Ecophysiological responses of three evergreen woody Mediterranean species to water stress

Acta Oecologica 19 (4) (1998) 377-387 / 0 Elsevier, Paris

Ecophysiological responses of three evergreen woody Mediterranean species to water stress Mireia Abril ‘*, Ralph Hanano 2 r Dqbartament d’Ecalagia, Facultai de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain. ‘Julius von Sachs Institutfiir Biowissenschafien, L. Botanik I, Universittit Wiirzburg 97082 Wiirzburg, Germany. * Corresponding author wax: +34-3 411 14 38; e-mail: [email protected]) Received July 28, 1997; accepted April 15, 1998

Abstract - The ecophysiological response to drought in three different evergreen Mediterranean species were compared. For a better interpretation of the mechanisms regulating physiological processes, the choice of species was based on evident differences in morphological and structural features (leaf size, leaf specific weight, water-conducting system). Seedlings of Ceanothus thyrsiforus, Quercus agrifolia and Bums microphylla grown in pots were subjected to natural stressing conditions during late spring in Southern California. Gas exchange, xylem water potential and abscisic acid concentration in xylem sap were measured in control (irrigated) and water-stressed plants, from predawn to sunset. Environmental, hydraulic and hormonal effects on water control and limitations to photosynthesis were analyzed. Q. agrifolia had the highest maximums of net photosynthesis, stomata1 conductance and transpiration, which were significantly different from C. thyrsijZoms and B. microphylla. B. microphylla had the lowest values. Stressed individuals of C. thyrsiflorus and B. microphylla reached absolute minimum water potentials during the day and at predawn. Q. agrifolia plants had a water conservative behaviour and did not show these low values. Control plants from Q. agrifolia had the lowest values of hydraulic resistance with high maximum stomata1 conductance, while B. microphylla control plants had the lowest maximum stomatal conductance due to higher hydraulic resistance. Changes in plant hydraulic resistance during soil drying were found, which differed among the species. In general, water-use efficiency was reduced during the day by water stress but increased as seasonal drought proceeded. On a longterm basis, Q. agrifolia was the most efficient species in water use. The results support the hypothesis that information on abscisic acid concentration in xylem sap may be one of the most important physiological keys when modelling stomata1 conductance and canopy gas exchange over seasons as drought develops on Mediterranean vegetation, Nevertheless, we believe it is necessary to integrate all known factors that control stomatal behaviour in order to construct general models of the vegetation response to environmental changes. 0 Elsevier, Paris

Bums microphylla I Ceanothus thyrsijlorus I Quercus agrifolia I abscisic acid I stomata1 conductance I photosynthesis I sclerophyllous I water relations / water-use efficiency ABA, abscisic acid / ABAxyl, xylem sap ABA concentration I Ci, internal CO, I G, stomata1 conductance / Gmax, maximum stomata1 conductance I HR, hydraulic resistance / LA, leaf size / LSW, leaf specific weight / NP, net photosynthesis / NPmax, maximum net photosynthesis /PAR, incident photosynthetically active radiation / RH, relative humidity / Ta, chamber air temperature / Tleaf, leaf temperature / TR, transpiration I VPD, air vapour pressure deficit / WP, xylem water potential I WPmin, minimum xylem water potential during the day / WPpd, predawn xylem water potential I WUE, water-use efficiency

1. INTRODUCTION Long periods of drought during summer along with high temperatures impose severe stress, which limits plant growth in the Mediterranean climate areas. Mediterranean species- mainly evergreensclerophyllous plants - have, among other adaptive mechanisms, strong control over gas exchange. Survival during dry periods usually implies that stomata maximize the rate of carbon gain while minimizing water loss. Thus, in the long term, efficient stomata1regulation allows the

plant to optimize growth despite environmental variability. Differences in the ability to regulate stomata1 conductancemay determine individual speciespersistence on a changing environment. There are several hypotheses to explain the role of the stomata in regulating gas exchange for different types of vegetation. However, there has been much discussion about the role and relative importance of the factors involved in regulation of stomata1conductance in speciesthat grow on periodically drying soils [19, 25, 29, 401. Such regulation occurs as the inte-

378 grated effect of simultaneous changes in environmental factors, above-ground (PAR, CO, concentration, RH, Ta) and below-ground (mainly soil water availability) [28, 38, 491. Endogenous effects on plant water status and leaf turgor, growth hormones, water flux and others have recently been studied as important elements determining stomata1 control of gas exchange under stress [ 12,4 I]. Modelling of Mediterranean species has to describe how these factors interact to regulate stomata1 behaviour as environmental stress develops. Recent papers convey the importance of integrating many of these factors to better understand stomata1control and provide a basis for further models [23, 421. Following the approach of Ball et al. [6], Tenhunen et al. [48] and Sala and Tenhunen [36] showed that leaf gas exchange for the Mediterranean oak species Quercus coccifera and Quercus ilex could be described over annual cycles assuming a fixed mesophyll photosynthetic capacity and time-dependent changes in the relationship between NP versus G, expressed as changes in the slope factor Gf. Gf was postulated to represent the integrated expression of various control mechanisms such as storage and release of ABA from the leaf mesophyll, apoplast pH and ionic composition of the xylem sap. Thus, Gf is viewed as a lumped endogenous factor that changes in responseto the water stressundergone by the plant and summarizes the sensitivity to stress by reflecting the degree to which stomata may open for a given rate of carbon fixation. However, evidence from experiments in laboratory [54] and field [44] led us to believe that the relationship between G and ABAxyl could be used in modelling [42]. The aim of this study is to identify the main mechanisms of water control and photosynthesis limitations in Mediterranean species under drought by analyzing how gas exchange and xylem water potential are related to xylem sap abscisic acid concentration. A key question addressed is whether the synthetic variable Gf used to simulate the complex influence of different factors over stomata1control as drought develops can be described as a function of the hormone (ABAxyl). By studying three different species, we attempted to relate the physiological behaviour to the inherent structural and morphological plant characteristics. 2. MATERIALS AND METHODS 2.1. Study area The study was carried out in spring 1991 at the Santa Margarita Ecological Reserve (San Diego State University), in California at the southern extreme of the Santa Ana Mountains and south of the Santa Rosa Plateau (San Diego, Riverside County Line, 33”26’ N -

