assimilation and spatiotemporal dynamics of photosynthesis in leaves ...

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Correlation between photorespiration, CO2-assimilation and spatiotemporal dynamics of photosynthesis in leaves of the C3-photosynthesis/crassulacean acid ...
Trees (2007) 21:531–540 DOI 10.1007/s00468-007-0146-y

ORIGINAL PAPER

Correlation between photorespiration, CO2-assimilation and spatiotemporal dynamics of photosynthesis in leaves of the C3-photosynthesis/crassulacean acid metabolism-intermediate species Clusia minor L. (Clusiaceae) H. M. Duarte Æ U. Lu¨ttge

Received: 3 January 2006 / Revised: 3 January 2007 / Accepted: 11 May 2007 / Published online: 19 June 2007  Springer-Verlag 2007

Abstract The tree Clusia minor L. (Clusiaceae) operates with different modes of photosynthesis in response to different combinations of environmental parameters. Here plants were subjected to experimental conditions eliciting performance of C3-photosynthesis and crassulacean acid metabolism (CAM), respectively. A combination of instruments was used to determine CO2 and water vapour gas exchange, relative quantum use efficiency of photosynthesis (FPSII) and for the first time in such studies also photorespiration simultaneously with the other parameters. In the C3-mode photorespiration was constant during the light period, where oxygenase activity of ribulose-bisphosphate carboxylase/oxygenase (RubisCO) ðJO2 Þ was ranging between 32.1 and 35.7% of total RubisCO activity. In the CAM-mode photorespiration depended on the CAM phases. In phase II in the morning JO2 was 15.6%. In phase IV in the afternoon initially it was 37.9% and then declined to 17.6% of total RubisCO activity towards the evening. Anatomically leaves of C. minor are differentiated in palisade and spongy parenchyma with an internal air space of 9.3% of the total volume and therefore could be structurally homobaric. However, heterogeneity of FPSII under both non-photorespiratory and photorespiratory conditions in the C3- and CAM-mode indicated that lateral diffusion

Communicated by M. Ball. H. M. Duarte Rua Botucato 460, Aptdo. 803, bl. 4, Grajau´, 20541-340 Rio de Janeiro, Brazil e-mail: [email protected] U. Lu¨ttge (&) Institut fu¨r Botanik, Darmstadt University of Technology, Schnittspahnstr. 3–5, 64287 Darmstadt, Germany e-mail: [email protected]

of CO2 and O2 were subject to limitations showing that leaves are functionally heterobaric. Keywords Clusia  Crassulacean acid metabolism  Heterobaric leaf  Homobaric leaf  Photorespiration  Photosynthesis

Introduction Clusia is the only genus of dicotyledonous trees and shrubs expressing crassulacean acid metabolism (CAM). Many of the species are C3-photosynthesis/CAM-intermediate, but Clusia minor L. appears to be the most flexible species in its photosynthetic metabolism and ecophysiological behaviour (Lu¨ttge 2007a, 2007b). It switches back and forth reversibly between various modes, such as (1) full C3-photoynthesis, (2) full CAM, (3) CAM-idling, where stomata remain closed night and day and internal CO2 is recycled via synthesis/remobilization of organic acids and (4) CAM-cycling, where stomata remain closed in the night and respiratory CO2 is recycled via malate, and stomata open in the day for CO2 uptake from the atmosphere in addition to the CO2 remobilized from nocturnally stored malate (Lu¨ttge 2006, 2007a). A variety of environmental factors, mainly water, irradiance, temperature and nitrogen nutrition, can affect switches between these modes (Franco et al. 1991; Haag-Kerwer et al. 1992; Lu¨ttge 2006). In the present study we applied simultaneous measurements of several parameters of photosynthesis with a unique online combination of instruments to characterize its behaviour in the C3- and CAM-mode. Gas exchange measurements were used to analyse net CO2 exchange ðJCO2 Þ and leaf conductance for water vapour ðgH2 O Þ and to calculate internal CO2 partial pressure ðpiCO2 Þ: For an

