Stomatal and non-stomatal limitations of bell pepper (Capsicum annuum L.) plants under water stress and re-watering: Delayed restoration of photosynthesis during recovery

Environmental and Experimental Botany 98 (2014) 56–64

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Stomatal and non-stomatal limitations of bell pepper (Capsicum annuum L.) plants under water stress and re-watering: Delayed restoration of photosynthesis during recovery b ˜ Huitziméngari Campos a,∗ , Carlos Trejo b , Cecilia B. Pena-Valdivia , Rodolfo García-Nava b , b c F. Víctor Conde-Martínez , M.R. Cruz-Ortega a Posgrado en Recursos Genéticos y Productividad-Fisiología Vegetal, Colegio de Postgraduados, Carretera México-Texcoco, km 36.5, Montecillo, México 56230, Mexico b Posgrado en Botánica, Colegio de Postgraduados, Carretera México-Texcoco, km 36.5, Montecillo, México 56230, Mexico c Departamento de Ecología Funcional y Aplicada, Universidad Nacional Autónoma de México, Instituto de Ecología, Circuito Exterior, Ciudad Universitaria, México, DF 04510, Mexico

a r t i c l e

i n f o

Article history: Received 29 August 2013 Received in revised form 8 October 2013 Accepted 20 October 2013 Keywords: Drought stress Gas exchange Water relations Prompt fluorescence Photosystem I Photosystem II

a b s t r a c t Low soil water availability is the major environmental factor limiting plant growth and yield. The objective of this study was to elucidate the mechanisms underlying photosynthesis inhibition during water stress and recovery in Capsicum annuum L. cv. Cannon by evaluating soil and plant water relations, gas exchange and the prompt fluorescence rise OJIP. The soil ( S ) and leaf ( L ) water potential decreased from −0.16 and −0.53 to −1.1 and −1.7 MPa, respectively, and recovered after re-watering. The stomatal conductance (gs ) decreased to 114 and 13 mmol m−2 s−1 under moderate and severe water stress, respectively. Similarly, the CO2 assimilation (A) and transpiration (Tr) rates decreased during water stress but recovered after re-watering. During severe water stress, photosynthesis decreased due to stomatal closure and to both slower maximum carboxylation rate (Vcmax ) and ribulose 1,5-bisphosphate (RuBP) regeneration capacity mediated by maximum electron transport rate (Jmax ). In fact, the fluorescence parameters reflecting the electron flow from the intersystem carriers to final reduction of photosystem I (PSI) end electron acceptors declined throughout water deficit development. In conclusion, water stress mainly damaged the electron transfer from the plastoquinone (PQ) pool to the PSI terminal acceptors; this, along with constraints to both stomatal and non-stomatal components of photosynthesis, limited carbon assimilation. Photosynthesis recovery after re-watering was mainly restricted by both stomatal conductance and the gradual recovery of the electron transport chain. Finally, JIP-test parameters that quantifying electron transfer from the PQ pool to the PSI end acceptors are effectives for monitoring water stress in crop plants. © 2013 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: A, CO2 assimilation rate; A-Ci , CO2 assimilation rate-intercellular CO2 concentration; Chl a, chlorophyll a; gm , mesophyll conductance; gs , stomatal conductance; Jmax , the rate of ribulose 1,5-bisphosphate regeneration mediated by electron transport; NADH, nicotinamide-adenine dinucleotide; OEC, oxygen evolving complex; OJIP transient, fluorescence rise defined by its steps; PAR, photosynthetic active radiation; PSI, photosystem I; PSII, photosystem II; PQ, oxidized plastoquinone; QA , primary quinone electron acceptor of PSII; Rd , dark respiration; RuBP, ribulose 1,5-bisphosphate; Rubisco, ribulose 1,5-bisphosphate carboxylaseoxygenase;  L , leaf water potential;  S , soil water potential; Tr, transpiration rate; Vcmax , maximum carboxylation rate of Rubisco. ∗ Corresponding author. Tel.: +55 58 04 59 42; fax: +55 58 04 59 00x1254. E-mail addresses: [email protected], [email protected] ˜ (H. Campos), [email protected] (C. Trejo), [email protected] (C.B. Pena-Valdivia), [email protected] (R. García-Nava), [email protected] (F.V. Conde-Martínez), [email protected] (M.R. Cruz-Ortega). 0098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.envexpbot.2013.10.015

The majority of climate change scenarios predict an increase in drought incidence throughout different regions of the world (IPCC, 2007). Because agricultural activities are water intensive, the increase in arid and semi-arid cropland, along with increases in population, will produce greater water demands and exploitation that will directly affect crop growth, survival and yield (Chaves et al., 2009). The limitation of plant growth due to low water availability is mainly due to reductions of plant carbon balance, which is largely dependent on photosynthesis (Flexas et al., 2009). However, there is an on-going debate about whether the restrictive factor for photosynthesis during water stress is stomatal closure and diffusive resistance or metabolic uncoupling (Flexas and Medrano, 2002; Lawlor and Cornic, 2002; Flexas et al., 2009; Pinheiro and Chaves, 2011). A decrease in the diffusion of atmospheric CO2 to

