Responses of gas exchange, photosynthesis, nocturnal acid accumulation and water relations of Aptenia cordifolia to short-term drought and rewatering

J. Plant Physiol. \t0L 150. pp. 467-474 (1997)

Responses of Gas Exchange, Photosynthesis, Nocturnal Acid Accumulation and Water Relations of Aptenia cordifolia to Short-Term Drought and Rewatering W. B.

HERPPICH

and K.

PECKMANN

Inst. £ Oko!. der Pflanzen, WWU Munster, Hindenburgplatz 55, D-48143 Munster, Germany Received April 30, 1996 . Accepted September 1, 1996

Summary

In a controlled environment chamber study, changes of diurnal gas exchange, malic and citric acid accumulation, water relations and chlorophyll fluorescence in response to a short-term drought (10 days) and to rewatering were examined in Aptenia cordifolia. This all-cell leaf succulent Mesembryanthemaceae is native to summer rainfall regions of the eastern parts of the coastal deserts in South Africa and Namibia. When well-watered, A. cordifolia showed all attributes of CAM, i.e. nocturnal accumulation of malate (~-malate) and, to a lesser extent, of citrate (~-citrate; 24 % that of ~-malate) and nighttime carbon uptake. During drought, daytime CO 2 uptake ceased within 2 days due to stomatal closure. All changes in light period CO 2 exchange and transpiration were highly positively correlated. Nocturnal carbon uptake gradually diminished but malate and citrate accumulation was slighdy enhanced and the ratio of ~-citrate to ~-malate increased to almost 0.5. While water potential was reduced with decreasing water content, bulk leaf pressure potential was constant due to active osmotic adjustment. Changes in tissue osmotic content, which were fully reversible upon rewatering, mainly (== 50%) resulted from variations in citrate content. However, during prolonged drought even the constancy of bulk leaf pressure potential and CAM could not prevent reduction of maximum photochemical efficiency, as estimated from dark-adapted fluorescence parameters (Fy/FM). This indicated a photoinhibitory disturbance of photosynthesis, which probably not only resulted from CO 2 deprivation due to stomatal limitations. Partial non-stomatal restrictions at the biochemical level may be deduced from the close relationship between changes in Fy/FM and tissue osmolarity. Kinetics of recovery after rewatering showed that daytime and nighttime gas exchange may be regulated differentially. The former was independent of water content and pressure potential as was the diurnal rhythmicity of organic acids.

Key words: Apunia cordifolia, chbJrophyU fluorescence, citric acid, Crassulacean acid metabolism, drought stms, gas exchange, malic acid, osmotic adjustment, water relations. Abbreviations: CAM = Crassulacean acid metabolism; Fo, FM = initial, maximum fluorescence yield; Fy = variable fluorescence; NCG D/N = net carbon gain during the day/night; PPFD = photosynthetically active photon flux density; WUE =molar water use efficiency.

Introduction Crassulacean acid metabolism (CAM) is considered to be

~ adaptive mechanism allowing many terrestrial and epiphy-

tic succulents to survive in periodically droughted habitats (Kluge and TIng, 1978; von Willert et al., 1992). It has now C> 1997 by Gustav Fischer Verlag, Jena

become evident that succulents with different morphological and physiological characteristics inhabiting very contrasting places exhibit different patterns of CAM (Griffiths, 1988). It is thus conceivable that drought responses of plants differing in form and function may be diverging. Many perennial desert succulents take up CO 2 at night, leading to the accumula-

