dodd et al. 1997; Borland et al. 2000; Sayed 2001b; Luttge 2002; Masrahi et al. 2011; Masrahi et al. 2012a, 2012b). Reports on photosynthesis in the genus Euphorbia from different parts of the world indicated that leaf succulent Euphorbia species exhibited C₄ photosynthesis (Pearcy & Troughton 1975), whereas leafless stem succulent Euphorbia species were CAM plants (Pearcy & Troughton 1975; Webster et al. 1975; Ziegler et al. 1981; Willert et al. 1985; Martin et al. 1990; Van Damme 1991; Mies et al. 1996; Ameh 2006; Feakins and Sessions 2010). However, work on photosynthesis of the genus Euphorbia in the Middle East is scarce. A report on photosynthesis in the genus Euphorbia in Egypt indicated that all leaf succulent species were C₄ plants, whereas leafless stem succulent species were CAM plants (Batanouny et al. 1988).

The leafless stem succulent Euphorbia fractiflexa S. Carter & J.R.I.Wood is a common stem succulent in arid regions of the Arabian Peninsula (Collenette 1999). Work presented in this paper aimed at investigating the presence of CAM in E. fractiflexa. Investigations involved field studies of seasonal and diurnal changes in stomatal diffusive conductance, and cell sap titratable acidity. Moreover, plants with the CAM pathway exhibit different CAM physiotypes namely; obligate CAM, facultative CAM, CAM-cycling, and CAM-idling (Luttge 2004; Herrera 2009). Of these modes, CAM-idling has early been recognized (Ting & Sipes 1985), and is considered to be a very strong modification of CAM occurring under extreme environmental conditions (Cushman 2001; Sayed 2001b; Dodd et al. 1997). This CAM-idling mode is characterized by stomatal closure during the entire day and night, no net CO₂ uptake, and acidification fed by internal recycling of nocturnally re-fixed respiratory CO₂ (Luttge 2004; Lüttge 2008; Herrera 2009). Work presented in this paper also aimed at describing possible modifications CAM in E. fractiflexa. Furthermore, during Phase III of CAM occurring under closed stomata, high irradiance, and high temperature, Photosystem II (PSII) becomes over-energized (Niewiadomska & Borland 2008) and plants are thought to invoke photoprotection of PSII by excess energy dissipation via non-radiative mechanisms and the xanthophyll cycle (Tallman et al. 1997; Tallman 2004; Herrera 2009). Therefore, our work also involved using pulse amplitude modulated chlorophyll fluorescence to investigate the involvement of CAM-idling in mitigating deleterious effects of water stress on the photosynthetic machinery.

Materials and methods

The study site southwest of Saudi Arabia (17°19′N–42°48′E) is characterized by sand-loam soil, high temperature, high irradiance, scarce water, erratic rainfall, and a climate influenced by tropical maritime air mass (Fisher & Membery 1998). Climatic records of mean monthly maximum air temperature, precipitation, and evaporation for the past 40 years (1970–2010) were obtained from the Ministry of Electricity and Water (Riyadh, Saudi Arabia).

Stomatal diffusive conductance was measured in intact stem using a porometer (AP4, Delta-T Devices, Cambridge, UK). Pulse amplitude modulated chlorophyll fluorescence was measured using a chlorophyll fluorescence monitoring system (FMS2, Hansatech Instruments, Norfolk, UK). Measured chlorophyll fluorescence parameters included Fv/Fm, and Φ PSII, reflecting efficiency of PSII antenna and the quantum yield of PSII, respectively (Bolhàr-nordenkampf & Öquist 1993; Sayed 2003). The value qNP reflecting non-photosynthetic quenching of chlorophyll fluorescence was calculated using standard fluorescence nomenclature (Sayed 2003) and Equation (1)

where Fo is the minimal fluorescence level emitted by antenna chlorophyll molecules, Fm is the maximal fluorescence level emitted as all PSII traps become closed, Fv is the variable chlorophyll fluorescence, and Fm’ is the light-adapted fluorescence maximum.