H. Campos et al. / Environmental and Experimental Botany 98 (2014) 56–64

the carboxylation site in the leaves is generally considered the main cause of the decrease in photosynthesis under water stress conditions (Flexas et al., 2004a; Grassi and Magnani, 2005; Chaves et al., 2009). Moreover, when a decrease in the stomatal conductance is combined with sustained irradiance, the leaves are subjected to an excess of incident energy relative to the intercellular availability of CO2 because the reducing power production rate exceeds its consumption rate during the Calvin cycle (Pinheiro and Chaves, 2011). Under these circumstances, down-regulation and even photoinhibition of photosynthesis can become a potent defence mechanism in C3 plants. This protection can be afforded by thermoregulated dissipation that occurs in the light harvesting complexes and involves the xanthophyll (Demmig-Adams et al., 2006) and lutein cycles (García-Plazaola et al., 2003). These photoprotection mechanisms compete with photochemistry for the absorbed energy, causing down-regulation of photosynthesis, as revealed by the decreased quantum yield of photosystem II (PSII) (Genty et al., 1989). However, reports regarding the effects of water stress on the functionality of PSII are contradictory, and the exact site and the mechanisms for the PSII damage have not yet been elucidated (Georgieva et al., 2005; Sperdouli and Moustakas, 2012). Several in vivo studies have shown that water stress damages the PSII oxygen-evolving core complexes and reaction centers (Scotnica et al., 2000; Colom and Vazzana, 2003), while other studies have shown that PSII is not affected at all or is only affected under severe water stress conditions (Massacci et al., 2008; Flexas et al., 2009). In addition to the plant metabolism response, the carbon balance during water stress and recovery can depend on the velocity and the development of the photosynthetic recovery because the latter depends on the degree and rate of decrease in photosynthesis during water deficit (Flexas et al., 2006). In general, plants subjected to severe water stress exhibit 40–60% of their maximum rate of photosynthesis 1 day after irrigation, and this recovery continues during the following days. However, the maximum photosynthesis rate is not always restored (Sofo et al., 2004; Flexas et al., 2009). The influence of water stress severity on the speed and degree of recovery has been demonstrated in Phaseolus vulgaris (Miyashita et al., 2005) and in Vitis hybrid Richter-110 (Flexas et al., 2009). However, photosynthetic recovery depends not only on the stress severity and the species studied but also on the complex interactions with the plant, leaf age, light intensity and water deficit cycles, among others (Flexas et al., 2004b). In Mexico, approximately 5800 hectares are planted with bell pepper (Capsicum annuum L.), with a production of up to 50 t ha−1 year−1 (Reséndiz-Melgar et al., 2010). In 2006, 240,000 tons of bell pepper were exported to the United States and Canada alone (Castellanos and Borbón, 2009), making this a profitable crop. Moreover, bell pepper adds not only flavor and aroma but also medicinal value to foods; it is an excellent source of vitamins A and C, with a low caloric content (Villalón, 1981; Andrews, 1995). However, this crop is especially sensitive to soil water deficit (González-Dugo et al., 2007), and drought stress during the initial developmental stages can reduce the size and number of buds and fruits (Rylski and Spigelman, 1982). Currently, little is known about the physiological limitations of photosynthesis during the acclimation and development of water stress and the recovery after re-watering, but this information is essential to improve the understanding of the plant response to drought and to develop appropriate irrigation practices. Additionally, it would be useful to evaluate the relative importance of the distinct processes that limit photosynthesis at different water stress levels to add to the on-going debate regarding stomatal vs. non-stomatal limitations. The objective of the present study was to quantify the responses of the stomatal, biochemical and photochemical factors involved in photosynthesis regulation to water deficit and recovery after

57

re-watering in C. annuum plants, to better characterize the metabolic changes in the leaves during these processes. We hypothesized that (i) when the excitation pressure exceeds the CO2 assimilation capacity during water stress it will cause a general decline of the photosynthetic plant performance, with possible damages to PSII functionality, electron transport chain and carboxylation; and (ii) even though the CO2 assimilation rates are re-established after re-watering, some of the electron transport limitations will persist. 2. Materials and methods 2.1. Plant material and treatments Seeds of C. annuum cv. Cannon (Zeraim Gedera Ltd., Israel) were germinated in germination trays using peat moss as a substrate. Twenty-seven-day old seedlings were transferred to 250 mL containers filled with soil and grown in a semi-automatic growth chamber (Thermo Scientific, USA) with a 12 h photoperiod and 382 ␮mol photons m−2 s−1 of photosynthetically active radiation (PAR) at the plant level. The plants were grown at the Colegio de Postgraduados, México. A nutrient solution (UltrasolTM Multipurpose 18-18-18 with micro-elements; Chilean Chemical and Mining Society S.A., Chile) was applied directly in the irrigation water. At 23 days after transplanting (DAT), the plants were assigned randomly to two different groups. In the control group, the volumetric water content ( v ) was kept between 45% and 30%; in the other group, water was withheld until the  v fall to 20% (medium stress) and 5% (severe stress). Afterwards, the treated plants were watered to field capacity, and their recovery was evaluated. 2.2. Soil and leaf water status During the experiment, the soil volumetric water content ( v ) was measured on a daily basis within the first 8 cm from the substrate surface using a time-domain reflectometer (HH2 handheld moisture meter adapted with a WET sensor; Delta-T Devices, England) for 6 plants for each treatment. At the same time, the soil water potential ( S ) values of the different moisture levels were obtained from psychrometer readings from a C-52 sample chamber (Wescor Inc., Utah, USA). To determine the water status of the leaf tissue, 0.5 cm diameter leaf discs were placed into C-52 psychrometric chambers (Wescor Inc., Utah, USA) and equilibrated for 2 h at a constant temperature, with 6 replicates per treatment. Both leaf ( L ) and  S were measured using the dew point technique with a HR 33 Dew Point Microvoltmeter (Wescor Inc., Utah, USA). 2.3. Gas exchange and A-Cc curves Carbon dioxide (CO2 ) assimilation rate (A) in response to the increase of internal CO2 (Ci ) in the leaf were measured in leaves from 5 plants per treatment using an open portable infrared gas analyser system (Ciras-1, PP Systems, UK) and a Parkinson automatic leaf chamber (PLC-B, PP Systems, UK) set at a PAR of 1050 ␮mol photons m−2 s−1 at a leaf temperature of 25 ◦ C and relative humidity of 70%. The A-Ci response curves were generated using the protocol described by Long and Bernacchi (2003). The leaf chamber was initially set at a CO2 concentration of 370 ␮mol mol−1 for 5 min to ensure the activation of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) under steady-state conditions. Subsequently, the A-Ci curves were constructed by recording the response of A to different CO2 concentrations in the chamber (Ca ). The CO2 concentration inside the chamber decreased to 300, 250, 200, 150, 100 and 50 ␮mol mol−1 . Subsequently, 370 ␮mol mol−1

