Differential responses of stomatal morphology to partial root-zone drying and deficit irrigation in potato leaves under varied nitrogen rates

Scientia Horticulturae 145 (2012) 76–83

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Differential responses of stomatal morphology to partial root-zone drying and deficit irrigation in potato leaves under varied nitrogen rates Fei Yan a,b,c,d , Yanqi Sun c,e , Fengbin Song a,b,∗ , Fulai Liu c,∗∗ a

Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130012, China Graduate University of Chinese Academy of Sciences, Beijing 100049, China c University of Copenhagen, Faculty of Science, Department of Agriculture and Ecology, Højbakkegaard Allé 13, DK-2630 Taastrup, Denmark d College of Plant Science, Jilin University, Changchun 130062, China e College of Water Resources and Architectural Engineering, Northwest A&F University, Yangling, Shaanxi 712100, China b

a r t i c l e

i n f o

Article history: Received 7 June 2012 Received in revised form 26 July 2012 Accepted 26 July 2012 Keywords: ABA Carbon isotope discrimination Partial root-zone drying Nitrogen Stomatal aperture Stomatal density

a b s t r a c t The effects of partial root-zone drying (PRD) as compared with deficit irrigation (DI) on stomatal morphology of potato (Solanum tuberosum L.) under varied nitrogen (N) rates were investigated. The plants were grown in split-root pots under three N rates, viz., 70 (N1), 125 (N2), and 200 (N3) mg N kg−1 soil, respectively. For each N rate, PRD and DI plants received the same amount of water, which allowed refilling one half of the PRD pot to 100% water holding capacity. Across the three N rates, guard cell size was larger in DI than in PRD, whereas stomatal pore aperture area (SA) was similar between the two irrigation treatments. Stomatal density (SD) was affected by both N rate and irrigation treatment and was lower in PRD than in DI under N2 and N3, whereas the reverse was the case under N1. Plant leaf area increased with increasing N rate, but was unaffected by the irrigation treatment. SD positively correlated with leaf N concentration and xylem sap ABA concentration for the DI plants, but not for the PRD plants. Nonetheless, negative linear relationships of SD to the mean soil water content in the pots and the carbon isotope discrimination in the leaves were found across all treatments. Regression analyses showed that it was SA rather than SD positively correlated with the stomatal conductance and the transpiration rate per unit leaf area in the DI; however such relationships were not evident in the PRD. In conclusion, compared to DI, PRD led to a more conservative control in plant water use via modulating stomatal morphology; the smaller stomata combined with a lower SD in the plants had efficiently reduced plant water use under high N rate, which maintained a better soil water moisture condition in the PRD pots. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Worldwide shortage of freshwater resource has stimulated research into development of water-saving agricultural practices aiming at producing ‘more crop per drop’ (Davies et al., 2011). In the last two decades, biological water-saving irrigation strategies such as deficit irrigation (DI) and partial root-zone drying (PRD) irrigation that exploit the plant drought adaptation mechanisms have been developed and have shown great potential to enhance crop water use efficiency (WUE) (Bacon, 2004; Davies and Hartung, 2004). The physiological basis for improving WUE under both PRD and DI involves utilizing the ABA-based root-to-shoot signaling system decreasing stomatal conductance (gs ) thereby curtailing

∗ Corresponding author at: Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130012, China. ∗∗ Corresponding author. Tel.: +45 3533 3392; fax: +45 3533 3478. E-mail addresses: [email protected] (F. Song), fl@life.ku.dk (F. Liu). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.07.026

transpiration rate during moderate soil drying (Stoll et al., 2000; Liu et al., 2006a,b; Dodd, 2009). Accumulated evidence indicates that, by alternately drying and wetting part of the root system, PRD induces much stronger ABA signal than does DI under a similar soil water deficit, which may result in a fine-tune stomatal control over plant water use and thus an increase in WUE (Dodd et al., 2008; Liu et al., 2009). Stomatal conductance (gs ) is determined predominantly by the aperture of the stomatal pores (SA) as well as the density of stomata (SD) on the leaf surface. It can be computed by physically based formulas that take both SA and SD into account (Brown and Escombe, 1900; Parlange and Waggoner, 1970; Franks and Farquhar, 2001). Therefore, manipulation of both SA and SD may modify gs thereby bringing about an increase of WUE (Wang et al., 2007). To date, research into the mechanisms by which water-saving irrigation strategies such as PRD and DI improve plant WUE has mainly been focused on the significance of the xylem-borne ABA signaling in regulating SA and thus gs in a short-time scale (Stoll et al., 2000). However, little attention has been paid to the plasticity in SD in