468

w. B. HERPPICH and K. PECKMANN

2 tion of large amounts of malate when well-watered (Hans- jected leaf area 3.75 ± 1.01 cm ), entire, heart-shaped to ovate, allcell succulent (degree of succulence, S = 3.8 ± 0.3 g dm -2) leaves. It com and Ting, 1978; von Willert et al., 1992). In these plants is found in the eastern parts of the coastal deserts of the Cape prostomata are tightly closed day and night and CAM is subvince in South Africa (Namaland to Namibia; Jacobsen, 1981). stantially reduced during prolonged drought (Kluge and Plants were raised from seeds collected in the Namib desert and Ting, 1978). In contrast, the expression of this metabolic further propagated by cuttings. The latter were rooted in earthen pathway may be more flexible in succulents from less arid or pots (diameter 12 cm) containing a sandy soil and grown in a green epiphytic habitats (Griffiths, 1988). CAM may be enhanced house under controlled conditions. Temperature and humidity were or induced in response to photoperiod, water shortage and/or 27 ± 3 ·C and 40 ± 10 % during the day and 18 ± 3 ·C and 80 ± salt stress (Winter, 1985). However, it has now been realized 10 % at night. Natural light was supplemented by high-pressure that induction processes are often closely linked to the devel- mercury lamps (Power Star HQI-TS 250 WID; Osram, MUnchen, active photon flux opmental stages of plants or leaves (Herppich et al., 1992; Germany) to give a minimum photosynthetically density (PPFD) of about 200 !lmol m- 2 s-I. Plants were watered Holthe et al., 1987). Diurnal changes in tissue malate content (~-malate) are twice a week. One week before the experiments began, plants were transferred often accompanied by smaller fluctuations of citrate (~-cit­ into a phytobox (Ecophyt Model VEPHQ 5/1350, Heraeusrate; Liittge, 1988; von Willert et al., 1992). ~-citrate may Voetsch, Balingen, Germany) and grown at temperatures of 30·C at also be larger than (Haag-Kerwer et al., 1992) or even replace day and 15·C at night at a dewpoint of 12 ·C and a PPFD of about ~-malate under some circumstances (von Willert et al., 400 !lmol m- 2 S-I (12 h; Osram HQI-R 250W/NDL; Osram; 1992). However, physiological and ecophysiological relevance MUnchen, Germany). During the first week of the investigation of nocturnal accumulation of citrate is by far less clear than plants were sufficiently watered daily. Drought was then imposed by that of malate (Liittge, 1988; Franco et al., 1992). While cit- withholding watering for 10 days. Thereafter, plants were again warate accumulation does not contribute to net acquisition of tered every day. CO2 at night, it may provide reducing equivalents without loss of carbon. Additionally, during periods of drought its Gas o:change and fluoromeur measurnnents break-down at day may help to minimize photoinhibition Gas exchange was measured on branches (4 leaf pairs) with an more effectively (Franco et al., 1992) than that of malate. open system (Minicuvette, H. Walz GmbH, Effeltrich, Germany). The increased internal CO 2 concentration during decarbo- CO exchange and transpiration were monitored with a differential 2 xylation of stored malate and citrate may thus stabilize pho- infrared gas analysor (BINOS 4b.2, Leybold-Heraeus, now Fishertosynthetic activity in CAM plants. Recently, it has been Rosemount GmbH u. Co., Hanau, Germany) and humidity of the shown that drought stress inhibition of photosynthesis in C 3 air entering or leaving the assimilation chamber by dewpoint mirand C 4 plants may simply reflect reduced CO 2 availability rors (H. Walz GmbH, Effeltrich, Germany). Data were continudue to high stomatal resistance (Cornie et al., 1992). Never- ously recorded by a computer at 5-min intervals. Gas exchange patheless, reversible inhibition of photosynthetic activity as well rameters, calculated after von Caemmerer and Farquhar (1981), were as photodamage may be serious in C 3 and in CAM plants based on leaf surface area (approximated as two times2 thel projected when excitation energy becomes excessive during prolonged leaf area). PPFD was maintained at 400 ± lO!lmolm- s- , and temperature and humidity corresponded to that in the growth chamber. drought (Adams et al., 1988). Chlorophyll fluorescence was monitored simultaneously with gas Control and active regulation of leaf water relations, i.e. exchange using a PAM fluorometer (PAM 101, H. Walz GmbH, Efosmotic adjustment (Turner and Jones, 1980), during large feltrich, FRG). Leaves were enclosed in a small, black plastic clamp periods of water shortage may, therefore, also be essential for and dark-adapted for 30 min. After measuring the initial fluoresCAM plants, although diurnal acid metabolism is highly wa- cence (Fo ), actinic white light (1210 !lmol m- 2 s-I) was provided by ter conserving per se (Kluge and Ting, 1978). However, while a KL1500 cold light source (Schott, Wiesbaden, Germany) and drought induced changes in water relations are well estab- transients of fluorescence were monitored until terminal steady state lished for several CAM plants with separated water storage fluorescence (FT ) was attained. Immediately after switching off actitissue (Goldstein et al., 1991), information about all-cell suc- nic light, initial fluorescence in the light adapted state (Fo ') was measured. Maximum photochemical efficiency of photosystem II, culents are rare (Herppich, 1989; Sinclair, 1983; Smith and i.e., the ratio of variable (F y = FM-FO) to maximum fluorescence Liittge, 1985) and less complete. (F M), was calculated according to von Willert et al. (1995) from the Young seedlings of Aptenia cordifolia, a perennial Mesem- transient registered with a line recorder. Quenching of the initial bryanthemaceae native to the coastal deserts of southern Af- fluorescence was estimated as [1 - Fo'/Fol (Bilger and Schreiber, rica, have been reported to induce CAM in response to salt 1986). stress (Treichel, 1975). We have used cuttings of this species, at least 9 months old, to establish the responses of CAM, Determination oftissue malau and citrau concmtrations and leaf photosynthesis and leaf water relations to short-term drought water relations under controlled environmental conditions.

Materials and Methods

Plant material and growth conditions Aptmia cordifolia Schwant. (Subtribus Aptenieae, Jacobsen, 1981) is a perennial low, prostrating herb with medium sized (mean pro-

About 1 and 9 h, respectively, after illumination leaf samples (3n! to 5th leaf pair, numbered from the shoot tip) were taken, immediately copied on paper, weighed and dried to constant weight at 85·C. Leaf water content was calculated from the difference between leaf fresh and dry weight and related to either dry weight or to leaf surface. The projected leaf area was obtained from the paper traces with an area meter (Delta T Devices, Cambridge, UK). In hot water extracts, malate and citrate concentrations were determined enzymatically (Mollering, 1974; Mollering and Gruber, 1966) and

469

Drought response of CAM in Aptmia cordifolia osmolarity cryoscopically (Osmomat 030, Gonotec, Berlin, Germany). Water potential of single leaves was estimated from leaf discs (diameter = 1 cmr with a psychrometer (SC-1O-A, Decagon Devices Inc., Pullman, USA). Pressure potential was derived from the difference between the means of leaf water potential and osmotic potential, the la((er calculated from osmolarity using the van't Hoff law (von Willert et a1., 1995).