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assessment of photorespiration non-photorespiratory conditions were established by short-term application of air with only 1% O2. Hence, this is the first study of Clusia where photorespiration was recorded online with other parameters of photosynthesis so that carboxylase and oxygenase activities of ribulose-bis-phosphate carboxylase/ oxygenase (RubisCO) could be compared. A chlorophyll fluorescence imaging system provided spatiotemporal resolution of quantum use efficiency of photosystem II (FPSII) over entire leaves. It is a relative measure of energy use in photosynthesis, and together with the gas exchange measurements it allowed assessment of the energy use relations of the carboxylase and oxygenase functions of RubisCO, respectively. The FPSII-imaging also allowed studying patchiness of photosynthetic activity in the leaves because using a special algorithm the heterogeneity of FPSII over the leaf could be calculated. Heterogeneity is related to constraints of lateral diffusion of CO2 within leaves. It is primarily known from stomatal patchiness, where patchy opening/ closing over a leaf causes internal differences of piCO2 ; and hence, of photosynthetic activity in the leaf. Such differences of piCO2 may be equalized by lateral diffusion of CO2 in the leaves. Heterogeneity is also seen in photosynthetic dynamics caused by partial shading of leaves, e.g. in light fleck regimes, where lateral CO2 diffusion mediates source/ sink relationships of CO2 within the leaf (Pieruschka et al. 2006). Another example is artefacts occurring in gas exchange measurements when the clip attaching leaves to porometers is partially darkening the leaves (Jahnke and Krewitt 2002; Pieruschka et al. 2005; Jahnke and Pieruschka 2006). In relation to CAM in the obligate CAM-plant Kalanchoe¨ daigremontiana Hamet et Perrier it has been noted that in the daytime high internal CO2-concentration gradients may be built up because of a non-synchronized patchy spatiotemporal interplay of internal CO2 enrichment due to decarboxylation of organic acids accumulated in the night and exhaustion of the organic acid pool and CO2 depletion, respectively (Duarte et al. 2005). This occurs particularly in the transitions between CAM phases, i.e. between phase II where stomata are still open and phase III where stomata are closed and organic acid remobilization and decarboxylation behind closed stomata dominates, and between phase III and IV, when stomata begin to open again (Rascher et al. 2001; Rascher and Lu¨ttge 2002). In phase III when internal CO2 concentration is high lateral diffusion in K. daigremontiana is substantial (Duarte et al. 2005). In the endogenous circadian rhythm of CAM in K. daigremontiana synchronization and desynchronization dynamics of individual oscillators in cells and patches of the leaves are observed and lateral CO2 diffusion was thought to be involved in a CO2-signalling function for

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synchronization (Rascher et al. 2001; Rascher and Lu¨ttge 2002). The question of lateral diffusion has led to the concepts of homobaric and heterobaric leaves (Terashima 1992). Where rapid diffusion prevents heterogeneity and piCO2 is homogenous within the leaves, the leaves are called homobaric. Where anatomical constraints hinder diffusion, the leaves are heterobaric. However, it has also been noted that leaves may be physiologically or functionally heterobaric as it is the case with K. daigremontiana (Maxwell et al. 1997). The internal airspace of the functionally heterobaric K. daigremontiana is only 3% of the total volume (Duarte et al. 2005) but in C. minor it is 9.6% (Duarte 2006). This has led us to the hypothesis that the C3/CAMintermediate C. minor might be comparatively more homobaric than the obligate CAM species K. daigremontiana. In addition to lateral diffusion of CO2 related to the carboxylase function of RubisCO O2 distribution within the leaves related to the oxygenase function of RubisCO may also be important. Photorespiration has a particularly high demand for energy (Osmond and Grace 1995; Heber et al. 2001; Heber 2002) and since FPSII is a measure of photosynthetic energy use a further hypothesis tested was that photorespiration would reduce FPSII heterogeneity.

Materials and methods Plants of C. minor were vegetatively propagated from a mother plant of the Clusia collection of the Botanical Garden of the Technical University of Darmstadt and grown in the phytotron at Darmstadt potted in the soil Fruhstorfer-Einheitserde LD-80 (Industrie Erdenwek, Lauterbach, Germany) under a day/night temperature regime of 28/20C and a relative air humidity of 65–80%. During winter the daily photoperiod was extended to 12 h using 400 W halogen lamps (Scatto-TS and HQI-TS WLD lamps, Osram, Frankfurt/M, Germany). Leaves of the third leaf pair from the top of plants that had developed at least ten leaf pairs were used for the experiments. Prior to the measurements of photosynthesis parameters plants were kept for at least 7 days at a constant temperature of 21 or 25 or 31C day and night and an irradiance of 120 lmol m–2 s–1 (k = 400–750 nm). The performance of C3-photosynthesis and CAM was induced under these conditions by regularly watering and withholding water for 3–4 days, respectively, and checked via enzymatic tests of night/day changes of malate and citrate concentration in leaf sap according to Mo¨llering (1974, 1985). Gas exchange of CO2 ðJCO2 Þ and water vapour ðJH2 O Þ was measured using a mini cuvette system (CMS400) with climate regulation (GK-020) (H. Walz, Effeltrich, Germany) according to Lange et al. (1984) with an infrared