Changes in CAM acidification and deacidification were studied in chlorenchyma cell sap extracted by grinding a known weight of tissue in distilled water, expressing cell sap through two layers of muslin, and determining cell sap titratable acidity (Osmond et al. 1991). Studies were conducted in mid-May and Mid-July for the dry and wet season, respectively. All experiments were routinely repeated in samples taken from 10 different individual plants and the standard deviation was calculated.

Results

Climatic records indicated that the study site is characterized by a long dry season and a short wet season (June–August) during which air temperature, precipitation, and evaporation markedly increased (Table 1). These climatic data also showed that the mean annual maximum air temperature is 36.7°C and that the total annual precipitation is 93.4 mm.

Table 1. Climatic records at the site for the period 1970–2010.

	Time of year (months)
Parameter	January	February	March	April	May	June	July	August	September	October	November	December
Max. air temperature (°C)	30.8	34.7	35.4	35.5	38.5	39.0	40.0	40.3	38.3	37.3	36.0	34.9
Precipitation (mm)	5.0	4.5	5.0	4.5	5.0	4.4	20.0	8.0	9.0	9.0	9.0	10.0
Evaporation (mm)	4.9	5.6	7.5	8.9	10.2	9.6	10.0	8.6	8.8	9.3	6.3	4.9

Measurements of stomatal diffusive conductance during the wet season indicated that E. fractiflexa exhibited high night-time values and low day-time values (Figure 1). On the other hand, measurements during the dry season indicated that E. fractiflexa exhibited markedly low values of stomatal conductance in the range of 3–5 mmol m⁻² s⁻¹ during both night and day (Figure 1). Moreover, determinations of chlorenchyma cell sap titratable acidity indicated that E. fractiflexa exhibited diurnal acidity changes during the wet season, and that these acidity changes were markedly dampened during the dry season (Figure 2). Measurement of chlorophyll fluorescence during the dry season showed reduction in the value of the parameters Fv/Fm, and Φ PSII compared to those measured during the wet season (Table 2). This reduction of the value of chlorophyll fluorescence parameters during the dry season occurred in concomitance with a marked increase in the value of the parameter qNP (Table 2).

Figure 1. Diurnal changes in stomatal diffusive conductance of E. fractiflexa during the wet season (○) and the dry season (•) (±SD, n=10).

Figure 2. Diurnal changes in titratable acidity of chlorenchyma in E. fractiflexa during the wet season (○) and the dry season (•) (±SD, n=10).

Table 2. Seasonal changes in chlorophyll fluorescence parameters of E. fractiflexa (±SD, n=10).

	Season
Parameters	Wet	Dry
Fv/Fm	0.85±0.05	0.70±0.0.7
Φ PSII	0.80±0.03	0.65±0.05
qNP	0.02±0.003	0.07±0.005

Discussion

Climatic records reflected the extreme aridity prevailing at the study site. This aridity was manifested by a nine-month-long dry season and a short wet season characterized by water scarcity, high temperature and high evaporation (Table 1). In such arid habitats, perennial stem succulents survive periods of protracted drought by morphoanatomical traits and physiological adaptations that enable them to tolerate harsh environmental conditions (Sayed 2001a, 2001b; Masrahi et al. 2011; Masrahi et al. 2012a, 2012b).