-

U,

~

E

(5

E

~ N

..., 0

Results

U

Under the given experimental conditions well-watered, 9-month-old plants of A. cordifoiia exhibited a pronounced diurnal variation in tissue malate content (Fig. 1), which increased with leaf maturation (leaf pairs 1 to 3, numbered from the shoot tip) and decreased with the onset of senescence (leaf pairs 8 and higher, data not shown). Gas exchange was typically that of an unstressed CAM plant (Fig. 2A, open symbols). Daytime net CO 2 uptake made up 62 % (± 3 %) of the total daily (24 h) carbon gain. Mean nocturnal carbon gain (NCG N ) was 21.3 ± 4.0 mmol m -2 (n = 9) while mean nocturnal accumulation of malate (~-malate) was 22.0 ± 4.0 mmol m -2 (n = 24). Thus, at the low nighttime temperature of 15 ·C relative recycling of respiratory COb calculated as [(~-malate - NCG N )/ ~-malate], was only about 3 %. Withholding watering led to a near cessation of daytime gas exchange within 2 days (Fig. 2 B). This was due to stomatal closure as can be seen from the close relationship between changes in the integrated daytime carbon uptake and transpiration at the beginning of the drought period and after rewatering (Fig. 3). After relief from drought stomata quickly reopened but the initial gas exchange pattern was restored only after a lag phase of about 4 days (Fig. 2B). Mean daily water economy was highly improved (almost fivefold) at the beginning of the drought (Table 1). However, after prolonged drought it was again reduced by nearly 50 % because nocturnal CO 2 gain (NCG N) also gradually decreased (Fig. 2 A, B). NCGN was reduced by about 65 % after 8 days of drought.

-

~

35

0 E

30

E

--

2.0 1.5 1.0 0.5

~

-

60

(5

50

E

E E

( !)

()

Z

30 20 10 0 0

-

40 30

E

20

!! as

10

-

o

8 z 14

16

18

20

Time of day (h) Fig. I: Diurnal changes in leaf surface related malate content (means ± SO, n=3) of mature leaves (leaf classes 4 to 5, numbered from the rip) of Apttnia cordifolia. Leaf temperature and air humidity were 30'C and 30 % at da(' and 15'C and 80 % at night; PPFD was about 400l1molm-2s- at mid-plant level.

20

15

Fig. 2: (A) Diurnal changes in CO 2 uptake rates Gco) of wellwatered (open symbols; last day of watering) and drought-stressed (closed symbols; day 10 of drought cycle) plants of Apttnia cordifolia. Dark periods are indicated by black bars. (B) Changes in net carbon gain (NCG) at day (open circles), at night (closed circles) and over the whole day (squares) during the course of the experiment. Drought was imposed by withholding watering. Duration of drought is indicated by dotted lines and a do((ed bar. Climatic conditions were as given for Fig. 1.

E

12

10

5

Days of experiment (d)

E

10

B

40

(5

8

8

24

70

20

~

20

Time of day (h)

c: !! c:

5

12

8

25

.!!!

.~""'+-4~"""""""'

-0.5

E

15

.....-........................-.

0.0

~

8

A

2.5

r2= 0.930

10

o o

5

10

15

20

Daytime waterJoss (mol

25

30

m-2 )

Fig. 3: Correlation between changes in net daytime carbon gain (NCGo) and total daytime transpirational waterloss during the course of the experiment. Arrows indicate desiccation and rewatering, the solid line the linear fit.

w. B. HERPPICH and K. PECKMANN

470

Table 1: Changes in daytime, nighttime and 24 h molar water-useefficiency (WUE). Climatic conditions were as given in legend of Fig. I.

before drought beginning of drought end of drought after drought

WUE(day)

WUE (night)

WUE (24 h)

2.0±0.2 -0.3± 1.0 0 1.5±0.2

13.7 ±1.0 28.9 ±6.5 12.0 ±7.0 6.25±1.3

2.9 ±0.2 13.8 ±3.7 6.2 ±2.5 2.02±0.3

-E

C)I

(5

E E c:

.! c:

8 u

;

0

E II) 0

-

C)I

E

(5

E E

-c:

.! CO a;

10

-E

"tJ

.! c:

8 .s

30

-

20

t!

0

4.0

.!

3.0 0

B

50

10 15 5 Days of experiment (d)

20

Fig. 5: Changes of (A) total osmotic content and (B) tissue water content before and during a drought period and after rewatering. Given are means (± SD; n=3) of samples taken 1h (closed circles) or 9 h (open circles) after the beginning of each light phase. All values were related to leaf surface. Water content is given as gdm- 2 to allow easy comparison with values of the degree of succulence published in the literature. Duration of drought is visualized by the dotted lines (first to last day without watering) and the dotted bar.

10 0

B

3.5

~

60 40

c:

4.5

.! c:

8...

