Citrus rootstock responses to water stress

Citrus rootstock responses to water stress

Scientia Horticulturae 126 (2010) 95–102 Contents lists available at ScienceDirect Scientia Horticulturae journal homepage:

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Scientia Horticulturae 126 (2010) 95–102

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage:

Citrus rootstock responses to water stress Juan Rodríguez-Gamir, Eduardo Primo-Millo, Juan B. Forner, M. Angeles Forner-Giner ∗ Centro de Citricultura y Producción Vegetal, Instituto Valenciano de Investigaciones Agrarias, Apartado Oficial, 46113 Moncada, Valencia, Spain

a r t i c l e

i n f o

Article history: Received 8 March 2010 Received in revised form 22 June 2010 Accepted 24 June 2010 Keywords: Cleopatra mandarin Poncirus trifoliata Water potential Stomatal conductance Transpiration Forner–Alcaide 5

a b s t r a c t Tolerance to drought-stress (DS) of the citrus rootstock Forner–Alcaide no. 5 (FA-5) was tested and compared with that of its parents, Cleopatra mandarin (CM) and Poncirus trifoliata (PT). Nine-monthold seedlings of CM, PT and FA-5 and 15-month-old grafted trees of ‘Valencia’ orange scions on these three rootstocks were cultivated in sand under glasshouse conditions and irrigated with a nutrient solution. Plants were drought-stressed by withholding irrigation until leaves were fully wilted. Survival time of both seedlings and grafted trees under DS was linked to the water extraction rate from the soil, which depended mainly on leaf biomass and on transpiration rate. Seedling responses to DS affecting leaf water relationships and gas exchange parameters varied among genotypes. FA-5 seedlings survived longer than the other seedlings, maintaining the highest levels of water potential, stomatal conductance, transpiration rate and net CO2 assimilation towards the end of the experiment, when water stress was most severe. Thus, FA-5 was more resistant to DS than its parents (CM and PT). Moreover, rootstock affected the performance of grafted trees under water stress conditions. The higher drought tolerance induced by FA-5 rootstock could be related to the greater osmotic adjustment (OA), which was reflected by smaller reductions in leaf relative water content (RWC) and in higher turgor potentials and leaf gas exchange than the other rootstocks. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Water deficit in citrus diminishes vegetative growth and yield, and reduces fruit size, and sometimes quality, causing important economic losses in orchards (Hilgeman and Sharp, 1970; Levy et al., 1978, 1979; Castel and Buj, 1990; Ginestar and Castel, 1996; Gonzalez-Altozano and Castel, 1999, 2000; Romero et al., 2006). Additionally, drought-stress reduces CO2 assimilation, stomatal conductance and transpiration (Sinclair and Allen, 1982; Syvertsen et al., 1988; Gomez-Cadenas et al., 1996; Arbona et al., 2005; Perez-Perez et al., 2007; Garcia-Sanchez et al., 2007). Root systems can respond to soil drying by sending signals to the leaves, where stomatal closure is induced to reduce water loss (Davies and Zhang, 1991). A drought-induced signaller can be abscisic acid (ABA), which is synthesized in the roots and transported through the transpiration stream to the leaves (Zeevaart and Boyer, 1984; Zhang et al., 1987; Zhang and Davies, 1989a,b, 1990a,b; Gomez-Cadenas et al., 1996). Moreover, plants have developed other mechanisms to resist drought, such as increased root development or leaf mass reduction (Zhang and Davies, 1989b, 1990b;

∗ Corresponding author at: Departamento de Citricultura y otros frutales, Instituto Valenciano de Investigaciones Agrarias, Apartado Oficial, 46113 Moncada, Valencia, Spain. Tel.: +34 963 424 000; fax: +34 963 424 001. E-mail address: forner [email protected] (M.A. Forner-Giner). 0304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.06.015

Yin et al., 2005; Lei et al., 2006). Also, osmotic adjustment enables plants to maintain the leaf turgor necessary for stomatal opening, thus sustaining photosynthesis and growth (Syvertsen and Smith, 1983; Morgan, 1984; Delauney and Verma, 1993; Nolte et al., 1997; McNeil et al., 1999; Perez-Perez et al., 2007; Garcia-Sanchez et al., 2007). In addition, certain traits of citrus rootstocks have a marked influence on drought-stress response, including differences in root distribution (Castle and Krezdorn, 1975), water uptake efficiency (Castle and Krezdorn, 1977) and root hydraulic conductivity (Sinclair and Allen, 1982; Syvertsen and Graham, 1985; Zekri and Parsons, 1989). Also, rootstocks affect vigour, crop load and fruit characteristics (Albrigo, 1977; Syvertsen et al., 2000; Barry et al., 2004; Romero et al., 2006). Thus, the characteristics of the rootstock may be a key consideration when determining the response of citrus plants to water deficit. In 1974, the Valencian Institute for Agricultural Research (IVIA) began a program to breed citrus rootstocks by hybridization, and more than 500 hybrids were evaluated to determine their horticultural performance. From among these, a hybrid of Cleopatra mandarin (Citrus reshni Hort ex Tan) × Poncirus trifoliata (L.) Raf., specifically ‘Forner–Alcaide no. 5’ (FA-5) was selected for its characteristics, which include high productivity and good fruit quality of the scion cultivar (Forner et al., 2003; Forner-Giner et al., 2003). Furthermore, FA-5 seems to be tolerant to salinity (LopezCliment et al., 2008; Forner-Giner et al., 2009) and to calcareous