F. Yan et al. / Scientia Horticulturae 145 (2012) 76–83

2. Materials and methods 2.1. Plant material and growth condition The experiment was conducted from March to May, 2011 in a climate controlled greenhouse at the experimental farm of the Faculty of Life Sciences, University of Copenhagen, Taastrup, Denmark. Potato tubers (S. tuberosum L. cv. Folva) were planted in split-root pots (25.2-cm diameter and 40-cm tall) on 11th March. The pots were evenly separated into two compartments with plastic sheets such that water exchange between the two compartments was prevented. A section of the plastic sheet (width (5 cm) × height (10 cm)) was removed to allow a seed tuber to be planted in the top-center of the pots (see Fig. 1 – the split-pot system in Wang et al. (2010b)). The pots were filled with 20.2 kg of naturally dried soil with a bulk density of 1.14 g cm−3 . The soil was classified as sandy loam, having a pH of 6.7, total C 12.9 g kg−1 , total N 1.4 g kg−1 , NH4 + -N 0.7 mg kg−1 , NO3 − -N 19.1 mg kg−1 . The soil was sieved passing through a 2 mm mesh and had a volumetric soil water content (vol.%) of 30.0% and 5.0% at water holding capacity and permanent wilting point, respectively. Before planting, the seed potatoes were exposed to 12–14 ◦ C with constant dim overhead light (a PAR of 100–150 ␮mol s−2 s−1 ) for sprouting. During planting only one sprout was retained. The roots from this sprout were evenly distributed between the two separated compartments. The average soil water contents in the pots were monitored by a time domain reflectometer (TDR, TRASE, Soil Moisture Equipment Corp., CA, USA) with probes (35 cm in length) installed in the middle of each soil compartment. The climate conditions in the greenhouse were set at: 16/14 ± 2 ◦ C day/night air temperature, 15 h photoperiod and >500 ␮mol m−2 s−1 photosynthetic photon flux density (PPFD) supplied by sunlight plus metal-halide lamps. 2.2. N and irrigation treatments Three N rates, i.e., 70, 125, and 200 mg N kg−1 soil, denoted as N1, N2 and N3, respectively, were included in the experiment. The N fertilizer supplied as NH4 NO3 was mixed thoroughly with the soil before filling the pots. In addition, P and K were also applied

30 PN1-L

PN1-R

DN1

(a)

PN2-L

PN2-R

DN2

(b)

25

20

15

10

Soil water content (vol.%)

response to the irrigation treatments and its significance in controlling plant water use in a long-term perspective (Shimada et al., 2011). Even though both SA and SD can be modified by environmental cues, it is widely accepted that SA is more dynamic while SD is more static in response to changing environments (Franks and Farquhar, 2007). Thus, a temporal (minutes to hours) change of gs for a leaf under changing environments will be due mainly to a change of SA; whereas plants exposed to varied environments for a longer period (e.g., weeks to months), alterations in both SA and SD may account for the change of gs . Early evidence has shown that crop grown under deficit irrigation may modify both SA and SD to optimize gs thereby improving WUE (Zhang et al., 2006). However, until now the underlying mechanisms of modifying SD in response to soil water deficits are not fully understood. In the present study, potato plants (Solanum tuberosum L.) were grown in split-root pots under three N rates and were subjected to PRD and DI during tuber initiation and tuber bulking stages. Stomatal morphology in the abaxial leaf surface, transpiration rate per unit leaf area, gs , N concentration and 13 C in the leaf biomass, ABA concentration in the xylem sap were determined. The objective was to investigate the relative significance of changes in SA and SD in controlling gs under PRD and DI regimes with varied N rates. The possible mechanisms modifying SD across different water and N environments are discussed.

