Photosynthetic characteristics in leaves of ‘Valencia’ orange (Citrus sinensis (L.) Osbeck) grown under high and low temperature regimes

Environmental and Experimental Botany, Vol. 27, No. 3, pp. 27~287, 1987 printed in Great Britain.

0098-8472/87 $3.00 + 0.00 Pergamon Journals Ltd.

P H O T O S Y N T H E T I C CHARACTERISTICS IN LEAVES OF 'VALENCIA' ORANGE (CITRUS SINENSIS (L.) OSBECK) GROWN U N D E R HIGH AND LOW T E M P E R A T U R E REGIMES* J. 121. V. VU~ and G. Y E L E N O S K Y U.S. Department of Agriculture, Agricultural Research Service, Orlando, FL 32803, U.S.A.

(Received 5 August 1986; acceptedin revisedform 13 October 1986) Vu J. C. V. and YELENOSKY G. Photosynthetic characteristics in leaves of'Valencia' orange (Citrus sinensis (L.) Osbeck) grown under high and low temperature regimes. ENVIRONMENTALAND EXPERIMENTAL BOTANY 27, 279--287, 1987. The photosynthetic activities of leaves of 1-year-old 'Valencia' orange (Citrus sinensis (L.) Osbeck) were determined after trees were maintained for 30 consecutive days in controlled growth chambers under high temperature (HT, 32.2°C day/21.1 °C night) and low temperature (LT, 15.6°C day/4.4°C night). Leaf CO2 exchange rates (CER), stomatal conductance (C,), transpiration (E), chlorophyll (Chl), soluble protein, and proline of the LT treatment were 48, 55, 26, 78, 113 and 265%, respectively, of the H T treatment. The water potentials (0) of leaves from H T treatment were about 0.5 MPa more negative than those from LT leaves. Activities of ribulose-l,5-bisphosphate carboxylase (RuBPCase) and phosphoenolpyruvate carboxylase (PEPCase) of the HT treatment, expressed on a leaf ti'esh weight basis, were 91 and 49%, respectively, of the LT treatment. PEPCase of both temperature treatments, however, showed no differences in affinities for HCO3 and PEP, having a Km (HCO:~) of 0.61 mM and a Km (PEP) of 0.15 raM. The ratio of RuBPCase/PEPCase was 6.6 for the H T treatment, compared to 3.5 for the LT treatment. When the 30-day HT-treated trees were transferred to the LT chamber, CER, C~ and ~O, determined 24 hr after transfer, were comparable to values of 30-day LT-treated trees; the RuBPCase activity decreased about 18°/~, and PEPCase activity increased about 31% after 96 hr in the LT chamber. These relative changes decreased the RuBPCase/PEPCase ratio from 6.6 to 3.7. Switching trees from the 30day LT treatment to the HT chamber resulted in increases in CER, C, and E and decreases in ~; the RuBPCase activity decreased about 10%, but PEPCase activity was relatively unaffected after 96 hr following the transfer to the HT chamber. The LT-induced changes in the ratio of carboxylase activities would indicate that some alteration in the photosynthetic carbon metabolism might occur in 'Valencia' orange leaf tissues subjected to cold acclimation conditions.

* Mention of a trademark, warranty, proprietary product or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. ~"Name and address for correspondence: Dr Joseph C. V. Vu, U.S. Horticultural Research Laboratory, 2120 Camden Road, Orlando, FL 32803, U.S.A. Abbreviations: CER, CO2 exchange rate; Cs, stomatal conductance; E, transpiration; 0, water potential; Chl, chlorophyll; HT, high temperature; LT, low temperature; RuBPCase, ribulose-l,5-bisphosphate carboxylase; RuBP, ribulose-l,5-bisphosphate; PEPCase, phosphoenolpyruvate carboxylase; PEP, phosphoenolpyruvate; PVP, polyvinylpyrrolidone;DTT, dithiothreitol; DNPH, dinitrophenylhydrazine;PPFD, photosynthetic photon flux density; VPD, vapor pressure deficit. 279