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H. Campos et al. / Environmental and Experimental Botany 98 (2014) 56–64

CO2 was re-established to verify that the original assimilation was recovered; if that was the case, Ca was increased stepwise to 450, 550, 650, 800, 1000, and 1200 ␮mol mol−1 . The A-Ci curves were used to estimate the mesophyll conductance (gm ), the velocity of the maximum carboxylation of Rubisco (Vcmax ), the electron transport rate (Jmax ) and the CO2 release rate (Rd ), using a utility developed by Sharkey et al. (2007) based on an alternative A-Ci curve fitting method (Ethier and Livingston, 2004) that accounts for CO2 conductance transfer through a non-rectangular hyperbola version of the model of Farquhar et al. (1980). The chloroplast CO2 concentration (Cc ) is estimated using the equation [Ci − (A/gm )] and a reasonable estimate of gm , can be made directly from A-Ci data (Sharkey et al., 2007).

2.4. Measurement of leaf OJIP transients The chlorophyll a fluorescence transients were performed using a Plant Efficiency Analyzer (PEA, Hansatech Instruments Ltd., UK) on young fully expanded leaves at room temperature. Leaves were dark adapted for 1 h, and then exposed to a saturating red light pulse (650 nm, 3000 ␮mol m−2 s−1 ) provided by the PEA through an array of six light-emitting diodes. The fluorescence signal was recorded for 1 s, at an acquisition rate of 10 ␮s for the first 2 ms, and every 1 ms thereafter (Strasser et al., 1995). The chlorophyll a fluorescence transients were analyzed according to the JIP-test (Strasser et al., 2004, 2010). The following data from the original measurements were used: fluorescence intensity at 50 ␮s (considered as minimum fluorescence, F0 ), 300 ␮s (F300 ␮s ) required for calculation of the initial slope (M0 ) of the relative variable fluorescence (V) kinetics, 2 ms (J-step, FJ ), 30 ms (I-step, FI ) and P-step (considered as maximum fluorescence, Fm ). The biophysical parameters derived from the OJIP transients were calculated, and the following parameters, which refer to time 0 (onset of fluorescence induction) were used: (a) flux ratios or yields; i.e. maximum quantum yield of primary photochemistry (ϕPo = TR0 /ABS = Fv /Fm ), quantum yield for electron transport (ϕEo = ET0 /ABS) and quantum yield for the reduction of end acceptors of PSI per photon absorbed (ϕRo = RE0 /ABS); (b) the efficiency with which a trapped exciton can move an electron into the electron transport chain further than QA − ( Eo = ET0 /TR0 ), and the efficiency with which an elecacceptors to tron can move from the reducedintersystem electron  the PSI end electron acceptors ıRo = REO/ETO ; (c) the fraction of PSII Chl a molecules that function as reaction centers (RC/ABS); (d) the performance index on an absorption basis (PIABS ), which measures the potential for energy conservation from photons absorbed by PSII to the reduction of the intersystem electron acceptors (PIABS = (RC/ABS) × (ϕPo /(1 − ϕPo )) × ( Eo /(1 − Eo ))); and (e) the performance index total (PItotal ), which describes the potential for energy conservation from photons absorbed by PSII to the reduction of PSI end acceptors (PItotal = (PIABS ) × (ıRo /(1 − ıRo )). The formulae showing how each of the above-mentioned biophysical parameters can be calculated from the original fluorescence measurements have been described previously in detail (Yusuf et al., 2010; Strasser et al., 2010). Additionally, an extended OJIP trace study was performed by estimating fluorescence within the individual exchange rate range (i.e., from F0 , measured at 50 ␮s, to FJ , measured at 2 ms) and visualizing the L band (between 100 and 200 ␮s) and the K band (between 200 and 400 ␮s) by subtracting or estimating the differences between the fluorescence traces for the control and the treated samples (Strasser et al., 2004; Yusuf et al., 2010). Also, the width of the bands presented graphically were calculated as follow (Desotgiu et al., 2013): V0K 100 = (F100 ␮s − F0 )/(F300 − F0 ) and V0J 300 = (F300 ␮s − F0 )/(FJ − F0 ) for the L and K band, respectively.