M. Abril, R. Hanano

ll7”lO’ W). It lies about 29 km inland from the Pacific Ocean and has a typical Mediterranean climate. It is about 500 m a.m.s.1.Annual precipitation varies from 125 to 967 mm with a yearly mean of 409 mm. The mean annual temperature is 16.4 “C. The summer dry period typically extends from April to midOctober [49]. 2.2. Plant species The mesophyllous evergreen shrub Ceanothus thyrsifzorus Esch., the sclerophyllous evergreen tree Quercus agrifolia NeC and the sclerophyllous evergreen shrub Buxus microphylla, were all obtained from a local nursery. C. thyrsiflorus and Q. agrifolia are native chaparral species,while B. microphylla is an ornamental cultivar closely related to B. sempervirens, a native species from the western Mediterranean Basin. 2.3. Methods Six seedlings per species were planted outside in pots. Forty litre pots were used for Q. agrifolia (1.52 m tall) while B. microphylla and C. thyrsiflorus (0.5-0.75 m tall) were planted in 20 L pots. The soil mix had high concentrations of all nutrients and a pH of about 5.6. To avoid excessive soil temperature, the pots were placed in holes in the ground. Prior to the experiments, plants were well watered for 4 weeks and subjected to ambient conditions. When drought experiments started, three plants of each species were watered every day after sunset and three remained unwatered throughout the drying treatment. The whole set of measurementsdescribed were performed 1 day prior to the drying cycle, about the mid-point of the drying cycle, and when stressed plants were severely dry (n = 10 f 4 days). Xylem water potential and leaf gas exchange measurements were taken simultaneously in terminal twigs from intact plants using a pressure chamber [37] and an IRGA-porometer (LI-6200/6250; LiCor, Lincoln, NE, USA) in time intervals of about 2 h from sunrise to sunset. Predawn xylem water potentials were determined before sunrise. Precautions outlined by Turner [51] were followed. While gas-exchange measurements were done, twigs inside the cuvette were maintained as close as possible to the natural position (orientation, inclination, etc.). Transpiration, net photosynthesis, stomata1conductance, leaf temperature, internal CO, and chamber air conditions were always obtained from the same twig throughout the whole day, and from three twigs per plant. For xylem water potentials and xylem sap extraction we used the same twig, one per time and plant. Xylem sap was collected with a cannula after xylem water potential (WP) determination. Extracted sap was frozen immediately Actn Oecologicu

379

Ecophysiology of three sclerophyllous species

with liquid nitrogen. ABA was determined using the ELISA immunoassaytest as describedby Tenhunenet al. [49]. Leaf area of all collected twigs was determinedwith an area meter (LI-3000; LiCor, Lincoln, NE, USA) and leaf size was calculated. Leaves were oven-dried for 48 h at 70 “C and weighed to calculate leaf specific weight. 2.4. New variables Soil-plant hydraulic resistancewas calculated from the relationship between the water potential changes associatedwith transpirational water loss per unit leaf area when transpiration increases. It is the absolute value of the slope of that relationship [24, 341, which would be expectedto increaseas the soil dries [27,391 (HR = MPa.mmol-‘.m2+). Gf was calculated as the slope of the relationship

[61: G=Go+GfxNPxhs/Cs

(1:) where G is the stomata1conductance(mol.m-2.s-1),Go is the intercept of the linear regression (representing the stomatal conductance when NP = 0 at the light compensation point), NP is the net photosynthesis (ymol.m-2.s-‘), hs is relative humidity at leaf surface (as decimal fraction) and Cs is the carbon dioxide concentration at the leaf surface (ymol.mol-‘).

showed an asymmetric curve, with highest values during the morning. Curves of stressed plants were almost flat for all variables, with highest values in the early morning. Mean values for maximum NP, G and TR in control plants (spring weather) were highest in Q. agrifoliu and lowest in B. m icrophyllu (table II). WPpd and WPmin in table II are mean values while, when considering absolute individual values, plants whose WPpd fell below -2.5 MPa on C. thyrsiflorus and -3.5 MPa on B. m icrophyllu reached the permanent wilting point during the day with WPmin values of about -3.5 MPa for C. thyrsiflorus and -4.0 MPa for B. m icrophyllu. Q. ugrifoEia plants did not reach values under -2.5 MPa due to its water conservative behaviour during the experiment. Referencesto other evergreenQuercus species13-5, 16, 18, 32, 351 indicate the m inimum for WPpd between -3 and -4 MPa, which we assume would be similar for Q. agrifolia seedlingsif stresslasted a few more days. Xylem sap ABA contents were available only for C. thyrsijlorus and B. m icrophylla and were found to be similar. Average daily concentrationswere between O-500 nmolK’ for control and 500-6 000 nmolK’ for stressedplants. Neither control nor stressedplants showed a regular diurnal pattern on ABAxyl but stressedplants showed an increasing tendency during the day (as reported for C. thyrsiflorus by Tenhunenet al. [49]). 3.2. Stomata1 regulation

3. RESULTS

3.2.1. Hydraulic lim itations to the gas exchange

There were significant differences in leaf size and leaf specific weight in the three species (table I). Q. agrifoliu had the largest leaves, almost twice as large as the other two species.B. m icrophyllu had the highest LSW, while C. thyrsiflorus with the smallest leaves had the lowest LSW. Table I. Mean and standard error (SE) of leaf size (LA) and leaf specific weight (LSW) for each species. Differences among species in both variables are all significant (P < 0.05). LA+SE (cm’)

LSW + S E (mgkn?)