77

25

20

15

10 PN3-L

PN3-R

(c)

DN3

25

20

15

10 0

5

10

15

20

25

30

35

DAT Fig. 1. Daily average soil water content (vol.%) in the pots of potato plants under PRD and DI irrigation in combination with three N rates (N1, a; N2, b; N3, c). PN1, PN2, PN3, DN1, DN2, and DN3 denote the combinations of PRD and DI with the three N-fertilization rates, respectively; PRD-L and PRD-R indicate the left and right sides of the PRD pots. Error bars indicates the standard error of the means (S.E.) (n = 4).

as KH2 PO4 (380 mg kg−1 soil) and K2 SO4 (130 mg kg−1 soil) into the soil to meet the nutrients requirement for plant growth. From tuber initiation to tuber bulking stages (30–64 days after planting), the plants were exposed to PRD and DI treatments. In PRD, one soil compartment was watered daily at 18:00 h to an average soil water content of 28% while the other was allowed to dry until the average soil water content in the pots reached ca. 12–15%, then the irrigation was shifted to the dry compartment; in DI, the same amount of water used for the PRD plants was irrigated evenly into the whole pot. Here, we did not include a fully irrigated control since the main purpose of the current study was to exploit the possible mechanisms by which the PRD treatment over-performs DI in terms of improving WUE as reported in earlier studies (Liu et al., 2006a,b). The experimental set-up was a complete factorial design comprising six treatments and each treatment had four replicates. The irrigation water was tap water with negligible concentration of nutrients. The irrigation treatments lasted for 5 weeks, during which each soil compartment of the PRD pots had experienced 3 drying/wetting cycles. The changes of daily average soil water content in the soil compartments/pots during the treatment period are shown in Fig. 1. Since different N rates significantly affected

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plant size and the water consumption; thus, during the treatment period plant water uses were 22.3, 24.9, and 26.9 l for N1, N2, and N3, respectively.

Mean soil water content in PRD (vol.%)

26

2.3. Stomatal conductance (gs ) and diurnal transpiration rate measurements On 9th May, 2011 (a sunny clear day, 29 days after onset of treatments), gs of the upper canopy fully expanded leaves were measured by a leaf porometer (Decagon Devices, Inc., Pullman, WA, USA) at 11:00 am. The measurements were done on the adaxial leaf surface with three leaves per plant. On the same day, diurnal course of plant water use was determined by weighing the pots at every 2 h interval from 7:00 am to 17:00 pm. Transpiration rate per unit leaf area was then calculated by dividing the plant water use with plant leaf area (measured on 14th May, here it was assumed that the leaf expansion growth had been ceased at that stage according to Shahnazari et al. (2007)). 2.4. Stomatal morphology observation On the same day immediately after gs measurement, epidermal impressions were taken from three leaves in each plant. Fingernail polish imprints were obtained halfway from the leaf tip to the base from the abaxial surface of each leaf, and using clear cellophane tape to transfer the ‘impression’ to a microscope slide. The imprints were observed under a LEITZ DMRD microscope camera system (Leica Microscope and System GmbH, D 35530, Wetzlar, Germany) equipped with a digital camera, and the images were presented using image-editing software (Leica Microsystems, version 2.5.0, CMS GmbH (Switzerland) Limited) on a computer screen. Stomatal density (SD) and stomatal size parameters including guard cell length (Ls ), guard cell pair width (Ws ), stomatal pore aperture length (La ), and stomatal pore aperture width (Wa ) were measured with the images using UTHSCSA ImageTool software (UTHSCSA ImageTool for Windows version 3.00). Stomatal aperture (SA) was then calculated according to Doheny-Adams et al. (2012) using the following equation: SA =

 × Wa × La 4

(1)

where Wa is the aperture width and La is the aperture length. 2.5. Xylem sap collection, xylem sap ABA concentration ([ABA]xylem ), and leaf area measurements The plants were harvested on 14th May. First, the plants were detopped at ca. 5 cm from the stem base at 8:30 am, immediately the cut surface was cleaned with pure water and dried with blotting paper, and the pots with the stem stump were covered with black plastic bags. After 15–20 min, the bags were removed and the xylem bleeding sap was collected using a pipette from the cutting surface into an Eppendorf-vial wrapped with aluminum foil. All sap samples were frozen immediately in liquid N after sampling and stored at −80 ◦ C until analysis. ABA concentration in the xylem sap was determined by ELISA using the protocol of Asch (2000). Plant leaf area was measured by a leaf area meter (Li-3100, Li-Cor Inc., Lincoln, NB, USA). 2.6. Leaf N concentration and carbon isotope discrimination (13 C) analyses Leaves used for N concentration and carbon isotope discrimination (13 C) determination were dried in an oven at 70 ◦ C for 72 h. Dry samples of leaves were ground to a fine powder for N and 13 C analysis using the Dumas dry combustion method in a system