J. Rodríguez-Gamir et al. / Scientia Horticulturae 126 (2010) 95–102

soils (Forner et al., 2003; Gonzalez-Mas et al., 2009). FA-5 has been used as a commercial rootstock in the European Union since 2005. The objective of this study was (1) to determine the water stress responses of three genotypes: Cleopatra mandarin (CM), Poncirus trifoliata (PT) and their hybrid FA-5; and (2) to test the performance of FA-5 rootstock under drought-stress conditions, as compared with its parents, CM and PT. The experiments were performed using seedlings of the three rootstocks onto which trees of Valencia orange scions were grafted. Resistance to drought-stress was tested by withholding irrigation and determining: (a) the maintenance of gas exchange functions in the time-course of the drought-stress period, and (b) the time that both seedlings and grafted trees can survive without irrigation. To gain insight into the causes underlying drought-stress resistance, the responses of seedlings, rootstocks and grafted trees to increasing levels of water deficit were determined. In addition, the relationships between morphological traits of each type of plant and drought-stress resistance were considered. 2. Materials and methods 2.1. Plant material and growing conditions Nine-month-old seedlings of CM, PT and FA-5 and 15-monthold trees of ‘Valencia’ orange (C. sinensis (L.) Osb.) grafted on these three rootstocks were used in this experiment. Rootstocks were budded when seedlings were 6-month-old, to approximate the size of seedlings and grafted trees as far as possible. Plants were grown under glasshouse conditions with supplementary light (50 ␮mol m−2 s−1 , 400–700 nm) to extend the photoperiod to 16 h. Temperature ranged between 16–18 ◦ C at night and 24–28 ◦ C during the day. Relative humidity was maintained at approximately 80%. Plants were grown individually in 3 L pots filled with coarse sand. All plants were irrigated twice weekly until the beginning of the experiment with the following nutrient solution: 3 mM Ca (NO3 )2 , 3 mM KNO3 , 2 mM MgSO4 , 2.3 mM H3 PO4 , 17.9 ␮M FeEDDHA and trace elements as prescribed by Hoagland and Arnon (1950). Nutrient solution pH was adjusted to 6.0 with 1 M KOH or 1 M H2 SO4 . One litre of solution was used per pot at each watering event. Excess solution drained out of the pot, thereby avoiding salt accumulation in the sand. Plants growing as a single shoot were selected for uniformity of size at the beginning of the experimental treatments. In all experiments plants were randomized over the experimental area and analysed individually. A row of plants, not included in the experiment, was placed around the perimeter as a buffer row.

Gravimetric soil water (Wg) content was also determined at the end of the drought period in all pots. After plant removal, soil samples from each pot were weighed (Ww), dried in a forced-draft oven at 65 ◦ C to a constant weight and reweighed again (Wd). Wg was calculated as Wg = (Ww − Wd) × 100/Wd and expressed in g H2 O/100 g soil (Perez-Perez et al., 2007). This experiment was repeated three times. 2.3. Leaf water relations In another independent experiment, 12 plants of each type were used, half of which were allowed to become drought-stressed by withholding irrigation while the other half was irrigated with nutrient solution every 2–3 days. To determine leaf water potential components two leaves per plant were sampled every 2–3 days. For each type of plant, the last sample was taken when leaves showed visible symptoms of leaf turgor loss (still reversible with re-watering), as later measurements were very difficult because of leaf dryness. This was between 1 and 3 days before the end of the survival time established in each case. All leaf tissue evaluations were performed using uniform fully expanded mature leaves from the mid-stem area of each of the six replicates per plant type and treatment. The average of the measurements taken for two leaves was considered representative of each individual plant. Leaf water potential (« s ) was measured at daybreak (6.30–7.30 h) with a Scholander-type pressure chamber (Soilmoisture equipment Corp, Santa Barbara, CA, USA) (Scholander et al., 1965). After measurement, leaves were tightly wrapped in aluminium foil, frozen in liquid nitrogen and stored in a freezer at −18 ◦ C. After thawing, leaf osmotic potential (« ␲ ) was measured in expressed cell sap collected from a syringe at 25 ± 1 ◦ C and placed in an osmometer (Digital Osmometer, Wescor, Logan, UT, USA). Leaf osmotic potential at full turgor («␲100 ) was determined in similar leaves also collected in the last sampling. After hydration overnight, fully turgid leaves were then frozen in liquid nitrogen, and osmotic potential was measured as above. Osmotic adjustment (OA) was calculated as the difference between «␲100 of control and stressed plants (Garcia-Sanchez et al., 2007). Also, two other similar leaves were collected in the last sampling, immediately weighed and leaf fresh weight (FW) was recorded. Then, leaf petioles were blotted dry with paper towels and placed in a beaker of water overnight in the dark so that leaves could become fully hydrated. Leaves were reweighed to obtain turgid weight (TW) and dried at 80 ◦ C for 24 h to obtain dry weight (DW). The relative water content (RWC) of the leaves was calculated as RWC = (FW − DW) × (TW − DW) − 1 × 100 according to Morgan (1984). 2.4. Gas exchange