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gas analyser (BINOS 100 4P, Rosemount, Hanau, Germany). The cuvette temperature was regulated via a thermistor so that the leaf temperature measured via a NiNiCr-thermo element could be kept constant. Relative air humidity in the cuvette was regulated via a cold trap at the inlet of the mini cuvette. Irradiance was measured via a silicon photo-element calibrated against a LI-COR quantum sensor LI 190 SZ (LI-COR Inc., Lincoln, NE, USA). The leaf was enclosed in the gas tight cuvette and remained attached to the plant in the controlled climate chamber of the phytotron. Measurements were recorded every 60 s. Leaf conductance for water vapour, (gH2 O ) and internal CO2 partial pressure ðpiCO2 Þ were calculated from JCO2 and JH2 O according to Eqs. 1 and 2: gH 2 O ¼ piCO2

¼

JH 2 O VPD paCO2

pixels and 14 bit intensity steps (16,384 grey tone steps). The camera had a macro lens (Nikkor, Nikon, Japan) with a k < 655 nm filter (RG655, 5 mm thickness, Schott, Mainz, Germany) eliminating light reflected from the leaf. A relative measure of quantum use efficiency of electron transport of photosystem II, FPSII, was then obtained as UPSII ¼ ðHIGH  LOWÞ=HIGH

corresponding to effective quantum yield of photosystem II, 0 0 DF ¼ ðFm  FÞ=Fm (Schreiber and Bilger 1993). Values are depicted using a colour code. Heterogeneity of FPSII over the leaves was calculated in relation to Hu¨tt and Neff (2001) using a nearest neighbour matrix algorithm, where the measure of heterogeneity gives the distance to the nearest neighbour in the matrix B at the time t:

ð1Þ 1:6  JCO2  gH2 O

I½B ¼ ð2Þ

where VPD is leaf to air water vapour pressure difference and paCO2 is atmospheric CO2 partial pressure. Very low gH2 O causes errors in the calculations of piCO2 ; and as it is in the denominator in Eq. 2 these errors may result in an important underestimation of piCO2 : This is a particular problem in phase III of CAM. However, due to the CO2concentrating effect of phase III (Lu¨ttge 2002) piCO2 is always very high and conclusions based on observations of increased piCO2 in this phase are not affected qualitatively as piCO2 may even be higher quantitatively than calculated by Eq. 2. Chlorophyll fluorescence of the leaf enclosed in the gas exchange cuvette was recorded by the chlorophyll fluorescence camera system constructed and described by Rascher et al. (2001) and Rascher and Lu¨ttge (2002) after Siebke and Weis (1995). The illumination system consisted of eight halogen lamps (ENH 120 V 250 W, Sylvania, Japan) where light filters KG-1, 5 mm thickness (Schott, Mainz, Germany) and blue–green no. 9782 4-96, 5 mm thickness (Corning Inc. Jamaica, USA) provided that only light of k < 650 nm could pass. Pictures were taken at low ambient irradiance (120 lmol m–2 s–1) called LOW related to steady-state fluorescence of chlorophyll a of PSII (F), and under light pulses of 1,200 lmol m–2 s–1 of 800 ms duration, which were saturating in the plants grown at 120 lmol m–2 s–1 and called HIGH related to maxi0 mum fluorescence (Fm ). A constant voltage element assured constant irradiance for the total time of experiments. The detection system consisted of a computer regulated digital camera (AP-1, Apogee Instruments Inc., Tucson, AZ, USA) with a cooled CCD-chip (KAF-0401M, Eastman Kodak, Rochester, NY, USA) and a field of 768 · 512

ð3Þ

 1 1 X 1 X    aij  b   AB jV j ði;jÞ2A Nij b2N ij

ð4Þ

B

where |V| = number of elements in the space V according to the maximum difference of two states, a, b = entries in the picture matrix, i,j = matrix indices, Nij = neighbourhood around the matrix position (i,j), |Nij| = number of neighbours, AB = leaf area considered. To suppress photorespiration and establish non-photorespiratory conditions in the cuvette a computer regulated device was constructed (Duarte 2006) allowing online switches between atmospheric air and an artificial air mixture of 1% O2, 0.04% CO2 and 98.96% N2 (Messer, Griesheim, Germany). The mixture with 1% O2 was applied for 20 min at intervals. Immediately after the application of 1% O2 net CO2 exchange increased rapidly but several minutes were required to replace the atmospheric air with the 1% O2 gas mixture in the system after which net CO2 exchange under 1% O2 could be determined. The rate of photorespiration was then obtained by the value of net CO2 uptake under 1% O2 minus CO2 uptake under 21% O2 of the atmospheric air, i.e. the maximum carboxylation rate of RubisCO under 1% O2 minus the normal carboxylation rate of RubisCO under 21% O2 was taken as the oxygenation rate of RubisCO or the rate of photorespiration ðJO2 Þ: Relative photorespiration (rel:JO2 Þ was calculated as per cent of maximum carboxylation rate.

