Abstract
Isohydric plants maintain constant water potential through rapid stomatal closure, whereas anisohydric plants only close their stomata at very low water potentials. However, distinctions between isohydric and anisohydric behaviors among different cultivars of the same species are unclear. This study compared the physiological response to prolonged drought stress in the isohydric Grenache and the anisohydric Shiraz cultivars of the Vitis vinifera species. Plants were exposed to 60-day periods of deficit irrigation (25% of plant water consumption under well-watered conditions) during the summers of 2011 and 2012. Physiological measurements, water potential, leaf gas exchange, canopy area, leaf senescence, stem characteristics, and morphological characteristics were analyzed. Stomatal conductance was consistently lower in Grenache than in Shiraz at all values of midday stem and predawn leaf water potentials, respectively. The Shiraz plants exhibited greater vegetative growth and less defoliation than the Grenache plants in response to water deficit. Anatomical architecture analyses revealed that Grenache plants had greater xylem vessel diameter, hydraulic conductivity, and stomatal density than the Shiraz plants. These results suggest isohydric and anisohydric behaviors may be well-defined, time-regulated responses rather than distinct mechanisms that plants use to cope with drought stress. The rapid response to water deficit exhibited by isohydric plants may be because they are more vulnerable to fatal xylem embolisms than anisohydric plants. Thus, the accelerated response allows isohydric plants to avoid drought stress and minimize risk of xylem cavitation, but may lower the plant’s ability to survive moderate stress of prolonged drought.
Higher plants respond to water stress with a variety of physiological and molecular mechanisms, and several processes affect the ability of a plant to adapt to drought stress. Stomatal closure is the primary physiological adaptation used by plants to prevent a decrease in water potential by limiting gas exchange and transpiration during periods of water deficit (Breda et al. 2006, Chaves 1991, Sperry et al. 2002). When the soil is very dry, the water potential may fall below the permanent wilting point at which the plant is unable to recover, and critically negative xylem pressures may cause excessive xylem cavitation and embolism.
Stomatal function is closely associated with plant hydraulics under drought conditions (Sperry et al. 2002). Mediterranean summers are characterized by drought, high air temperatures, and high evaporative demand. Supra-optimal leaf temperatures combined with a water deficit lead to a severe decrease in photosynthesis and thereby a dramatic reduction in carbon assimilation, which is often accompanied by a partial loss of canopy leaf area (Chaves et al. 2003).
Embolism formation is also among the major physiological factors contributing to reduced primary forest productivity and drought-induced mortality in woody plants (Anderegg et al. 2012, Choat et al. 2012). Thus, xylem organization is critically important to the isolation of drought-induced embolism in grapes (Brodersen et al. 2013).
Plants respond to water deficit with isohydric or anisohydric behaviors. Isohydric behavior is more conservative and involves maintenance of a relatively constant leaf water potential to avoid loss of conducting vessels and maintain overall plant water potential to prevent wilting. These plants often have highly sensitive stomatal regulation, characterized by immediate closure in response to water deficit. Anisohydric behavior involves maximizing carbon production in the short term at the cost of decreased water potential in the leaf and xylem. Stomatal regulation under drought conditions is slower in anisohydric plants than in isohydric plants, which leads to more efficient carbon fixation (i.e., greater assimilation of carbon per unit of available water) but decreased water potential (Jones and Sutherland 1991).
As drought conditions push soil water potential (Ψpredawn) below its Ψmidday set-point (which represents Ψleaf), isohydric plants can no longer extract water for gas exchange. Anisohydric behavior in plants allows Ψmidday to respond more dynamically to changes in Ψpredawn and to decrease with rising evaporative demand. Drought-affected plants reach a much lower Ψmidday than do well-watered ones, which produces a gradient between Ψpredawn and Ψmidday that allows gas exchange to continue over a greater range of Ψmidday values. However, the isohydric and anisohydric behaviors are not always distinguishable, and plants that typically exhibit anisohydric behavior may show constrained stomatal conductance (gs) under certain conditions (Beis and Patakas 2010).