C. thyrs(florus

1.33 + 0.04

I I .7 + 0.2

B. microphylla

1.87 + 0.04

20.9 + 0.8

Q. agrifolia

3.12~0.25

16.2 f 0.7

3.1. Description of diurnal courses The shape of the daily gas exchange patterns (figure I) dependedon the variable plotted (G, TR or NP) and the degreeof stress.Bell-shapedcurves were found for control plants only in TR, while G and NP Vol. 19 (4) 1998

Maximum values of G (figure 2) and NP (figure 3) varied in relation to WPpd. The decline in maximum exchange rate as stress developed depended on the species, as reflected by the exponents of the fitted curves to a negative power function [5, 30, 47, 491. Although the highest maximum stomata1conductance values occurred in Q. ugrifolia (table ZZ), a marked decrease(0.77 slope) at relatively small potentials was observed.This was not as steep in B. m icrophyllu (0.57) and C. thyrsiflorus (0.32), which started from lower maximum conductancevalues (table ZZ). Maximum net photosynthesisinitial values and the rate of decreasewere almost the same in Q. ugrifolia and C. thyrsiflorus (0.32, 0.31, respectively), while B. m icrophyllu had lower initial values (table ZZ)and a more rapid decrease(0.41). Hydraulic resistance (figure 4) differed among the three speciesin control plants: B. m icrophyllu had the highest value (0.70), C. thyrsiflorus lower (0.28) and Q. agrifolia the lowest (0.24). Values for stressed plants were significant only for Q. ugrifoliu (1.26); B. m icrophylla and C. thyrsiforus gave a non-significant regression.

M. Abril, R. Hanano

380

h

A

r Quercusa “-“*

rifdia

8000

.. .I

..

L ..*:-

‘.o”-’

l

6000 g ID 40002 0 2000~

?i/

0 5 4

2

3

z 0

2 1

5 E

0 400

300

200

0 -. g .g

100

time of day Figure 1. Diurnals of the last day of one experiment for each species, all typical spring days in Southern California: (a) 3 June for Ceanothus thyrsiforus, (b) 22 May for Buxus microphyllu and (c) 6 June for Querc~.s ugrifolia. Upper panel: Water potentials (WP, circles) (measured simultaneously with gas exchange) and xylem sap abscisic acid concentration (ABA, squares). Middle panel: Stomata1 conductance (G, circles) and transpiration (TR, squares). Lower panel: Net photosynthesis (NP, circles) and internal CO? concentration (Ci, squares). Open symbols and continuous lines correspond to the control plants and filled symbols and dotted lines correspond to the stressed plants. For data obtained with the IRGA-porometer each value is the average of nine measurements (three plants and three twigs per plant). Water potential values are represented as the mean of three measurements (three plants and one twig per plant).

3.2.2. ABA: above- or below-ground signalling? To discern how the environmentinfluenced ABAxyl increaseduring the day a stepwiseregressionwas performed. As predictors, Ta, RH, VPD and WP were used. WP explained a 77.4 % of the ABAxyl variation in C. thyrsiflorus and an 89 % in B. microphylla, while

RH explained only a 4.6 % in C. thyrsiforus. Others are not significant at P < 0.05. An exponential pattern was found when relating ABAxyl depending on WP (not shown): ABAxyl did not increase significantly while WP remained above -1.5 MPa. Below these values, the ABAxyl increase was rapid, especially for C. thyrsiflorus, which

Table II. Values of the means (mean & standard error) and statistical significance of the differences among species (P ) for net photosynthesis (NP), stomata1 conductance (G) and transpiration (TR) maximums reached by the control plants (n = 9), and xylem water potential minimums reached at predawn (WPpd) and during the day (WPmin) by the stressed plants (n = 9). C. thyniflorus

B. microphyk~

Q. agrifolia

Mean + S E

Mean + S E

Mean f S E

Npmax (umol.m-‘.s-‘)

7.97 k 0.39

4.57 * 0.63

9.17 + 1.44

Gmax (mmol.m-2.s~‘) TRmax (mmol.m-‘.s-‘)

145.4 2 5.94

75.5 -+ 3.6

WPpd (MPa) WPmin (MPa)

-2.33 2 0.17

-3.13

f 0.23

-3.62 k 0.14

-3.97

2 0.20

3.9 2 0.2

1.4kO.l

172.4?

P

Differences

0.03 15

b#q,b#c

35.5

0.0404

4.7 f 1.0

0.0204

b#q,bf#c

-0.50 ?z 0.00

0.000 I

-2.32 k 0.11

0.0007

c=b#q c=b#q

w

Acta Oecologica

381

Ecophysiology of three sclerophyllous species

o

ct

----.

Qa

n

-

. .--

Gma = 68.4 x (-WPpd)“.32

f* = 0.373 n = 30

Gmax = 33.6 x (-WPpd)“,57

r*=0.645

G~=33.2~(-WPp@~

r2=0.690n=12

n=30

Figure 2. Maximum stomata1 conductance (Gmax) as a negative power function of the predawn xylem water potential (WPpd) on the last day of the drying cycle for the six plants of each species (all significant, P < 0.01). Ct: Ceanothus thyrsiflorus, Bm: Buxus microphylla, Qa: Quercus agrifolia.

0

-1

-2 WPpd (MPa)

-3

-4

12 0

ct

----_ NPmax = 4.00 x (-WPpd)“.3’ r2=0.586 .-.-.-.-- NPmax= 2.02 ~(-Wpptj)~~~’r*=0.632

n=30

-

n=12

NPmax=4,07~(-WPpd)~.~

?=a.446

n=30

Figure 3. Maximum net photosynthesis rate (NPmax) reached during the day as a negative power function of the predawn xylem water potential (WPpd) on the last day of the experiment for the six plants of each species (all significant, P < 0.01). Ct: Ceanothus thyrsiflorus, Bm: Buxus microphylla, Qa: Quercus agrifolia.