N1

N2

N3

24 22 20 18 16

N3

14

N1

N2

12 10 10

12

14

16

18

20

22

24

26

Mean soil water content in DI (%, vol.) Fig. 2. The relationship between the daily mean soil water contents (vol.%) of the PRD pots and that of the DI pots during the treatment period. The dash line indicates the 1:1 relationship, and the three ovals denote the data points for N1, N2, and N3, respectively.

consisting of an ANCA-SL Elemental Analyzer coupled to a 20-20 Tracer Mass Spectrometer (Europa Scientific Ltd., Creve, UK). 13 C composition (␦13 C) of leaf dry biomass was calculated as:



13

␦ C = 1000

Rsample − Rstandard



Rstandard

(2)

Rsample and Rstandard are the 13 C:12 C ratios of the leaf sample and Pee Dee Belemnite (PDB) calcium carbonate. 13 C of the leaf samples was calculated using Eq. (3): 13 C =

ıa − ıp 1 + ıp

(3)

where the subscripts a and p refer to air and the plant, respectively (Farquhar et al., 1989). The ıa value for the ambient atmosphere was taken as −7.7‰. 2.7. Statistical analysis Two-way analysis of variance (ANOVA) of the statistical programme SPSS 13.0 for Windows (SPSS Inc., Chicago, IL) was conducted to determine the effects of the irrigation regimes and N rates on the leaf 13 C, leaf N concentration, [ABA]xylem , and the stomatal morphological parameters observed on the leaf imprint images. Linear regressions (SPSS) were used to evaluate the relationships between the observed variables across different irrigations and N rates. 3. Results 3.1. Relationship of the mean soil water contents between the PRD and the DI pots Fig. 2 shows the relationship of the mean soil water contents between the PRD and the DI pots under the three N rates. It was found that, as indicated by the 1:1 line, the mean soil water contents in the pots were lower for PRD than for DI under N1, particularly when the soil water content >20% in the DI pots. Under N2, the mean soil water contents in the pots were similar for the two irrigation treatments as the data points were mostly followed the 1:1 line. However, under N3 on most of the occasions the mean soil water contents in the pots were greater for PRD (15–20%) than for DI (12–16%).

F. Yan et al. / Scientia Horticulturae 145 (2012) 76–83

(a)

PRD

DI

150

N: P = 0.735 I: P = 0.013 N × I: P = 0.075

32

30

(a)

PRD

DI

120 SA ( μm2)

L s (μm)

34

79

N: P = 0.001 I: P = 0.373 N × I: P = 0.457

90 60 30

28

0 N: P = 0.376 I: P = 0.002 N × I: P = 0.900

(b)

SD (mm -2)

W s (μm)

18 16 14

(b)

N: P < 0.001 I: P = 0.036 N × I: P = 0.004

(c)

N: P = 0.137 I: P = 0.137 N × I: P = 0.002

260 200 140

12 80 N: P = 0.009 I: P = 0.289 N × I: P = 0.893

(c)

L a (μm)

23 21 19

1.0 g s (mol m -2 s -1)

10

0.8 0.6 0.4

17 0.2

15

N: P = 0.002 I: P = 0.151 N × I: P = 0.068

(d)

W a (μm)

8 6 4 2 0 N1

N2

N3

N fertilization Fig. 3. Guard cell length (Ls ) (a), guard cell pair width (Ws ) (b), stomatal pore aperture length (La ) (c) and stomatal pore aperture width (Wa ) (d) in the abaxial leaf surface of tomatal plants exposed to different irrigation and N treatments. The data were determined based on 12 leaf impression images (each from one individual leaf and three leaves per plant of four plants per treatment), and of which 10 stomata for each image were measured for determining the average La , Wa , Ls , and Ws . Statistical comparisons (two-way ANOVA) between the irrigation and N treatments are presented for each variable. Error bars indicate S.E. (n = 120).

3.2. Stomatal morphology and stomatal conductance (gs ) The effects of the irrigation and N rate on stomatal morphological characteristics are shown in Figs. 3 and 4. When analyzed across three N rates, guard cell length (Ls ) and guard cell pair width (Ws ) were significantly larger for DI than for PRDI; whereas stomatal pore aperture length (La ), stomatal pore aperture width (Wa ), and the stomatal pore aperture (SA) were similar for PRD and DI plants.