0

-E E

A

c:

C)I

(5

E

CJ)

20

::E

C)I

30

.! c:

8

-

A

40

220 200 180 160 140 120 100 80

0

5

10

5

20

Days of experiment (d) Fig.4: Variation of malate (A) and citrate (B) content of leaves before and during drought and after rewatering. Leaves were harvested 1 h after illumination (closed symbols) and in the late afternoon (open symbols). Values (means ± SD; n=3) were related to total surface area. Beginning and end of drought, which was imposed by withholding watering, is indicated by dotted lines. Contrastingly, diurnal fluctuation of malate content increased by about 30 % during the drought cycle (Fig. 4 A). This mainly resulted from an increase (13 %) in nighttime malic acid accumulation and to a minor extent from a more complete depletion of the malate pool (compare morning and evening values, Fig. 4A) . .6-malate was always accompanied by a diurnal fluctuation in citrate content (Fig. 4 B, .6-citrate). When well-watered, .6-citrate was about 25 % that of .6-malate. This proportion increased to almost 50 % during prolonged drought. Contrasting to malate, basic levels (i.e. evening tissue contents) of citrate were almost doubled during drought (Fig. 4 B) and declined again to pre-stress values within 5 days after rewatering. At the beginning of the experiment a similar reaction was observed (Fig. 4) when

plants had been watered sufficiently every day, while water was given only twice a week during growth in the greenhouse. A. cordifolia, thus, showed pronounced osmotic adjustment during drought, i.e. a net accumulation of osmotically active solutes (Fig. 5A). Total osmotic content, calculated on a leaf surface area basis, increased by about 50 %. Almost half of these changes were due to citrate (c£ Figs. 4 B and 5 A). This organic anion also constituted a major organic osmolyticum, making up 15 to 22 % of tissue osmotic content (wellwatered and drought-stressed, respectively). Furthermore, both citrate and osmotic content closely followed any variation in leaf water content (Fig. 5 B). Because water content substantially decreased during the course of the drought, the increase in osmotic concentration, i.e. osmolarity, and, thus, the reduction of the osmotic potential was even more prominent (Fig. 6, triangles). The latter decreased by almost 100%, as did leaf water potential (Fig. 6, squares). Bulk pressure potential seemed to be even higher during drought, but this may not be significant (Fig. 6, circles). During prolonged drought maximum photochemical efficiency (Fy/F M) and, thus, maximum quantum yield of photosynthesis were increasingly depressed (Fig. 7 A). This mainly resulted from a large reduction of FM while mean Fa

Drought response of CAM in Aptmia cordifolia

was more or less constant (Fig. 7 B). In contrast to net CO 2 gain, photosynthetic competence was quickly restored after rewatering (compare Fig. 2 B and Fig. 7 A). Daily means of

471

r2= 0.954

0.80 0.78

0.76

111 Q.

::E

0.74

0.5

.

.

0.72

........ -----,.--------------------_ .... _--------... -----------------------

0.0

0.70 0.68

-0.5

A 200

-1.0

300 400 500 600 Osmolarity (mol m-3)

-1.5

o

5

10

15

0.80

20

0.78

Days of experiment (d) Fig. 6: Means (± SD) of bulk leaf water potentials (squares; n =6), osmotic potentials (triangles; n = 3) and pressure potentials (circles) during progression of drought and after rewatering. Duration of the drought period (day 5 to day 14 of me experiment) is indicated by dotted lines and me dotted bar. Pressure potential was calculated from the difference between water potential and osmotic potential. Samples were harvested about 1 h after illumination.

::E

0.76

~

0.74

u..

0.72

0.70

r2= 0.689

0.68 2.5

0.80

A

Fig. 8: Correlation between Fy/FM and (A) osmolarity of leaf sap and (B) leaf water content of samples measured during drought and after rewatering. Values are given as means ± SD.

0.78

u..::E

"> u..

0.76 0.74

Fo-quenching were only slightly higher at the end of the drought period (0.217 ± 0.018 versus 0.153 ± 0.031), indicating that non-photochemical quenching was not substantially higher under these conditions. No significant diurnal changes of Fy/FM were detectable in well-watered and in droughtstressed plants, while in well-watered A. cordifolia quenching of the initial fluorescence was relevant only in the afternoon (data not shown). Levels of Fy/FM were highly negatively correlated with tissue osmolarity (Fig. 8A) and, to a much smaller degree, positively correlated with leaf water content (Fig.8B).

0.72 0.70

20

3.0 3.5 4.0 Water content (9 dm-2 )

B

Discussion

5

BlBI-m

5

10

15

20

Days of experiment (d) FIg.7: (A) Changes of the ratio of variable (Fy) to maximum (F M ) fluorescence and (B) of the initial (Fo, squares; relative units) and the maximum fluorescence (triangles) of mature leaves (leaf classes 3 t? 5, numbered from the shoot tip) during progressive soil desiccatIon and after rewatering. The first (day 5) and me last day (14) of the drought period are indicated. Given are the means (± SD) of all results obtained during the course of a day (n between 6 and 12).

