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Cladode growth dynamics in Opuntia ficus-indica under drought
Environmental and Experimental Botany 122 (2016) 158–167
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Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot
Cladode growth dynamics in Opuntia ficus-indica under drought Alessio Scalisia,* , Brunella Morandib , Paolo Inglesea , Riccardo Lo Biancoa a b
Department of Agricultural and Forest Sciences, University of Palermo, Viale delle Scienze 11, Palermo 90128, Italy Department of Agricultural Sciences, University of Bologna, Viale Fanin 44, Bologna 40127, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 September 2015 Received in revised form 16 October 2015 Accepted 18 October 2015 Available online 3 November 2015
Cactus pear (Opuntia ficus-indica L. Miller) is a CAM plant with an extraordinary capacity to store water in its succulent stems (cladodes). However, the daily variations of cladode thickness is unknown. Studying cladode thickness fluctuations may be useful for the early prediction of plant dehydration stress. The objective of this study was to determine if age, water availability and temperature influence diel cladode shrinkage and enlargement dynamics in cactus pear. The experiment was conducted in a greenhouse from April to July 2014, using cactus pear plants, equally split into irrigated and unirrigated treatments, and unrooted cladodes detached from mother plants. Soil moisture content (SMC), soil water depletion over 24-h, cladode relative water content (RWC), cladode thickness, stomatal conductance (gs), cladode growth rates (area increase) and nocturnal malic acid accumulation were monitored in plants of various ages. Cladode shrinkage and enlargement dynamics were assessed using stem gauges and expressed as absolute growth rate (AGRthickness, mm min1). In unirrigated pots, drought decreased SMC, RWC,cladode thickness, cladode growth rate and gs. Younger cladodes lost water later than older ones. Detached cladodes exhibited gs activity 3–4 months after detachment. Trends of AGRthickness showed that there was a progressive reduction of diel swelling and shrinkage fluctuations as cladodes aged. Such fluctuations were minimized under severe drought when 1-year-old cladodes reached 8 mm thickness and a RWC of about 45%. A positive correlation was found between SMC and AGRthickness. Temperatures were also directly correlated with AGRthickness, although this relationship was gradually lost as SMC decreased. Overall, cactus pears were able to maintain some growth at very low hydration levels, and cladode growth was highly responsive to rehydration after long periods of drought. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Cactus water status Cladode age Cladode thickness Prickly pear Malic acid Stem gauge
1. Introduction Cactus pear (Opuntia ficus-indica L. Miller) is known to have an extraordinary capacity to store water. Over other cacti, scientists have often given much emphasis to cactus pear due to its variety of uses as food and agricultural product (ornamental, fruit and fresh cut, forage and fodder, pest management). Like other CAM plants, cactus pears open their stomata during the dark period (Ranson and Thomas, 1960; Ting, 1985; Nobel, 2001, 2003, 2010). At night, CO2 is fixed and oxaloacetate and malate accumulate in the cytosol to be subsequently moved and stored in the vacuoles of the
Abbreviations: AGRarea, absolute growth rate in terms of cladode area; AGRthickness, absolute growth rate in terms of cladode thickness; CAM, crassulacean acid metabolism; DW, dry weight; FC, field capacity; FW, fresh weight; gs, stomatal conductance; RGRarea, relative growth rate in terms of cladode area; RWC, relative water content; SMC, soil moisture content; TW, weight at full turgor. * Corresponding author at: Department of Agricultural and Forest Sciences, University of Palermo, Viale delle Scienze 11, Palermo 90128, Italy. E-mail addresses: [email protected], [email protected] (A. Scalisi). http://dx.doi.org/10.1016/j.envexpbot.2015.10.003 0098-8472/ ã 2015 Elsevier B.V. All rights reserved.
chlorenchyma cells (Nobel, 2003). In spite of occurring at night, CO2 uptake and acid accumulation are influenced by light and plant water status (Nobel and Hartsock, 1983). During daytime, CO2 is in fact released again (internally) and Rubisco can now work in the same way as in C3 metabolism, with the only exception that stomata are closed and prevent the escape of CO2 and water vapor. The result is a tremendous increase of water use efficiency. Many studies have focused on diel variations of tissue acidity in cladodes of cactus pear (Nobel, 1982, 1983; Nobel and Hartsock, 1983; Acevedo et al., 1983). Differences in titratable acidity of cladode tissues at dusk and dawn are commonly expressed in terms of malic acid equivalents (Adams et al., 1989; Goldstein et al., 1991; Meraz-Maldonado et al., 2012) and are indicative of daily assimilation rates. In cactus pear, many of the net photosynthetic parameters are measured on individual cladodes, even though studies with the use of open gas exchange chambers are available (Liguori et al., 2013). As expected, net CO2 uptake is enhanced by high atmospheric CO2 concentration, but also by increasing temperatures (Cui et al., 1993; Drennan and Nobel, 2000). However, optimal temperatures
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for cladode nocturnal net CO2 uptake are reduced if plants are under drought conditions (Nobel and Hartsock 1984), especially when water deficit is prolonged (Nobel, 2001). The same declining pattern is shown in detached unrooted cladodes (Raveh and Nobel, 1999). Also, the presence of young cladodes is an additional factor reducing the amount of net CO2 uptake by mother cladodes (Pimienta-Barrios et al., 2005). In young cladodes, water initially moves through the phloem, while xylematic water uptake starts after about a month from their appearance (Nobel, 1997; Wang et al., 1997). Sink to source transition is also accompanied by an inversion of water potential difference between young and mother cladodes. Specifically, water potential shifts from being higher in the very young than in the mother cladodes, to being lower in the former after four weeks of growth (Luo and Nobel, 1992; Nobel, 1997; Wang et al., 1997). Under optimal soil moisture conditions, cactus pears sequester water into their parenchyma tissue, which becomes a recognizable thick white layer; on the contrary, parenchyma consistently narrows down during drought until it is hardly distinguished from the surrounding chlorenchyma tissue (Barcikowski and Nobel, 1984 Liguori et al., 2013). Overall cladode thickness is also reduced under drought (Nerd and Nobel, 2000). The ability of the parenchyma tissue to store/release water towards the chlorenchyma provides an efficient buffer effect on many physiological responses to drought (Nobel, 2003), whereas chlorenchyma thickness of mother cladodes shows little changes between dry and wet periods (Nobel, 2006; Liguori et al., 2014). Long drought periods have several physiological effects on cactus pear. For instance, after two/three months without irrigation, nocturnal stomatal conductance and acid accumulation are reduced, but respectively the highest rate and the highest concentration are always found at pre-dawn (Acevedo et al., 1983; Goldstein et al., 1991). After prolonged drought, also photosynthesis of single cladodes is reduced, probably due to reductions in relative water content, parenchyma thickness and chlorophyll content (Pimienta-Barrios et al., 2007). Parenchyma and chlorenchyma osmotic pressure is little affected after three months of drought, whereas turgor pressure is reduced by 86% compared to well-watered conditions (Goldstein et al., 1991). On the contrary, Nobel (2006) found very high increases of parenchyma osmotic pressure in pitahaya (Hylocereus undatus). In saguaro (Carnegiea gigantea), growth dynamics using dendrometers have shown shrinkage at night and enlargement during the day, probably due to stomatal opening patterns (MacDougal, 1924; Nobel, 2003). In this species, even the distance
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among rib crests may be taken as an index of plant water status. Saguaro plants however have cylindrical stems and might show different diel stem dynamics compared to platyopuntias (flattened stems). A study on diel stem fluctuations of Opuntia occidentalis measured with a mechanic dial gauge showed an association between cladode temperature and thickness gain (Schroeder, 1975). To date, the effect of drought stress on the physiological mechanisms leading to cladode growth in cactus pear has not been reported in the literature. The opportunity to study cladode enlargement and shrinkage and the relationship between cladode growth and environmental parameters is crucial to better understand the functioning of succulents. Diel fluctuations of cladode thickness may also represent an early indicator of the occurrence of dehydration stress. Eventually, acquisition of continuous measurements over a period of 24 h will allow the development of models useful for preventing the negative effects of drought on plant growth by real-time automated environmental control. This work investigated the effect of soil water deficit and temperature on the daily mechanisms of cladode growth in cactus pear. The experimental model included also cladode age as a secondary factor affecting growth dynamics in response to the environment. It was hypothesized that the youngest cladodes could show more pronounced diel thickness fluctuations compared to older cladodes, and therefore serve as a suitable model for assessing plant responses to environmental factors. It was also assumed that drought would reduce cladode diel thickness fluctuations and interfere with other environmental factors. 2. Materials and methods 2.1. Plant material and experimental setup The experiment was conducted in a greenhouse at the Department of Agricultural and Forest Sciences, University of Palermo (38 60 31.78900 N and 13 210 2.27100 E, 40 m a.s.l.) from April to July 2014, using cactus pear plants of the cultivar Gialla, cultivated in Sicily for fruit production (Barbera et al., 1992). Different types of plants were selected: (a) six 2-year-old plants, (b) two 3-year-old plants, (c) four detached cladodes, (d) six rooted cuttings. Within each age category, plants were uniform in size and number of cladodes. The first two groups had already been growing in 40-liter containers with sandy-loam soil. For rooted cuttings, 1year-old cladodes were collected from adult mother plants about a month before drought treatment was imposed and, after one week
Fig. 1. Schematic representation of various-age cladodes on a 3- and a 2-year-old Opuntia ficus-indica plant.
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of drying the cut surface to prevent rotting and improve establishment, they were potted (half cladode buried) in 40-liter containers with sandy-loam soil. Rooting was evident by the end of June. Two weeks before drought treatment was imposed, all plants were moved inside the greenhouse and fertilized with 50 g/pot of a NPK fertilizer (20-10-10). Greenhouse internal air temperature and relative humidity were recorded using HOBO H-08 sensors (Onset Computer Corporation, Bourne, MA, USA). Four 1-year-old cladodes were removed from 3-year-old plants (detached) and left on a bench in the greenhouse until the end of the experiment to study growth dynamics and compare a soilisolated system to fully functional plants. On April 15 (beginning of drought treatment) all plants were watered to FC by providing water to the point of runoff, and then randomly split into irrigated and unirrigated plants. Irrigated plants were watered to FC once per week during April and May and twice per week in June and July, as temperatures increased. Unirrigated plants received no irrigation for the same period. On July 17 (end of treatment), a final rewatering of unirrigated pots was carried out and a final set of measurements was taken to study cladode responses to sudden soil rehydration after three months of drought treatment. Three months of drought can be considered extreme under Mediterranean climates. 2.2. Soil moisture Twice a month SMC was measured in pots of 2- and 3-year-old plants by the gravimetric method. In each pot, soil samples were collected from the top 25 cm, where cactus pear roots typically lie. FW was immediately recorded, and samples were oven-dried at 60 C until constant weight (72–96 h) to obtain DW. SMC (%) was calculated as (FW–DW) 100/DW. In addition, between July 4 and 5, a day after irrigating to FC, 24h soil water depletion was monitored in irrigated 2-year-old plant and cladode cutting pots. In those pots, evaporation was avoided by covering the soil with aluminum foil for the entire 24-h period. No further loss of water occurred after initial runoff, therefore water loss was mainly due to plant uptake. Measurements were taken every two hours in proximity of the roots with a portable HH2 moisture meter connected to a WET sensor (Delta-T Device LTD., Cambridge, UK) and expressed as percentage of FC (FC = 100 %).