Results Acclimation of plants for performance of C3-photosynthesis or crassulacean acid metabolism Analyses of night/day changes (D) of organic acids and net exchange of CO2 integrated ðJCO2 -integr) for the dark

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Table 1 Night/day changes of organic acids (D malate and D citrate) and net exchange of CO2 integrated for the dark period and light period, respectively, of plants of C. minor well watered and droughted for 3–4 days under three different constant temperatures Watered

Droughted

21C

25C

30C

21C

25C

30C

D Malate (mM m–2)



–1





+50

D Citrate (mM m–2)



–6





+96



JCO2 -Integr (mM m–2) light period

268

411

304

112

246

88

JCO2 -Integr (mM m–2) dark period

–18

–16

–22

57

47

23

30

piCO [Pa] 2

Fig. 1 Relation of internal CO2 partial pressure, piCO2 ; and leaf conductance for water vapour at three different temperatures in the C3- and CAM-mode. Open circles indicate 21C, closed triangles indicate 25C and open squares indicate 30C

C3



CAM

20

10

0 0

20

40

60

80

100 120 140

0

20 -2

40

60

80

100 120 140 160

-1

gH O [mmol m s ] 2

period and light period, respectively, are shown in Table 1. Plants droughted for 3–4 days performed CAM at the three temperatures checked as indicated. Hence, acclimation to performance of the two respective modes of photosynthesis appeared to be successful. CO2-uptake was higher at 25C in both modes of photosynthesis. Night/day changes of organic acids measured at 25C show that D malate matched night JCO2 -integr. Therefore, most of the malate synthesized was derived from external CO2, and there was no internal recycling of respiratory CO2 via phosphoenolpyruvate carboxylase (PEPC) and malate. However, there was substantial cycling of carbon via citrate, which is futile in terms of carbon gain (Lu¨ttge 1988, 2006, 2007a). Figure 1 shows the relations between internal CO2partial pressure ðpiCO2 Þ and leaf conductance for water vapour ðgH2 O Þ derived from the gas exchange curves recorded at the three temperatures. For the C3-mode measurements at all temperatures follow the same curvilinear relationship, suggesting that under all conditions tested stomatal opening determines piCO2 : For the CAM-mode the pattern was quite different reflecting the three light period phases of JCO2 and gH2 O of CAM, i.e. stomatal opening in the early morning (phase II) followed by stomatal closure (phase III) and then stomatal opening again in the later afternoon (phase IV) (Osmond 1978). The pattern strongly depended on temperature. At 30C the gH2 O =piCO2 -curve was Ushaped, where the higher piCO2 levels at low gH2 O relate to phase III, when stomata close in the light period and the CO2-concentrating mechanism of malate remobilization

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and decarboxylation increases piCO2 (Lu¨ttge 2002). The lowest piCO2 levels at medium gH2 O and the medium piCO2 levels at the highest gH2 O relate to phases IV in the later afternoon and II in the early morning, respectively. However, both piCO2 and gH2 O were lower at 30C than at the other two temperatures, where gas exchange was higher in both the dark and the light period (Table 1). The U-shape was less pronounced although also seen at the two lower temperatures. As for net CO2 exchange (Table 1) the highest values of piCO2 and gH2 O were obtained at 25C. Clearly, in the CAM-mode piCO2 is controlled by both the state of stomatal opening and the CO2-concentrating mechanism of CAM. Diurnal dynamics of photosynthesis The temperature of 25C, which yielded the best performance among the three temperatures considered above in both the C3- and the CAM-mode, was used for a more detailed analysis. In the dark period curves of JCO2 and gH2 O showed the simple pattern of close to zero gas exchange and stomatal closure in the C3-mode and CO2 uptake via open stomata in the CAM-mode (Table 1). In the light period in the C3-mode (Fig. 2) the curves for JCO2 ; gH2 O and piCO2 closely followed each other confirming stomatal control of gas exchange and photosynthesis. In the interveinal lamina tissue FPSII followed the curves of JCO2 and gH2 O and was related to bulk piCO2 of the leaf. In the major vein the increase of FPSII was delayed in the

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160

40

6

80

4 40

30

20

i

120 8

p CO2 [Pa]

-2

10

-2

-1

-1

JCO2 [µmol m s ]

CAM

gH2O [mmol m s ]

C3

12

10

2

0.8

rel. φPSII

0 1.0

c lamina

0.8

b

0.6

0.6 vein

0.4

d

0.2

0.4 0.2

0.0

1 0

2 3

3

6

4 9

a1 12 0

2

3

3

6

4

0.0

5

9

0

Heterogeneity (rel.)