Potential drawbacks associated with lower Ψmidday in anisohydric plants include a decrease in xylem conductivity caused by embolism. Xylem embolism in relation to stomatal control and Ψleaf threshold values is species-dependent (Cochard et al. 1996, Cochard et al. 2002, Cruiziat et al. 2002, Froux et al. 2005, Jones and Sutherland 1991, Meinzer et al. 2009, Poggi et al. 2007, Salleo et al. 2000, Tyree and Ewers 1991). Hacke and Sauter (1995) found that Populus is extremely vulnerable to cavitation and loses functional vessels due to embolism at water potentials lower than −0.7 MPa. By contrast, the plant species Fagus experiences embolism when water potential falls below −1.9 MPa.
Drought resistance is agronomically defined as enhanced productivity under less than optimal water availability (Blum 2011). Thus, any factor that leads to increased crop productivity under drought stress may be regarded as a valuable agronomic trait. An anisohydric crop is likely to improve its yield under moderate water stress because it has higher gs and CO2 assimilation than isohydric plants under the same conditions. However, anisohydric behavior is not beneficial under conditions of prolonged water stress. The soil moisture threshold below which anisohydric plants lose this agronomic advantage is species specific and depends on the plant’s ability to avoid embolism and return to its optimal productivity rate after recovering from water stress (Sade et al. 2012).
Bean, maize, and poplar are examples of plant species that display isohydric behavior, whereas cauliflower, almond, peach, and sunflower display anisohydric behavior (Limpus 2009, Tardieu and Simonneau 1998). Different cultivars of grapes exhibit a range of hydraulic conductance and stomatal behavior (Chaves et al. 2010, Chouzouri and Schultz 2005, Schultz 2003). Vitis vinifera L. is characterized by considerable genetic variability, as several thousand varieties are cultivated worldwide. In general, this type of grape is considered a ‘drought-avoiding’ species, as it contains a large, deep root system and relatively efficient stomatal control over transpiration and xylem embolism. However, some genotypes are classified as isohydric, whereas others are considered anisohydric (Chaves et al. 2010). The Shiraz and Grenache varieties vary drastically in their water management, which has important consequences for their survival, growth, and yield in different environments. Grenache exhibits isohydric behavior, with rapid regulation of gs in response to decreasing soil water, whereas Shiraz exhibits anisohydric behavior (Schultz 2003, Soar et al. 2006). Grenache also shows more rapid induction of ABA in the xylem sap in response to water deficit than does Shiraz (Soar et al. 2006). However, the distinctions between isohydric and anisohydric strategies are not always clear, and plants may switch strategies depending on the environmental conditions and severity of drought stress (Chaves et al. 2010, Domec and Johnson 2012, Rogiers et al. 2012, Zhang et al. 2012).
The aim of this study was to investigate the mechanisms responsible for the variability in the drought response strategies of the two grape cultivars Grenache and Shiraz. We chose these cultivars because they demonstrate isohydric (Grenache) and anisohydric (Shiraz) behaviors in extreme forms (Soar et al. 2006). We compared the physiological and anatomical parameters of the two cultivars in response to various irrigation regimes, including drought stress.
Materials and Methods
Plant material and experiment design
The experiment was conducted over 60-day periods (July–August) during the 2011 and 2012 seasons using the V. vinifera cultivars Grenache and Shiraz grafted onto Richter or Paulsen rootstocks. A total of 160 plants (40 plants of each cultivar and rootstock) were examined. The plants were 1.5 years old in the summer of 2011 and grown in 10-L pots with a tuff-peat-coir mix under greenhouse conditions using a completely randomized four-block design. Fertilization with N, P, and K at a ratio of 4:2:6, respectively, was supplied through the irrigation system. Plants were pruned down to one central stem and 60 leaves one week prior to the start of the experiment. During the first week of the experiments, each plant received 37 mg of fertilizer and 0.52 L of water. Fertilization was continued once per week at a concentration of 0.5% of the water supplied for the duration of the experiment.
Water deficit treatments
Irrigation water was supplied at a rate of 2.0 L/hr through drip emitters. Plant water consumption was evaluated twice per week during the experimental period, and these values were set as the control treatment (100%). Water deficit treatments were set at 75, 50, and 25% of the plant water consumption (100%). Daily water consumption was measured in three plants from each of the four experimental groups (cultivar × rootstock) using homemade lysimeters. These plants were placed in plastic containers for 24 hr and covered with aluminum foil. Water was supplied in excess, and the drained water was collected in the containers. Evapotranspiration (ET), was calculated by the equation ET(Kg) = I − D − ∆W, where I represents total water supplied (kg), D represents the drained water (kg) and ∆W represents the difference in the plant weight over 24 hr (kg).