0

reachedconcentrationsof about 8 000 nmol.L-’ within a WPpd range of -2.5 to -3.5 MPa. If instead of relating momentary values we consider xylem sap ABA concentrations at the moment of maximum conductance (ABAatGmax) as a function of WPpd, a linear fit is found (figure 5). Thus, 86-77 % of the variance of the xylem sap hormone concentration can be explained by the WPpd. Vol. 19 (4) 1998

-1

-2 WPpd (MPa)

-3

-4

3.2.3. Hormonal influence on stomata1function An inverse relationship was found between G and ABAxyl (momentary values). However, the best fit between conductanceand the hormone occurred when we related these variables on a long-term basis (seasonal developmentof the stress) as Gmax and ABAatGmax cfigure 6). It is important to note that, in

M. Abril, R. Hanano

382

-ABAatGmax

=-25.6-717.2

WPpd

~=0.859

ABAatGmax =-g&3-534.7 WPpd ?=0.769

E

-2

-

IO

E

:i

e3

'i

.

Ceanothus thyrsiflorus

-4-

CO~~~~d ~-y

-5

-1 -2

E g

-3

-0.5

-1

Ceanothu,s thyrs@rus.

c

.

/;

-1.5

-2 -2.5 WPpd (MPa)

-3

-3.5

-4

Figure 5. Relationship between the predawn xylem water potential (WPpd) and the concentration of the hormone abscisic acid in the xylem sap at the time of maximum conductance during the day (ABAatGmax). Regressions are all significant (P < 0.01). Cr:

0

-4 1

Buxus microphylla

Bm: Buwus microph.ylla.

200

controls stressed -ssmsmssmsy = - 0.31 - 0.70x r*= 0.815 not significant

i--c

o l

Cr

Q -Gmax= 221.3-50.6log(AEAatGmax) P~O.433 -Gmax%139.3-37.4lq(ABAatGmax)

- -Bm--

150

0

0

0

I

0

10

100 1 000 ABAatGmax (nmol/L)

** . *...

z k$

0

00 ~b

2

I

I

=-0.28 - 0.28x r* =0.945 not significant I I I I

-3

-4t 1 ~J I 0

Quercus agrifolia controls y =- 0.45 -0.24x stressed ----.----y=0,50- 1.2~~

;

;

;

i

;

r’=0.493 r2z0,w5

6

‘/

TR (mmol/rr?.s) Figure 4. Relationship between xylem water potential (WP) and transpiration (TR) with increasing transpiration. The slope of the linear regression is considered as the hydraulic resistance (P < 0.05 when significant). Open circles are control plants, filled circles correspond to the stressed plants (pooled data from three plants per treatment the last day of the drying cycle - several points per day).

C. thyrsiforus, ABA explained more of Gmax variance (43 %) than WPpd (37 %) (see jgure 2).

3.2.4. ABA and Gf Gf values (equation 1) obtained per treatment and species are shown in table III. Although differences are not significant between B. microphylla and Q. agrifolia control plants and between C. thyrsifzorus

lo4

Figure 6. Maximum stomata1 conductance (Gmax) in relation to the momentary abscisic acid concentration in the xylem sap (ABAatGmax). Regressions are all significant (P < 0.01). Ct: Ceunothus

thyrsijlorus,

Bm: Buxus microphyllu.

and B. microphylla stressed plants, differences among treatments for each species are significant (P < O.Ol), and indicating a drought adjustment. Q. agrifolia B. microphylla control plants had a higher Gf than C. thyrsiflorus. Stressed Q. agrifolia plants had higher values because they were not as stressed as the other two species. The bigger shift in Gf occurred on B. microphylla.

Considering Gf as a function of maximum xylem sap ABA concentration (ABAmax) we found: C. thyrsiflorus

Gf = 12.4 - 2.0 1ogABAmax ? = 0.637 (n = 6) (2) B. microphylla

Gf = 23.1 - 5.0 1ogABAmax ? = 0.983 (n = 6) (3)

383

Ecophysiology of three sclerophyllous species

Table III. Slope factor (Gf) (mean + standard error) of the relationship between stomata1 conductance and net photosynthesis, relative humidity and CO, concentration at the leaf surface (equation I, explanation in the text).

When ABA results are not available, we used WPpd on the regression: Q. agrifolia

Gf = 11.9 - 6.2 log(-WPpd) ? = 0.863 (n = 6) (4) Gf*SE controls C. thq’rs$i’orus

GfkSE stressed

7.84 c 0.11

3.3. Photosynthetic limitations

4.63 20.53

B. microphylkz

12.83 T 0.90

4.56 + 0.15

Q. ugrifolia

Il.47

7.40 c 0.2 1

+ 0.44

The best fit of the net photosynthesisversus stomata1 conductance(figure 7) always showed a positive direct relationship between the two variables, indicating strong dependenceof the assimilation rate on stomata1 aperture, with a small difference among the species. For C. thyrsiflorus and B. microphylla, the best tit was linear without significant differences tested on the slope. For Q. agrifolia, the best fit was a power function. To elucidate possible nonstomatal effects, NP was related to leaf internal CO, partial pressure(Ci) in controls and stressedplants of each species (not shown). Ci should changein the same direction as NP to establish that stomata1responseis dominant over the assimilation rate [15]. If the changes are opposite, as we found in our data (a negative slope in all regressions, significant with P < 0.01 for C. thyrsiflorus - both treatments - and stressedB. microphylla and Q. agrifolia plants), the most important change must have occurred in the mesophyll cells, involving the metabolism associatedwith CO, assimilation. Daily values of Ci (figure I, lower panels) support this interpretation. Mean Ci of stressedplants were higher than controls for B. microphylla (P < 0.01) and C. thyrsiflorus (not significant) and equal for Q. agrifolia. There was a significant rise of Ci at the end of the day (P < 0.01 for all speciesand treatments except control B. microphylla) when severewater stress occurred, at the same time when G and NP reached their minimum in each species.Only B. microphylla control plants maintained an almost constant Ci throughout the day.