0 1.0

12

Time [h]

Fig. 2 Light period dynamics of photosynthetic parameters in the C3and CAM-mode. JCO2 ; net exchange of CO2 ; gH2 O ; leaf conductance for water vapour, piCO2 internal CO2 partial pressure, rel. FPSII, relative quantum use efficiency of photosystem II and its heterogeneity on the leaf. Numbers on the abscissa indicate the times when the

Fig. 3 Relation of relative quantum use efficiency of photosystem II (rel. FPSII) and piCO2 in the C3- and CAM-mode. Small letters refer to time points in Fig. 2

corresponding figures depicted in Fig. 4 were taken by the chlorophyll fluorescence camera. Lower case letters refer to time points in Fig. 3. The curves depict one representative experiment each for the C3- and the CAM-mode, respectively. Two additional experiments showed qualitatively and quantitatively the same pattern

C3 1.0

CAM 1.0

vein

0.8

b

vein

0.5 r2=0.83 lamina

0.8

rel. φPSII

rel. φPSII

0.6 0.4 1.0

c

d 0.0 1.0

a b

lamina

c

d

0.5

0.6 r2=0.52 0.4 15

20 i

p CO2 [Pa]

morning and the decrease was accelerated in the afternoon and higher values were reached at midday. The correlation between FPSII and piCO2 was much steeper in the vein than in the lamina (Fig. 3). Heterogeneity was very low and constant throughout the day (Fig. 2) as also shown by the representative pictures obtained with the chlorophyll fluorescence camera (Fig. 4). Only at the very beginning of the light period there was a small indication of heterogeneity which was probably due to the abrupt dark/light transition. In the light period in the CAM-mode (Fig. 2) the three light period CAM phases of JCO2 and gH2 O were clearly expressed. In the interveinal lamina tissue FPSII increased

25

a

0.0 10

15

20

25

30

35

i

p CO2 [Pa]

rapidly in the early light period at the beginning of phase II, together with JCO2 ; gH2 O and piCO2 : The latter parameter showed a small transient decline (Fig. 2). At this time maximum heterogeneity of FPSII was observed (see also Fig. 4). Towards the end of phase II JCO2 and gH2 O declined to minimum values but piCO2 remained constant showing that internal CO2 levels were not supported by CO2 uptake via the stomata but that internal malate decarboxylation began to supply CO2 for photosynthesis. Concomitantly FPSII continued to rise indicating increased energy demand of photosynthesis during phase II. Heterogeneity declined to minimum values. Subsequently with the beginning of

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Fig. 4 Pictures of relative quantum use efficiency of photosystem II (rel. FPSII) taken by the chlorophyll fluorescence camera during the light period in the C3-mode and CAM-mode under normal air (21% O2) as indicated in Fig. 2 and during applications of air with 1% O2 as indicated in Fig. 5

phase III piCO2 rose up to its maximum in the middle of the light period when JCO2 and gH2 O were at their minimum. The UPSII did not change much during this time and heterogeneity remained low as in the C3 -mode. As stomata opened in phase IV, piCO2 declined again, CO2 was taken up from the atmosphere, FPSII declined to lower values and heterogeneity rose progressively towards the end of the light period. On the major vein FPSII rose more slowly at the beginning, reached higher values in the middle and declined more rapidly at the end of the light period. The relation between FPSII and piCO2 was more complex than in the C3-mode, and it reflected the CAM phases (Fig. 3). In phase III piCO2 was high and the increase of FPSII indicates a progressively increased energy demand. On the vein this phase was also slower than on the lamina. While on the lamina FPSII was stable in phase III it still increased in the vein and reached its maximum values. The dependency of FPSII on piCO2 was particularly pronounced in phase IV and was stronger in the vein than in the lamina. Diurnal dynamics of photorespiration During the light period 1% O2 was applied for 20 min four times in the C3-mode and five times in the CAM-mode to cover the CAM phases. In the first 4 min of the application

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of 1% O2 there was always an instrumental measuring artefact. These data were deleted from the curves presented in Fig. 5, where JCO2 is depicted with the 21% O2 background and the 1% O2 applications for 20 min as indicated by the arrows, together with gH2 O ; piCO2 ; FPSII and heterogeneity for one representative run out of three replicates each for the C3- and CAM-mode, respectively. In the C3-mode immediately after switching to 1% O2, i.e. non-photorespiratory conditions, JCO2 increased and piCO2 decreased indicating increased RubisCO carboxylation activity due to a rapid reduction of its oxygenase activity. Stomata responded more slowly and gH2 O increased more slowly suggesting that the response of stomata was not related to oxygen itself but to decreased piCO2 due to increased carboxylation activity of RubisCO. The gradual increase of gH2 O led to a further increase of CO2 uptake so that JCO2 increased during the 1% O2-pulse, which is a good illustration of the interaction of photorespiratory and stomatal control. This is also reflected in FPSII (see also Fig. 4 in addition to Fig. 5) indicating the energy demand which was lower under 1% O2 than under 21% O2. The colour code in Fig. 4 indicates reduced FPSII, i.e. increased fluorescence as expected when photochemical energy use by photorespiration is eliminated. This is due to the particular energy demand of photorespiration