Fig. 2. Gravimetric soil moisture content of potted 3- (A) and 2-year-old (B) Opuntia ficus-indica plants. Error bars indicate standard errors of the means for 2-year-old plants. Panel A shows the moisture content of one irrigated and one unirrigated 3year-old plant. The vertical dashed line indicates the last irrigation before drought treatment started. Data were analyzed by analysis of variance.
2.3. Plant water status and cladode thickness Plant water status was estimated by measuring cladode RWC (%) according to Barrs and Weatherley (1962) on 1-year-old and new developing irrigated and unirrigated cladodes. Cladode cores were collected with a cork borer (13-mm diameter) on April 24 and July 4, the beginning and the end of the drought treatment, respectively. Individual samples were sealed in plastic bags and quickly transported to the laboratory for FW determination. Subsequently, the disks were immersed in deionized water for 24 h at 8 C to obtain the TW. The DW was measured after oven drying samples at 60 C until constant weight (2–4 days). RWC was calculated as (FW–DW)/(TW–DW) 100. Changes of 1-year-old cladode thickness were monitored with a digital caliper (at an intermediate portion of the cladode) in both irrigated and unirrigated plants as an additional information regarding plant water status. Six cladodes were chosen as replicates from irrigated and unirrigated plants. Thickness was firstly measured on May 9 and then once per week in the last month of treatment (from beginning of June to beginning of July). In addition, plant water status was also estimated by measuring the thickness of fresh cladode cores extracted with a cork borer on June 6 and July 4. Six cylinders mother cladodes (1-year-old) and six from new developing cladodes of irrigated and unirrigated
Fig. 3. Daily trends of soil water content in irrigated pots of 2-year-old Opuntia ficus-indica plants and rooted cuttings (from 8:00 am of July 4 until 6:00 am of July 5). Error bars indicate standard errors of the means. Data were analyzed by analysis of variance.
2-year-old plants. Samples were laid on a white board including a reference measuring tape and disk sections were photographed with a Fujifilm FinePix F600EXR digital camera (Fujifilm Holdings
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Corporation, Tokyo, Japan). Subsequently, thickness of total, parenchyma and chlorenchyma tissues was estimated to the tenth of a millimeter with the software ImageJ (Wayne Rasband, National Institute of Health, Bethesda, MD, USA). Water storage parenchyma was identified as the whitish tissue in the inner part of the cladode; the chlorenchyma was identified as the surrounding green tissue including the cuticle layer.
Table 1 Parenchyma, chlorenchyma and total thickness (means standard errors) of cores extracted from irrigated and unirrigated 1-year-old (old) and new developing cladodes (new) on June 6 and July 4. No differentiation was observed between parenchyma and chlorenchyma in new developing cladodes. Date
Cladode type
Thickness (mm) Parenchyma
Chlorenchyma
Total
June 6
Irrigated new Irrigated old Unirrigated new Unirrigated old
– 15.42 1.91 – 4.33 0.56
– 5.87 0.41 – 4.83 0.64
5.82 0.26 21.29 2.19 4.88 0.27 9.17 1.05
July 4
Irrigated new Irrigated old Unirrigated new Unirrigated old
– 10.72 1.30 – 2.44 0.32
– 6.10 0.29 – 1.83 0.45
6.91 0.33 16.82 1.50 4.00 0.23 4.27 0.30
2.4. Cladode stomatal conductance and malic acid accumulation A Delta-T AP4 dynamic porometer (Delta-T Devices LTD., Cambridge, UK) was used to measure gs on June 6 and July 4, at 4-h intervals, from 8 pm to 8 am due to nocturnal stomatal opening. Measurements were taken on young and mother cladodes from 2-year-old plants, and cladode cuttings of both irrigated and unirrigated plants. Carbon assimilation was estimated by measuring nocturnal accumulation of malic acid on mother and new developing cladodes of 2-year-old plants. Cladode cores were extracted with a cork borer at dusk and dawn on April 30-May 1 and July 4–5 and immediately frozen. Afterwards, samples were thawed, the cuticle layer was peeled off with a scalpel and chlorenchyma tissue was separated from the remaining parenchyma. Titratable acidity was measured on chlorenchyma of 1-year-old cladodes and on whole peeled tissue of developing cladodes, due to non-visible differences between chlorenchyma and parenchyma in the latter. Samples were weighed and then ground with a mortar and pestle and gradually added with 10 ml of deionized water. Homogenates were transferred into a 50ml beaker, titrated to pH 8.1 with a Crison Compact titrator (Crison Instruments, SA, Barcelona, Spain), and acidity was expressed as grams of malic acid per gram of tissue. 2.5. Cladode growth Cladode growth rate was determined manually, using a measuring tape to measure width and length. Changes of cladode area were monitored at 10-day intervals from May to July 2014. Cladode area was estimated as (width/2) (length/2) p (Tiznado-Hernández et al., 2010). AGRarea and RGRarea were estimated as changes in cladode area per day and changes in cladode area per day and per initial area, respectively. Continuous cladode diel swelling/shrinkage were assessed using stem gauges described by Morandi et al. (2007). The same devices
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have been adopted for several studies on fruit growth patterns. Briefly, the stem gauge is composed by a linear potentiometer which is connected to a mobile metal plunger surrounded by a light spring. The movement of the plunger sends signals in terms of mV to a data logger, which can be easily converted in mm. The gauges lightweight and frame shape make them particularly suitable for studying growth in cactus pear cladodes. Gauges were connected to a CR1000 data logger (Campbell Scientific Ltd., Leicestershire, UK) and data were recorded at 15-min intervals. The growth of 1-year-old cladodes from 2-year-old potted plants was continuously monitored for periods of 7 days/month in April, May, June, and July in order to study daily growth patterns. Each of the seven days of a week was taken as a replicate of the 24-h AGRthickness trend. Furthermore, in April, gauges were used for comparing growth dynamics of 1-, 2- and 3-year-old cladodes from 3-year-old plants, and proximal and distal portion of 1-year-old cladodes from 2-year-old plants (Fig. 1). Due to lack of significant differences between the two portions of 1-year-old cladodes, subsequent gauge measurements were only taken from the central portion of cladodes. The last series of gauge measurements was carried out in July 2014 using 1-year-old cladodes from 2year-old plants and the four detached cladodes. All collected data were converted in cladode AGRthickness and expressed in mm min1. Maximum and minimum diel AGRthickness were used as parameters of growth for comparisons of cladode ages and drought treatments in April and only drought treatments in July. Maximum AGRthickness was defined as the highest enlargement rate of cladodes during the 24 h, whereas minimum AGRthickness was the highest shrinkage rate (i.e. the most negative AGRthickness in each day). 2.6. Statistical analysis SYSTAT procedures (Systat software Inc., Chicago, Illinois, USA) were used to carry out analysis of variance on all data, and, when appropriate, means were compared by Tukey’s multiple range test. Linear regression analysis was performed to test correlations between soil water depletion (% of FC) and AGRthickness, and between temperatures and AGRthickness using Sigmaplot procedures (Systat software Inc., Chicago, Illinois, USA). Slopes of regressions were compared by analysis of variance using slope coefficients and standard errors. 3. Results and discussion 3.1. Soil moisture
Fig. 4. Changes of 1-year-old cladode thickness in irrigated and unirrigated 2-yearold Opuntia ficus-indica plants. Error bars indicate standard errors of the means. Analysis of variance was followed by Tukey’s test (n.s., non-significant; **significant for P < 0.001; *significant for P < 0.05).
Drought treatments and plant age and size generated significant differences in the SMC (Fig. 2). Specifically, SMC of older plants (late April, Fig. 2A) started to decrease earlier in unirrigated than in
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mechanism of root water uptake is not only driven by the transpiration stream, but it may be the consequence of a delayed and gradual rehydration of cladode cells after nocturnal transpiration, possibly driven by the accumulation of osmotically active compounds (Lee, 2010). 3.2. Plant water status and cladode thickness As expected, no RWC difference was observed in April between 1-year-old cladodes of irrigated (84.4% 3.05) and unirrigated (84.8% 3.05) plants. A lower RWC was observed in unirrigated (44.9% 2.78) compared to irrigated (78.5% 2.78) cladodes by the
Fig. 5. Stomatal conductance of new developing cladodes (A), mother cladodes (B) and rooted cuttings (C) in irrigated and unirrigated Opuntia ficus-indica plants on July 4. Error bars indicate standard errors of the means. Analysis of variance was followed by Tukey’s test (n.s., non-significant; **significant for P < 0.001; * significant for P < 0.05).
irrigated treatments compared to younger plants (beginning to late May, Fig. 2B). By the first week of July, 2-year-old irrigated plants generated a significant soil water depletion (24.5%) during the 24 h after irrigation, while irrigated rooted cuttings showed no change during the same time period (Fig. 3). These results are probably due to the 2-year-old plant larger size, more developed root systems and higher water need compared to rooted cuttings. Despite stomata being closed during the day, and preventing evaporation by covering the soil with aluminum foil, a decrease of SMC during daylight was shown in 2-year-old plants. This suggests that the
Fig. 6. Cladode surface area, absolute growth rate (AGRarea) and relative growth rate (RGRarea) of new developing cladodes for irrigated and unirrigated Opuntia ficusindica plants. Error bars indicate standard errors of the means. Analysis of variance was followed by Tukey’s test (n.s., non-significant; **significant for P < 0.001; *significant for P < 0.05).
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Fig. 7. Twenty-four-hour trend (A) and daily maximum and minimum (B) absolute thickness growth rates (AGRthickness) in 1-, 2-, and 3-year-old cladodes from 3-yearold Opuntia ficus-indica plants. Mean values of seven days in April are shown. Error bars indicate standard errors of the means. Analysis of variance was followed by Tukey’s test (different letters indicate significant differences for P < 0.001).