Ceanothus thyrsiflorus

12 _ 6uxu.s microphylla 3

-----NP=0.17+0.057G~=0.876

lo-

i';

8-

5

6-

4 42-

0/i

0+* g&*0

I

I

I

I

1 2 _ Quercus agrifolia g

-----

108-

NP = 0.12xG”.= 0

SC6 *-

0 .--

0

50

r2= 0.654 a* /- -’ __-I 0

C- .e

3.4. Water-use efficiency

0

OO

I I 100 150 G (mmolhf . s)

1 200

250

Figure 7. Net photosynthesis (NP) versus stomata1 conductance (Gi for the three species studied. All regressions are significant (P < 0.01). Data from the last day of the experiment (three control and three stressed plants). Open symbols are control plants and filled symbols are stressed plants. Vol. 19 (4) 1998

Water-use efficiency (WUE), the amount of carbon acquired per unit of water lost, can be measuredand calculated by different methods [50]. Using gas exchangedata from the last day of the experiments,we calculated WUE for each species as follows (results are summarized in table IV): - An instantaneous measureof WUE as the ratio of NP to TR, in pmol CO, fixed per mol H,O used per unit leaf area and unit time. Diurnal patterns described a symmetrical curve, with high values during favourable conditions (morning and afternoon) and low values at midday (not shown). Values in table IV are means of all daily measurements.Overall water stress resulted in an increase in WUE although differences were only significant for B. microphylla.

384

M. Abril, R. Hanano

Table IV. Water-use efficiency (mean + standard error) calculated as explained in the text. Instantaneous: in mm01 CO, fixed per mol H,O used per second (mean from four to six points per day in three plants). Daily: in mm01 CO, fixed per mol H,O used per day during the last day of each experiment (mean from three plants). Long-term: slope of the relationship between GatNPmax and NPmax (n = 6, ? = 0.923, ? = 0.919, ? = 0.862. respectively). Method

WUE + SE controls

WUE f SE stressed

Instantaneous

2.67 + 0.62

3.25 + 0.80

Daily

I .98 + 0. I I

Long term

2.00 + 0.64 20.37

Instantaneous

1.61 kO.19

5.23 + 1.41

Daily

2.88 + 0.29

5.04 + 0.86

Long term

17.71

Instantaneous

4.05 * 0.94

4.03 f 0.77

Daily

2.25 2 0.20

3.79 + 0.32

Long term

-Analyzing WUE as the CO, fixed by water used during a whole day (daily integration) we found daily values using table IV. Among control plants, B. microphylla showed significantly higher values (no significant differences found among Q. agrifolia and C. thyrsiflorus). Stressedplants of B. microphylla and Q. agrifolia had significantly higher values than controls. The most conservative species was B. microphyllu in both treatments, and the least was C. thyrsiflorus (significant differences found on stressed Q. agrifolia, B. microphylla and C. thyrsiflorus). In general, these results seem similar to the instantaneous values. - A long-term indicator of WUE is the slope of the regression between maximum assimilation rate (NPmax) and stomata1conductance(GatNPmax). We pooled data from control and stressedplants to calculate the relationship shown in table IV On a long-term basis, Q. agrifolia is the most efficient species on water use, while B. microphylla is the least and C. thyr$orus is in between. 4. DISCUSSION Some morphological traits of evergreensclerophyllous leaveshelp to explain the different strategiesused to reduce water loss - when water is a limiting resource- without reducing net photosynthesisin the long term. The leaf surface boundary layer of small leavesis thinner and helps leaf refrigeration (B. microphylla and C. thyrsiflorus); leaveswith thicker cuticles adsorb less radiation (Q. agrifolia and B. microphylla) (table Z). Other biological features such as root depth or xylem structure determine the efficiency of the use of water resources.On plants grown in pots, as in our experiment, root growth was restricted, eliminating a

24.1 I

source of variability on water use. At high soil moisture (control plants in$gure 4), the soil resistancewas low and the HR should be largely attributable to differences in plant resistance. The structureof the water-conductingsystem(vessel size and hydraulic architecture) determines hydraulic resistance and influences water use [27, 32, 34, 521. Wood structure at a genus level is described ([lo], Abril, unpubl. obs.): while Buxus has relatively thin vesselsand ‘true’tracheids (typical of a mesic climate), Quercus and Ceanothus have wide vessels and vasicentric tracheids, like many evergreen drought-resistant shrubs, which indicate xeric habitats. These characteristics explain differences in HR and TRmax among species (table II and figure 4, control plants): Q. agrifolia had a low HR that allowed highest maximum stomata1conductance.At the other extreme, B. microphylla had the highest HR and low maximum conductance. Changes in HR during the soil drying as shown in figure 4, may be attributable to variations in root:leaf surfacesratio [9], or a variation in the inherent absorption capacity and root permeability [27, 32, 341. These changes suggest a mechanism of adaptation to xeric environmentsthat is important for drought toleranceof Mediterranean evergreen plants. While Reich and Hinckley [34] reported that for two deciduous oaks maximum stomata1conductance varies according to changes in soil-plant hydraulic resistance, Sala and Tenhunen [35] concluded that long-term variation of maximum leaf conductancein sun leaves of Quercus ilex was not related to changesin soil-plant hydraulic resistance.For the three speciesstudied here to discern and conclude on that question would require more significant regressionsand data on the long term. Acta Oecologica