Trees (2007) 21:531–540 C3

CAM -1

40

80

piCO2 piCO2

4

40 2

30 20

i

6

p CO2 [Pa]

-2

-2

gH2O [mmol m s ]

120

-1

JCO2 [µmol m s ]

8

10

0 1.0

0 1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

1/2 0

3/4 3

5/6 6

7/8 9

1/2 12 0

3/4 3

5/6 6

7/8 9

9/10 12

0

Heterogeneity (rel.)

rel. φPSII

Fig. 5 Light period dynamics of photorespiratory activity as revealed by applications of air with only 1% O2 (arrows) causing non-photorespiratory conditions so that the values of the photosynthetic parameters shown, viz. JCO2 ; gH2 O ; piCO2 ; rel. FPSII and heterogeneity (for explanation of symbols see caption of Fig. 2), under 1% O2 minus the values under 21% O2 indicate the effects of photorespiration. Numbers on the abscissa indicate the times when the corresponding pictures depicted in Fig. 4 were taken by the chlorophyll fluorescence camera

537

0.0

Time [h]

(Osmond and Grace 1995; Heber et al. 2001; Heber 2002). Moreover, under the non-photorespiratory conditions with 1% O2, i.e. under the lower energy use by carboxylase activity of RubisCO alone, the spatial distribution of FPSII over the leaf was much more heterogeneous than in normal air with 21% O2 when the high energy demand of photorespiration contributed to energy use by photochemical work. Thus, the strong energy demand of photorespiration in addition to that of the carboxylase activity of RubisCO led to a more homogenous energy use over the entire leaf. All effects were reversible when 1% O2 was replaced again by 21% O2. Quantitatively the effects were larger in the middle than at the beginning and at the end of the light period. The relative photorespiratory activity, rel: JO2 ; revealed by the four subsequent applications of 1% O2 in Fig. 5 was 33.7 ± 2.2, 35.0 ± 3.2, 35.7 ± 2.5 and 32.1 ± 3.5% at the times indicated. In the CAM-mode the effects of 1% O2 were determined by the CAM phases. 1% O2 was first applied in phase II when JCO2 and gH2 O were still rising. When photorespiration was eliminated JCO2 and gH2 O continued to rise. In stark contrast to the C3-mode also piCO2 continued to increase, which shows that organic acid remobilization and decarboxylation must have begun to contribute to piCO2 : The FPSII declined which showed that notwithstanding this increase of piCO2 due to organic acid decarboxylation photorespiration prior to the 1% O2-pulse had used irradiance energy so that the beginning CO2-concentrating effect of acid decarboxylation did not hinder photorespiratory activity. In this phase photorespiratory activity was 15.6 ± 6.2% of total. Heterogeneity decreased during the transition from phase II to phase III and the application of 1% O2 had not affected this tendency. In phase III stomata were

largely closed and gH2 O was very low. Application of 1% O2 had no effect in phase III. This may have been due to the large resistance to diffusion of external air into the leaves. On the other hand at high piCO2 in phase III photorespiration may have already been suppressed without application of 1% O2, but as we note in the discussion below, also piO2 is increased in phase III and therefore the assessment of photorespiration is very difficult in this phase of the CAM cycle. There were very small responses of heterogeneity (Figs. 4, 5) which were related to the lateral edges of leaves and possibly these locations were also responsible for the minimal JCO2 and gH2 O still observed in phase III (Fig. 5). The two applications of 1% O2 in phase IV showed a pattern very similar to that of the C3-mode, i.e. JCO2 ; gH2 O and heterogeneity increased while piCO2 decreased. Relative photorespiratory activity, rel: JO2 ; was 37.9 ± 2.9 and 17.6 ± 4.2% as revealed by these subsequent 1% O2-pulses. This may indicate that RubisCO was gradually down regulated towards the end of phase IV and PEPC was taking over driving CO2-uptake.