Fig. 1. Effects of the irrigation interruption regime on the soil water potential (A) and leaf water potential (B) in C. annuum plants. Data points and error bars represent the means and corresponding ±SE (n = 6).

2.5. Experimental design and statistical analysis This study was performed using a completely randomized design, and the data were subjected to repeated measures analysis of variance (ANOVA) using the PROC MIXED procedure in SAS (SAS 9.1, SAS Institute Inc., NC) and Tukey’s mean separation test with ˛ = 0.05. Normality and variance homogeneity tests were conducted prior to the analysis of variance using the Anderson–Darling and Levene tests, respectively; for cases in which the hypothesis was rejected, the data set was transformed using the Johnson transformation (Chou et al., 1998). 3. Results 3.1. Soil and plant water status At the beginning of the water deficit treatment (Fig. 1A), the soil water potential ( S ) was approximately zero; it decreased to −1.1 MPa with the development of the water deficit and remained, on average, at −0.19 MPa in the control. After re-watering and during the recovery period, the  S reached values similar to those of the control. On the other hand, the leaf water potential ( L ) in the control plants remained, on average, at −0.61 MPa throughout the duration of the experiment (Fig. 1B). In the stressed plants,  L decreased to −1.7 MPa on day 10 and reached control levels after re-watering. 3.2. Gas exchange responses during water stress and recovery The water stress caused a significant reduction in A, gs and Tr after 4 and 9 days of water deficit (Fig. 2). The A values of the leaves in the stressed plants were 65% and 11% of that of the control plants after 4 and 9 days, respectively. The gs value decreased to 60% and 95% compared to control plants, while the Tr was reduced by 46%

H. Campos et al. / Environmental and Experimental Botany 98 (2014) 56–64

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Fig. 3. Net CO2 assimilation rate (A) relative to the internal partial pressure of CO2 in the chloroplast (Cc ) in C. annuum leaves at three water stress levels defined by the leaf water potential: Control at −0.45 MPa (closed circles), −0.85 MPa (open circles), and −1.24 MPa (closed triangles). Data shown are five replicates for each water potential (n = 5).

3.3. CO2 assimilation rate in response to CO2 concentration at the chloroplast during water stress and recovery

Fig. 2. Effect of water stress and recovery on gas exchange variables. (A) CO2 assimilation rate; (B) stomatal conductance; and (C) transpiration rate. The data points and error bars represent the mean ± SE (n = 10).

and 90% during the same experimental period. The gas exchange variables gradually recovered during the first 2 days of irrigation (Fig. 2), with different degrees of recovery observed on days 1 and 2. Leaf A was restored to 54% and 93% of the control values on days 1 and 2, respectively, after re-watering; the gs value recovered to 29% and 64% and the Tr value recovered to 36% and 77% of the level in the control plants.

Changes in A as a function of increased CO2 concentrations at the chloroplast were used to determine the biochemical limitations of photosynthesis under stress conditions and after re-watering. Water stress reduced the initial portion of the A vs. Cc curves, as well as A at the highest concentrations of Cc (Fig. 3). The values of Vcmax , Jmax and Rd are presented in Table 1. No significant reduction in Vcmax was observed after 4 days of stress ( L equal to −0.85 MPa). However, at day 9 ( L equal to −1.24 MPa), Vcmax was reduced by 52% compared with the control plants. The values of Jmax decreased 36% and 57% compared to the control at days 4 and 9, respectively. The Rd variable was significantly higher than that of control plants after 9 days of water stress. After re-watering, Vcmax and Jmax gradually increased to levels equivalent to those of the control plants and even surpassed the controls after day 2 (Table 1). Similarly, the Rd decreased to values similar to those of control plants on day 2 after irrigation (Table 1). 3.4. Changes in chlorophyll a fluorescence during water stress and recovery The chlorophyll a fluorescence transients showed that the progressive increase of water stress decreased the amplitude of the OJIP curves, mainly that at the P level (FP ), with a  L value of

Table 1 Rubisco maximum carboxylation rate (Vcmax ), RuBP regeneration capacity mediated by the maximum rate of electron transport (Jmax ), CO2 release under light conditions (Rd ) by C. annuum plants with water stress and after re-watering. All variables were obtained from CO2 response curves. Numbers in brackets on the first part show elapsed days after stress imposition whereas in the second part indicates elapsed days after re-watering. Variable

 L (MPa)

Vcmax (␮mol m−2 s−1 )

Jmax (␮mol m−2 s−1 )

Rd (␮mol m−2 s−1 )

Treatments after water withheld −0.45 Control −0.85 Stress (4) −0.54 Control −1.24 Stress (9)

40.3 27.0 51.5 24.6

± ± ± ±

3.1ab 2.3b 15.6a 7.0b

67.4 43.1 65.8 28.1

± ± ± ±

3.6ab 5.0cde 13.4ab 8.1e

0.6 0.4 0.7 2.2

± ± ± ±

0.3bc 0.1c 0.3bc 0.7a

Treatments after re-watering Control Day (1) Control Day (2)

32.4 23.4 33.4 48.4

± ± ± ±

4.2ab 10.4b 3.1ab 4.5ab

55.4 45.2 57.9 78.7

± ± ± ±

8.0abc 9.8bcd 6.2abc 8.2a

0.2 1.5 0.2 0.6

± ± ± ±

0.1c 0.5ab 0.1c 0.2bc

−0.58 −1.08 −0.54 −0.67

Different letters within the same column represent significant differences at P < 0.05 by Tukey test. Values are means with ±SE, n = 5.