2.2. Survival time of plants under drought conditions, whole plant transpiration and soil water content An experiment was performed to determine how long plants can survive without irrigation. The term “survival time” refers to the time it took for all the leaves of 50% of drought-stressed plants to become irreversibly wilted, i.e. to the point when recovery after rewatering was not possible. Irrigation was withheld from six plants of each type until this occurred. At the beginning of the experiment pots were irrigated, left for about 1 h to drain excess water, and weighed. Then, pots were covered with black plastic bags, sealing the base of the stem to prevent evaporative water loss. Pots were weighed at least twice a week throughout the drought period to determine whole plant transpiration, prior to which they were drawn out of the bags to aerate roots.

In a further experiment, six plants were irrigated normally and six others had irrigation withheld, as above. Stomatal conductance (gs), photosynthesis (A) and transpiration (E) of single attached leaves were measured outdoors between 10.30 and 11.30 h on sunny days, under relatively stable environmental conditions. Determinations were performed 1–2 times a week during the experimental period using two leaves for each of the six plants per type and treatment. The average of the measurements taken for these two leaves was considered representative of each individual plant. Photosynthetically active radiation (PAR) at the leaf surface was adjusted to 1000 ␮mol m−2 s−1 , which exceeds the saturating value for citrus (Iglesias et al., 2002). A closed gas exchange CIRAS-2 (PP-systems, Hitchin, UK) was used to take the measurements. Leaf laminae were fully enclosed within a PLC 6(U) universal leaf

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Table 1 Dry weight of organs and shoot/root ratio of drought-stressed seedlings of Poncirus trifoliata (PT), Cleopatra mandarin (CM) and Forner–Alcaide 5 (FA-5) and Valencia orange trees grafted on these three rootstocks (V/PT, V/CM and V/FA-5). Values are means of six replicates. Within each column, different letters indicate significant differences at P < 0.05 (LSD test). Ns, *, ** and *** indicate non-significant or significant differences at P < 0.05, 0.01 or 0.001, respectively. Seedlings and grafted trees were analysed independently. Dry weight (g)

PT CM FA-5 V/PT V/CM V/FA-5 ANOVA Seedlings Trees

DW leaves

DW shoots

DW roots

S/R ratio

4.06 b 15.20 a 2.45 c 7.13 x 5.25 y 7.09 x

12.94 b 21.75 a 11.07 b 4.72 y 6.97 x 6.73 x

10.43 8.58 8.49 8.30 10.56 10.7

1.62 b 4.30 a 1.59 b 1.42 x 1.15 y 1.29 xy

*** *

*** *

ns ns

*** *

autocuvette in a closed circuit mode and kept at 25 ± 0.5 ◦ C, with a leaf-to-air vapour deficit of about 1.7 Pa. The air flow rate through the cuvette was 0.5–1.5 L min−1 . The CO2 analyser was calibrated daily with a series of standard CO2 /air mixtures. Ten consecutive measurements were taken at 3-s intervals. 2.5. Harvesting For each tree type, the experiment concluded when leaves of 50% of the drought-stressed plants became irreversibly wilted. Plants were then harvested, divided into roots, shoots and leaves, washed and weighed. These organs were then dried in a forced-draft oven at 65 ◦ C for 48 h and re-weighed.