A. cordifolia is an obligate CAM plant. Under all conditions, i.e. well-watered or drought-stressed, all attributes typical of CAM plants (Kluge and Ting, 1978) were found. Furthermore, even the green stems showed both nocturnal acid accumulation and nighttime CO 2 uptake (Herppich and Peckmann, unpublished data). This contradicts the findings of Treichel (1975) that this taxon exhibits C 3 mode of photosynthesis and no diurnal fluctuation in tissue acid content when well-watered and CAM when salt-stressed. However, these discrepancies may be explained by the fact that this author investigated very young seedlings. In our study at least

472

w. B. HERPPICH and K. PECKMANN

9-month-old cuttings were used. It is well established that ported for several other but not all members of the MesemCAM is enhanced with plant and leaf ageing in many species bryanthemaceae investigated in a large number of field studof the Mesembryanthemaceae (Herppich et al., 1992) and ies in the Namib desert (von Willert et al., 1992). In contrast other families (cf. Kluge and Ting, 1978). Although it has re- to M. crystallinum, where organic anions comprise only a cently been shown that CAM-flexibility is widespread in the little more than 2 % (Herppich et al., 1995), citrate substanMiddayflowers (Herppich, Midgley, von Willert, Veste, sub- tially contributes to the total osmotic content even in wellmitted) it is reasonable that CAM is constitutive in fully de- watered plants of A. cordifolia. Similar or even higher proporveloped leaves of mature plants of A. cordifolia. Nevertheless, tions have also been reported for several tropical trees of the seasonal changes in CAM capacity as found e.g. in Portulaca- genus Clusia (Franco et al., 1994). This emphasizes that citrate may be an important regulatory factor in stabilizing ria afra (Guralnick and Ting, 1987) could not be excluded. Drought slightly enhanced both nocturnal accumulation leaf water status in several CAM plants during drought. of malate as well as its diurnal fluctuation. On the other However, this function has not been systematically investihand, nighttime CO 2 uptake gradually diminished. Thus, the gated up to now. Formation of this osmolyticum from storrelative recycling of respiratory CO 2 at night (Griffiths, 1988) age sugars as well as its remobilisation after relief from stress substantially increased from about 3 % to 70 %. This has also provides the plants with additional reducing power (Liittge, been found in several (Griffiths, 1988), but not in all CAM 1988). As summarized by Franco et al. (1992), reversible plants (Franco et al., 1992). According to Martin et al. (1988) accumulation of citrate during stress also restores carbon it is possible to estimate the amount of water potentially skeletons. saved with carbon cycling by dividing the total nocturnally Additionally, overnight accumulation of this organic acid stored CO 2 by the actual water use efficiency. During pro- and its proportion relative to 6-malate increased substangressing drought a mean of 17 mmol m -2 of CO 2 was recy- tially and reversibly during drought. These results confirm cled. Thus, at a mean daily WOE of 6.15 mmolmol-I about earlier reports by von Willert (c£ von Willert et al., 1992, and 2.75 mol m- 2 H 20 can be assessed as saved, assuming that literature cited therein) that 6-citrate may become more imthe CO2 has otherwise to be taken up during the light phase. portant during prolonged water shortage (Liittge, 1988). In This amount of water equals twice the mean daily transpira- well-watered plants of A. cordifolia a 6-citrate/6-malate ratio tional water loss during the drought period. This points out of 0.25 was found. This lies well in the range reported for the efficiency of the water and carbon conserving function of several members of the Mesembryanthemaceae (Herppich et CAM in A. cordifolia. al., 1995; von Willert et al., 1992) and CAM plants from Nevertheless, despite this effective water conserving stra- other families (c£ Liittge, 1988; and literature cited therein). tegy (von Willert et al., 1992) tissue water content gradually During drought this ratio rose in A. cordifolia and e.g. in saltdecreased by ca. 20 % as soil desiccated. In spite of this, pres- stressed M crystallinum plants (Herppich et al., 1995). Fursure potential was more or less constant during the whole ex- thermore, in several CAM plants from the tropics the 6-citperiment. It was even slightly higher during drought. In rate/6-malate ratio was higher during the dry season (Grifmany other studies it had been shown that it declines with fiths et al., 1989; Popp et al., 1987). decreasing water content (Ruess et al., 1988; Herppich, 1989; Although several hypotheses have been extensively disSchafer and Liittge, 1986). Thus, this constancy indicates cussed (Franco et al., 1992; Liittge, 1988), the physiological that A. cordifolia was able to perform effective osmotic adjust- and ecophysiological roles of diurnal fluctuations in citrate ment, i.e. the reversible net accumulation of osmotically ac- contents as well as their increase in response to drought are tive solutes in response to water shortage (Turner and Jones, by far less clear than those of malic acid. As pointed out by 1980). In addition to the increase in the concentration of the Franco et al. (1992) citrate is a very effective buffer with a cell sap due to decreasing water content, osmotic adjustment maximum buffering capacity within the pH-range of 3-6. led to metabolically controlled decreases in osmotic potential Reduction of tissue water and, thus, the enhanced concentraand, thus, leaf water potential. This should help to maintain tion of cell-sap with progressive desiccation should increase water uptake at steadily falling soil water potential. Investiga- the need of buffering the vacuolar content. This might maintions on osmotic adjustment in CAM plants are rare (Golds- tain high levels of malate accumulation in this compartment tein et al., 1991; Herppich, 1989; Sinclair, 1983). However, as found for several members of the genus Clusia (Franco et Mesembryanthemum crystallinum is known to maintain pres- al.,1992). Additionally, 6-citrate may play an important role in carsure potential during salt stress (Winter and Gademann, 1991) mainly resulting from an increase in NaCl concentra- bon recycling. The breakdown of citrate to pyruvate provides tion in the vacuoles (Herppich et al., 1995). three times more CO 2 than that of malate (Franco et al., In A. cordifolia osmotic adjustment was largely due to the 1992). Assuming a hypothetical constant decarboxylation of reversible accumulation of citrate. In contrast to malate, the both organic acids during the 12 h light period the amount of accumulation of this organic anion was significantly ampli- 12.7 mmol m -2 of citrate accumulated overnight yielded 42 % fied during the progression of drought. Furthermore, both more internal CO 2 than 26.6 mmol m -2 malate. So, even a the morning and the evening content decreased again after smaller 6-citrate should be much more effective in preventrewatering. Thus, citrate made up about half of all changes in ing photoinhibition than 6-malate. Both acids together osmotic content. To our knowledge, this is the first direct evi- could maintain more than 55 % of the maximum late afterdence that this organic acid can play the essential role in os- noon carbon uptake rates in well-watered plants of A. cordifomotic adjustment in a CAM plant. Nevertheless, increased lia. Additional CO2 may be provided if these organic acids citrate contents in response to drought have also been re- were totally oxidized in plant mitochondria (Winter, 1985).