Fig. 8. Daily maximum and minimum of absolute thickness growth rates (AGR thickness) in irrigated, unirrigated, and detached 1-year-old cladodes of Opuntia ficusindica. Mean values of seven days in July and their standard errors are shown. Analysis of variance was followed by Tukey’s test (different letters indicate significant differences for P < 0.001).
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end of the drought treatment. The slight decrease of RWC observed in irrigated cladodes from April to July was probably due to the production of a high number of daughter pads, which may compete for water with mother cladodes (Pimienta-Barrios et al., 2005). However, this reduction was not significant because irrigated plants had enough SMC to maintain hydration of both mother and daughter cladodes. An interaction was found between cladode age on the same plant and drought effects, with unirrigated cladodes showing a sharper difference between developing (62.5% 1.29) and mother cladodes (44.9% 1.46) than irrigated cladodes (85.2% 1.22 and 78.5% 2.34, respectively). Differences found between mother and daughter cladodes are the proof that cactus pear, particularly under drought conditions, tends to store water in newly formed structures, probably as a survival strategy and to provide more water to the more photosynthetically active organs. Significant differences of thickness between irrigated and unirrigated 1-year-old cladodes were observed starting on June 5 (Fig. 4). Thickness of irrigated cladodes was stable throughout the trial, while those on unirrigated cladodes consistently decreased, becoming as much as 57.5% smaller than in irrigated by the end of treatment period. In cladode cores sampled from June 6 to July 4 (Table 1), thickness increased by 1.8% in new cladodes and a simultaneous 30.8% decrease occurred in the mother cladodes (P = 0.002). Irrigated cladodes were thicker than unirrigated ones (P < 0.001), with sharper differences in mother cladodes (64.7%) than in new daughter cladodes (30.2%). This is a further indication that plants under drought tend to allocate more water to the most photosynthetically active organs. Furthermore, after 75 days, drought caused a reduction of the thickness of parenchyma and chlorenchyma tissues by 75.0% and 55.8%, respectively (Table 1). This suggests that the parenchyma loses water more readily than chlorenchyma, confirming that in CAM plants, parenchyma water is the most prone to be lost under drought conditions (Nobel, 2006; Pimienta-Barrios et al., 2007). Reduction of the parenchyma was therefore responsible for most of the reduction in thickness of unirrigated cladodes observed on July 4 (Fig. 4). 3.3. Cladode stomatal conductance and malic acid accumulation Analysis of gs data showed no difference between the two dates (June 6 and July 4), and no difference between irrigation treatments or organ type or age in June, so only data from July 4 were considered. In this case, a significant (P < 0.001) interaction between irrigation and organ age and type was found. Specifically, in the case of new developing cladodes and rooted cuttings, conductance was significantly higher in irrigated than in unirrigated treatments at midnight and 4 am (Fig. 5). Also, new developing cladodes of irrigated plants maintained a constant high rate of stomatal conductance (about 150 mmol m2 s1) in the middle of the night, consistent with the results reported by Nobel and De la Barrera (2000). In contrast, rooted cuttings experienced the highest conductance at 4 am. This can be explained by a high RWC in growing cladodes (Section 3.2), which allows for relatively high gs during most of the night. Differences between mother and daughter cladodes in this study contrast with the results of Cui et al. (1993), who recorded higher nocturnal conductance in mother cladodes. It is possible that cladodes measured by Cui et al. (1993) were too young (within 4 weeks of age) and still doing diurnal rather than nocturnal transpiration, as reported by Acevedo et al. (1983). This would explain the differences with our observations. When gs was measured in 1-year-old irrigated, unirrigated and detached cladodes, no significant difference was found between June and July, while the mean nocturnal gs of irrigated cladodes (40.2 mmol m2 s1) was higher than that of unirrigated
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(5.37 mmol m2 s1) and detached (2.39 mmol m2 s1) cladodes. As expected, gs was highly influenced by plant water status, but in the case of O. ficus-indica, detached photosynthetic organs were able to overcome many months without soil contact and under stressful summer greenhouse conditions during summer. The detached cladodes must have maintained a minimal photosynthetic activity as they produced daughter cladodes for up to two months after detachment (personal observations). The latter has been previously documented, especially in the presence of high light intensity and high tissue concentration of indole-acetic acid and kinetin (Nobel, 1996). At the end of the imposed stress (4–5 July), new developing cladodes of irrigated plants had a consistently higher nocturnal malic acid content (7.75 mg g1) compared to either their mother cladodes (2.05 mg g1) or to developing unirrigated cladodes (1.24 mg g1), which instead did not significantly differ from unirrigated mother cladodes (3.77 mg g1). Thus, similar levels of nocturnal malic acid in daughter and mother cladodes may be indicative of drought stress in cactus pear. 3.4. Cladode growth dynamics Drought also affected cladode surface area, AGRarea and RGRarea from May to July (Fig. 6). In particular, mean cladode areas were greater in irrigated plants compared to unirrigated ones starting on June 15 (Fig. 6A), with young unirrigated cladodes having a surface area 58.6% smaller than young irrigated ones by the end of the drought treatment. This is expected as area is a cumulative result of
the growth process. Similar reductions in size (52%) were found by Luo and Nobel (1993). On the other hand, drought significantly reduced AGRarea just on June 5 and 16 (Fig. 6B). The final decrease of AGRarea and lack of significant differences between irrigated and unirrigated treatments in July are likely due to the fact that cladodes were reaching their final size. Conversely, drought reduced RGRarea only at the beginning (Fig. 6C), when cladodes were rapidly growing. The data clearly indicates that unirrigated mother cladodes could not supply enough water and assimilates to support the normal development of new organs. Toward the end of the drought treatment, slightly negative RGRarea were recorded, indicating some shrinkage of organs due to tissue dehydration. In April, trends of thickness fluctuations (AGRthickness,mm min1) in cladodes of different ages were similar in irrigated and unirrigated plants. This lack of difference was possibly due to early measurements taken before any water deficit in the soil. Nevertheless, different AGRthickness trends were observed among 1, 2- and 3-year-old cladodes (Fig. 7A). Cladode age affected also maximum and minimum daily AGRthickness, with older cladodes showing the lowest and younger cladodes the highest daily shrinkage (Fig. 7B). Maximum daily AGRthickness of 3-year-old cladodes was higher than max daily AGRthickness of both 1- and 2year-old stems, which were similar. Differences among cladodes of various ages may be in part explained by the degree of lignification, particularly evident at the base of 3-year-old cladodes. In cactus pear, accumulation of fiber and lignin in old stems is directly correlated with age (Oliveira Ribeiro et al., 2010; Liguori et al., 2014). Cells of lignified organs are characterized by low elasticity,
Fig. 9. Twenty-four-hour trends of temperature and absolute growth rate (AGRthickness) of irrigated and unirrigated 1-year-old cladodes from 2-year-old Opuntia ficus-indica plants. Mean values of seven days in the months of April, May, June and July 2014 are shown. Data were analyzed by analysis of variance; error bars indicate standard errors of the means.
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decreasing AGRthickness fluctuations. Moreover, 2-year-old cladodes showed less night shrinkage than 1-year-old cladodes, but similar max AGRthickness (Fig. 7B). This may indicate that a greater amount of biomass gained during the day is lost at night in 1-year-old than in 2-year-old cladodes. In July, daily max AGRthickness of 1-year-old cladodes was significantly greater in irrigated than in unirrigated and detached cladodes, while no differences were observed between irrigated and unirrigated cladodes (Fig. 8). On the other hand, daily min AGRthickness was similar for all three cladode types, in spite of gs being highest in irrigated cladodes. Hence, high SMC in irrigated treatments allowed higher transpiration rates, resulting in a nocturnal shrinkage rate similar to the other treatments. In detached cladodes, fluctuations of AGRthickness were due to climatic factors such as light intensity, relative humidity and temperature rather than root water uptake. Indeed, Schroeder (1975) showed that the enlargement of detached cladodes of O. occidentalis is negatively correlated with air temperature but positively correlated with light intensity. Daily trends of AGRthickness in April, May, June and July were compared using 1-year-old cladodes from 2-year-old potted plants (Fig. 9). Data analysis revealed significant effects of month, time of the day and drought, but also a significant interaction among these three factors, suggesting that AGRthickness responses to drought may vary depending on time of the day and drought severity. Specifically, in April, the peak of AGRthickness occurred in early morning and in early afternoon for unirrigated and irrigated cladodes, respectively. Differences in this period were probably unrelated to SMC. Irrigated plants showed daily max AGRthickness between 1 and 3 pm, with the highest daily max AGRthickness being in June. In unirrigated plants, AGRthickness fluctuations decreased with the advancement of drought, with an almost flat daily pattern in June and particularly July, when cladode thickness (Fig. 4) and RWC (Section 3.2) were low. In particular, our data showed that when 1-year-old cladodes reach a minimum thickness of about 8 mm and a RWC of about 45%, AGRthickness daily fluctuations were almost canceled. When mean daily AGRthickness were calculated for all sampling dates, no differences were found between irrigated and unirrigated treatments in April (Fig. 10), in absence of soil moisture deficit (Fig. 2B). In May and June, mean daily AGRthickness was greater in
Fig. 10. Trend (April to July) of daily average absolute growth rate (AGRthickness) of irrigated and unirrigated 1-year-old cladodes from 2-year-old Opuntia ficus-indica plants. Mean values of seven days in each month and their standard errors are shown. Rewatering (dashed gray line) was carried out on July 17. Analysis of variance was followed by Tukey’s test (n.s., non-significant; **significant for P < 0.001; *significant for P < 0.05).