Fig. 1. Time elapsed until leaves of 50% of drought-stressed plants became fully wilted (mean survival time). Plants tested were: seedlings of Cleopatra mandarin (CM), Poncirus trifoliata (PT) and Forner–Alcaide 5 (FA-5), and Valencia orange trees grafted on these three rootstocks (V/CM, V/PT and V/FA-5). Values are means of three experiments ± SD.

reduced in all plants to below 4.5%, which was near wilting point in sand. 3.2. Leaf water relations Leaves from well-watered control plants of the seedlings (PT, CM and FA-5) had similar values of « s (Fig. 3). By day 2, leaf « s of drought-stressed plants began to decrease in the three species, reaching the lowest values at the end of the experiment (−3.95 and −3.70 MPa in PT and CM, respectively, on day 11, and −3.52 MPa in FA-5 on day 18). Leaf « ␲ values were also similar in control plants

2.6. Statistical analyses The data presented correspond to the mean of at least six independent plants. Parameters were statistically tested by analyses of variance and averages were compared with LSD test P ≤ 0.05. Statistical analyses were performed with Statgraphics Plus version 5.1 (Statistical Graphics, Englewood Cliffs, NJ, USA). 3. Results 3.1. Growth, survival time of plants under drought conditions and whole plant transpiration Regarding the seedlings, CM had the highest leaf dry weight and FA-5 the lowest (Table 1), and CM also had the greatest shoot/root (S/R) ratio. Grafted trees were more homogeneous in size and S/R ratio, although V/CM had a slightly lower leaf DW than the other plants. At the end of the experiment, there were no significant differences in weight between control and drought-stressed plants (data not shown). Fig. 1 shows the time lapse required for leaves of 50% of droughtstressed plants to become fully wilted (mean survival time). This time was 11 and 13 days in PT and CM seedlings, respectively; whereas in FA-5, mean survival time was 20 days. Fifty percent of trees grafted on PT (V/PT) survived 21 days of stress, whereas V/CM and V/FA-5 survived 26 and 29 days, respectively. Water extraction during these periods varied between 232.9 and 273.3 g of water per pot. In seedlings, the fastest water consumption corresponded to CM plants and the slowest to FA-5. In contrast, trees grafted on CM consumed water more slowly than those on PT and FA-5 (Fig. 2). Soil water content (Wg) in pots of well-watered treatments was 15.5 ± 0.5%. At the end of the drought-stress treatment, Wg was

Fig. 2. Cumulative water transpiration by the whole plant (Tp) by (A) seedlings of Cleopatra mandarin (CM), Poncirus trifoliata (PT) and Forner–Alcaide 5 (FA-5) and (B) Valencia orange trees grafted on these three rootstocks (V/PT, V/CM and V/FA-5) during the drought-stress treatment. Values are means of 18 replicates ± SD.


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Fig. 3. Effect of drought-stress (DS) on leaf water (« s ), osmotic (« ␲ ) and turgor (« T ) potentials in seedlings of Cleopatra mandarin (A, CM), Poncirus trifoliata (B, PT) and Forner–Alcaide 5 (C, FA-5) and Valencia orange trees grafted on these three rootstocks (D, V/CM; E, V/PT and F, V/FA-5). Values are means of six replicates ± SD.

of all seedlings. After 2 days of DS, « ␲ decreased sharply in CM, whereas only a small reduction was observed in PT on day 8. In FA5 plants, a progressive decrease in « ␲ was observed between days 2 and 18. At the end of the experiment, « ␲ values were −2.75 MPa in PT, −4.14 MPa in CM and −3.76 MPa in FA-5 on days 8, 11 and 18, respectively. After 5 days, reductions in DS-related leaf « s in PT and CM were not fully compensated for by reductions in leaf « ␲ , therefore leaf turgor decreased in both plant species. By contrast, the DS-induced decrease in leaf « ␲ in FA-5 seedlings was sufficient to offset the reduction in « s , maintaining turgor at values similar to control leaves until day 15 (Fig. 3A–C). In grafted trees (V/PT, V/CM and V/FA-5), there was little or no difference in either « s or « ␲ values between Valencia leaves on the different rootstocks from control (not-stressed) plants (Fig. 3D–F). Leaf « s values declined in the time-course of the DS period, reaching lower values in V/PT (−3.27 MPa) and FA-5 (−3.28 MPa) than in V/CM (−1.56 MPa). Also, in the second half of the drought period, when water stress became more severe, there was a decrease in leaf « ␲ , which was more extreme in V/FA-5 (−4.51 MPa) and V/PT (−3.38 MPa) than in V/CM (−2.51 MPa). The reduction in « s in V/PT leaves was not compensated by the decrease in « ␲ , and, consequently, leaf turgor dropped considerably after day 8. In contrast,