N1

N2 N fertilization

N3

Fig. 4. Stomatal pore aperture (SA) (a), stomatal density (SD) (b) and stomatal conductance (gs ) (c) in the abaxial leaf surface of tomatal plants exposed to different irrigation and N treatments. The data were determined based on 12 leaf impression images (each from one individual leaf and three leaves per plant of four plants per treatment), and of which 10 stomata for each image were measured. SA was calculated by using Eq. (1); SD was calculated by counting stomatal number of the 12 images and each image with an area of 0.1125 mm−2 . Statistical comparisons (two-way ANOVA) between the irrigation and N treatments are presented for each variable. Error bars indicate S.E.

N rate had no effect on Ls and Ws but significantly influenced La and Wa . Stomatal density (SD) was significantly affected by both irrigation and N treatments. Across the two irrigation treatments, there was a tendency that SD increased with increasing N rate; and a significant interactive effect between the irrigation and N rate on SD was observed being that under N1, PRD plants had significantly greater SD than DI plants; whereas under N2 and N3 the opposite was true. The N rate and the irrigation treatment also interactively affected gs being that it was significantly lower in the PRD than in DI under N1, and was similar under N2 and N3. 3.3. Leaf area, leaf carbon isotope discrimination (13 C), leaf N concentration, and xylem sap ABA concentration ([ABA]xylem ) Leaf area was increased with increasing N rate. When analyzed crossing the three N rates, leaf area was identical between the two irrigation treatments (Fig. 5a). Under DI, leaf 13 C decreased with increasing N rate, while this was not the case under PRD (Fig. 5b). There was a significant interactive effect between the irrigation

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[ABA] xylem (pmol ml -1) Leaf N concentration (mg g-1)

13 Δ C of leaf biomass (‰)

Leaf area (m2 plant-1)

1.5

(a)

PRD

DI

1.2

N: P < 0.001 I: P = 0.261 N × I: P = 0.131

0.9

3.4. Diurnal course of plant transpiration rate

0.6 0.3 0

N: P = 0.003 I: P = 0.418 N × I: P = 0.001

(b)

20 19

N: P < 0.001 I: P = 0.005 N × I: P < 0.001

(c)

60

In the DI plants, both the transpiration rate per unit leaf area and gs were positively correlated with SA but negatively correlated with SD; For PRD plants, however, no clear relationships between those variables were observed (Fig. 7). 3.6. Relationships of stomatal density (SD) to leaf N concentration, [ABA]xylem , soil water content, and leaf 13 C

40 20 0

Fig. 6 shows the effects of the irrigation treatment and N rate on the diurnal course of transpiration rate of potato plants. Across the three N rates, PRD plants transpired less water per unit leaf area from 9 to 13 h but transpired much more water per unit leaf area from 13 to 17 h than DI plants. When comparing the day-time (i.e., 7 am to 17 pm) transpiration rate per unit leaf area between the two irrigation treatments, it was found that PRD plants transpired significantly less water than did DI across the three N rates. 3.5. Relationships of stomatal aperture (SA) and stomatal density (SD) to transpiration rate per unit leaf area and stomatal conductance (gs )

18 17

treatment alone had not significantly affected on [ABA]xylem , the two factors interactively influenced the [ABA]xylem being that the PRD plants had significantly higher [ABA]xylem than DI under N1, while the opposite was true under N3; under N2 [ABA]xylem was similar for the two irrigation treatments.

N: P = 0.402 I: P = 0.685 N × I: P = 0.024

(d)

150 100 50 0 N1

N2

N3

N fertilization Fig. 5. Plant leaf area (a), carbon isotope discrimination (13 C) in leaf biomass (b), leaf nitrogen (N) concentration (c), and xylem sap ABA concentration ([ABA]xylem ) (d) of potato plants exposed to different irrigation and N treatments. Error bars indicate the standard error of the means (S.E.) (n = 3–4). Statistical comparisons (two-way ANOVA) between the irrigation and N treatments and their interactions are presented for each variable.

treatment and N rate on the leaf 13 C being that PRD plants had significantly lower 13 C than did DI under N1 and N2, while under N3 the opposite was true. As expected, leaf N concentration increased with increasing N rate; however the increase was more pronounced under PRD than under DI (Fig. 5c). Compared to DI plants, PRD plants had significantly higher leaf N concentration under N3; while under N1 and N2 leaf N concentration was similar for the two irrigation treatments. Fig. 5d shows the effects of the irrigation treatment and N rate on [ABA]xylem of potato plants. Even though irrigation and N