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the irrigated than in unirrigated treatments, whereas in July, it was again similar, probably due to larger plants and generally higher water consumption. After rewatering of all pots to FC, mean AGRthickness was higher in unirrigated than irrigated cladodes. This demonstrates the extraordinary ability of cactus pear mucilages to sequester water and to retain it for long periods. Multiple factors are probably involved in the regulation of AGRthickness dynamics. Nocturnal shrinkage could be due to loss of water when transpiration is faster than root water uptake. Specifically, stomata are open at night and the transpiration causes reductions of stem water potential and stem contractions (Nobel, 2003). Also, the nocturnal accumulation of malate in chlorenchyma vacuoles and the diurnal synthesis of carbohydrates leads to an increase of osmotic pressure. This, in turn, drives water into the chlorenchyma and results in cladode swelling during the day. Therefore, SMC is partially responsible for the differences between irrigated and unirrigated cladode dynamics. Yet, path resistances and time lags may mask a direct relationship between SMC and fluctuations in stem thickness. The gs differences among cladode types found in this study suggested that water loss by transpiration could be related to nocturnal shrinkage of cladodes. Nevertheless, no significant association was found between cladode gs and nocturnal shrinkage. This indicates that some other factor must play a major role in the 24-h growth dynamics. A direct linear relationship was found between SMC (% of FC) and AGRthickness during the 24 h (Fig. 11), suggesting that AGRthickness of 1-year-old cladodes (on 2year-old plants) may respond to changes of SMC. AGRthickness was minimal when SMC reached 57.3% of FC, indicating a soil moisture threshold above which cactus pears grow. In addition, direct linear relationships were found between diel temperatures and AGRthickness of irrigated and unirrigated 1-yearold cladodes (Fig. 12). A similar relationship was observed in O. occidentalis (Schroeder, 1975). In April and May, both AGRthickness of irrigated and unirrigated cladodes were directly related to temperature with similar slopes, indicating analogous responses to temperature. This is expected since little soil or plant water deficit was found at that time. Similar effects of temperature on stem daily growth dynamics were also found in Scots pine (Antonova and Stasova, 1993), eucalyptus (Downes et al., 1999), balsam fir (Deslauriers et al., 2003), Mexican mountain pine (Biondi and Hartsough, 2010) and other species, whereas no
Fig. 11. Correlation between soil water content and absolute growth rate (AGR thickness) of 1-year-old cladodes from 2-year-old Opuntia ficus-indica plants. Measurements were carried out in irrigated plants a day after irrigating to field capacity (FC). AGRthickness fluctuations are canceled at a soil water content threshold of 57.3% of FC. Data were analyzed by linear regression.
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Fig. 12. Correlations between temperature and absolute growth rate (AGRthickness) of irrigated (black circles) and unirrigated (white circles) 1-year-old cladodes from 2-yearold Opuntia ficus-indica plants during the drought treatment. Temperature and AGRthickness records are hourly data averaged from seven days in April, May, June and July. Data were analyzed by linear regression.
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relationship was found in peach and plum (Gènard et al., 2001). In June, AGRthickness was also correlated to temperature, but the slope was greater in irrigated than in unirrigated cladodes (Fig. 12). In contrast, in July (severe drought), AGRthickness of unirrigated cladodes was not related to temperature (P = 0.39). This can be explained by the lack of AGRthickness fluctuations due to cladode dehydration (i.e. reduced cladode thickness and RWC) at severe drought stages. 4. Conclusions This study demonstrated that drought stress reduced cladode thickness, RWC, malic acid accumulation, gs, size and growth rates of developing cladodes. Under well-watered conditions, diel fluctuations of cladode thickness are directly related to temperature variations, while under severe drought stress, SMC has higher influence than temperature. This suggests differential regulation of cladode growth depending on plant water status. Our data also showed that cactus pears are able to maintain some growth and assimilation at RWC above 45%, an extremely low limit compared to non-CAM plants. In addition, cladode growth was extremely responsive to rehydration after long periods of drought, suggesting that a regulated reduction of irrigation would not significantly affect plant biomass. These results indicate that a variety of factors are involved in cladode growth dynamics. Thus, a model that takes into account temperature, light, soil moisture content, plant water status, and concentration of osmotica in the cladode tissue needs to be considered in order to predict cladode growth under different conditions (e.g. different age, drought stress, etc.). References Acevedo, E., Badilla, I., Nobel, P.S., 1983. Water relations, diurnal acidity changes, and productivity of a cultivated cactus, Opuntia ficus-indica. Plant Physiol. 72 (3), 775–780. doi:http://dx.doi.org/10.1104/pp.72.3.775. Adams, W.W., Díaz, M., Winter, K., 1989. Diurnal changes in photochemical efficiency, the reduction state of Q, radiationless energy dissipation, and nonphotochemical fluorescence quenching in cacti exposed to natural sunlight in northern Venezuela. Oecologia 80 (4), 553–561. doi:http://dx.doi.org/10.1007/ bf00380081. Antonova, G.F., Stasova, V.V., 1993. Effects of environmental factors on wood formation in Scots pine stems. Trees 7 (4), 214–219. doi:http://dx.doi.org/ 10.1007/bf00202076. Barbera, G., Carimi, F., Inglese, P., 1992. Past and present role of the Indian-fig prickly-pear (Opuntia ficus-indica (L.) Miller, Cactaceae) in the agriculture of Sicily. Econ. Bot. 46 (1), 10–20. doi:http://dx.doi.org/10.1007/bf02985249. Barcikowski, W., Nobel, P.S., 1984. Water relations of cacti during desiccation: distribution of water in tissues. Bot. Gaz. 145, 110–115. doi:http://dx.doi.org/ 10.1086/337433. Barrs, H., Weatherley, P., 1962. A re-examination of the relative turgidity technique for estimating water deficits in leaves. Aust. J. Biol. Sci. 15 (3), 413–428. doi: http://dx.doi.org/10.1071/BI9620413. Biondi, F., Hartsough, P., 2010. Using automated point dendrometers to analyze tropical treeline stem growth at Nevado de Colima, Mexico. Sensors 10 (6), 5827–5844. doi:http://dx.doi.org/10.3390/s100605827. Cui, M., Miller, P.M., Nobel, P.S., 1993. CO2 exchange and growth of the crassulacean acid metabolism plant Opuntia ficus-indica under elevated CO2 in open-top chambers. Plant Physiol. 103, 519–524. doi:http://dx.doi.org/10.1104/ pp.103.2.519. Deslauriers, A., Morin, H., Urbinati, C., Carrer, M., 2003. Daily weather response of balsam fir (Abies balsamea (L.) Mill.) stem radius increment from dendrometer analysis in the boreal forests of Québec (Canada). Trees 17 (6), 477–484. doi: http://dx.doi.org/10.1007/s00468-003-0260-4. Downes, G., Beadle, C., Worledge, D., 1999. Daily stem growth patterns in irrigated Eucalyptus globulus and E. nitens in relation to climate. Trees 14 (2), 102–111. doi: http://dx.doi.org/10.1007/pl00009752. Drennan, P.M., Nobel, P.S., 2000. Responses of CAM species to increasing atmospheric CO2 concentrations. Plant Cell Environ. 23 (8), 767–781. doi:http:// dx.doi.org/10.1046/j1365-3040.2000.00588.x. Gènard, M., Fishman, S., Vercambre, G., Huguet, J.G., Bussi, C., Besset, J., Habib, R., 2001. A biophysical analysis of stem and root diameter variations in woody plants. Plant Physiol. 126, 188–202. doi:http://dx.doi.org/10.1104/pp.126.1.188. Goldstein, G., Ortega, J.K.E., Nerd, A., Nobel, P.S., 1991. Diel patterns of water potential components for the crassulacean acid metabolism plant Opuntia ficusindica when well-watered or droughted. Plant Physiol. 95 (1), 274–280. doi: http://dx.doi.org/10.1104/pp.95.1.274.
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Lee, J.S., 2010. Stomatal opening mechanism of CAM plants. J. Plant Biol. 53, 19–23. doi:http://dx.doi.org/10.1007/s12374-010-9097-8. Liguori, G., Inglese, G., Pernice, F., Sortino, G., Inglese, P., 2013. CO2 uptake of Opuntia ficus-indica (L.) Mill. whole trees and single cladodes, in relation to plant water status and cladode age. Ital. J. Agron. 8, 14–20. doi:http://dx.doi.org/10.4081/ ija.2013e3. Liguori, G., Inglese, P., Sortino, G., Inglese, G., 2014. Dry matter accumulation and seasonal partitioning in mature Opuntia ficus-indica (L.) Mill. fruiting trees. Ital. J. Agron. 9, 44–47. doi:http://dx.doi.org/10.4081/ija.2014.537. Luo, Y., Nobel, P.S., 1992. Carbohydrate partitioning and compartmental analysis for a highly productive CAM Plant, Opuntia ficus-indica. Ann. Bot. 70 (6), 551–559. Luo, Y., Nobel, P.S., 1993. Growth characteristics of newly initiated cladodes of Opuntia ficus-indica as affected by shading, drought and elevated CO2. Physiol. Plant. 87 (4), 467–474. doi:http://dx.doi.org/10.1111/j1399-3054.1993.tb02495.x. MacDougal, D.T., 1924. Dendrographic measurements. In: MacDougal, D.T., Shreve, F. (Eds.), Growth in Trees and Massive Organs of Plants. Carnegie Institution of Washington, Washington, pp. 1–88. Meraz-Maldonado, N., Valle-Guadarrama, S., Hernández-Morales, J., Anaya-Rosales, S., Rodríguez-Maciel, J.C., Leyva-Ruelas, G., 2012. Quality of three sizes of prickly pear cactus stems (Opuntia ficus-indica L. ATLIXCO). African J. Agric. Res. 7 (32), 4512–4520. doi:http://dx.doi.org/10.5897/ajar12.956. Morandi, B., Manfrini, L., Zibordi, M., Noferini, M., Fiori, G., Corelli Grappadelli, L., 2007. A low-cost device for accurate and continuous measurements of fruit diameter. HortSci 42 (6), 1380–1382. Nerd, A., Nobel, P.S., 2000. Water relations during ripening for fruit of well-watered versus water-stressed Opuntia ficus-indica. J. Am. Soc. Hortic. Sci. 125 (5), 653– 657. Nobel, P.S., 1982. Orientation, PAR interception, and nocturnal acidity increases for terminal cladodes of a widely cultivated cactus, Opuntia ficus-indica. Am. J. Bot. 69 (9), 1462–1469. doi:http://dx.doi.org/10.2307/2443107. Nobel, P.S., 1983. Nutrient levels in cacti-relation to nocturnal acid accumulation and growth. Am. J. Bot. 70 (8), 1244–1253. doi:http://dx.doi.org/10.2307/ 2443294. Nobel, P.S., 1996. Shading, osmoticum, and hormone effects on organ development for detached cladodes of Opuntia ficus-indica. Int. J. Plant Sci. 157 (6), 722–728. doi:http://dx.doi.org/10.1086/297394. Nobel, P.S., 1997. Recent ecophysiological findings for Opuntia ficus-indica. J. PACD 2, 89–96. Nobel, P.S., 2001. Ecophysiology of Opuntia ficus-indica. In: Mondragón-Jacobo, C., Pérez-González, S. (Eds.), Cactus (Opuntia spp.) as Forage. FAO Plant. FAO, Rome, IT, pp. 13–20. Nobel, P.S., 2003. Environmental Biology of Agaves and Cacti. Cambridge University Press, Cambridge, UK. Nobel, P.S., 2006. Parenchyma-chlorenchyma water movement during drought for the hemiepiphytic cactus Hylocereus undatus. Ann. Bot. 97 (3), 469–474. doi: http://dx.doi.org/10.1093/aob/mcj054. Nobel, P.S., 2010. Desert Wisdom/Agaves and Cacti: CO2, Water, Climate Change. iUniverse Inc., New York. Nobel, P.S., Hartsock, T.L., 1983. Relationships between potosynthetically active radiation, nocturnal acid accumulation, and CO2 uptake for a crassulacean acid metabolism plant, Opuntia ficus-indica. Plant Physiol. 71, 71–75. doi:http://dx. doi.org/10.1104/pp.71.1.71. Nobel, P.S., Hartsock, T.L., 1984. Physiological responses of Opuntia ficus-indica to growth temperature. Physiol. Plant. 60, 98–105. doi:http://dx.doi.org/10.1111/ j.1399-3054.1984.tb04257.x. Nobel, P.S., De la Barrera, E., 2000. Carbon and water balances for young fruits of platyopuntias. Physiol. Plant. 109 (2), 160–166. doi:http://dx.doi.org/10.1034/ j.1399-3054.2000.100208.x. Oliveira Ribeiro, E.M., de Silva, N.H., da Lima Filho, J.L., de Brito, J.Z., de Paz Carvalho da Silva da, M., 2010. Study of carbohydrates present in the cladodes of Opuntia ficus-indica (fodder palm), according to age and season. Ciência e Tecnol. Aliment. 30 (4), 933–939. doi:http://dx.doi.org/10.1590/s010120612010000400015. Pimienta-Barrios, E., Zañudo-Hernandez, J., Rosas-Espinoza, V.C., Valenzuela-Tapia, A., Nobel, P.S., 2005. Young daughter cladodes affect CO2 uptake by mother cladodes of Opuntia ficus-indica. Ann. Bot. 95, 363–369. doi:http://dx.doi.org/ 10.1093/aob/mci034. Pimienta-Barrios, E., Castillo-Cruz, I., Zañudo-Hernández, J., Méndez-Morán, L., Nobel, P.S., 2007. Effects of shade, drought and daughter cladodes on the CO2 uptake by cladodes of Opuntia ficus-indica. Ann. Appl. Biol. 151 (2), 137–144. doi: http://dx.doi.org/10.1111/j1744-7348.2007.00160.x. Ranson, S.L., Thomas, M., 1960. Crassulacean acid metabolism. Annu. Rev. Plant. Physiol. 11, 81–110. doi:http://dx.doi.org/10.1146/annurev. pp.11.060160.000501. Raveh, E., Nobel, P.S., 1999. CO2 uptake and water loss accompanying vernalization for detached cladodes of Opuntia ficus-indica. Int. J. Plant Sci. 160, 92–97. doi: http://dx.doi.org/10.1086/314111. Schroeder, C.A., 1975. Diurnal size fluctuation in Opuntia stem. Bot. Gaz. 136, 94–98. doi:http://dx.doi.org/10.1086/336787. Ting, I.P., 1985. Crassulacean acid metabolism. Annu. Rev. Plant Physiol. 36, 595–622. Tiznado-Hernández, M.E., Fortiz-Hernández, J., Ojeda-Contreras, Á.J., RodriguezFélix, A., 2010. Use of the elliptical mathematical formula to estimate the surface area of cladodes in four varieties of Opuntia ficus-indica. J. PACD 12, 98–109. Wang, N., Zhang, H., Nobel, P.S., 1997. Phloem-xylem water flow in developing cladodes of Opuntia ficus-indica during sink-to-source transition. J. Exp. Bot. 48 (3), 675–682. doi:http://dx.doi.org/10.1093/jxb/48.3.675.