in V/FA-5 leaves, turgor was maintained till day 26 because the reduction of « s was lower than that of « ␲ . In V/CM leaves, the small decline of « s was accompanied by a slight reduction in « ␲ , and, therefore, only moderate turgor loss was observed at the end of the DS period (Fig. 3D–F). Table 2 shows the osmotic adjustment and relative water content of leaves from both seedlings and grafted trees compared independently. The values correspond to the last sampling date for each tree type. Further samples could not be taken because leaves wilted irreversibly from 1 to 3 days later. In these conditions, leaves from FA-5 and CM seedlings had the highest OA value, whereas those from PT had the lowest (Table 2). Likewise, leaf RWC values from stressed seedlings were lower in PT than in CM and FA-5. In grafted trees, the highest OA occurred in leaves from V/FA5, followed by those of V/PT, whereas V/CM leaves presented the lowest OA level. At the same time, RWC of leaves from droughtstressed V/FA-5 and V/CM plants were significantly higher than in V/PT (Table 2). 3.3. Gas exchange parameters In seedlings, leaf gas exchange parameters (gs, E and A) of non-stressed PT and FA-5 were significantly higher than those

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Table 2 Leaf osmotic potential at full turgor («␲100 ; c: control plants, s: drought-stressed plants), osmotic adjustment (OA) and relative water content (RWC; c: control plants, s: drought-stressed plants) in seedlings of Poncirus trifoliata (PT), Cleopatra mandarin (CM) and Forner–Alcaide 5 (FA-5) and Valencia orange trees grafted on these three rootstocks (V/PT, V/CM and V/FA-5). Values are means of six replicates. Within each column, different letters indicate significant differences at P < 0.05 (LSD test). Ns, *, ** and *** indicate non-significant or significant differences at P < 0.05, 0.01 or 0.001, respectively. Seedlings and grafted trees were analysed independently.

PT CM FA-5 V/PT V/CM V/FA-5 ANOVA Seedlings Trees

Days of DS

«␲100 (c) (MPa)

«␲100 (s) (MPa)

OA (MPa)

Leaf RWC (c) (%)

Leaf RWC (s) (%)

8 11 18 20 24 26

−2.29 −2.19 −2.35 −1.75 −2.15 −2.07

−1.87 b −3.19 a −3.55 a −2.88 y −2.37 z −4.43 x

−0.42 b 1.00 a 1.20 a 1.14 y 0.22 z 2.36 x

92.4 88.7 90.8 90.3 89.1 93.4

37.18 b 75.03 a 66.92 a 15.96 y 70.85 x 79.55 x

ns ns

* *

** ***

ns ns

*** ***

corresponding to CM (Fig. 4). The values of all these parameters decreased gradually in CM with the time-course of drought treatment, reaching minimum values on day 11, just before leaves wilted irreversibly. PT seedlings underwent a marked reduction in their gas exchange between days 5 and 8, reaching a similar level to that of CM on day 11. Reductions were less pronounced for all param-

eters in FA-5 seedlings, which maintained the highest gs, E and A levels from days 8 to 18 (Fig. 4A–C). Based on the data recorded on day 11 without irrigation, the decrease in values of gs, E and A in PT and CM seedlings represented below 20% of the values recorded for controls. In contrast, the values of these parameters in FA-5 seedlings

Fig. 4. Effect of drought-stress (DS) on leaf stomatal conductance (gs), transpiration rate (E) and net CO2 assimilation (A) in seedlings (A–C) of Cleopatra mandarin (CM), Poncirus trifoliata (PT) and Forner–Alcaide 5 (FA-5) and Valencia orange trees (D–F) grafted on these three rootstocks (V/CM, V/PT and V/FA-5). Values are means of six replicates ± SD.


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Table 3 Gas exchange parameters [(gs) leaf stomatal conductance, (E) transpiration rate and (A) net CO2 assimilation] and water-use efficiency (WUE) in seedlings of Poncirus trifoliata (PT), Cleopatra mandarin (CM) and Forner–Alcaide 5 (FA-5) and Valencia orange trees grafted on these three rootstocks (V/PT, V/CM and V/FA-5). Values are means of six replicates. Within each column, different letters indicate significant differences at P < 0.05 (LSD test). Ns, *, ** and *** indicate non-significant or significant differences at P < 0.05, 0.01 or 0.001, respectively. Seedlings and grafted trees were analysed independently.