During the wet season, E. fractiflexa exhibited values of stomatal conductance that were high during the night and low during the day (Figure 1), a stomatal behavior typical of CAM plants (Sayed 2001b; Luttge 2004; Herrera 2009). Determination of chlorenchyma cell sap titratable acidity during both the wet and the dry seasons indicated existence of diurnal acidification–deacidification cycles denoting operation of the obligate CAM. This CAM physiotype has previously been reported in some stem succulent Euphorbia species (Pearcy & Troughton 1975; Willert et al. 1985; Martin et al. 1990; Van Damme 1991; Mies et al. 1996; Elhaak et al. 1997; Winter et al. 2005; Ameh 2006; Feakins and Sessions 2010). Nocturnal CO₂ uptake and daytime stomatal closure characteristic of CAM imply avoidance of gas exchange when environmental conditions favor transpirational water loss and hence enhanced plant water economy (Walker & Leegood 1996; Winter & Smith 1996; Borland et al. 2000; Sayed 2001b; Luttge 2004; Lüttge 2008; Herrera 2009). However, during the long dry season, E. fractiflexa exhibited very low values of stomatal conductance during both day and night (Figure 1) and markedly dampened diurnal oscillation of cell sap titratable acidity (Figure 2). These results indicated that E. fractiflexa shifted from the obligate CAM physiotype to a CAM-idling mode in response to protracted drought. The CAM-idling mode is a strong modification of CAM that greatly enhances plant water economy by stomatal closure during the entire day and night and sustaining diurnal fluctuations in cell sap titratable acidity by nocturnally re-fixing respiratory CO₂ (Sayed 2001b; Dodd et al. 1997; Nobel & De La Barrera 2002; Luttge 2004; Lüttge 2007; Herrera 2009).

Comparison of chlorophyll fluorescence parameters measured during the wet and the dry seasons in E. fractiflexa indicated reduction of PSII activity manifested by reduction of Fv/Fm and Φ PSII (Table 2). Reduction of Fv/Fm and Φ PSII denotes reduction of the efficiency of PSII antenna and PSII quantum yield, respectively (Bushmann 1999; Sayed 2003). Similar reduction of PSII activity has previously been shown for other CAM plants under protracted drought (Mattos et al. 1999) and was attributed to an over-energized PSII during Phase III of CAM that takes place behind closed stomata under high irradiance and high temperature (Mattos et al. 1999; Niewiadomska & Borland 2008). However, our observed reduction in PSII activity occurred in concomitance with a markedly increased qNP (Table 2) denoting increased non-photochemical quenching of chlorophyll fluorescence and hence increased non-photochemical excess energy dissipation (Bushmann 1999; Sayed 2003). Similar increased qNP in Clusia minor performing CAM-idling (Lüttge 2007) was attributed to a relationship between zeaxanthin, a precursor of abscisic acid, and the onset of CAM-idling. Moreover, inhibition of zeaxanthin accumulation in guard cells of CAM-performing Mesembryanthemum crystallinum was also suggested to prevent stomatal opening in response to light (Tallman et al. 1997). In this case, daytime decarboxylation and high rate of respiration due to high temperature imply high intercellular CO₂ concentration that ensures daytime stomatal closure by favoring Calvin cycle activity in guard cell chloroplasts, consumption of NADPH, and prevention of the destruction of endogenous guard cell abscisic acid (Tallman 2004). Furthermore, reports the literature suggested that CAM-idling plays a role in photoprotection by non-radiative excess energy dissipation via the xanthophyll cycle (Robinson & Osmond 1994; Tallman et al. 1997; Tallman 2004; Herrera 2009). The CAM-idling mode is thought to result in reduction of oxidative stress by processing reactive oxygen species that appear when PSII becomes over-energized during Phase III of CAM and hence confer photoprotection under protracted drought (Lüttge 2007; Niewiadomska & Borland 2008).

It can, therefore, be concluded that E. fractiflexa is an obligate CAM plant exhibiting stomatal behavior typical of CAM plants during both the wet and the dry seasons. Low stomatal conductance during the entire day and night and dampened acidification–deacidification cycles indicated that E. fractiflexa shifts from the obligate CAM to CAM-idling in response to protracted water stress during the long dry season. Under severe drought, PSII activity in E. fractiflexa was reduced due to oxidative stress during CAM-idling. This oxidative stress appears to be mitigated by operation of photoprotective non-radiative excess energy dissipation as reflected by increased non-photochemical quenching of chlorophyll fluorescence.