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Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot
Cladode growth dynamics in Opuntia ficus-indica under drought Alessio Scalisia,* , Brunella Morandib , Paolo Inglesea , Riccardo Lo Biancoa a b
Department of Agricultural and Forest Sciences, University of Palermo, Viale delle Scienze 11, Palermo 90128, Italy Department of Agricultural Sciences, University of Bologna, Viale Fanin 44, Bologna 40127, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 September 2015 Received in revised form 16 October 2015 Accepted 18 October 2015 Available online 3 November 2015
Cactus pear (Opuntia ficus-indica L. Miller) is a CAM plant with an extraordinary capacity to store water in its succulent stems (cladodes). However, the daily variations of cladode thickness is unknown. Studying cladode thickness fluctuations may be useful for the early prediction of plant dehydration stress. The objective of this study was to determine if age, water availability and temperature influence diel cladode shrinkage and enlargement dynamics in cactus pear. The experiment was conducted in a greenhouse from April to July 2014, using cactus pear plants, equally split into irrigated and unirrigated treatments, and unrooted cladodes detached from mother plants. Soil moisture content (SMC), soil water depletion over 24-h, cladode relative water content (RWC), cladode thickness, stomatal conductance (gs), cladode growth rates (area increase) and nocturnal malic acid accumulation were monitored in plants of various ages. Cladode shrinkage and enlargement dynamics were assessed using stem gauges and expressed as absolute growth rate (AGRthickness, mm min1). In unirrigated pots, drought decreased SMC, RWC,cladode thickness, cladode growth rate and gs. Younger cladodes lost water later than older ones. Detached cladodes exhibited gs activity 3–4 months after detachment. Trends of AGRthickness showed that there was a progressive reduction of diel swelling and shrinkage fluctuations as cladodes aged. Such fluctuations were minimized under severe drought when 1-year-old cladodes reached 8 mm thickness and a RWC of about 45%. A positive correlation was found between SMC and AGRthickness. Temperatures were also directly correlated with AGRthickness, although this relationship was gradually lost as SMC decreased. Overall, cactus pears were able to maintain some growth at very low hydration levels, and cladode growth was highly responsive to rehydration after long periods of drought. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Cactus water status Cladode age Cladode thickness Prickly pear Malic acid Stem gauge
1. Introduction Cactus pear (Opuntia ficus-indica L. Miller) is known to have an extraordinary capacity to store water. Over other cacti, scientists have often given much emphasis to cactus pear due to its variety of uses as food and agricultural product (ornamental, fruit and fresh cut, forage and fodder, pest management). Like other CAM plants, cactus pears open their stomata during the dark period (Ranson and Thomas, 1960; Ting, 1985; Nobel, 2001, 2003, 2010). At night, CO2 is fixed and oxaloacetate and malate accumulate in the cytosol to be subsequently moved and stored in the vacuoles of the
Abbreviations: AGRarea, absolute growth rate in terms of cladode area; AGRthickness, absolute growth rate in terms of cladode thickness; CAM, crassulacean acid metabolism; DW, dry weight; FC, field capacity; FW, fresh weight; gs, stomatal conductance; RGRarea, relative growth rate in terms of cladode area; RWC, relative water content; SMC, soil moisture content; TW, weight at full turgor. * Corresponding author at: Department of Agricultural and Forest Sciences, University of Palermo, Viale delle Scienze 11, Palermo 90128, Italy. E-mail addresses: [email protected], [email protected] (A. Scalisi). http://dx.doi.org/10.1016/j.envexpbot.2015.10.003 0098-8472/ ã 2015 Elsevier B.V. All rights reserved.
chlorenchyma cells (Nobel, 2003). In spite of occurring at night, CO2 uptake and acid accumulation are influenced by light and plant water status (Nobel and Hartsock, 1983). During daytime, CO2 is in fact released again (internally) and Rubisco can now work in the same way as in C3 metabolism, with the only exception that stomata are closed and prevent the escape of CO2 and water vapor. The result is a tremendous increase of water use efficiency. Many studies have focused on diel variations of tissue acidity in cladodes of cactus pear (Nobel, 1982, 1983; Nobel and Hartsock, 1983; Acevedo et al., 1983). Differences in titratable acidity of cladode tissues at dusk and dawn are commonly expressed in terms of malic acid equivalents (Adams et al., 1989; Goldstein et al., 1991; Meraz-Maldonado et al., 2012) and are indicative of daily assimilation rates. In cactus pear, many of the net photosynthetic parameters are measured on individual cladodes, even though studies with the use of open gas exchange chambers are available (Liguori et al., 2013). As expected, net CO2 uptake is enhanced by high atmospheric CO2 concentration, but also by increasing temperatures (Cui et al., 1993; Drennan and Nobel, 2000). However, optimal temperatures
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for cladode nocturnal net CO2 uptake are reduced if plants are under drought conditions (Nobel and Hartsock 1984), especially when water deficit is prolonged (Nobel, 2001). The same declining pattern is shown in detached unrooted cladodes (Raveh and Nobel, 1999). Also, the presence of young cladodes is an additional factor reducing the amount of net CO2 uptake by mother cladodes (Pimienta-Barrios et al., 2005). In young cladodes, water initially moves through the phloem, while xylematic water uptake starts after about a month from their appearance (Nobel, 1997; Wang et al., 1997). Sink to source transition is also accompanied by an inversion of water potential difference between young and mother cladodes. Specifically, water potential shifts from being higher in the very young than in the mother cladodes, to being lower in the former after four weeks of growth (Luo and Nobel, 1992; Nobel, 1997; Wang et al., 1997). Under optimal soil moisture conditions, cactus pears sequester water into their parenchyma tissue, which becomes a recognizable thick white layer; on the contrary, parenchyma consistently narrows down during drought until it is hardly distinguished from the surrounding chlorenchyma tissue (Barcikowski and Nobel, 1984 Liguori et al., 2013). Overall cladode thickness is also reduced under drought (Nerd and Nobel, 2000). The ability of the parenchyma tissue to store/release water towards the chlorenchyma provides an efficient buffer effect on many physiological responses to drought (Nobel, 2003), whereas chlorenchyma thickness of mother cladodes shows little changes between dry and wet periods (Nobel, 2006; Liguori et al., 2014). Long drought periods have several physiological effects on cactus pear. For instance, after two/three months without irrigation, nocturnal stomatal conductance and acid accumulation are reduced, but respectively the highest rate and the highest concentration are always found at pre-dawn (Acevedo et al., 1983; Goldstein et al., 1991). After prolonged drought, also photosynthesis of single cladodes is reduced, probably due to reductions in relative water content, parenchyma thickness and chlorophyll content (Pimienta-Barrios et al., 2007). Parenchyma and chlorenchyma osmotic pressure is little affected after three months of drought, whereas turgor pressure is reduced by 86% compared to well-watered conditions (Goldstein et al., 1991). On the contrary, Nobel (2006) found very high increases of parenchyma osmotic pressure in pitahaya (Hylocereus undatus). In saguaro (Carnegiea gigantea), growth dynamics using dendrometers have shown shrinkage at night and enlargement during the day, probably due to stomatal opening patterns (MacDougal, 1924; Nobel, 2003). In this species, even the distance
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among rib crests may be taken as an index of plant water status. Saguaro plants however have cylindrical stems and might show different diel stem dynamics compared to platyopuntias (flattened stems). A study on diel stem fluctuations of Opuntia occidentalis measured with a mechanic dial gauge showed an association between cladode temperature and thickness gain (Schroeder, 1975). To date, the effect of drought stress on the physiological mechanisms leading to cladode growth in cactus pear has not been reported in the literature. The opportunity to study cladode enlargement and shrinkage and the relationship between cladode growth and environmental parameters is crucial to better understand the functioning of succulents. Diel fluctuations of cladode thickness may also represent an early indicator of the occurrence of dehydration stress. Eventually, acquisition of continuous measurements over a period of 24 h will allow the development of models useful for preventing the negative effects of drought on plant growth by real-time automated environmental control. This work investigated the effect of soil water deficit and temperature on the daily mechanisms of cladode growth in cactus pear. The experimental model included also cladode age as a secondary factor affecting growth dynamics in response to the environment. It was hypothesized that the youngest cladodes could show more pronounced diel thickness fluctuations compared to older cladodes, and therefore serve as a suitable model for assessing plant responses to environmental factors. It was also assumed that drought would reduce cladode diel thickness fluctuations and interfere with other environmental factors. 2. Materials and methods 2.1. Plant material and experimental setup The experiment was conducted in a greenhouse at the Department of Agricultural and Forest Sciences, University of Palermo (38 60 31.78900 N and 13 210 2.27100 E, 40 m a.s.l.) from April to July 2014, using cactus pear plants of the cultivar Gialla, cultivated in Sicily for fruit production (Barbera et al., 1992). Different types of plants were selected: (a) six 2-year-old plants, (b) two 3-year-old plants, (c) four detached cladodes, (d) six rooted cuttings. Within each age category, plants were uniform in size and number of cladodes. The first two groups had already been growing in 40-liter containers with sandy-loam soil. For rooted cuttings, 1year-old cladodes were collected from adult mother plants about a month before drought treatment was imposed and, after one week
Fig. 1. Schematic representation of various-age cladodes on a 3- and a 2-year-old Opuntia ficus-indica plant.