PT CM FA-5 V/PT V/CM V/FA-5 ANOVA Seedlings Trees

Days of DS

gs (mmol m−2 s−1 ) Control Stress

E (mmol m−2 s−1 ) Control Stress

A (␮mol m−2 s−1 ) Control Stress

WUE (mmol) Control

11 11 11 20 20 20

154.00 a 26.55 c 120.20 b 84.14 x 49.81 y 65.79 xy

16.40 b 3.26 c 49.93 a 27.70 y 26.83 y 38.71 x

2.22 a 0.79 b 2.15 a 1.49 x 1.10 y 1.20 xy

0.38 b 0.05 c 1.32 a 0.31 y 0.30 y 0.50 x

10.40 a 4.23 b 9.53 a 6.82 x 4.05 y 5.62 xy

0.22 b 0.27 b 5.72 a 0.49 z 0.91 y 2.31 x

4.68 5.35 4.43 4.57 3.68 4.68

*** ***

*** *

* *

*** *

** *

*** **

ns ns

represented around 40% of that corresponding to the controls (Table 3). Gas exchange parameters of ‘Valencia’ orange scions grafted on CM, PT and FA-5 rootstocks also decreased during the drought period, although differences between them were less pronounced than in the case of seedlings. At the beginning of the experiment, gs, E and A were higher in V/PT trees than in V/CM, whereas V/FA5 presented intermediate values. All these parameters decreased during the course of the experimental period, but dropped more quickly in scions on PT than on the other rootstocks. After 12 days, scions on FA-5 maintained the highest levels of all values until the end of the experiment (Fig. 4D–F). In grafted trees, on day 20, gs values were lower in stressed V/PT, V/CM and V/FA-5 plants than in the control (irrigated) plants by 67.1, 46.1 and 41.2%, respectively; for E values by 79.2, 72.7 and 58.3% respectively and for A values by 92.8, 77.5 and 58.9%, respectively (Table 3). Near the end of the experimental period (day 11 in seedlings and day 20 in grafted trees), WUE was not affected by DS treatment in CM, FA-5, V/CM and V/FA-5. However, in stressed PT and V/PT plants, WUE decreased by over 65% with respect to control plants (Table 3). 4. Discussion It has been reported (Yin et al., 2005; Lei et al., 2006) that morphological differences among plants, may influence their resistance to DS. In this study, CM seedlings showed the highest leaf biomass, whereas FA-5 presented the lowest. Both, FA-5 and PT seedlings had similar S/R ratios, with lower values than those of CM (Table 1). On the other hand, in non-stressed plants, E values were higher in PT and FA-5 than in CM (Fig. 4B). Lower E values in CM with respect to other rootstocks have been reported previously (Moya et al., 1999; Garcia-Sanchez et al., 2007). However, during the drought period, E progressively decreased in all stressed plants. After day 5, as stress became more severe, E decreased rapidly in PT seedlings, reaching values close to those of CM, whereas FA-5 maintained the highest E levels. Regarding water-use curves (Fig. 2A) CM, which had the highest leaf mass but the lowest E, seemed to use water from the pots more quickly, and, consequently, the plants wilted in a relatively short time. PT, with a lower leaf mass but higher E values than CM, displayed slower water consumption rates. However its survival time was slightly less prolonged, when compared with CM, probably because of its deficient osmoregulation. FA-5, with the lowest leaf mass and relatively high E values, had the slowest water consumption rate, and therefore the longest survival time. Our results indicate that total water transpiration in seedlings was more dependent on leaf biomass than on E values. This may

Stress 0.57 b 5.40 a 4.33 a 1.58 z 3.03 y 4.62 x *** **

be explained by considering that the E values reported here were obtained from specific measurements, which can change during the day or according to varying external conditions and, therefore, cannot be fully representative of whole plant transpiration. However, obviously, leaf mass (or total leaf surface area) and transpiration rate are closely linked in determining water extraction velocity, as reported by Moya et al. (1999, 2003). The latter authors demonstrated that when leaves are removed, transpiration rates increase proportionally to the magnitude of the leaf removal and, therefore, whole plant transpiration does not change. An alternative explanation for rapid water uptake in CM plants is a more effective root system. Davies and Albrigo (1994) indicated that this occurs mainly due to higher root density or a deeper and more densely branched root system compared to other rootstocks. Moreover, in a field experiment, Romero et al. (2006) studied differences between rootstocks CM and Carrizo citrange (Camellia sinensis × P. trifoliata) in response to deficit irrigation strategies, they concluded that trees on CM were more efficient in soil water-use and exploited more of the available soil water resources. Nevertheless, in the conditions of our experiment, leaf biomass was probably the major driving force for water extraction and, therefore, was a crucial factor determining citrus response to DS. This is supported by certain reports indicating that tree size may influence the rate of water uptake in trees on different rootstocks (Castle and Krezdorn, 1977; Romero et al., 2006). The fact that the highest leaf mass corresponded to CM seedlings could be due to the character of its leaves, which are larger on the whole than the smaller trifoliate leaves of both PT and FA5 seedlings. The high biomass most likely determined the short survival time of CM seedlings under severe DS. Water-use curves of ‘Valencia’ orange scions grafted on the three rootstocks (V/CM, V/PT and V/FA-5) varied markedly with respect to those of seedlings. This is probably because the leaf mass values and S/R ratios were similar in grafted trees, in contrast with the pronounced differences in these parameters found among seedlings (Table 1). Thus, V/PT displayed the fastest water consumption, whereas V/CM presented the slowest, and V/FA-5 demonstrated intermediate behaviour (Fig. 2B). As expected, V/CM and V/FA-5 plants survived under severe DS for a longer time than V/PT (Fig. 1). Also, in grafted trees, water extraction from pots would appear to depend more on E values (Fig. 4, D–F) and, therefore, E was a main factor in determining plant survival under DS. The results show that water relations in drought-stressed citrus plants differed depending on rootstock. DS treatment resulted in a decline in leaf « s , although this effect was more pronounced in CM, PT and V/PT than in FA-5, V/FA-5 and V/CM, coinciding with the speed of water uptake by plants, during the first half of the experiment. During the early period of treatment, DS was apparently insufficient to alter leaf « ␲ , but later, when stress became more severe, « ␲ decreased in leaves (Fig. 3).