60

Variable

L

TR0 /ABS

ET0 /ABS

RE0 /ABS

ET0 /TR0

RE0 /ET0

RC/ABS

PIABS

PItotal

Treatments after water withheld Control −0.57 0.790 ± 0.002ab 0.790 ± 0.002ab Stress (1) −0.60 Control −0.62 0.791 ± 0.002ab 0.792 ± 0.003ab Stress (3) −0.77 −0.64 0.788 ± 0.004ab Control 0.785 ± 0.003ab Stress (7) −1.27 −0.63 0.794 ± 0.002ab Control Stress (10)−1.72 0.772 ± 0.004bc

0.492 0.501 0.503 0.510 0.489 0.482 0.495 0.445

± ± ± ± ± ± ± ±

0.006ab 0.007a 0.006a 0.008a 0.010ab 0.008ab 0.004a 0.011bc

0.167 0.174 0.181 0.187 0.175 0.166 0.191 0.146

± ± ± ± ± ± ± ±

0.009cdef 0.009bcdef 0.010abcdef 0.011abcde 0.009bcdef 0.009cdef 0.009abcd 0.008ef

0.623 0.634 0.635 0.644 0.620 0.613 0.623 0.577

± ± ± ± ± ± ± ±

0.006abc 0.007ab 0.006ab 0.008a 0.010ab 0.008abc 0.005abc 0.011cd

0.340 0.346 0.358 0.365 0.356 0.344 0.386 0.327

± ± ± ± ± ± ± ±

0.015cd 0.014cd 0.016bc 0.017bc 0.013bc 0.015cd 0.016abcd 0.014d

0.511 0.516 0.529 0.515 0.532 0.489 0.538 0.473

± ± ± ± ± ± ± ±

0.013abc 0.015abc 0.014ab 0.010abc 0.010ab 0.008bc 0.007ab 0.015cd

Treatments after re-watering −0.70 0.802 ± Control −1.40 0.696 ± Day (1) −0.66 0.798 ± Control −0.90 0.702 ± Day (2) −0.55 0.804 ± Control Day (3) −0.88 0.727 ± Control −0.64 0.803 ± −0.90 0.755 ± Day (4) −0.66 0.799 ± Control 0.783 ± −0.86 Day (7) −0.62 0.801 ± Control 0.792 ± Day (10) −0.81

0.513 0.349 0.505 0.364 0.509 0.374 0.510 0.416 0.501 0.464 0.499 0.472

± ± ± ± ± ± ± ± ± ± ± ±

0.001a 0.017e 0.004a 0.013e 0.001a 0.017de 0.003a 0.015cd 0.006a 0.007abc 0.004a 0.008ab

0.199 0.135 0.203 0.139 0.212 0.150 0.220 0.167 0.191 0.173 0.210 0.189

± ± ± ± ± ± ± ± ± ± ± ±

0.005abc 0.009f 0.010abc 0.008f 0.003ab 0.009def 0.002a 0.010cdef 0.006abcd 0.003bcdef 0.006abc 0.005abcde

0.640 0.500 0.632 0.518 0.633 0.512 0.635 0.549 0.627 0.592 0.623 0.596

± ± ± ± ± ± ± ± ± ± ± ±

0.002ab 0.016f 0.004ab 0.013ef 0.001ab 0.015ef 0.003ab 0.013de 0.007ab 0.005abcd 0.005abc 0.007bc

0.388 0.386 0.401 0.381 0.416 0.401 0.431 0.400 0.380 0.373 0.420 0.399

± ± ± ± ± ± ± ± ± ± ± ±

0.009abcd 0.010abcd 0.017abc 0.012abcd 0.007ab 0.011abc 0.003a 0.012abc 0.008abcd 0.008abcd 0.013ab 0.005abc

0.553 0.398 0.537 0.406 0.549 0.402 0.543 0.427 0.516 0.455 0.510 0.500

± ± ± ± ± ± ± ± ± ± ± ±

0.004a 4.00 ± 0.05a 0.010e 0.996 ± 0.16f 0.006ab 3.69 ± 0.12ab 0.010e 1.07 ± 0.11f 0.005a 3.91 ± 0.03a 0.016e 1.23 ± 0.18f 0.006ab 3.89 ± 0.11a 0.014de 1.72 ± 0.20ef 0.008abc 3.50 ± 0.18ab 0.013cde 2.43 ± 0.18cde 0.005abc 3.43 ± 0.12ab 0.010abc 2.87 ± 0.19bcd

0.001a 0.012f 0.002ab 0.007ef 0.001a 0.013de 0.001a 0.011cd 0.001ab 0.007abc 0.001ab 0.004ab