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of drying the cut surface to prevent rotting and improve establishment, they were potted (half cladode buried) in 40-liter containers with sandy-loam soil. Rooting was evident by the end of June. Two weeks before drought treatment was imposed, all plants were moved inside the greenhouse and fertilized with 50 g/pot of a NPK fertilizer (20-10-10). Greenhouse internal air temperature and relative humidity were recorded using HOBO H-08 sensors (Onset Computer Corporation, Bourne, MA, USA). Four 1-year-old cladodes were removed from 3-year-old plants (detached) and left on a bench in the greenhouse until the end of the experiment to study growth dynamics and compare a soilisolated system to fully functional plants. On April 15 (beginning of drought treatment) all plants were watered to FC by providing water to the point of runoff, and then randomly split into irrigated and unirrigated plants. Irrigated plants were watered to FC once per week during April and May and twice per week in June and July, as temperatures increased. Unirrigated plants received no irrigation for the same period. On July 17 (end of treatment), a final rewatering of unirrigated pots was carried out and a final set of measurements was taken to study cladode responses to sudden soil rehydration after three months of drought treatment. Three months of drought can be considered extreme under Mediterranean climates. 2.2. Soil moisture Twice a month SMC was measured in pots of 2- and 3-year-old plants by the gravimetric method. In each pot, soil samples were collected from the top 25 cm, where cactus pear roots typically lie. FW was immediately recorded, and samples were oven-dried at 60 C until constant weight (72–96 h) to obtain DW. SMC (%) was calculated as (FW–DW) 100/DW. In addition, between July 4 and 5, a day after irrigating to FC, 24h soil water depletion was monitored in irrigated 2-year-old plant and cladode cutting pots. In those pots, evaporation was avoided by covering the soil with aluminum foil for the entire 24-h period. No further loss of water occurred after initial runoff, therefore water loss was mainly due to plant uptake. Measurements were taken every two hours in proximity of the roots with a portable HH2 moisture meter connected to a WET sensor (Delta-T Device LTD., Cambridge, UK) and expressed as percentage of FC (FC = 100 %).
Fig. 2. Gravimetric soil moisture content of potted 3- (A) and 2-year-old (B) Opuntia ficus-indica plants. Error bars indicate standard errors of the means for 2-year-old plants. Panel A shows the moisture content of one irrigated and one unirrigated 3year-old plant. The vertical dashed line indicates the last irrigation before drought treatment started. Data were analyzed by analysis of variance.
2.3. Plant water status and cladode thickness Plant water status was estimated by measuring cladode RWC (%) according to Barrs and Weatherley (1962) on 1-year-old and new developing irrigated and unirrigated cladodes. Cladode cores were collected with a cork borer (13-mm diameter) on April 24 and July 4, the beginning and the end of the drought treatment, respectively. Individual samples were sealed in plastic bags and quickly transported to the laboratory for FW determination. Subsequently, the disks were immersed in deionized water for 24 h at 8 C to obtain the TW. The DW was measured after oven drying samples at 60 C until constant weight (2–4 days). RWC was calculated as (FW–DW)/(TW–DW) 100. Changes of 1-year-old cladode thickness were monitored with a digital caliper (at an intermediate portion of the cladode) in both irrigated and unirrigated plants as an additional information regarding plant water status. Six cladodes were chosen as replicates from irrigated and unirrigated plants. Thickness was firstly measured on May 9 and then once per week in the last month of treatment (from beginning of June to beginning of July). In addition, plant water status was also estimated by measuring the thickness of fresh cladode cores extracted with a cork borer on June 6 and July 4. Six cylinders mother cladodes (1-year-old) and six from new developing cladodes of irrigated and unirrigated
Fig. 3. Daily trends of soil water content in irrigated pots of 2-year-old Opuntia ficus-indica plants and rooted cuttings (from 8:00 am of July 4 until 6:00 am of July 5). Error bars indicate standard errors of the means. Data were analyzed by analysis of variance.
2-year-old plants. Samples were laid on a white board including a reference measuring tape and disk sections were photographed with a Fujifilm FinePix F600EXR digital camera (Fujifilm Holdings
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Corporation, Tokyo, Japan). Subsequently, thickness of total, parenchyma and chlorenchyma tissues was estimated to the tenth of a millimeter with the software ImageJ (Wayne Rasband, National Institute of Health, Bethesda, MD, USA). Water storage parenchyma was identified as the whitish tissue in the inner part of the cladode; the chlorenchyma was identified as the surrounding green tissue including the cuticle layer.
Table 1 Parenchyma, chlorenchyma and total thickness (means standard errors) of cores extracted from irrigated and unirrigated 1-year-old (old) and new developing cladodes (new) on June 6 and July 4. No differentiation was observed between parenchyma and chlorenchyma in new developing cladodes. Date
Cladode type
Thickness (mm) Parenchyma
Chlorenchyma
Total
June 6
Irrigated new Irrigated old Unirrigated new Unirrigated old
– 15.42 1.91 – 4.33 0.56
– 5.87 0.41 – 4.83 0.64
5.82 0.26 21.29 2.19 4.88 0.27 9.17 1.05
July 4
Irrigated new Irrigated old Unirrigated new Unirrigated old
– 10.72 1.30 – 2.44 0.32
– 6.10 0.29 – 1.83 0.45
6.91 0.33 16.82 1.50 4.00 0.23 4.27 0.30
2.4. Cladode stomatal conductance and malic acid accumulation A Delta-T AP4 dynamic porometer (Delta-T Devices LTD., Cambridge, UK) was used to measure gs on June 6 and July 4, at 4-h intervals, from 8 pm to 8 am due to nocturnal stomatal opening. Measurements were taken on young and mother cladodes from 2-year-old plants, and cladode cuttings of both irrigated and unirrigated plants. Carbon assimilation was estimated by measuring nocturnal accumulation of malic acid on mother and new developing cladodes of 2-year-old plants. Cladode cores were extracted with a cork borer at dusk and dawn on April 30-May 1 and July 4–5 and immediately frozen. Afterwards, samples were thawed, the cuticle layer was peeled off with a scalpel and chlorenchyma tissue was separated from the remaining parenchyma. Titratable acidity was measured on chlorenchyma of 1-year-old cladodes and on whole peeled tissue of developing cladodes, due to non-visible differences between chlorenchyma and parenchyma in the latter. Samples were weighed and then ground with a mortar and pestle and gradually added with 10 ml of deionized water. Homogenates were transferred into a 50ml beaker, titrated to pH 8.1 with a Crison Compact titrator (Crison Instruments, SA, Barcelona, Spain), and acidity was expressed as grams of malic acid per gram of tissue. 2.5. Cladode growth Cladode growth rate was determined manually, using a measuring tape to measure width and length. Changes of cladode area were monitored at 10-day intervals from May to July 2014. Cladode area was estimated as (width/2) (length/2) p (Tiznado-Hernández et al., 2010). AGRarea and RGRarea were estimated as changes in cladode area per day and changes in cladode area per day and per initial area, respectively. Continuous cladode diel swelling/shrinkage were assessed using stem gauges described by Morandi et al. (2007). The same devices
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have been adopted for several studies on fruit growth patterns. Briefly, the stem gauge is composed by a linear potentiometer which is connected to a mobile metal plunger surrounded by a light spring. The movement of the plunger sends signals in terms of mV to a data logger, which can be easily converted in mm. The gauges lightweight and frame shape make them particularly suitable for studying growth in cactus pear cladodes. Gauges were connected to a CR1000 data logger (Campbell Scientific Ltd., Leicestershire, UK) and data were recorded at 15-min intervals. The growth of 1-year-old cladodes from 2-year-old potted plants was continuously monitored for periods of 7 days/month in April, May, June, and July in order to study daily growth patterns. Each of the seven days of a week was taken as a replicate of the 24-h AGRthickness trend. Furthermore, in April, gauges were used for comparing growth dynamics of 1-, 2- and 3-year-old cladodes from 3-year-old plants, and proximal and distal portion of 1-year-old cladodes from 2-year-old plants (Fig. 1). Due to lack of significant differences between the two portions of 1-year-old cladodes, subsequent gauge measurements were only taken from the central portion of cladodes. The last series of gauge measurements was carried out in July 2014 using 1-year-old cladodes from 2year-old plants and the four detached cladodes. All collected data were converted in cladode AGRthickness and expressed in mm min1. Maximum and minimum diel AGRthickness were used as parameters of growth for comparisons of cladode ages and drought treatments in April and only drought treatments in July. Maximum AGRthickness was defined as the highest enlargement rate of cladodes during the 24 h, whereas minimum AGRthickness was the highest shrinkage rate (i.e. the most negative AGRthickness in each day). 2.6. Statistical analysis SYSTAT procedures (Systat software Inc., Chicago, Illinois, USA) were used to carry out analysis of variance on all data, and, when appropriate, means were compared by Tukey’s multiple range test. Linear regression analysis was performed to test correlations between soil water depletion (% of FC) and AGRthickness, and between temperatures and AGRthickness using Sigmaplot procedures (Systat software Inc., Chicago, Illinois, USA). Slopes of regressions were compared by analysis of variance using slope coefficients and standard errors. 3. Results and discussion 3.1. Soil moisture
Fig. 4. Changes of 1-year-old cladode thickness in irrigated and unirrigated 2-yearold Opuntia ficus-indica plants. Error bars indicate standard errors of the means. Analysis of variance was followed by Tukey’s test (n.s., non-significant; **significant for P < 0.001; *significant for P < 0.05).