Ecophysiology of three sclerophyllous

species

Physiological regulation via stomata1conductanceis the mechanism that allows the leaves an effective control over water loss in the short term. B. microphylla is the only species that significantly increased WUE on an instantaneousand daily basis (table ZV). However, the meaning of increased WUE with drought must be interpreted carefully: high WUE under drought may be linked to reduced stomata1 aperture under wellwatered conditions, limiting potential CO, assimilation and dry-weight gain. This may be the case of B. microphylla, where stomata1 limitations (see low values for G on B. microphyla control plants in $gure 1) led to a substantial reduction in NP compared to other speciesstudied (figure 3). In the context of the hydraulic restrictions to the gas exchange we considered Q. agrifolia and C. thyrsijlorus as more xeric spe,ties and B. microphylla as more mesic (see [21]). Buxus is a sub-Mediterraneangenus, and although we found it in dry habitats, its best physiological adjust ment and growth is probably developedin more mesic conditions. C. thyrsijorus and Q. ugrifoliu showed small differences in instantaneous and daily WUE values between stressedand control plants. On a longterm basis WUE increased as drought proceeded and, by relatingfigures 2 and 3, we deducethat Q. agrifoliu is the most efficient speciesin gas exchangeand water use, becauseof the high CO, gain with simultaneous low water loss as soon as WPpd started to decrease. The fact that WP is the variable that best explains ABAxyl on a daily basis and considering WPpd as a plant variable related to the prevailing water conditions in the soil, the good fit found when relating WPpd to the ABAatGmax (figure 5) suggests an important role for the ABAxyl in signalling, from roots to shoots, seasonalchangesoccurring in a belowground environment. Results shown in$gure 6 give us evidence of the role of the hormone in adjusting stomatal conductanceaccording to these conditions in the root surroundings. The decrease in photosynthetic rates as WPpd becomesmore negative, as observedinjgure I (lowei panels) and Jigure 3, for the three species can be explained by a stomata1dependence(limited CO, dif-. fusion through the stomata,figure 7) and by a nonsto-, matal limitation (negative NP-C, relationship, not shown) due to different components that affect the assimilation rate. Many data sets collected under natural or experimental conditions reveal a linear relationship between G and NP as we found on C. thyrsiflorus and B. microphylla (figure 7) ([7] for Arbutus unedo, [20] for many herbaceous species reported by different authors). However, as we found in Q. agrifolia, Lin et al. [22] reported curvilinear correlations for Mediterranean Pistuciu species, Schulze and Hall [39] for Prunus armeniaca and Pereira et al. [30] for Eucalyptus gloVol. 19 (4) 1998

385 b&s. These increases in G are not associated with increases in NP because, most likely, a nonstomatal factor limits carbon assimilation. On the other hand, linear relationships can be found when experimental data (usually from the field) belong only to the initial portion of the responsecurve, where the stomata1limitation of NP is quite significant [17]. However, these results (fisure 7) do not necessarily imply that G is the primary factor regulating NP, although stomata1aperture certainly does have an effect on CO, diffusion into the leaf mesophyll, and therefore, carbon assimilation. Photosynthesis is sensitive, for example, to changes in osmotic pressure and cell volume rather than to changes in water potential [38]. The mechanisms of these limitations and their relative importance in natural environments are not well known, and probably depend on the species [21, 311. The nonstomatal photosynthetic limitation has also been deduced by observation of the Ci patterns with stress. The behaviour of leaves with respect to the Ci level may differ greatly between species that are differently adapted to dry habitats [47]. For Mediterranean evergreen sclerophylls, there is evidence that, despite strong decreasesin G and NP with drought, Ci remains surprisingly constant, which has been interpreted as indicating a change in carboxylation efficiency [7, 17, 45, 461. The mechanisms responsible for such coordinated regulation of stomata1 conductance and mesophyll photosynthetic activity are still under discussion. It is difficult to discriminate between the relative importance of stomata1and nonstomatal regulation, since the same metabolic factor acts on stomata and the photosynthetic apparatusin the mesophyll. Plant hormones, such as ABA, can play an important role [26, 491. In fact, Raschke [33] found that ABA has a stomata1 effect on guard cells (closing stomata and limiting CO, supply) and mesophyll cells, over both carboxylation and RuBP regenerationcapacity. Our results suggestthat the high increasein ABAxyl during the day (1 000-8 000 nmol.L-‘) in stressed plants (figure 1, upper panels) may affect the stomata1 movement and the mesophyll activity on a daily basis (explaining the diurnal patterns and especially the lack of recovery during the afternoon) while the low basal increase in ABAxyl on a long-term basis (5& 500 nmol.L-‘), related to a decrease in WPpd (figure 5), applies for the seasonal control of water savings and gas exchange [43]. If Gf was postulated to representan integrated expressionof different internal control mechanisms on G, it can be explained as a function of maximum xylem sap ABA concentration (equations 2 and 3). In the species for which ABA results are available, ABAmax is the variable that best explains Gf, although when ABA analysesare not pos-

386 sible, WPpd can be used to set Gf [36] as shown for Q. agrifolia (equation 4). Other authors concluded from experimental data [13] and modelling [53] that ABA is not an inhibitor of the photosynthetic capacity and probably only causes the patchy stomata1 closure. Recently, by experimental observation of patchiness on stomata1 behaviour in the whole leaf of various species,caution has been suggestedin the calculation of gas exchange results [8, 11, 141.Basically, the effects are the underestimation of maximum photosynthetic capacity and, from the NP-C, relationship, an erroneous carboxylation efficiency calculation when patchiness occurs, mainly on stressed leaves, although steady-state patchy stomata1closure can occur at low humidities in well watered plants [26]. Some authors [ 1 I] argued that even considering patchiness, the whole change in apparent mesophyll activity cannot be explained by it. The correction algorithms that have been published [53] are only applicable if the ‘closed’ leaf area is known. Although standard techniques are developing [8, 531 and alternative experimental analysis such as chlorophyll fluorescence has been suggested [ 141, it has not been possible to apply them in our experiments. Acknowledgements Funds for this study were provided by the Spanish Ministerio de Education y Ciencia and by the Deutsche Forschungsgemeinschaft (SFB 25 1,TP3). We thank Dr John D. Tenhunen for his support during the development of the field study and Mr Tu Chung for maintaining plant materials at SMER. We appreciate the use of research facilities at the SMER of San Diego State University and the CREAF in the Universitat Autbnoma de Barcelona. Comments by A. Sala, D. Hilbert and E. Gutierrez on early versions of this manuscript are greatly appreciated.