3.21 3.42 3.53 3.62 3.32 2.89 3.46 2.26

± ± ± ± ± ± ± ±

0.18abc 0.21abc 0.20ab 0.21ab 0.23abc 0.18abcd 0.12ab 0.20de

L-band

K-band

1.71 1.85 2.04 2.16 1.90 1.56 2.23 1.13

± ± ± ± ± ± ± ±

0.20cdef 0.19bcde 0.24bcd 0.25abc 0.21cde 0.17cdef 0.21abc 0.14efg

0.223 0.227 0.224 0.225 0.223 0.221 0.220 0.221

± ± ± ± ± ± ± ±

0.001cde 0.002abcd 0.002cd 0.002bcde 0.002cd 0.001def 0.001def 0.001def

0.640 0.642 0.637 0.645 0.631 0.649 0.626 0.653

± ± ± ± ± ± ± ±

0.006defg 0.007defg 0.006defg 0.004defg 0.003defg 0.003cdef 0.002efg 0.007cde

2.55 0.64 2.54 0.68 2.80 0.84 2.95 1.18 2.17 1.44 2.51 1.92

± ± ± ± ± ± ± ± ± ± ± ±

0.09abc 0.13g 0.22abc 0.09g 0.09ab 0.13fg 0.09a 0.16cdefg 0.16abc 0.09cdefg 0.15abc 0.16bcde

0.217 0.239 0.215 0.236 0.214 0.233 0.213 0.225 0.215 0.220 0.211 0.215

± ± ± ± ± ± ± ± ± ± ± ±

0.001def 0.002a 0.001ef 0.002a 0.001ef 0.005abc 0.001f 0.003bcde 0.001def 0.003def 0.001f 0.002ef

0.621 0.699 0.627 0.698 0.618 0.690 0.622 0.675 0.634 0.660 0.633 0.634

± ± ± ± ± ± ± ± ± ± ± ±

0.002fg 0.006a 0.003defg 0.005a 0.002g 0.011ab 0.002fg 0.010abc 0.002defg 0.009bcd 0.001defg 0.006defg

Quantities are expressed as dimensionless ratios (PIABS and PItotal is expressed as arbitrary units) except  L expressed in MPa. Different letters within the same column represent significant differences at P < 0.05 by Tukey test. Values are means with ±SE, n = 8.

H. Campos et al. / Environmental and Experimental Botany 98 (2014) 56–64

Table 2 Leaf water potential ( L ) and JIP-test parameters of dark-adapted leaves of C. annuum plants under water stress and after re-watering. For parameter description, see Section 2.4. Numbers in brackets on the first part show elapsed days after stress imposition whereas in the second part indicates elapsed days after re-watering.

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Fig. 4. Chlorophyll a fluorescence transients OJIP of dark adapted leaves of C. annuum plants under normal irrigation at −0.57 MPa (closed circles) and different water stress conditions at −0.77 (open circles), −1.27 (closed triangles), and −1.72 MPa (open triangles) respectively. Each curve represents the mean value from eight curves measured in leaves (n = 8).

−1.72 MPa, while F0 (step O) remained unchanged (Fig. 4). To further investigate the mechanisms underlying the observed changes, the JIP-test was applied to the fluorescence induction transients. The effects of water stress on several parameters of the JIP-test are presented in Table 2. The parameters reflecting the quantum yields for electron transport in PSI and between both photosystems (RE0 /ABS and ET0 /ABS, respectively), were sensitive to water stress and decreased as the  L became more negative. In contrast, water stress had a little effect on the maximum quantum yield of the primary photochemistry of PSII (TR0 /ABS = Fv /Fm ), as confirmed by the high stability of this parameter during water deficit (Table 2). Similarly, the composed parameters PIABS and PItotal changed significantly during water stress (Table 2). The rewatering of C. annuum plants at the end of the stress treatment gradually restored the  L (Fig. 1). With the complete rehydration of leaves, all the fluorescence signals were recovered, i.e., the OJIP transient both as amplitude and shape, and hence all the JIP-test parameters (Table 2). Additionally, the fluorescence traces of Chl a of the different treatments and the control samples were double normalized at F0 (0.05 ms) and FK (0.3 ms), and expressed as WOK = (Ft − F0 )/(FK − F0 ). Subsequently, the traces of the control samples were subtracted from the traces of the treated leaves (WOK ). This difference makes the L band visible and its response was distinct at the different  L and after re-watering (Fig. 5A and B), showing a high amplitude in days 1–4 after re-watering (Table 2), and similar to control on days 7 and 10 (Table 2). In Fig. 5C and D, the Chl a fluorescence transients were double normalized between F0 and FJ (=1 ms), and expressed as WOJ = (Ft − Fo )/(FJ − Fo ), and the difference among different levels of stress and the control were determined (WOJ ). This allowed the visualization of the K band with a peak between 0.25 and 0.30 ms. An increasing positive deviation was also observed, indicating that the K band becomes more pronounced with more severe water stress (Fig. 5C). This limitation lasted from days 1 to 4 after rewatering (Table 2 and Fig. 5D). 4. Discussion 4.1. CO2 assimilation, biochemical and stomatal limitations to photosynthesis In plants under water stress, a rapid increase in A after rewatering indicates that the biochemical mechanisms were not affected by water deficit, which suggests that the decrease in A is