Discussion Due to their very homogenous mesophyll anatomy with isodiametric almost spherical cells and no differentiation of palisade and spongy parenchymas leaves of the obligate CAM plant K. daigremontiana were considered anatomically homobaric. However, the dense packing of the cells with an internal air space of the leaves of only 3% of the total volume (Duarte et al. 2005) was shown to cause high resistance to internal gas diffusion, and therefore the leaves must be considered to be functionally heterobaric (Max-

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well et al. 1997). This was not expected for C. minor which has a C3-leaf like anatomy with well separated palisade and spongy parenchymas, large intercellular air spaces in the latter and a larger total volume of the internal air space of 9.3% (Duarte 2006). Locally different stomatal resistances for CO2 diffusion and piCO2 in the leaf lamina in the C3mode can cause photosynthetic patchiness in anatomically and functionally heterobaric leaves (Beyschlag and Eckstein 1997). The low degree of heterogeneity of FPSII over the entire leaf (Fig. 2) and the continuous curvilinear relation between gH2 O and piCO2 in the C3-mode leaves of C. minor (Fig. 1) would be consistent with a homobaric nature of the leaves. In the C. minor leaves in the CAM-mode, however, functionally heterobaric performance became evident. In contrast to the C3-mode leaves there was high heterogeneity of FPSII in phases II and IV of CAM, i.e. when both carboxylases (PEPC and RubisCO) competed for internal CO2 (Borland et al. 1996). This was influenced by changes in stomatal resistance and the onset of organic acid decarboxylation during phase II or the decline of decarboxylation at the end of phase III and in the early phase IV. Heterogeneity clearly shows that these processes were not synchronized over the whole leaves at these times of the light period. Synchronization over the entire leaf was restored when piCO2 rose at the transition from phase II to phase III but it was lost again at the transition to phase IV when piCO2 declined and heterogeneity reached its maximum levels. Maximum heterogeneity was also observed at this time of the CAM cycle in the obligate CAM plant K. daigremontiana (Rascher et al. 2001; Rascher and Lu¨ttge 2002). The difference between low heterogeneity at high internal piCO2 and high heterogeneity at low internal piCO2 during the light period of CAM was explained by higher and lower CO2-concentration gradients, respectively (Rascher et al. 2001; Rascher and Lu¨ttge 2002). Concentration differences of CO2 in the air spaces within a leaf cause lateral diffusion of CO2 in the lamina (Jahnke and Krewitt 2002; Pieruschka et al. 2005, 2006; Jahnke and Pieruschka 2006). Therefore, CO2 can exert a signalling function in spatiotemporal synchronization/desynchronization of photosynthetic activities (Lu¨ttge and Hu¨tt 2006). This was verified by applying patches of silicon grease to leaves of K. daigremontiana locally preventing stomatal gas exchange, and thus, causing artificial CO2 gradients in the leaves which varied during CAM phases (Duarte et al. 2005). Thus, the high heterogeneity in phases II and IV and in the transitions between phases in C. minor shows that there is a fair extent of functionally heterobaric behaviour. This assessment is supported by considering the dynamics of photorespiration during the light period. In the C3-mode

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leaves a change from photorespiratory (21% O2) to nonphotorespiratory (1% O2) conditions had the expected effects of increased JCO2 and gH2 O and decreased piCO2 (Fig. 5). Photorespiration was rather constant and there was only a small increase in the middle of the light period which we can not explain at present. In the CAM-mode photorespiration depended on the CAM phases. In phase II the relative contribution of oxygenase activity to total RubisCO activity was about half that observed in the C3mode leaves at the beginning of the light period but still 15.6%. This shows that the onset of organic acid decarboxylation and internal CO2 concentrating associated with it still allowed photorespiration to continue to some extent. The CO2-concentrating mechanism also leads to an O2concentrating effect of up to 40% in Clusia performing CAM as already shown by Alexander von Humboldt in 1800 (Faak 2000; see Lu¨ttge 2002), which, however, did not lead to an increase of photorespiration here in phase II of C. minor. Unfortunately, because of stomatal closure piCO2 could not be calculated precisely and photorespiration could not be measured in phase III. The absence of an effect of 1% O2 in phase III can have two reasons, (1) due to stomatal closure application of the air mixture with 1% O2 did not penetrate and was not effective, (2) due to very high piCO2 as observed in many CAM plants in phase III (Lu¨ttge 2002) including Clusia (Sternberg et al. 1987) photorespiration might have been already suppressed at 21% external O2. With respect to the second argument, we must bear in mind, however, that also internal O2 partial pressure ðpiO2 Þ is highly increased in phase III (see above) and photorespiration may well have been affected by a changed balance of high internal CO2 and O2 quite strongly then. At the beginning of phase IV photorespiratory activity was highest in all measurements and even higher than in the C3-mode (37.9%) perhaps due to still high internal O2 levels. Under photorespiratory conditions at 21% O2 heterogeneity of FPSII was only high in the CAM-mode and in relation to the dynamics of the CAM phases. Under nonphotorespiratory conditions during the pulses of 1% O2 heterogeneity of FPSII, however, was also strongly increased in the C3-mode leaves. Since FPSII is an indicator of overall energy demand it is evident that the use of both of its substrates, CO2 and O2, under photorespiratory conditions has led to a homogenous energy use over the leaf and this was lost when photorespiration was suppressed. Thus, a functionally heterobaric behaviour became apparent when only lateral diffusion of one of the two substrates of RubisCO, i.e. CO2, was relevant and O2 was not involved. Thus, the hypothesis that photorespiration reduces FPSII heterogeneity is confirmed. Conversely, the hypothesis of a heterobaric nature of C. minor leaves is not confirmed