Drought response of CAM in Aptenia cordifolia Despite this internal carbon source maximum photochemical efficiency substantially decreased in drought-stressed plants, as estimated from the lower levels of Fy/FM' The time used for dark adaptation of the leaves (20 min) was sufficient to allow complete relaxation of the fast relaxing component of total non-photochemical quenching, mainly due to non-radiative energy dissipation processes (Bilger and Bjorkman, 1994). Thus, the lasting depression of Fy/FM levels, relative to those in non-stressed plants, should indicate a certain degree of photoinhibition, i.e. the reversible down regulation of photosynthetic performance in response to excessive excitation energy (Osmond, 1994). This was relevant despite of the relative stability of bulk leaf pressure potential due to osmotic adjustment. Fy/FM changed only slightly if at all in detached leaves of C3 plants desiccated in the dark (Epron and Dreyer, 1992). Thus, leaf photosynthetic metabolism seems to be rather resistant to drought stress (Kaiser, 1987). Any short-term reversible reduction of CO 2 uptake may then simply result from increased stomatal resistance (Cornie et al., 1992). Nevertheless, it has been repeatedly shown that in CAM plants ribulose-1,5-bisphosphate carboxylase (RuBisCo) activity declined during the initial phase of drought treatment while that of phosphoenolpyruvate carboxylase (PEPC) may temporarily increase (Guralnick and Ting, 1987; Smith and Eickmeier, 1983). Changes in pigment composition, also potentially leading to a decrease in Fy/FM' were not detected in P. afra during short-term drought (Guralnick and Ting, 1987). Furthermore, investigations on drought responses of detached leaves of Plectranthus marrubioides (Herppich, 1989) and shoots of two Mesembryanthemaceae, Delosperma tradescantioides and Prenia sladeniana (Tiiffers et al., 1995), also indicated pronounced reduction of maximum photochemical efficiency ofPSII with desiccation. Thus, CAM was not able to prevent photoinhibition (Osmond, 1994) in A. cordifolia and in these plants during progression of drought even in moderate light. However, this inhibition was completely reversed overnight after rewatering. Serious damage to the photosynthetic apparatus could therefore be excluded. This may be further substantiated by the constancy of Fo throughout the experiment (von Willert et al., 1995). Nevertheless, the close relationship between changes in Fy/FM and total tissue osmotic concentration or leaf water content may indicate that inhibition of photochemistry did not exclusively result from deprivation of CO 2 and thus orderly photosynthetic energy dissipation, due to stomatal closure. While water content and Fy/FM regain prestress values overnight total recovery of daytime CO 2 uptake and leaf conductance lasted several days. Thus, both photochemistry and dark reactions may be partially regulated differently. In contrast to the findings of Guralnick and Ting (1987) restriction and restoration of daytime gas exchange may be rather independent of leaf water relations in A. cordifolia. It is now accepted that in C 3 plants gas exchange is mainly governed by soil water content during a drought, most probably via the concentration of abscisic acid in the leaf apoplast (Schulze, 1986). Thus, the fast initial step of recovering may indicate stomatal opening due to the relief from this control. The second, slower phase may result from restoration of prestress capacity of photosynthetic enzymes

473

(Guralnick and Ting, 1987) and/or tissue solute concentrations. It is noticeable that a very close positive correlation between leaf water content and nocturnal CO 2 uptake (r2 = 0.854) but not nighttime transpiration could be found in A. cordifolia. There was only a very poor and negative correlation between water relations and 6-malate (? = 0.12). Furthermore, nocturnal malate accumulation was substantially decreased after rewatering, as was 6-citrate. This is in contrast with the finding that CAM is magnified after irrigation in many plants (Hanscom and Ting, 1978; Herppich, 1989; von Willert et al., 1992). However, results presented here may show transients with a low steady state not being reached at the end of the drought period. Experiments under steady state conditions (Herppich, 1989) or long-term drought stress in the field might then yield quite different responses (Hanscom and Ting, 1978; von Willert et al., 1992). Another explanation was outlined by Guralnick and Ting (1987) who proposed that reactions of plants with less CAM plasticity may be different from those with highly flexible CAM, which display partial enhancement of CAM with drought. Nevertheless, our data indicate only limited CAM plasticity in A.

cordifolia.