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In FA-5 and V/FA-5 plants, the reduction in « s caused by DS was compensated for by a reduction in leaf « ␲ during most of the experimental period (Fig. 3C and F). Turgor was maintained at values similar to, or greater than, those in control leaves, indicating that, in these plants, osmotic adjustment was sufficient to offset the reduction in leaf « s caused by DS. In drought-stressed PT and V/PT plants, « s decreased to a greater extent than « ␲ (Fig. 3B and E), and consequently turgor was reduced as water stress progressed. These plants seem to have a limited osmotic adjustment capacity given the moderate « ␲ decrease in response to severe water deficit. However, there were important differences between CM and V/CM plants with respect to the changes in the water potential components in response to DS treatment. Stressed CM seedlings experienced a rapid decline in « s , probably due to rapid water extraction from the pots (Fig. 3A and D). This decline was compensated, at least until day 8, by a reduction in « ␲ , which maintained turgor and allowed these plants to survive during the final days (8–13 days) of the experiment. By contrast, in DS-treated V/CM trees, which presented the slowest water extraction rates, leaf « s and « ␲ only dropped slightly during the experimental period. Thus, turgor stayed almost constant till day 24. The behavioural patterns described above were observed in the time-course of DS experiment, up until the last determination recorded for each type of plant (Fig. 3). After that, water availability in the substrate became practically depleted within 1 and 3 days (Wg < 4.5%), leading to a total loss of leaf turgor and irreversible wilting. It is known that OA improves water status in plants under severe drought-stress conditions. FA-5 and V/FA-5 plants osmotically adjusted to DS, as shown by «␲100 and OA values, whereas PT and V/PT did so to a far lesser extent (Table 2). Furthermore, CM either grafted or ungrafted, seemed to induce OA in leaves, only when soil dryness was extreme, at least under these experimental conditions. Leaf « ␲ decline in stressed plants is believed to be the result of solute accumulation, enabling positive leaf turgor to be maintained, which is necessary to keep stomata open and sustain gas exchange and growth (Garcia-Sanchez and Syvertsen, 2006). During water stress, the accumulation of compatible solutes in leaves can include amino acids (e.g. proline) and soluble sugars (Syvertsen and Smith, 1983; Garcia-Sanchez et al., 2007; Perez-Perez et al., 2007). The increase in osmolyte concentrations could have been due to a passive process by leaf dehydration and/or an active process caused by their synthesis (Bray, 1997; Lazcano-Ferrat and Lovatt, 1999). It has also been suggested (Garcia-Sanchez et al., 2007) that under drought conditions, citrus plants could enhance net uptake of certain inorganic ions that contribute to OA. Our results indicate that the increase in solute concentrations responsible for OA was not only a passive process resulting from the decreased water content, since «␲100 differed in leaves from wellwatered and drought-stressed plants. FA-5, V/FA-5, CM and V/CM plants maintained a high RWC during the experiment. In contrast, at the end of the experiment, PT and V/PT leaves had lower RWC values than leaves from the other plants (Table 2). Thus, to avoid high water loss by transpiration, stronger stomatal regulation of gas exchange in leaves was induced by PT rootstock than CM and FA-5. Data also showed a decrease in A that paralleled gs reduction as water stress progressed. The DS-related reductions in A were much lower in FA-5 than in either CM or PT (Fig. 4C). Grafted rootstocks behaved similarly, although differences were not so pronounced (Fig. 4F). It is generally accepted that water stress-related A reduction is induced by a hormonal intermediary, such as abscisic acid (ABA) which regulates stomatal conductance (Hartung and Davies, 1991). A previous work reported that ABA was generated by citrus roots and leaves in response to soil dryness (Gomez-Cadenas