Physiological measurements
To determine the optimal time of day to measure the various physiological parameters, samples were taken from well-watered plants every four hours from early morning to late evening. A day curve was constructed for the following parameters: leaf temperature (Tl), gs, stem water potential (Ψs), and leaf water potential (Ψl) (Supplemental Figure 1). At noon, Tl and gs were highest and Ψs and Ψl were lowest in both genotypes, and the differences between genotypes were greatest at this time point. Therefore, all measurements except for predawn Ψl were taken at noon.
Water potential
The Ψl and Ψs measurements were obtained weekly using an ARIMAD 3000 pressure chamber (M.R.C., Holon, Israel). The Ψl measurements were taken during the predawn hours (0300–0500 hrs) or around noon (1100–1300 hrs). Midday stem water potentials (Ψs) were measured in non-transpiring mature leaves by bagging them with a plastic sheet covered with aluminum foil for one hour before measurement. All water potential measurements were carried out in four replicates (four plants) for each of the 16 experimental groups (2 cultivars × 2 rootstocks × 4 irrigation regimes.
Leaf gas-exchange
Individual leaf gas-exchange measurements, including leaf temperature (Tl), photosynthetic rate (A), transpiration rate (E), and gs were conducted weekly at noon in four replicates on mature, well-exposed canopy leaves using an open-system gas-exchange module (LI-6400; LI-COR, Lincoln, NE). The LI-6400 parameters were set according to manufacturer’s recommendations. Internal photosynthetically active radiation (PARin) was set to equal the external PAR (PARout). Reference CO2 was set at 400 mg/L, and the temperature and humidity were set to reflect the outside temperature and humidity. However, the LI-6400 did not exceed 35°C even if the outside temperature was greater than 35°C.
Canopy area and leaf senescence
Four plants from each cultivar × rootstock × irrigation treatment group were photographed at the beginning of the experiment and every two weeks thereafter (a total of five photos per plant). These plants were not used for water potential or gas exchange measurements and were photographed on a white background with a ruler to provide a scale. The green area (leaves) was measured using Photoshop CS5 (Adobe, San Jose, CA). At the end of the experimental period, all leaves were removed from the plants and screened in a leaf scanner (Ll-3100C area meter; LI-COR). The ratio between the two-dimensional photos and the real foliage area (scanning) was calculated from photos taken at the end of the experiment and was used to evaluate the foliage area in other stages of the experiment. These plants were also used to evaluate leaf senescence. Yellow and/or senescent leaves were counted and removed once per week.
Stem anatomy and hydraulics
Stem characterization
Stems from four plants of each cultivar and irrigation treatment on the Paulsen rootstock were analyzed. The 20th stem segment of each plant was cut into 2-mm pieces and fixed in an FAA solution with a 10:5:85 ratio of formaldehyde, acetic acid, and 70% ethanol, respectively. Fixation was followed by a series of ethanol dilutions and subsequent stepwise exchange of ethanol with histoclear. Samples were embedded in paraffin and cut into 12-μm sections with a microtome (Leica RM2245; Leica Biosystems, Buffalo Grove, IL). Sections were stained with safranin and fast green staining (Ruzin 1999) and examined with a light microscope (Olympus BX50; Olympus, Tokyo, Japan) at 50× and 100× magnifications.
The cross-sectional area of all xylem vessels in every fourth segment (250–500 vessels) was evaluated in 16 plants using NIS elements software (Nikon, Melville, NY).
Theoretical hydraulic conductance (kh; kg m/MPa s) was calculated with the modified Hagen–Poisseuille’s law described by Tyree and Ewers (1991): where d is the radius of the vessel in meters, ρ is the fluid density (assumed to be 1000 kg/m3 or equal to that of water at 20°C), and η is the viscosity (assumed to be 1 × 10−9 MPs·s or equal to that of water at 20°C) (Tombesi et al. 2010).
Morphological characterization of stoma
Morphological characterization was performed on five representative leaves from each of five well-watered plants from each cultivar. Nail polish was applied to three regions of the leaf surface and examined with a light microscope (Olympus BX50; Olympus) at 20× magnification. Stomata length and density were analyzed using NIS elements software (Nikon).