REFERENCES [I] Abacus Concepts, StatView II and SuperAnova software and manuals, Abacus Concepts Inc., Berkeley, CA, 1987. 121 Abelbeck Software, KaleidaGraph TM software and manual, 1993. [3] Abrams M.D., Adaptations and responses to drought in Quercus species of North America, Tree Physiol. 7 (1990) 227238. 141 Abril M., Diego V., Resultados preliminares de un experiment0 de riego y fertilizacibn con plantulas de encina: Efectos sobre el crecimiento y estructura de la planta, relaciones hidricas y fotosintesis, Stud. Oecologica 10-I 1 (1994) 349-358. [5] Acherar M., Rambal S., Quercus ibex facing water stress: a functional equilibrium hypothesis, Vegetatio 99/100 (1992) 177-184. [6] Ball J.T., Woodrow I.E., Berry J.A., A model predicting stomatal conductance and its contribution to the control of photosynthesis under different environmental conditions, in: Biggins

M. Abril, R. Hanano

I. (Ed.), Progress in photosynthesis research, vol. IV’S, Proceedings of the VII International Photosynthesis Congress, 1987, pp. 221-224. [7] Beyschlag W., Lange O.L., Tenhunen J.D., Photosynthesis and water relations of the Mediterranean evergreen sclerophyll Arbutus unedo L. throughout the year at a site in Portugal. II. Characteristic gas exchange parameters of CO, uptake and transpiration, Flora 179 (1987) 399420. IS] Beyschlag W., Pfanz H., Rye1 R.J., Stomata1 patchiness in Mediterranean evergreen sclerophylls. Phenomenology and consequences for the interpretation of the midday depression in photosynthesis and transpiration, Planta 187 (1992) 546-553. [9] Caldwell M.M., Richards J.H., Competing root systems: morphology and models of absorption, in: Givnish T.J. (Ed.), On the economy of plant form and function, Cambridge University Press, Cambridge, 1986, pp. 25 l-269. [IO] Carlquist S., Vasicentric tracheids as a drought survival mechanism in the woody flora of Southern California and similar regions; review of vasicentric tracheids, Aliso 11 (1985) 37-68. 1 I] Cheeseman J.M., PATCHY: simulating and visualizing the effects of stomata1 patchiness on photosynthetic CO, exchange studies, Plant Cell Environ. 14 (1991) 593-599. 121 Davies W.J., Mansfield T.A., Hetherington A.M., Sensing of soil water status and the regulation of plant growth and development, Plant Cell Environ. 13 (1990) 709-719. [ 131 Downton W.J.S.. Loveys B.R., Grant W.J.R., Stomata1 closure fully accounts for the inhibition of photosynthesis by abscisic acid, New Phytol. 108 (1988) 263-266. 1141 Downton W.J.S., Loveys B.R., Grant W.J.R., Non-uniform stomatal closure induced by water stress causes putative non-stomatal inhibition of photosynthesis, New Phytol. 110 (1988) 503-5 10. [ 151 Farquhar G.D., Sharkey T.D., Stomata1 conductance and photosynthesis, Ann. Rev. Plant, Physiol. 33 (1982) 3 15-327. [ 161 Griffin J.R., Xylem sap tension in three woodland oaks of central California, Ecology 54 (1973) 152-159. 1171 Harley P.C., Tenhunen J.D.. Lange O.L., Beyschlag W.. Seasonal and diurnal patterns in leaf gas exchange of Phyllirea an~ustifolia growing in Portugal, in: Tenhunen J.D., Catarino EM., Lange O.L., Oechel W.C. (Eds.), Plant response to stress functional analysis in Mediterranean ecosystems, NATO Advanced Science Institute Series. Springer-Verlag, Berlin, Heidelberg, 1987, pp. 3299337. [ 181 Hollinger D.Y., Photosynthesis, water relations and herbivory in co-occurring deciduous and evergreen California oaks, Ch. 3. PhD thesis, Standford University, CA, 1983. 1191 Kramer P.J., Changing concepts regarding plant water relations, Plant Cell Environ. 1 1 (1988) 565-568. [20] Lange O.L., Beyschlag W., Tenhunen J.D., B. Control of leaf carbon assimilation - Input of chemical energy into ecosystems, in: Schulze E.D., Zwolfer H. (Eds.), Ecological studies, vol. 61, Springer-Verlag, Berlin, Heidelberg, 1987. 1211 Larcher W., De Moraes J.A.P.V., Bauer H., Adaptative responses of leaf water potential, COz-gas exchange and water use efficiency of Olea europuea during drying and rewatering, in: Margaris N.S., Mooney H.A. (Eds.), Components of productivity of Mediterranean-climate regions. Basic and applied aspects, Dr W. Junk Publishers, The Hague, 1981, pp. 77-84. [22] Lin T.S., Crane J.C., Ryugo K., Polito V.S., DeJong T.M., Comparative study of leaf morphology, photosynthesis and leaf conductance in selected Pistacia species, J. Am. Sot. Hortic. Sci. 109 (1984) 325-330. Acta Oecologiw