Acknowledgements

The authors wish to thank Mrs. M. Herppich for very helpful discussion and for carefully and critically reading the manuscript. Furthermore, we are grateful to an unknown referee for many suggestions helping to improve the text. This investigation was supported by a grant of the Deutschen Forschungsgemeinschafi: to W. B. H. K. P. was supported by a Promotionsstipendium (GrFg NW).

References

ADAMS, W. W. III, I. TERASHIMA, E. BRUGNOLI, and B. DEMMIG: Comparison of photosynthesis and photo inhibition in the CAM vine Hoya australis and several C3 vines growing on the coast of eastern Australia. Plant Cell Environ. 11, 173-181 (1988). BILGER, W. and U. SCHREIBER: Energy-dependent quenching of dark-level chlorophyll fluorescence in intact leaves. Photosynth. Res. 10, 303-308 (1986). BILGER, W. and O. BJORKMAN: Relationships among violaxanthin deepoxidation, thylakoid membrane conformation, and nonphotochemical chlorophyll fluorescence quenching in leaves of cotton (Gossypium hirsutum L.). Planta 193,238-246 (1994). CORNIC, G.,]. GHASHGHAIE, B. GENTY, and ].-M. BRIANTAIs: Leaf photosynthesis is resistant to a mild drought stress. Photosynthetica 26, 295-308 (1992). EPRON, D. and E. DREYER: Effects of severe dehydration on leaf photosynthesis in Q;4ercus petraea (Matt.) Liebl.: Photosystem II efficiency, photochemical and non-photochemical quenching and electrolyte leakage. Tree Physiol. 19, 273-284 (1992). FRANCO, A. c., E. BALL, and U. LOTrGE: Differential effects of drought and light levels on accumulation of citric and malic acids during CAM in Clusia. Plant Cell Environ. 15,821-829 (1992). FRANCO, A. c., E. OLIVARES, E. BALL, U. LOTrGE, and A. HAAGKERWER: In situ studies of Crassulacean acid metabolism in several sympatric species of tropical trees of the genus Clusia. New Phythol. 126, 203-211 (1994).

474

w. B. HERPPICH and K. PECKMANN

GOLDSTEIN, G.,j. L. ANDRADE, and P. S. NOBEL: Differences in water relation parameters for the chlorenchyma and the parenchyma of Opuntia ficus-indica under wet versus dry conditions. Aust. j. Plant Physiol. 18, 95-107 (1991). GURALNICK, L. j. and I. P. TING: Physiological changes in Portulacaria afra (L.) jacq. during a summer drought and rewatering. Plant Physiol. 85,481-486 (1987). GRIFFITHS, H.: Crassulacean acid metabolism: A re-appraisal of physiological plasticity in form and function. Adv. Bot. Res. 15, 43-92 (1988). GRIFFITHS, H., j. A. C. SMITH, U. LOnGE, M. Popp, W. j. CRAM, M. DIAZ, H. S. j. LEE, E. MEDINA, C. ScHAFER, and K.-H. STIMMEL: Ecophysiology of xerophytic and halophytic vegetation of a coastal alluvial plain in northern Venezuela: Iv. Tillandsia jlexuosa Sw. and Schomburgkia humboldtiana Reichb., epiphytic CAM plants. New Phytol. 111,273-282 (1989). HMG-KERWER, A., A. C. FRANCO, and U. LOnGE: The effect of temperature and light on gas exchange and acid accumulation in the C 3-CAM plant Clusia minor L. j. Exp. Bot. 43, 345-352 (1992). HANSCOM, Z. III and I. P. TING: Irrigation magnifies CAM-photosynthesis in Opuntia basilaris (Cactaceae). Oecologia 33, 1-15 (1978). HERPPICH, M., w. B. HERPPICH, and D. j. VON WILLERT: Diurnal rhythm in citric acid content preceded the onset of nighttime malic acid accumulation during metabolic changes from C 3 to CAM in salt-stressed plants of Mesnnbryanthnnum crystal/inum. j. Plant Physiol. 147, 38-42 (1995). HERPPICH, W. B.: CAM-Auspragung in Pkctranthus marrubioides Benth. (Fam. Lamiaceae). EinfluB der Faktoren Licht, Blattemperatur, Lufrfeuchtigkeit, Bodenwasserverfiigbarkeit und Blattwasserzustand. Ph.D. Thesis, Westfalische Wilhelms-Universitlit, Miinster (1989). HERPPICH, W. B., M. HERPPICH, and D. j. VON WILLERT: The irreversible C3 to CAM shift in well-watered and salt-stressed plants of M~snnbryanthnnum crystalJinum is under strict ontogenetic control. Bot. Acta 105, 34-40 (1992). HOLTHE, P. A., L. S. L. STERNBERG, and I. P. TING: Developmental control of CAM in Pepnvmia scandms. Plant Physiol. 84, 743747 (1987). JACOBSEN, H.: Das Sukkulentenlexikon, pp. 381. Gustav Fischer Verlag, Stuttgart, New York (1981). KAISER, W. M.: Methods for studying the mechanism of water stress effects on photosynthesis. In: TENHUNEN, j. D., F. M. CATARINa, O. L. LANGE, and W. D. OECHEL (eds.): Plant Responses to Stress. Functional Analysis in Mediterranean Ecosystems, NATO ASI Ser., Vol. G 15, pp. 77-93. Springer-Verlag, Berlin, Heidelberg, New York (1987). KLUGE, M. and I. P. TING: Crassulacean Acid Metabolism. Analysis of an ecological adaptation. Ecological studies Vol. 30. SpringerVerlag, Berlin, Heidelberg, New York (1978). LOnGE, U.: Day-night changes of citric-acid levels in crassulacean acid metabolism: Phenomenon and ecophysiological significance. Plant Cell Environ. 11, 445-451 (1988). MARTIN, C. E., M. HIGLEY, and w.-Z. WANG: Ecophysiological significance of COz-recyding via Crassulacean acid metabolism in Talinum caiycinum Engelm. (Pottulacaceae). Plant Physiol. 86, 562-568 (1988). MOLLERING, H.: Malatbestimmung. In: BERGMEYER, H. U. (ed.): Methoden der enzymatischen Analyse, Vol. 2, pp. 1636-1639. Verlag Chemie, Weinheim (1974).