Data analysis
All statistical analyses were conducted using JMP software (SAS Institute, Inc., Cary, NC). The data collected from the different irrigation protocols of the four experimental groups were analyzed using one-way analysis of variance (ANOVA). Correlations between physiological parameters were evaluated by pairwise Pearson correlation coefficients, with p < 0.05. Because the physiological response in each of the two cultivars was not significantly different between the two rootstocks during both years, we combined the data and compared the Grenache to the Shiraz plants.
Results
Stomatal conductance and water potential
Plants that received 50 and 75% irrigation in 2011 showed the same physiological responses as the well-watered plants (Supplementary Table 1). Therefore, these treatments were not performed in 2012 and were excluded from our analysis.
The isohydric level of a plant can be determined by testing gs as a function of predawn Ψl (Figure 1A) or of Ψs (Figure 1B), under conditions of water deficiency (McDowell et al. 2008, Schultz 2003). Based on our physiological measurements, we found significant differences between the two cultivars (Supplementary Table 1). The Grenache exhibited a lower gs for every value of Ψ than the Shiraz (p < 0.05). This finding, based on data from well-watered as well as drought-stressed plants, clearly shows that the Grenache displayed an isohydric response to mild drought, whereas the Shiraz exhibited an anisohydric reaction under these conditions.
Correlation between various physiological parameters
Relationships between the various tested physiological parameters were analyzed by examining their correlation in all plants. Pairwise correlations between evapotranspiration, Ψl, Ψs, gs, and photosynthetic CO2 assimilation were evaluated. The noon Ψl was measured in the same plants (but not in the same leaves) as the other parameters. Because noon Ψl status changed rapidly and depended on a variety of parameters, it had a very low degree of correlation with the other parameters and was excluded from analysis (Supplementary Table 2). All other correlation coefficients ranged from 0.0005 to 0.95 (Figure 2A), and most coefficients were relatively high, especially in the deficiently irrigated plants. The average correlation coefficient was significantly higher in the plants undergoing deficit irrigation (r = 0.815) than in the well-watered plants (r = 0.565, p < 1.28×10−17, Figure 2B) and was significantly higher in the Grenache (r = 0.755) than in the Shiraz plants (r = 0.626, p < 5.18×10−5) (Figure 2C).
Canopy area and leaf senescence
The well-watered plants of both cultivars showed well-developed vegetative growth throughout the experimental period and ranged from 2900 to 3300 cm2 at the beginning of the experiments and from 3000 to 4300 cm2 at the end. Foliage area increased by 124.8 and 111.4% in the Grenache and Shiraz cultivars, respectively, over the two-month experimental period. In the drought-stressed Grenache and Shiraz plants, the foliage area decreased to 63.7 and 84.9%, respectively, of the original area by the end of the experiment. Although the well-watered Grenache plants had a higher foliage area than the Shiraz plants, the Grenache plants had smaller foliage area in drought stress conditions (1800 cm2 versus 2634 cm2 in the Shiraz cultivars). Leaf loss was greater in the Grenache plants during both well-watered and drought stress conditions (p < 9.41×10−5, Figure 3B). The Grenache plants showed an increase in the foliage area throughout the experiment, whereas the Shiraz increased during the first half of the experiment and decreased during the latter part.
Anatomical characteristics
The anatomical structure of the plant’s hydraulic system was characterized by recording stomata size and density in the leaves as well as xylem tissue area and vessel diameter in the stem. The average stomata length was similar between the cultivars (21 μm in the Grenache versus 19 μm in Shiraz). The stomata density was significantly higher in the Grenache than in the Shiraz (p < 0.035), and the Grenache leaf contained 175 stomatal pores per mm2 whereas the Shiraz leaf contained 161 pores per mm2 (Figure 4).
To characterize the stem architecture of both cultivars, we examined total xylem and xylem vessel area (Supplementary Figure 2). The average proportion of the xylem area relative to total stem area ranged between 0.7 and 0.72 and was not significantly different between cultivars (Figure 5A, B). The average Grenache xylem vessel area was 3350 μm2 in both the well-watered and the drought-stressed plants, whereas the Shiraz xylem vessel area was 2350 μm2 in plants under both irrigation regimes (p < 0.0025) (Figure 5C, D). The Grenache stem had significantly fewer small vessels (0–30 μm class) (p < 0.0083) and a greater number of large vessels (120–150 μm and >150 μm classes) than the Shiraz (p < 0.04) (Figure 5E). Hydraulic conductance was evaluated based on the number of vessels of each genotype in each diameter class. Grenache plants had significantly higher hydraulic conductance than Shiraz plants (p < 0.025) (Figure 5F).