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the result of stomatal closure (Cornic, 2000). The results showed that gs did not fully recover after 2 days of deficit irrigation, despite the almost complete recovery of A (Fig. 2). Under stress conditions, a reduction in stomatal conductance can have protective effects because it allows the plant to save water and to improve the water use efficiency (Chaves et al., 2009). However, although stomatal closure appears to be the main limiting factor of A in C. annuum plants subjected to water deficit, non-stomatal factors were also important for the regulation of photosynthesis during severe water stress ( L ≥ −1.24 MPa) (Fig. 3 and Table 1). In fact, the reduction of A at high Cc values (Fig. 3) was accompanied by reductions in Vcmax and Jmax (Table 1), which indicates that a decrease in CO2 assimilation occurs to adjust the mesophyll capacity to the lower CO2 supply caused by stomatal closure (Chaves et al., 2002; Lawlor and Cornic, 2002). The decrease in Vcmax values may result from a reduction in ˜ the amount of active Rubisco (Pena-Rojas et al., 2004). The reduction in Jmax is associated with a limited regeneration of RuBP due to an inadequate supply of ATP or NADPH or to a low enzymatic activity during the photosynthetic carbon reduction cycle, such as the sedoheptulose-1,7-biphosphatase and fructose-1,6-biphosphatase (Flexas et al., 2004b; Lawlor, 2002; Parry et al., 2002). The results also indicate that plants under severe water stress exhibited a greater Rd (Table 1) and therefore can contribute with more CO2 to that needed during photosynthesis, maintaining a positive carbon balance behind closed stomata (Hu et al., 2010). The above suggests that stomatal limitations in A were crucial during the initial stress stages, while non-stomatal mechanisms become limiting over prolonged periods of time. This response pattern is consistent with that described by Hu et al. (2010) under controlled experimental conditions. However, the reduction of A in stressed plants may also be the result of different effects on the efficiency of light capture and on the photosynthetic electron transfer system (Tezara et al., 2003). 4.2. Chlorophyll a fluorescence and leaf photochemistry In the present study, the fluorescence induction trace shape was sensitive to the development of water stress in the leaf tissue of the plants (Fig. 4). The analysis of several parameters of the JIP-test allowed us to locate the modifications induced by water stress on the electron transport chain (Table 2). The redox reactions were strongly affected by water stress; mainly the quantum yields of the reactions after QA − were less tolerant to water deficit (Table 2). In fact, the three JIP-parameters which reflect the quantum yields of photo-induced electron transfer from P680 to QA (ϕPo = TR0 /ABS), from QA − to PQ (ϕEo = ET0 /ABS) and from PQ to the PSI electron acceptors (ϕRo = RE0 /ABS), can be ranked according to their sensitivity to water stress in the sequence ϕRo > ϕEo > ϕPo . Moreover, the efficiency or probability of electron transport beyond QA − ( Eo = ET0 /TR0 ) and that of the intersystem electron carriers will reduce end electron acceptors at the PSI acceptor side (ıRo = RE0 /ET0 ) also decreased in plants under water stress (Table 2). The reduction of Eo and ϕEo along with the maintenance of ϕPo indicate that the water deficit inhibits not the primary light reactions but rather the redox reactions that follow QA − (Chen et al., 2011). This indicates that the water deficit causes the slowdown of the electron transfer and the reduction of end electron acceptors at the PSI acceptor side (Chen et al., 2011). Under normal conditions, the transfer of energy to the reaction centers of PSII and PSI results in the transport of the electron to ferredoxin (Fd) and the reduction of NADP+ . However, as A decreases gradually with the water deficit, the consumption of NADPH decreases as well, causing a decrease in the linear electron transport as the sinking capacity is reduced (Lawlor and Tezara, 2009). This leads to an impairment of the electron transport and a decline in the requirement of the reduction of end electron acceptors such as NADP+ and Fd, that in turn limits

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Fig. 5. Change in the shape of the Chl a fluorescence transient curves under waters stress (panels A and C) and recovery (panels B and D) revealing the L-band and Kband. Each result is average of eight repetitions. Panels A and B: WOK = (Ft − F0 )/(FK − F0 ); panels C and D: WOJ = (Ft − F0 )/(FJ − F0 ). In panel A and C the difference kinetics WOX = WOX(treatment) − WOX(control) are shown where treatment is control  L (dash line), −0.60 (closed circle), −0.77 (open circle), −1.27 (closed triangle), and −1.72 MPa (open triangle). In panels B and D the difference kinetics WOX = WOX(treatment) − WOX(control) are shown where treatment is control (dash line), day 1 (closed circle), day 2 (open circle), day 3 (closed triangle), day 4 (open triangle), day 7 (closed square) and day 10 (open square) after re-watering.

the synthesis of ATP and the regeneration of RuBP (Lin et al., 2009). The shortage of ATP causes the incomplete activation of Rubisco (Streusand and Portis, 1987), which partially explains why plants under water stress exhibit a lower Vcmax (Table 1). Thus, the limitations in photosynthetic electron transport capacity, along with the ATP shortage, limit the regeneration of RuBP (Jmax ) (Table 1) and are the main non-stomatal factors that contribute to the decreased CO2 assimilation under water stress conditions. The results showed that moderate  L had little effect on the maximum quantum yield of primary photochemistry (TR0 /ABS = Fv /Fm ) (Table 2) which reflects the efficiency of the light reactions (Strasser et al., 2004), consistent with previous reports (Lu and Zhang, 1999). The same observation has been reported by other researchers regarding the use of the Fv /Fm ratio to study the fluorescence in stressed plants (Oukarroum et al., 2007; Van Heerden et al., 2007). In contrast, a marked reduction in the performance index PIABS was observed along with the sudden decrease of  L in the leaf (Table 2). The reduction in PIABS as a response to water stress was mainly due to changes in the properties of the antenna (RC/ABS) and to the decrease in the photochemical efficiency in the transport of photosynthetic electrons beyond QA (ET0 /TR0 ) (Table 2), which indicates a reduction in the efficiency of the redox reactions in the electron transport chain (Chen et al., 2011). The composite parameter PItotal combines ϕPo , Eo , ıRo and RC/ABS and therefore summarizes all the partial conductive forces and their individual effects (Strasser et al., 2010). The PItotal was notably reduced as the  L value decreased due to the cessation of watering (Table 2); therefore, the reduction of this parameter during water deficit was mainly due to the added slowdown of the reduction of PSI final