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because of the great increase of heterogeneity observed under non-photorespiratory conditions in the C3-mode and the heterogeneity related to CAM phases under both photorespiratory and non-photorespiratory conditions. This showed that when only the carboxylase function of RubisCO was active photosynthesis was patchy, a functional feature of heterobaric leaves. Only the strong energy demand of photorespiration had led to a more homogenous energy use over the entire leaf under the photorespiratory conditions in the C3-mode. A further element of heterogeneity becomes evident by a closer look at the spatiotemporal resolution of the various photosynthetic parameters which reveals that C3-expression may not have been complete in the leaves called C3-mode leaves here. This is already suggested by the observation that gas exchange was almost zero in the dark period in the C3-mode leaves. That there was no respiratory loss of CO2 in the dark could be explained by refixation of respiratory CO2 behind closed stomata via PEPC. This is a characteristic feature of the CAM-cycling mode (Sipes and Ting 1985) which also can potentially be expressed in C. minor (Franco et al. 1991). However, this would be accompanied by nocturnal accumulation of malic acid, which was not seen in the present analyses perhaps because malic acid oscillations were too small to be detected in the bulk leaf cell sap. Conversely, the phenomenon could also be explained if only parts of the leaf were still performing CAM as it is revealed when one calculates FPSII from the chlorophyll fluorescence camera images separately for the interveinal lamina and the major leaf vein. For the lamina the light period traces of FPSII were different in the C3- and CAMmode. In the C3-mode there was a rapid early increase and values remained rather constant throughout the rest of the light period. In the CAM-mode the increase in the early light period was more gradual which could reflect the more complex relation of a gradual decline of CO2 fixation by PEPC and the increase in the carboxylation efficiency with a concomitant increase of the oxygenation efficiency of RubisCO. The FPSII remained constant in the middle of the day as energy demand was high in phase III, but unlike in the C3-mode it began to decline earlier in the later part of the light period reflecting the transition from phase III with high piCO2 to phase IV with lower piCO2 : Conversely, in the major leaf vein the light period traces of FPSII were rather similar in the C3- and CAM-mode leaves. In both cases they were much more CAM-like than C3-like when compared to the respective traces of the interveinal lamina. There was a gradual increase in the early light period, highest levels were reached during a pronounced peak at midday, which were even higher than those of the lamina at the time of phase III in the CAM-mode, and the decline at the time of phase IV in the afternoon was rapid. This can be

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best explained assuming that the green tissue of the major vein was still performing CAM in the C3-mode leaves. This implies that in the vein tissue RubisCO activity increased gradually in the morning, CO2 was concentrated in phase III and possibly could not diffuse away rapidly due to structural constraints and was consumed rapidly in the early afternoon. Anatomical constraints for gas diffusion between the lamina tissue and the green tissue above the major bundle may be given by the fact that in the green vein there is only a rather homogenous palisade parenchyma and no spongy parenchyma, and there are no conspicuous intercellular air spaces. In conclusion, we showed that heterogeneity of photosynthetic use of irradiance energy over the leaves as indicated by FPSII depended on the interaction of the carboxylase activities of PEPC and RubisCO and the oxygenase activity of RubisCO and anatomical differentiations such as interveinal lamina and major vein tissues and that heterogeneity was larger in the CAM-mode where both carboxylases were involved. It showed that in the C3-mode a residual CAM-activity was inherent in the leaves. It suggested that lateral diffusion of CO2 and CO2-signalling can be involved in providing information for synchronizing photosynthetic activities in the leaves of C. minor, which although anatomically homobaric were functionally heterobaric. This is an important basis for understanding the phenomenon of photosynthetic patchiness of leaves in the C3- and CAM-modes, the role of photorespiration in overall use of irradiance energy and the function of the biological clock using individual oscillators in each leaf cell in circadian rhythmicity of C. minor (Duarte and Lu¨ttge 2007). Acknowledgments We thank Professor Dr Ulrich Heber for many discussions and valuable comments on this work and Karl Schuller for his great engagement and help with construction of the system used for online switches of external O2 concentration.

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