Discussion
Many studies have compared isohydric and anisohydric behaviors in response to drought in a variety of plant species, and several reviews have been published recently (Buckley 2005, Chaves et al. 2010, Lovisolo et al. 2010, McDowell et al. 2008). Different varieties of V. vinifera vary in their response to drought stress, which makes this species optimal for comparing the two behaviors (Schultz 2003, Soar et al. 2006). In this study, we exposed the near-isohydric cultivar, Grenache and the anisohydric cultivar Shiraz to plentiful irrigation and drought-like conditions.
The response to intermediate-level irrigation treatments (50 and 75%) was not different from the 100% treatment, possibly because the 100% treatment involved irrigation with excess water. Thus, the water deficit may not have been severe enough in the 50 and 75% treatments to induce a drought stress response in the plants and likely explains why we only observed a drought stress response in plants undergoing 25% irrigation treatment.
The isohydric level (i.e., the differential response to drought stress) is often expressed by assessing gs as a function of water potential. The Ψs and predawn Ψl are early indicators of water deficiency in plants, whereas Ψl measured at noon is a much less reliable indicator (Chone et al. 2001). The Grenache had a lower gs for each value of Ψs and predawn Ψl, respectively, than the Shiraz did (Figure 1), which is similar to previous findings (Schultz 2003, Soar et al. 2006).
Drought stress experiments can be performed by reducing the amount of water supplied during each irrigation period or supplying a normal amount of water and lengthening the duration between irrigation periods. We simulated prolonged and moderate drought stress by reducing the quantity of water supplied during each irrigation period. Thus, drought stress simulated by lengthening the time periods between irrigation may yield results different from our findings. In addition, we used young potted plants rather than older plants in an open field, which may affect parameters such as root systems and water availability (Chaves et al. 2010, Lovisolo et al. 2010) Therefore, our conclusions are limited to potted plants, and it should be taken into account that plants may behave differently in the field.
Abiotic stresses such as drought cause a rapid decrease in plant hydraulic conductance and stomatal closure to reduce transpiration (Steudle 2000). However, stomatal closure also decreases photosynthesis and plant growth. Anisohydric plants, which regulate stomatal closure less efficiently than isohydric plants, are considered to be more tolerant to drought. A review by McDowell et al. (2008) indicates that anisohydric species tend to occupy more drought-prone habitats than do isohydric species because anisohydric species allow Ψl to approach the cavitation threshold. This results in a longer time period before the plant is unable to assimilate carbon from CO2 and experiences carbon starvation. Rapid carbon starvation in the isohydric plants leads to a cascade of downstream effects, such as reduced resistance to biotic agents. Grapevine varieties that display anisohydric behavior have a better response to, and recovery from, moderate water stress than those that exhibit isohydric behavior (Pou et al. 2012). In the present study, we similarly show that the anisohydric cultivar Shiraz had greater vegetative growth and less defoliation than the isohydric Grenache cultivar, indicating that the former coped with prolonged water stress more successfully (Figure 3). Although isohydric plants are often considered ‘drought avoiders’, our results and those of others suggest that the anisohydric plants may adapt to drought stress more effectively by reacting more slowly to low soil water content and continuing to produce energy even in a dry environment, thus enabling them to respond to other risk factors (McDowell et al. 2008, Sade et al. 2009, Pou et al. 2012, Sade et al. 2012).