Ecophysiology of three sclerophyllous species

[23] Meinzer F.C., Stomata1 control of transpiration, Trends Ecol. Evol. 8 (1993) 289-294. [24] Meinzer EC., Sharifi M.R., Nilsen E.T., Rrundel P.W., Effects of manipulation of water and nitrogen regime on the water rela tions of the desert shrub Larrea tridentam, Oecologia 77 (1988) 480486. [25] Monteith J.L., A reinterpretation of stomata1 responses to humidity, Plant Cell Environ. 18 (1995) 357-364. [26] Mott K.A., Cardon Z.G., Berry J.A., Asymmetric patchy stomatal closure for the two surfaces of Xunthium strumarium I,. leaves at low humidity, Plant Cell Environ. 16 (1993) 25-34. [27] Ni B.R., Pallardy S., Response of liquid flow resistance to soil drying in seedlings of four deciduous angiosperms, Oecologia 84 (1990) 260-264. [28] Nonami H., Schulze E.-D., Ziegler H., Mechanisms of stomata1 movement in response to air humidity, irradiance and xylem water potential, Oecologia 183 (1990) 57-64. [29] Passioura J.B., Response to Dr P.J. Kramer’s article, ‘Changing concepts regarding plant water relations’, 1 l(7): 565-568, Plant Cell Environ. 11 (1988) 569-57 1. [30] Pereira J.S., Tenhunen J.D., Lange O.L., Stomata1 control of photosynthesis of Eucalyptus globulus Labill. trees under field conditions in Portugal, J. Exp. Bot. 38 (1987) 1678-1688. [3 l] Quick W.P., Chaves M.M., Wendler R., David M., Rodrigues M.L., Passaharinho J.A., Pereira J.S., Adcock M.D., Leegood R.C., Stitt M., The effect of water stress on photosynthetic carbon metabolism in four species grown under field conditions, Plant Cell Environ. 15 (1992) 25-35. [32] Rambal S., Quercus ilex facing water stress: a functional equilibrium hypothesis, Vegetatio 99/100 (1992) 147-153. [33] Raschke K., Involvement of abscisic acid in the regulation of gas exchange: evidence and inconsistencies, in: Wareing P.F. (Ed.), Plant growth substances, Academic Press, London, 1982, pp. 581-590. [34] Reich P.B., Hinckley T.M., Influence of pre-dawn water potential and soil-to-leaf hydraulic conductance on maximum daily leaf diffusive conductance in two oak species, Funct. Ecol. 3 (1989) 7 19-726. [35] Sala A., Tenhunen J.D., Site-specific water relations and stomatal response of Quercus ilex in a Mediterranean watershed, Tree Physiol. 14 (1994) 601-617. [36] Sala A., Tenhunen J.D., Simulations of canopy net photosyn thesis and transpiration in Quercus ilex L. under the influence of seasonal drought, Agr. Forest. Meteorol. 78 (1996) 203-222. [37] Scholander PE, Hammel H.T., Bradstreet E.D., Hemmingsen E.A., Sap pressure in vascular plants, Science 148 (1965) 339246. [38] Schulze E.-D., Carbon dioxide and water vapor exchange in response to drought in the atmosphere and the soil, Ann. Rev. Plant Physiol. 37 (1986) 247-274. 1391 Schulze E.-D., Hall A.E., Stomata1 responses, water loss and CO, assimilation rates of plants in contrasting environments, in: Lange O.L., Nobel P.S., Osmond C.B., Ziegler H. (Eds.), Physiological plant ecology. II. Water relations and carbon assimilation. Encyclopedia of plant physiology, vol. 12B, SpringerVerlag, Berlin, Heidelberg, New York, 1982, pp. 181-230. [40] Schulze E.-D., Steudle E., Gollan T., Schurr U., Response to Dr PJ. Kramer’s article, ‘Changing concepts regarding plant water relations’, 1 l(7): 565-568, Plant Cell Environ. 1 1 (1988) 5733 576.

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387 [41] Tardieu F., Will increases in our understanding of soil-root relations and root signaling substantially alter water flux models? Phil. Trans. R. Sot. Lond. [B] 341 (1993) 57-66. [42] Tardieu F., Davies W.J., Integration of hydraulic and chemical signalling in the control of stomata1 conductance and water status of droughted plants, Plant Cell Environ. 16 (1993) 341349. [43] Tardieu F., Zhang J., Davies W.J., What information is conveyed by an ABA signal from maize roots in drying field soil? Plant Cell Environ. 15 (1992) 185-191. [44] Tardieu F., Zhang J., Katerji N., Bethenod 0.. Palmers S., Davies W.J., Xylem ABA controls the stomata1 conductance of field-grown maize subjected to soil compaction or soil drying, Plant Cell Environ. 15 (1992) 193-197. [45] Tenhunen J.D., Lange O.L., Gebel J., Beyschlag W., Weber J.A., Changes in photosynthetic capacity, carboxylation efficiency, and CO, compensation point associated with midday stomata1 closure and midday depression of net CO, exchange of leaves of Quercus suber, Planta 162 (1984) 193-203. [46] Tenhunen J.D., Lange O.L., Harley P.C., Beyschlag W., Meyer A., Limitations due to water stress on leaf net photosynthesis of Quercus coccifera in the Portuguese evergreen scrub, Oecologia (Berlin) 67 (1985) 23-30. [47] Tenhunen J.D., Beyschlag W., L.ange O.L., Harley P.C., Changes during summer drought in leaf CO, uptake rates of macchia shrubs growing in Portugal: limitations due to photosynthetic capacity, carboxylation efficiency, and stomata1 conductance, in: Tenhunen J.D., Catarino EM., Lange O.L., Oechel W.C. (Eds.), Plant response to stress - functional analysis in Mediterranean ecosystems, NATO Advanced Science Institute Series, Springer-Verlag, Berlin, Heidelberg, 1987, pp. 305-327. 1481 Tenhunen J.D., Sala Serra A., Harley PC., Dougherty R.L., Reynolds J.F., Factors influencing carbon fixation and water use by Mediterranean sclerophyll shrubs during summer drought, Oecologia 82 (1990) 381-393. [49] Tenhunen J.D., Hanano R., Abril M., Weiler E.W., Hartung W., Above- and below-ground environmental influences on leaf conductance of Ceanothus thyrsij7orus growing in a chaparral environment: drought response and the role of abscisic acid, Oecologia 99 (1994) 306-3 14. [50] Toft N.L., Anderson J.E., Nowak R.S., Water use efficiency and carbon isotope composition of planis in a cold desert environment, Oecologia 80 (1989) 11-18. [51] Turner N., The use of the pressure chamber in studies of plant water status, in: Proceedings of the International Conference on measurement of soil and plant water status, vol. 2, Plants, Utah State University, Logan, 1987, pp. 13-24. [52] Tyree M.T., Sperry J.S., Do woody plants operate near the point of catastrophic xylem dysfunction caused by dynamic water stress? Answers from a model, Plant Physiol. 88 (1988) 574580. [53] Van Kraalingen D.V.G., Implications of non-uniform stomata1 closure on gas exchange calculations, Plant Cell Environ. 13 (1990) 1001-1004. [54] Zhang J., Davies W.J., Changes in the concentration of ABA in xylem sap as a function of changing soil water status can account for changes in leaf conductance and growth, Plant Cell Environ. 13 (1990) 277-285.