When analyzed crossing the three N rates, it was found that only for DI plants SD was positively correlated with leaf N concentration and [ABA]xylem (Fig. 8a and b) Nonetheless, negatively linear relationships were found between SD and the mean soil water contents in the pots and the leaf 13 C across all the treatments (Fig. 8c and d). 4. Discussion Stomata play a pivotal role in balancing the rates of gas exchange (i.e., the uptake of CO2 and the loss of water) on the leaf surfaces. To do so, plants have evolved a suite of mechanisms to regulate stomatal aperture (SA) and stomatal density (SD) to suit the prevailing environmental conditions (Casson and Hetherington, 2010; Shimada et al., 2011). The mechanisms that regulate SA under soil water deficits have been well documented (Kim et al., 2010); whereas those for modulating SD remain largely elusive (Shimada et al., 2011), particularly for plants grown under deficit irrigation treatments such as PRD and DI. Therefore, the aim of this study was to investigate the effect of PRD and DI in combination with three N rates on SA and SD in potato leaves and to analyze their significance in controlling stomatal conductance (gs ) and plant transpiration rate. The results showed that, the responses of stomatal morphology to the irrigation and N treatment were complex due to significant interactions between the two factors on most of the variables investigated (see the statistics inserted in the figures). Early studies have shown that increase of SD is often accompanied by a decrease in stomatal size under water deficit conditions (Culter et al., 1977; Quarrie and Jones, 1977; Spence et al., 1986). In contrast, our results show that in relation to PRD, a greater SD did not associate with smaller stomata for the DI plants under N2 and N3 treatments (Figs. 3 and 4). It has also been suggested that small stomata respond to environmental changes more rapidly, allowing the leaf to attain high gs rapidly under favorable conditions, but then to rapidly reduce gs when evaporative conditions are unfavorable (Hetherington and Woodward, 2003). If this was true, one

F. Yan et al. / Scientia Horticulturae 145 (2012) 76–83

81

Fig. 6. Diurnal course of transpiration rates per unit leaf area of potato plants grown under different irrigation and N treatments on 9th May, 2011. Error bars indicate S.E. (n = 4). Statistical comparisons (two-way ANOVA) between the irrigation and N treatments on the day-time transpiration rates per unit leaf area are presented.

would have expected that the small stomata of the PRD plants would have been fully opening in the morning under a favorable soil water status (the pots were irrigated daily in the late afternoon) and would have transpired much more water per unit leaf area than the DI plants which had bigger stomata; while when the soil water become less available in the afternoon before the irrigation, the PRD plants would have transpired less water than did DI due to stomatal closure. This was obviously not the case in the present study as the PRD plants actually transpired much less water per unit leaf area in the morning but much more water per unit leaf area in the afternoon (under N1 and N2) in relation to the DI plants (Fig. 6). Even though the pattern in the diurnal course of transpiration rates differed between the two irrigation treatments, at each N rate the PRD plants transpired significantly less water per unit leaf area than did DI during the day time indicating that PRD plants largely avoided severe soil water deficits particularly under high N conditions (Fig. 2). A similar phenomenon has been reported

Transpiration rate (l m-2(2h)-1)

0.5 PRD

recently by Egea et al. (2011) who showed that PRD plants had a more rapid recovery of water status in the afternoon than DI plants and which was associated with increased WUE. Regression analyses of gs and transpiration rate per unit leaf area to SA and SD indicated only for the DI plants that there were significant correlations between the variables. Such kind of relationships indicate that it was SA rather than SD had controlled gs and thus transpiration rate in the DI leaves (Fig. 6); for PRD plants, however, no clear correlations between the variables were found. The negative correlations of the transpiration rate per unit leaf area and gs to SD are not expected as earlier studies have often assumed that greater SD would associate with higher gs and thus transpiration rate per unit leaf area (e.g., Franks and Farquhar, 2001). Nonetheless, our results are in good agreement with the findings by Green et al. (1987) who reported that a negative relationship was found between SD and leaf transpiration rate across 12 cool-season C3turfgrasses.