These findings raise questions regarding the parameters responsible for the basic differences between isohydric and anisohydric plants and evolutionary advantages of isohydric strategies in certain plants. Chouzouri and Shultz (2005) show that water use strategies of grape cultivars are correlated with plant architecture and anatomy. In our study, the Grenache plants had a larger vessel size and higher stomata density than the Shiraz plants. Xylem cavitation represents the major limit to water transport and drought tolerance in plants. Water in the xylem conduits must remain in liquid form even at pressures well below the vapor pressure, and the xylem water potential decreases dramatically under drought stress. Cavitation results in a primarily vapor-filled conduit that eventually fills with air (Tyree and Sperry 1989), which causes embolisms in the xylem and blocks hydraulic conductance (Sparks and Black 1999, Sperry et al. 1993). Hydraulic failure defines the point of death in water-stressed plants (Brodribb and Cochard 2009). Therefore, plants close their stomata when soil water decreases to avoid xylem embolism. The primary difference in isohydric and anisohydric behaviors lies in the speed of a plant’s reaction to decreased water availability. The results from this and other studies (McDowell et al. 2008, Sade et al. 2012, Sade et al. 2009) suggest that slow speed of reaction to water stress (i.e., anisohydric behavior) is more efficient for promoting plant survival under conditions of mild, prolonged drought.
Our results suggest that isohydric and anisohydric behaviors do not represent distinct strategies, but perhaps differences in the response time to drought caused by the plant’s “fear” of xylem embolism. Our study showed that at least in the tested grape cultivars, the isohydric plants (Grenache) have an anatomical architecture that increases their vulnerability to xylem embolism. Larger xylem vessels, higher hydraulic conductance, and a greater number of stomata affect water movement and transpiration, thus inducing xylem embolism under conditions of water stress. Chouzouri and Schultz (2005) show that grapevine cultivars of different geographic origins exhibit different hydraulic anatomy, cavitation susceptibility, and gas exchange mechanisms, which may account for differences in stomatal sensitivity to soil water availability in different grapevine cultivars. Anisohydric plants such as Shiraz, which have smaller xylem vessels, lower hydraulic conductance, and lower stomatal density, may respond more slowly to water deficit because xylem embolism only occurs under severely dry conditions. If this argument is correct, isohydric and anisohydric plants would respond similarly to a severe water deficit. Indeed, recent studies show that even anisohydric grape cultivars respond to extremely low soil water potential in an isohydric manner (Domec and Johnson 2012, Rogiers et al. 2012, Zhang et al. 2012). In another study (unpublished data), we found that lowering irrigation to 10% of its optimal level yielded similar responses in both the Grenache and the Shiraz plants (data not shown). Alsina et al. (2007) tested the xylem hydraulic characteristics and drought resistance of eight V. vinifera cultivars grown under field conditions without irrigation in a Mediterranean climate and show that Parellada, Tempranillo, and black Grenache are most vulnerable to xylem embolism whereas Chardonnay and Sauvignon blanc are the least vulnerable. They suggested that the differences in embolism rate found among the eight cultivars reflect differences in xylem structure. Tempranillo and Grenache are considered isohydric, whereas Chardonnay is considered anisohydric (Chaves et al. 2010).
Conclusion
In conclusion, our study suggests that variation in response to drought stress depends on the ability of the plant to avoid xylem embolism rather than on a predetermined drought-avoiding strategy. All plants respond to water stress by stomatal closure when they detect impending xylem cavitation to prevent xylem embolism and death. Plants characterized as anisohydric are characterized by an anatomical architecture that enables them to decrease their water potential without danger of xylem cavitation. In contrast, isohydric behavior is less efficient for dealing with moderate water stress, suggesting that plants adopt isohydric behavior because their stomata and xylem anatomy do not permit anisohydric behavior. However, as only two grapevine cultivars were used to represent isohydric and anisohydric strategies, and xylem cavitation was not measured, our conclusions must be accepted with caution.
Our findings may help inform growers on implementing appropriate cultivation strategies to accommodate differences in coping with drought stress among cultivars, which will help maximize crop productivity in drought conditions.
Acknowledgments
This work was supported by the Israeli Ministry of Agriculture and Rural Development Grant ( No. 20387911). The authors thank Yehuda Ben-Ari for valuable assistance with writing and editing this paper.
Footnotes
Supplemental data is freely available with the online version of this article at www.ajevonline.org.
- Received August 2014.
- Revision received January 2015.
- Revision received March 2015.
- Accepted April 2015.
- Published online December 1969
- ©2015 by the American Society for Enology and Viticulture
Literature Cited
Sign in for ASEV members
ASEV Members, please sign in at ASEV to access the journal online.
Sign in for Institutional and Non-member Subscribers
Log in using your username and password
Pay Per Article - You may access this article (from the computer you are currently using) for 2 day for US$10.00
Regain Access - You can regain access to a recent Pay per Article purchase if your access period has not yet expired.