Drought treatments and plant age and size generated significant differences in the SMC (Fig. 2). Specifically, SMC of older plants (late April, Fig. 2A) started to decrease earlier in unirrigated than in
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mechanism of root water uptake is not only driven by the transpiration stream, but it may be the consequence of a delayed and gradual rehydration of cladode cells after nocturnal transpiration, possibly driven by the accumulation of osmotically active compounds (Lee, 2010). 3.2. Plant water status and cladode thickness As expected, no RWC difference was observed in April between 1-year-old cladodes of irrigated (84.4% 3.05) and unirrigated (84.8% 3.05) plants. A lower RWC was observed in unirrigated (44.9% 2.78) compared to irrigated (78.5% 2.78) cladodes by the
Fig. 5. Stomatal conductance of new developing cladodes (A), mother cladodes (B) and rooted cuttings (C) in irrigated and unirrigated Opuntia ficus-indica plants on July 4. Error bars indicate standard errors of the means. Analysis of variance was followed by Tukey’s test (n.s., non-significant; **significant for P < 0.001; * significant for P < 0.05).
irrigated treatments compared to younger plants (beginning to late May, Fig. 2B). By the first week of July, 2-year-old irrigated plants generated a significant soil water depletion (24.5%) during the 24 h after irrigation, while irrigated rooted cuttings showed no change during the same time period (Fig. 3). These results are probably due to the 2-year-old plant larger size, more developed root systems and higher water need compared to rooted cuttings. Despite stomata being closed during the day, and preventing evaporation by covering the soil with aluminum foil, a decrease of SMC during daylight was shown in 2-year-old plants. This suggests that the
Fig. 6. Cladode surface area, absolute growth rate (AGRarea) and relative growth rate (RGRarea) of new developing cladodes for irrigated and unirrigated Opuntia ficusindica plants. Error bars indicate standard errors of the means. Analysis of variance was followed by Tukey’s test (n.s., non-significant; **significant for P < 0.001; *significant for P < 0.05).
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Fig. 7. Twenty-four-hour trend (A) and daily maximum and minimum (B) absolute thickness growth rates (AGRthickness) in 1-, 2-, and 3-year-old cladodes from 3-yearold Opuntia ficus-indica plants. Mean values of seven days in April are shown. Error bars indicate standard errors of the means. Analysis of variance was followed by Tukey’s test (different letters indicate significant differences for P < 0.001).
Fig. 8. Daily maximum and minimum of absolute thickness growth rates (AGR thickness) in irrigated, unirrigated, and detached 1-year-old cladodes of Opuntia ficusindica. Mean values of seven days in July and their standard errors are shown. Analysis of variance was followed by Tukey’s test (different letters indicate significant differences for P < 0.001).
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end of the drought treatment. The slight decrease of RWC observed in irrigated cladodes from April to July was probably due to the production of a high number of daughter pads, which may compete for water with mother cladodes (Pimienta-Barrios et al., 2005). However, this reduction was not significant because irrigated plants had enough SMC to maintain hydration of both mother and daughter cladodes. An interaction was found between cladode age on the same plant and drought effects, with unirrigated cladodes showing a sharper difference between developing (62.5% 1.29) and mother cladodes (44.9% 1.46) than irrigated cladodes (85.2% 1.22 and 78.5% 2.34, respectively). Differences found between mother and daughter cladodes are the proof that cactus pear, particularly under drought conditions, tends to store water in newly formed structures, probably as a survival strategy and to provide more water to the more photosynthetically active organs. Significant differences of thickness between irrigated and unirrigated 1-year-old cladodes were observed starting on June 5 (Fig. 4). Thickness of irrigated cladodes was stable throughout the trial, while those on unirrigated cladodes consistently decreased, becoming as much as 57.5% smaller than in irrigated by the end of treatment period. In cladode cores sampled from June 6 to July 4 (Table 1), thickness increased by 1.8% in new cladodes and a simultaneous 30.8% decrease occurred in the mother cladodes (P = 0.002). Irrigated cladodes were thicker than unirrigated ones (P < 0.001), with sharper differences in mother cladodes (64.7%) than in new daughter cladodes (30.2%). This is a further indication that plants under drought tend to allocate more water to the most photosynthetically active organs. Furthermore, after 75 days, drought caused a reduction of the thickness of parenchyma and chlorenchyma tissues by 75.0% and 55.8%, respectively (Table 1). This suggests that the parenchyma loses water more readily than chlorenchyma, confirming that in CAM plants, parenchyma water is the most prone to be lost under drought conditions (Nobel, 2006; Pimienta-Barrios et al., 2007). Reduction of the parenchyma was therefore responsible for most of the reduction in thickness of unirrigated cladodes observed on July 4 (Fig. 4). 3.3. Cladode stomatal conductance and malic acid accumulation Analysis of gs data showed no difference between the two dates (June 6 and July 4), and no difference between irrigation treatments or organ type or age in June, so only data from July 4 were considered. In this case, a significant (P < 0.001) interaction between irrigation and organ age and type was found. Specifically, in the case of new developing cladodes and rooted cuttings, conductance was significantly higher in irrigated than in unirrigated treatments at midnight and 4 am (Fig. 5). Also, new developing cladodes of irrigated plants maintained a constant high rate of stomatal conductance (about 150 mmol m2 s1) in the middle of the night, consistent with the results reported by Nobel and De la Barrera (2000). In contrast, rooted cuttings experienced the highest conductance at 4 am. This can be explained by a high RWC in growing cladodes (Section 3.2), which allows for relatively high gs during most of the night. Differences between mother and daughter cladodes in this study contrast with the results of Cui et al. (1993), who recorded higher nocturnal conductance in mother cladodes. It is possible that cladodes measured by Cui et al. (1993) were too young (within 4 weeks of age) and still doing diurnal rather than nocturnal transpiration, as reported by Acevedo et al. (1983). This would explain the differences with our observations. When gs was measured in 1-year-old irrigated, unirrigated and detached cladodes, no significant difference was found between June and July, while the mean nocturnal gs of irrigated cladodes (40.2 mmol m2 s1) was higher than that of unirrigated
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(5.37 mmol m2 s1) and detached (2.39 mmol m2 s1) cladodes. As expected, gs was highly influenced by plant water status, but in the case of O. ficus-indica, detached photosynthetic organs were able to overcome many months without soil contact and under stressful summer greenhouse conditions during summer. The detached cladodes must have maintained a minimal photosynthetic activity as they produced daughter cladodes for up to two months after detachment (personal observations). The latter has been previously documented, especially in the presence of high light intensity and high tissue concentration of indole-acetic acid and kinetin (Nobel, 1996). At the end of the imposed stress (4–5 July), new developing cladodes of irrigated plants had a consistently higher nocturnal malic acid content (7.75 mg g1) compared to either their mother cladodes (2.05 mg g1) or to developing unirrigated cladodes (1.24 mg g1), which instead did not significantly differ from unirrigated mother cladodes (3.77 mg g1). Thus, similar levels of nocturnal malic acid in daughter and mother cladodes may be indicative of drought stress in cactus pear. 3.4. Cladode growth dynamics Drought also affected cladode surface area, AGRarea and RGRarea from May to July (Fig. 6). In particular, mean cladode areas were greater in irrigated plants compared to unirrigated ones starting on June 15 (Fig. 6A), with young unirrigated cladodes having a surface area 58.6% smaller than young irrigated ones by the end of the drought treatment. This is expected as area is a cumulative result of
the growth process. Similar reductions in size (52%) were found by Luo and Nobel (1993). On the other hand, drought significantly reduced AGRarea just on June 5 and 16 (Fig. 6B). The final decrease of AGRarea and lack of significant differences between irrigated and unirrigated treatments in July are likely due to the fact that cladodes were reaching their final size. Conversely, drought reduced RGRarea only at the beginning (Fig. 6C), when cladodes were rapidly growing. The data clearly indicates that unirrigated mother cladodes could not supply enough water and assimilates to support the normal development of new organs. Toward the end of the drought treatment, slightly negative RGRarea were recorded, indicating some shrinkage of organs due to tissue dehydration. In April, trends of thickness fluctuations (AGRthickness,mm min1) in cladodes of different ages were similar in irrigated and unirrigated plants. This lack of difference was possibly due to early measurements taken before any water deficit in the soil. Nevertheless, different AGRthickness trends were observed among 1, 2- and 3-year-old cladodes (Fig. 7A). Cladode age affected also maximum and minimum daily AGRthickness, with older cladodes showing the lowest and younger cladodes the highest daily shrinkage (Fig. 7B). Maximum daily AGRthickness of 3-year-old cladodes was higher than max daily AGRthickness of both 1- and 2year-old stems, which were similar. Differences among cladodes of various ages may be in part explained by the degree of lignification, particularly evident at the base of 3-year-old cladodes. In cactus pear, accumulation of fiber and lignin in old stems is directly correlated with age (Oliveira Ribeiro et al., 2010; Liguori et al., 2014). Cells of lignified organs are characterized by low elasticity,
Fig. 9. Twenty-four-hour trends of temperature and absolute growth rate (AGRthickness) of irrigated and unirrigated 1-year-old cladodes from 2-year-old Opuntia ficus-indica plants. Mean values of seven days in the months of April, May, June and July 2014 are shown. Data were analyzed by analysis of variance; error bars indicate standard errors of the means.