(a)

DI

(b)

0.4 0.3 0.2 0.1

r DI 2 = 0.60 (P = 0.003)

0.0

r DI 2 = 0.64 (P = 0.002)

(c)

(d)

g s (mol m -2 s -1)

1.0 0.8 0.6 0.4 0.2

r DI 2 = 0.60 (P = 0.003)

r DI 2 = 0.60 (P = 0.003)

0.0 50

70

90 110 SA ( μm2)

130

50

100

150 200 SD (mm -2)

250

300

Fig. 7. Transpiration rate per unit leaf area and gs of potato leaves expressed as a function of stomatal aperture (SA) (a and c) and to tomatal density (SD) (b and d) under different irrigation and N treatments. The data were measured on 9th May, 2011. The regression lines were made on the data of DI plants only.

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F. Yan et al. / Scientia Horticulturae 145 (2012) 76–83

280 PRD

(b)

(a)

DI

SD (mm -2)

210

140

70 r DI 2 = 0.70 (P < 0.001)

r DI 2 = 0.75 (P < 0.001)

0 20

0

30 40 50 Leaf N concentration (mg g-1)

280

40

80

120

160

-1

[ABA] xylem (pmol ml ) (d)

(c)

SD (mm -2)

210

140

70 r 2 = 0.61 (P < 0.001 )

r 2 = 0.39 (P = 0.001)

0 10

13 16 19 22 Soil water content (vol.%)

17

18 19 20 13 Δ C of leaf biomass (‰)

21

Fig. 8. Stomatal density (SD) expressed as a function of leaf N concentration (a), xylem sap ABA concentration ([ABA]xylem ) (b), average soil water content in the pots (c), carbon isotope discrimination (13 C) in leaf biomass(d) of potato plants grown under different irrigation and N treatments. The dash regression lines in (a) and (b) were made for DI plants only, while the solid regression lines in c and d were made on the pooled data for PRD and DI plants.

In an eco-physiological context, earlier studies have shown that several environmental cues such as CO2 concentration, light intensity, and precipitation all could modulate SD. A long-distance signal generated from the mature leaves that sense the environmental cues has been suggested to be involved in regulating SD in the newly expanding leaves (Lake et al., 2001; Sekiya and Yano, 2008; Lake and Woodward, 2008). To explore the nature of the signaling system, correlations between SD and different physiological variables including gs (Miyazawa et al., 2006), leaf 13 C (Qiang et al., 2003; Sekiya and Yano, 2008), leaf N concentration (Franks et al., 2009), leaf transpiration rate, and leaf ABA concentration (Lake and Woodward, 2008) of plants grown under varied environments have been developed. Among these studies the most interesting finding is that, across different water, CO2 and phosphorus treatments, SD of cowpea (Vigna sinensis) is negatively correlated with leaf 13 C (Sekiya and Yano, 2008). In good agreement with this, here a similar correlation between SD and 13 C was found in potato plants exposed to different irrigation and N treatments (Fig. 8d). As 13 C is often used as good surrogate of long-term integrated WUE (Farquhar et al., 1989) such a relationship between SD and 13 C may indicate that increase of SD is associated with an improvement of WUE. However, it is noteworthy that in the present study the relationship between SD and 13 C was mainly resulting from the plants grown under N1 and N3 but not for those under N2, where greater SD did not correspond to lower 13 C

(Figs. 3b and 4a). In addition, here we also observed that, across all the treatments examined, there was a significant negative linear relationship between SD and the mean soil water contents in the pots (Fig. 8c) indicating that soil water deficits caused increase of SD in potato leaves. Such effect of soil water deficits on SD has been reported in a number of earlier studies across different plant species (Gindel, 1969a,b; Yang et al., 2007; Fraser et al., 2009). Moreover, we noticed that SD of the DI plants was positively related to leaf N concentration and [ABA]xylem ; whereas in PRD plants such relationships were not evident (Fig. 8a and b). A positive linear relationship between SD and leaf N concentration has previously been reported by Franks et al. (2009) in Eucalyptus globulus grown under both dry and wet environments; while the correlation between SD and [ABA]xylem is a novel finding and has never been reported previously. Nevertheless, Franks and Farquhar (2001) showed that exogenous ABA application could increase SD in Tradescantia virginiana leaves, and Lake and Woodward (2008) reported that leaf ABA concentration of Arabidopsis thaliana Col-0 accession positively correlated with SD; both findings are in good agreement with our results for the DI plants, indicating that xylemborne ABA or leaf ABA concentration may play a role in regulating SD on newly expanding leaves. Here, the differential responses of SD to leaf N concentration and to [ABA]xylem for the PRD as compared with the DI plants might indicate that the more dynamic soil water status under the PRD treatment had triggered differentadaptive