et al., 1996). This would suggest that ABA might act as a modulator of gas exchange responses to DS, mostly through its involvement in stomatal closure, which seems to be an adaptation mechanism to prevent leaf dehydration (Davies and Zhang, 1991). Nevertheless, lowered gs was not totally responsible for A reduction, since internal non-stomatal factors may be also important in limiting A (Farquhar and Sharkey, 1982). Moreover, at the end of the experiment, when water stress was very intense, cell turgor loss could have an additional negative effect on A. In these experimental conditions, FA-5 rootstocks induced greater OA in leaves than the other rootstocks, maintaining better photosynthetic capacity by preserving leaf turgor. Considering the overall results of this study, we can conclude that, under these experimental conditions, leaf biomass and transpiration rates were the main factors related with citrus plant survival under DS, since they determined water extraction speed from the pots and, consequently, the soil water depletion period. However, it is likely that at the end of the experiment, when water stress was most severe, the higher drought tolerance induced by FA-5 rootstock, compared to the other rootstocks, was related to greater OA, which was reflected by the smaller reductions in leaf RWC and better values of turgor potential and leaf gas exchange parameters. In conclusion, considering the characteristics of the rootstocks studied, FA-5 appears to be more resistant to DS than its parents (CM and PT). Also, the method used here appears to be suitable to test DS resistance of citrus rootstocks. Acknowledgements This work was funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and FEDER funds (RTA2008-00060). References Albrigo, L.G., 1977. Rootstocks affects ‘Valencia’ orange fruit quality and water balance. Proc. Int. Soc. Citriculture 1, 62–65. Arbona, V., Iglesias, D.J., Jacas, J., Primo-Millo, E., Talon, M., 2005. Hydrogel substrate amendment alleviates drought effects on young citrus plants. Plant Soil. 270, 73–82. Barry, G.H., Castle, W.S., Davies, F.S., 2004. Rootstocks and plant water relations affect sugar accumulation of citrus fruit via osmotic adjustment. J. Am. Soc. Hortic. Sci. 129, 881–889. Bray, E.A., 1997. Plant responses to water deficit. Trends Plant Sci. 2, 48–54. Castel, J.R., Buj, A., 1990. Response of Salustiana oranges to high frequency deficit irrigation. Irrigation Sci. 11, 121–127. Castle, W.S., Krezdorn, A.H., 1975. Effect of citrus rootstocks on root distribution and leaf mineral content of Orlando tangelo trees. J. Am. Soc. Hortic. Sci. 100, 1–4. Castle, W.S., Krezdorn, A.H., 1977. Soil water use and apparent root efficiencies of Citrus trees on four rootstocks. J. Am. Soc. Hortic. Sci. 102, 403–406. Davies, F.S., Albrigo, L.G., 1994. Citrus. Cab International, Wallingford, UK, 254 pp. Davies, W.J., Zhang, J., 1991. Root signals and the regulation of growth and development of plants in drying soil. Annu. Rev. Plant Phys. 42, 55–76. Delauney, A.J., Verma, D.P.S., 1993. Proline biosynthesis and osmoregulation in plants. Plant J. 4, 215–222. Farquhar, G.D., Sharkey, T.D., 1982. Stomatal conductance and photosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 33, 317–345. Forner, J.B., Forner-Giner, M.A., Alcaide, A., 2003. Forner–Alcaide 5 and Forner–Alcaide 13: two new citrus rootstocks released in Spain. HortScience 38, 629–630. Forner-Giner, M.A., Alcaide, A., Primo-Millo, E., Forner, J.B., 2003. Performance of Navelina orange on 14 rootstocks in Northern ‘Valencia’ (Spain). Sci. Hortic.: Amsterdam 98, 223–232. Forner-Giner, M.A., Primo-Millo, E., Forner, J.B., 2009. Performance of Forner–Alcaide 5 and Forner–Alcaide 13, hybrids of Cleopatra mandarin × Poncirus trifoliata, as salinity-tolerant citrus rootstocks. J. Am. Pomolog. Soc. 63, 72–80. Garcia-Sanchez, F., Syvertsen, J.P., 2006. Salinity tolerance of Cleopatra mandarin and Carrizo citrange rootstock seedlings is affected by CO2 enrichment during growth. J. Am. Soc. Hortic. Sci. 131, 24–31. Garcia-Sanchez, F., Syvertsen, J.P., Gimeno, V., Botia, P., Perez-Perez, J.G., 2007. Responses to flooding and drought stress by two citrus rootstocks seedlings with different water-use efficiency. Physiol. Plantarum 130, 532–542.


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