acceptors. This finding indicates that PItotal is sufficiently sensitive to characterize, quantify and detect water stress, even prior to the appearance of visible symptoms on the leaves of C. annuum. A more exhaustive analysis of the OJIP transients revealed more information about the effects of water stress on the photosynthetic units of C. annuum plants. The increase in the positive amplitude of the L band at  L = −1.72 MPa (Fig. 5A) and during the first 4 days of recovery (Table 2), implies that the PSII units are least grouped together, i.e., less energy is being exchanged among the independent PSII units (Yang et al., 2012), as a response to water stress. Grouping is sensitive to the unstacking of the thylakoid membranes, which can be induced by conditions such as high or low salinity (Strasser and Greppin, 1981; Strasser, 1981) and has been observed in Chlamydomonas reinhardtii in response to hyperosmotic stress (Cruz et al., 2001). The reduction in the amplitude of the L band during days 7 and 10 after re-watering (Fig. 5B) suggests that the grouping of the units of PSII is restored, resulting in a greater stability of the samples after stress (Yang et al., 2012). Moreover, the K step is exhibited in the fluorescence traces of several higher plants that are native to hot and dry climates i.e., Cycas revoluta, Permelia sp. and Juniperus sp., as reported by Srivastava et al. (1997). It is normally hidden by the O–J increase because the limitation of the electron transport through PSII is rarely strong enough to render the K step visible. However, as shown in Fig. 5C, water stress induces the positive appearance of the K band, and its amplitude increases as the  L value decreases (Table 2). The appearance of the K band is explained by an imbalance within PSII, among the electrons that leave the reaction centers in the electron acceptor side and the electrons donated by the donor side (Strasser, 1997),

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and it has been related to a dissociation of the oxygen-evolving complex (OEC) (Guissé et al., 1995; De Ronde et al., 2004). We also observed the persistence of the K band during the first 4 days after re-watering and its disappearance after 10 days (Table 2), possibly co-occurring with the restoration and normal functioning of OEC.

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Acknowledgement This work was supported by a scholarship (166340) from Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico. References

4.3. Photosynthetic limitations during recovery In contrast with the photosynthetic responses during the development of stress, the limitations after re-watering and recovery have received considerably less attention (Galmés et al., 2007a). Normally, the recovery of photosynthesis after a moderate water stress (gs > 0.15 mol m−2 s−1 ) is rapid (1 d) and almost complete (Flexas et al., 2006). In contrast, after severe water stress, the recovery of photosynthesis is progressive, slow (can take several days to weeks) and usually incomplete (De Souza et al., 2004; Miyashita et al., 2005). Our results showed that gs remained below the levels of the control plants 2 days after re-watering, but with similar CO2 assimilation rates (Fig. 2), and that the  L was slowly restored 10 days after re-watering (Fig. 1B). The limited recovery of hydraulic conductivity in the leaf is the apparent cause of the abatement of stomatal conductance after re-watering (Galmés et al., 2007b), and it was recently shown that the aquaporins play a determinant role in the regulation of dynamic changes in the variable hydraulic conductance in the leaves (Cochard et al., 2007). Metabolic damage and a corresponding restriction in the photosynthetic recovery of the leaves after irrigation is commonly assumed to occur (Flexas et al., 2004b); however, the values of Vcmax and Jmax were similar to those of control plants 2 days after re-watering (Table 1), consistent with reports by Galmés et al. (2007a) in 10 Mediterranean species that exhibited excellent recovery and few biochemical limitations after severe water stress. In addition, the efficiency of the photosynthetic electron transport (Table 2), the connectivity among the units of PSII (Fig. 5B) and the normal functioning of the donor side of PSII (Fig. 5D) recovered gradually after 10 days of re-watering. The recovery of such parameters suggests that the water stress did not irreversibly damage the light reactions and that the damage to the photosynthetic electron transport chain was gradually repaired during re-watering, promoting the recovery of photosynthesis. 5. Conclusion In conclusion, the results indicate that photosynthesis in C. annuum plants during water stress is inhibited by stomatal closure, which limits the diffusion of CO2 to the chloroplast and causes a reduction of the internal CO2 concentration, leading to a decrease in the enzymatic activity of Rubisco. At the same time, water stress reduces the efficiency of photosynthetic electron transport by uncoupling the electron transport chain mainly from the PSII acceptor side to the PSI end electron acceptors, limiting the regeneration of RuBP and the CO2 assimilation rate. Importantly, the decreased electron transfer efficiency from the plastoquinone pool to the PSI terminal acceptors showed to be a key regulatory mechanism underlying the photochemical limitations of C. annuum plants under water stress. These results emphasize that, early under water stress, the diffusive limitations are the main factor for the observed depression of photosynthesis and the photosynthetic metabolism is progressively down-regulated as water stress intensifies, whereas during recovery, photosynthesis is limited by stomatal closure and the gradual recovery of the electron transport chain activity. Our results also show that the JIP-test parameters (RE0 /ABS, RE0 /ET0 , and PItotal ) referring to the electron flow from the reduced intersystem electron acceptors to the PSI end-electron acceptors may represent good parameters for monitoring drought stress effects on photosynthesis.

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