F. Yan et al. / Scientia Horticulturae 145 (2012) 76–83

mechanisms in the potato plants in relation to the DI treatment which caused more static soil water status (Dodd, 2007; Liu et al., 2009). For instance, greater [ABA]xylem in PRD than in DI plants under similar soil water deficits had been reported previously in tomato (Lycopersicon esculentum) (Dodd, 2007; Wang et al., 2010a). However, the mechanisms underlying the differential responses of stomatal morphology to the two irrigation regimes under varied N rates remain larger obscure and which merit further investigations. 5. Conclusion In conclusion, our results showed that, in comparison to the DI treatment, PRD had caused a more conservative control over plant water use via modulating stomatal size, SA, and SD on the leaf surface. The small stomata combined with low SD in the PRD plants could have efficiently curtailed plant transpiration rates resulting in a more favorable soil moisture status especially under high N rates. In addition, the significant linear relationship between SD and soil water content across all the treatments indicates that soil water deficits may lead to an increase in SD on potato leaves. Acknowledgements Fei Yan thanks the Danish Government Scholarship for supporting his study stay at the Faculty of Life Sciences, University of Copenhagen, Denmark. Financial support from ViVa – Water Research Initiative at Faculty of Life Sciences, University of Copenhagen is also gratefully acknowledged. References Asch, F., 2000. Determination of abscisic acid by indirect enzyme linked immuno sorbent assay (ELISA). Technical Report. Laboratory for Agrohydrology and Bioclimatology, Department of Agricultural Sciences, The Royal Veterinary and Agricultural University, Taastrup, Denmark. Bacon, M.A., 2004. Water use efficiency in plant biology. In: Bacon, M.A. (Ed.), Water Use Efficiency in Plant Biology. Blackwell Publishing, Oxford, pp. 1–26. Brown, H.T., Escombe, F., 1900. Static diffusion of gases and liquids in relation to the assimilation of carbon and translocation in plants. Proc. R. Soc. Lond. 67, 124–128. Casson, S., Hetherington, A.M., 2010. Environmental regulation of stomatal development. Curr. Opin. Plant Biol. 13, 90–95. Culter, J.M., Rains, D.W., Loomis, R.S., 1977. The importance of cell size in the water relations of plants. Physiol. Plant. 40, 255–260. Davies, W.J., Hartung, W., 2004. Has extrapolation from biochemistry to crop functioning worked to sustain plant production under water scarcity? In: Proceeding of the Fourth International Crop Science Congress, Brisbane, Australia, September 26–October 1, 2004. Davies, W.J., Zhang, J., Yang, J., Dodd, I.C., 2011. Novel crop science to improve yield and resource use efficiency in water-limited agriculture. J. Agric. Sci. 149, 123–131. Dodd, I.C., Egea, G., Davies, W.J., 2008. ABA signalling when soil moisture is heterogeneous: decreased photoperiod sap flow from drying roots limits ABA export to the shoots. Plant Cell Environ. 31, 1263–1274. Dodd, I.C., 2007. Soil moisture heterogeneity during deficit irrigation alters root-toshoot signalling of abscisic acid. Funct. Plant Biol. 34, 439–448. Dodd, I.C., 2009. Rhizposphere manipulations to maximize ‘crop per drop’ during deficit irrigation. J. Exp. Bot. 60, 2454–2459. Doheny-Adams, T., Hunt, L., Franks, P.J., Beerling, D.J., Gray, J.E., 2012. Genetic manipulation of stomatal density influences stomatal size, plant growth and tolerance to restricted water supply across a growth carbon dioxide gradient. Philos. Trans. R. Soc. B: Biol. Sci. 367, 547–555. Egea, G., Dodd, I.C., González-Real, M.M., Domingo, R., Baille, A., 2011. Partial rootzone drying improves almond tree leaf-level water use efficiency and afternoon water status compared with regulated deficit irrigation. Funct. Plant Biol. 38, 372–385. Farquhar, G.D., Ehleringer, J.R., Hubick, K.T., 1989. Carbon isotope discrimination and photosynthesis. Ann. Rev. Plant Physiol. Plant Mol. Biol. 40, 503–537.

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