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decreasing AGRthickness fluctuations. Moreover, 2-year-old cladodes showed less night shrinkage than 1-year-old cladodes, but similar max AGRthickness (Fig. 7B). This may indicate that a greater amount of biomass gained during the day is lost at night in 1-year-old than in 2-year-old cladodes. In July, daily max AGRthickness of 1-year-old cladodes was significantly greater in irrigated than in unirrigated and detached cladodes, while no differences were observed between irrigated and unirrigated cladodes (Fig. 8). On the other hand, daily min AGRthickness was similar for all three cladode types, in spite of gs being highest in irrigated cladodes. Hence, high SMC in irrigated treatments allowed higher transpiration rates, resulting in a nocturnal shrinkage rate similar to the other treatments. In detached cladodes, fluctuations of AGRthickness were due to climatic factors such as light intensity, relative humidity and temperature rather than root water uptake. Indeed, Schroeder (1975) showed that the enlargement of detached cladodes of O. occidentalis is negatively correlated with air temperature but positively correlated with light intensity. Daily trends of AGRthickness in April, May, June and July were compared using 1-year-old cladodes from 2-year-old potted plants (Fig. 9). Data analysis revealed significant effects of month, time of the day and drought, but also a significant interaction among these three factors, suggesting that AGRthickness responses to drought may vary depending on time of the day and drought severity. Specifically, in April, the peak of AGRthickness occurred in early morning and in early afternoon for unirrigated and irrigated cladodes, respectively. Differences in this period were probably unrelated to SMC. Irrigated plants showed daily max AGRthickness between 1 and 3 pm, with the highest daily max AGRthickness being in June. In unirrigated plants, AGRthickness fluctuations decreased with the advancement of drought, with an almost flat daily pattern in June and particularly July, when cladode thickness (Fig. 4) and RWC (Section 3.2) were low. In particular, our data showed that when 1-year-old cladodes reach a minimum thickness of about 8 mm and a RWC of about 45%, AGRthickness daily fluctuations were almost canceled. When mean daily AGRthickness were calculated for all sampling dates, no differences were found between irrigated and unirrigated treatments in April (Fig. 10), in absence of soil moisture deficit (Fig. 2B). In May and June, mean daily AGRthickness was greater in
Fig. 10. Trend (April to July) of daily average absolute growth rate (AGRthickness) of irrigated and unirrigated 1-year-old cladodes from 2-year-old Opuntia ficus-indica plants. Mean values of seven days in each month and their standard errors are shown. Rewatering (dashed gray line) was carried out on July 17. Analysis of variance was followed by Tukey’s test (n.s., non-significant; **significant for P < 0.001; *significant for P < 0.05).
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the irrigated than in unirrigated treatments, whereas in July, it was again similar, probably due to larger plants and generally higher water consumption. After rewatering of all pots to FC, mean AGRthickness was higher in unirrigated than irrigated cladodes. This demonstrates the extraordinary ability of cactus pear mucilages to sequester water and to retain it for long periods. Multiple factors are probably involved in the regulation of AGRthickness dynamics. Nocturnal shrinkage could be due to loss of water when transpiration is faster than root water uptake. Specifically, stomata are open at night and the transpiration causes reductions of stem water potential and stem contractions (Nobel, 2003). Also, the nocturnal accumulation of malate in chlorenchyma vacuoles and the diurnal synthesis of carbohydrates leads to an increase of osmotic pressure. This, in turn, drives water into the chlorenchyma and results in cladode swelling during the day. Therefore, SMC is partially responsible for the differences between irrigated and unirrigated cladode dynamics. Yet, path resistances and time lags may mask a direct relationship between SMC and fluctuations in stem thickness. The gs differences among cladode types found in this study suggested that water loss by transpiration could be related to nocturnal shrinkage of cladodes. Nevertheless, no significant association was found between cladode gs and nocturnal shrinkage. This indicates that some other factor must play a major role in the 24-h growth dynamics. A direct linear relationship was found between SMC (% of FC) and AGRthickness during the 24 h (Fig. 11), suggesting that AGRthickness of 1-year-old cladodes (on 2year-old plants) may respond to changes of SMC. AGRthickness was minimal when SMC reached 57.3% of FC, indicating a soil moisture threshold above which cactus pears grow. In addition, direct linear relationships were found between diel temperatures and AGRthickness of irrigated and unirrigated 1-yearold cladodes (Fig. 12). A similar relationship was observed in O. occidentalis (Schroeder, 1975). In April and May, both AGRthickness of irrigated and unirrigated cladodes were directly related to temperature with similar slopes, indicating analogous responses to temperature. This is expected since little soil or plant water deficit was found at that time. Similar effects of temperature on stem daily growth dynamics were also found in Scots pine (Antonova and Stasova, 1993), eucalyptus (Downes et al., 1999), balsam fir (Deslauriers et al., 2003), Mexican mountain pine (Biondi and Hartsough, 2010) and other species, whereas no
Fig. 11. Correlation between soil water content and absolute growth rate (AGR thickness) of 1-year-old cladodes from 2-year-old Opuntia ficus-indica plants. Measurements were carried out in irrigated plants a day after irrigating to field capacity (FC). AGRthickness fluctuations are canceled at a soil water content threshold of 57.3% of FC. Data were analyzed by linear regression.
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Fig. 12. Correlations between temperature and absolute growth rate (AGRthickness) of irrigated (black circles) and unirrigated (white circles) 1-year-old cladodes from 2-yearold Opuntia ficus-indica plants during the drought treatment. Temperature and AGRthickness records are hourly data averaged from seven days in April, May, June and July. Data were analyzed by linear regression.
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relationship was found in peach and plum (Gènard et al., 2001). In June, AGRthickness was also correlated to temperature, but the slope was greater in irrigated than in unirrigated cladodes (Fig. 12). In contrast, in July (severe drought), AGRthickness of unirrigated cladodes was not related to temperature (P = 0.39). This can be explained by the lack of AGRthickness fluctuations due to cladode dehydration (i.e. reduced cladode thickness and RWC) at severe drought stages. 4. Conclusions This study demonstrated that drought stress reduced cladode thickness, RWC, malic acid accumulation, gs, size and growth rates of developing cladodes. Under well-watered conditions, diel fluctuations of cladode thickness are directly related to temperature variations, while under severe drought stress, SMC has higher influence than temperature. This suggests differential regulation of cladode growth depending on plant water status. Our data also showed that cactus pears are able to maintain some growth and assimilation at RWC above 45%, an extremely low limit compared to non-CAM plants. In addition, cladode growth was extremely responsive to rehydration after long periods of drought, suggesting that a regulated reduction of irrigation would not significantly affect plant biomass. These results indicate that a variety of factors are involved in cladode growth dynamics. Thus, a model that takes into account temperature, light, soil moisture content, plant water status, and concentration of osmotica in the cladode tissue needs to be considered in order to predict cladode growth under different conditions (e.g. different age, drought stress, etc.). References Acevedo, E., Badilla, I., Nobel, P.S., 1983. Water relations, diurnal acidity changes, and productivity of a cultivated cactus, Opuntia ficus-indica. Plant Physiol. 72 (3), 775–780. doi:http://dx.doi.org/10.1104/pp.72.3.775. Adams, W.W., Díaz, M., Winter, K., 1989. Diurnal changes in photochemical efficiency, the reduction state of Q, radiationless energy dissipation, and nonphotochemical fluorescence quenching in cacti exposed to natural sunlight in northern Venezuela. Oecologia 80 (4), 553–561. doi:http://dx.doi.org/10.1007/ bf00380081. Antonova, G.F., Stasova, V.V., 1993. Effects of environmental factors on wood formation in Scots pine stems. Trees 7 (4), 214–219. doi:http://dx.doi.org/ 10.1007/bf00202076. Barbera, G., Carimi, F., Inglese, P., 1992. Past and present role of the Indian-fig prickly-pear (Opuntia ficus-indica (L.) Miller, Cactaceae) in the agriculture of Sicily. Econ. Bot. 46 (1), 10–20. doi:http://dx.doi.org/10.1007/bf02985249. Barcikowski, W., Nobel, P.S., 1984. Water relations of cacti during desiccation: distribution of water in tissues. Bot. Gaz. 145, 110–115. doi:http://dx.doi.org/ 10.1086/337433. Barrs, H., Weatherley, P., 1962. A re-examination of the relative turgidity technique for estimating water deficits in leaves. Aust. J. Biol. Sci. 15 (3), 413–428. doi: http://dx.doi.org/10.1071/BI9620413. Biondi, F., Hartsough, P., 2010. Using automated point dendrometers to analyze tropical treeline stem growth at Nevado de Colima, Mexico. Sensors 10 (6), 5827–5844. doi:http://dx.doi.org/10.3390/s100605827. Cui, M., Miller, P.M., Nobel, P.S., 1993. CO2 exchange and growth of the crassulacean acid metabolism plant Opuntia ficus-indica under elevated CO2 in open-top chambers. Plant Physiol. 103, 519–524. doi:http://dx.doi.org/10.1104/ pp.103.2.519. Deslauriers, A., Morin, H., Urbinati, C., Carrer, M., 2003. Daily weather response of balsam fir (Abies balsamea (L.) Mill.) stem radius increment from dendrometer analysis in the boreal forests of Québec (Canada). Trees 17 (6), 477–484. doi: http://dx.doi.org/10.1007/s00468-003-0260-4. Downes, G., Beadle, C., Worledge, D., 1999. Daily stem growth patterns in irrigated Eucalyptus globulus and E. nitens in relation to climate. Trees 14 (2), 102–111. doi: http://dx.doi.org/10.1007/pl00009752. Drennan, P.M., Nobel, P.S., 2000. Responses of CAM species to increasing atmospheric CO2 concentrations. Plant Cell Environ. 23 (8), 767–781. doi:http:// dx.doi.org/10.1046/j1365-3040.2000.00588.x. Gènard, M., Fishman, S., Vercambre, G., Huguet, J.G., Bussi, C., Besset, J., Habib, R., 2001. A biophysical analysis of stem and root diameter variations in woody plants. Plant Physiol. 126, 188–202. doi:http://dx.doi.org/10.1104/pp.126.1.188. Goldstein, G., Ortega, J.K.E., Nerd, A., Nobel, P.S., 1991. Diel patterns of water potential components for the crassulacean acid metabolism plant Opuntia ficusindica when well-watered or droughted. Plant Physiol. 95 (1), 274–280. doi: http://dx.doi.org/10.1104/pp.95.1.274.
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