MOllElUNG, H. and W. GRUBER: Determination of citrate with lyase. Annal. Biochem. 17, 369-379 (1966). OSMOND, C. B.: What is photoinhibition? Some insights from comparison of shade and sun plants. In: BAKER, N. Rand j. R. BoWYER (eds.): Photoinhibition of Photosynthesis, pp. 1-24. BIOS Scientific Publishers, Oxford (1994). Popp, M., D. KRAMER, H. LEE, M. DIAZ, H. ZIEGLER, and U. LOnGE: Crassulacean acid metabolism in tropical dicotyledonous trees of the genus Clusia. Trees 1,238-247 (1987). RUESS, B. R, S. FERRARI, and B. M. ELLER: Water economy and photosynthesis of the CAM plant Sm~cio medley-woodii during increasing drought. Plant Cell Environ. 11, 583- 589 (1988). SCHAFER, C. and U. LOnGE: Effects of water stress on gas exchange and water relations of the succulent epiphyte, Kalanchoe uniflora. Oecologia 71, 127-132 (1986). SCHULZE, E. D.: Carbon dioxide and water vapour exchange in response to drought in the atmosphere and in the soil. Annu. Rev. Plant Physiol. 37, 247-274 (1986). SINCLAIR, R: Water relations of tropical epiphytes. II. Performance during droughting. j. Exp. Bot. 34, 1664-1675 (1983). SMITH, j. A. C. and U. LtiTTGE: Day-night changes in leaf water relations associated with the rhythm of crassulacean acid metabolism in Kalanchoed4igrnnontiana. Planta 163,272-282 (1985). SMITH, T. L. and W. G. EICKMEIER: Limited photosynthetic plasticity in Sedum puUch~l/um Michx. Oecologia 56, 374-380 (1983). TREICHEL, S.: Crassulaceensaurestoffwechsel bei einem salztoleranten Venreter der Aizoacea: Aptmia cordifolia. Plant Sci. Lett. 4, 141-144 (1975). TOFPERS, A., c. E. MARTIN, and D. j. VON WILLERT: Possible water movement from older to younger leaves and photosynthesis during drought stress in two succulent species ftom South Mrica, D~losp~ tradescantioides Bgr. and Prmia sladmiana L. Bot. (Mesembtyanthemaceae). j . Plant Physiol. 146, 177-182 (1995). TURNER, N. C. and M. M. JONES: Turgor maintenance by osmotic adjustment: A review and evaluation. In: TURNER, N. C. and P. J. KRAMER (eds.): Adaptation of Plants to Water and High Temperature Stress, pp. 87-104. j. Wiley and Sons, New York, Chichester, Brisbane, Toronto (1980). VON CAEMMERER, S. and G. D. FARQUHAR: Some relationships between the biochemistty of photosynthesis and the gas exchange of leaves. Planta 153,376-387 (1981). VON WILLERT, D. j., B. M. ELLER, M. j. A. WERGER, E. BRINCKMANN, and H.-D. IHLENFELDT: Life strategies of succulents in deserts. With special reference to the Namib desert. Cambridge Studies in Ecology. Cambridge University Press, Cambridge, New York, Port Chester, Sydney (1992). VON WILLERT, D. j., R MAlYSSEK, and W. B. HERPPICH: Experimentelle Pflanzenokologie, Gtundlagen und Anwendungen. Georg Thieme Verlag, Sturtgart (1995). WINTER, K.: Crassulacean acid metabolism. In: BARBER, j. and N. R BAKER (eds.): Photosynthetic Mechanisms and the Environment, pp. 329-387. Elsevier, Amsterdam (1985). WINTER, K. and R GADEMANN: Daily changes in CO 2 and water vapour exchange, chlorophyll fluorescence, and leaf water relations in the halophyte M~snnbryanthnnum crystallinum during the induction of Crassulacean acid metabolism in response to high NaCl salinity. Plant Physiol. 95, 768-776 (1991).