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INTRODUCTION

CITRUS is one of the most important tropical/ subtropical economic horticultural crops of the world. In the United States, citrus production surpasses the combined production of other fruit crops, including apples and peaches./l~/ Annual citrus gross revenues in Florida alone exceed one billion dollars. In spite of the importance of this species as a major international tree fruit crop, basic knowledge is lacking on citrus photosynthesis and effects of environmental variables on citrus photosynthetic reactions and processes. Citrus is considered an evergreen and continual replacement of 1- to 2-year-old leaves occurs as trees grow and develop./18/Like other higher plant species, temperature exerts a strong influence in determining growth and productivity of citrus trees. Also, like almost all other growth processes, photosynthesis of many plants has been reported to be strongly affected by temperature. !27 Different growth temperature regimes can result in different leaf photosynthetic characteristics and capacities. Particularly in citrus, growth temperature is one of the major environmental factors that determines how long a leaf remains on the tree./3°/The winter chillings and severe freezes in the past few years have had a major effect on Florida agriculture and, consequently, on the nature of the Florida citrus industry./16) Few studies, however, have been done on the carbon assimilation processes of citrus with respect to temperature, and nothing is known about the effects of chilling temperatures on component reactions of photosynthesis in citrus. In this report, young trees of'Valencia' orange were grown for 30 days in two controlled growth chambers simulating a high and low temperature regime. The temperature-induced changes in leaf photosynthetic capabilities and concentrations of Chl, soluble protein and proline were then investigated. In addition, short-term switchings of the 30-day acclimated trees to a thermally-contrasting growth regime provided some understanding of citrus photosynthesis in response to unexpected changes in ambient temperatures that occur under natural growing conditions. The determination of proline was partially incorporated in this study due to its reported high

accumulation in leaf tissues of citrus subjected to various environmental stress situations./33i This would provide a basis for future investigations of a probably important, but still unknown, role of this amino acid in citrus during cold hardening temperatures. MATERIALS AND METHODS

Plant materials and growth conditions Trees of'Valencia' orange (Citrus sinensis (L.) Osbeck), which were developed from buds grafted on rough lemon (C. jambhiri Lush.) rootstocks, were individually grown in 2.5-1 plastic pots containing a commercial mix of sphagnum peat moss, vermiculite and perlite. Single-stem trees were maintained under a natural-daylight greenhouse environment, watered daily, and fertilized monthly with a 1.6% solution of 15 7-7 ( N - P - K ) liquid fertilizer. One-year-old test trees were selected for uniformity and divided into two similar-appearing groups for high- and low-temperature treatments. Both groups, each consisting of 16 trees, were grown for 30 consecutive days in a walk-in, controlled environment chamber, 4 x 3 x 1.8 m. Growing conditions were maintained as tbllows: 12-hr light period, 375 #tool (quanta)/mT-sec of PPFD (400-700 nm) at tree terminal shoots, furnished by a combination of" 86% input wattage cool-white fluorescent and 14% incandescent lighting; 6 0 + 5 % relative humidity; day/night temperatures of 32.2/21.1 +0.5°C for H T treatment and 15.6/4.4+0.5°C for L T treatment. Trees were checked and watered daily to maintain adequate moisture. Details on controlled environment have been described previously. ~32i Thirty days after the temperature treatment period, CER, Cs, E, ~, proline, Chl, protein, and activities of RuBPCase and PEPCase were determined on fully developed top leaves of the trees. Following the determinations and at the beginning of the light period, trees kept tbr 30 days in the H T chamber were transferred to the L T chamber. Similarly, trees which had been maintained for 30 days in the L T chamber were transferred to the H T chamber. Following the transfer, CER, Cs and E were determined at 4, 24 and 96 hr; 0 and activities of RuBPCase and PEPCase were determined at 24 and 96 hr. Measurements

TEMPERATURE EFFECTS ON CITRUS PHOTOSYNTHESIS of leaf gas exchanges and collections of the leaf samples for enzyme studies and other analyses were made near the middle of the light period.

Determinations of leaf gas exchanges Measurements of leaf gas exchanges, including

CER, 6~ and E, were made on single attached leaves using the closed gas-exchange LI-6000 Portable Photosynthesis System ( L I C O R , Lincoln, Nebraska) as previously reported./29/ Measurements were made inside the controlled chamber where trees were maintained. The intensity of the cool-white fluorescent and incandescent lighting of the growth chamber was 375 /1tool (quanta)/m~'sec of PPFD at the measured leaf level. The values of CER and E were expressed on the basis of leaf area, determined after each measurement with the L I C O R LI-3000 Portable Area Meter.

Extraction and determination of RuBPCase and PEPCase activities Uppermost fully expanded leaves which were completely exposed to growing light were detached from trees, plunged immediately into liquid N2, ground to a powder with a mortar and pestle, and stored at liquid N~ temperature until analysis. For determinations of PEPCase and H C O ; - / M g 2+-activated RuBPCase, a portion of the leaf frozen powder, approximately 0.5 g fresh weight, was homogenized at 2°C in 10 ml of a prechilled extraction medium which contained 50 m M H e p e s - N a O H , 5 m M D T T , 10 m M MgCI~, 0.1 m M EDTA, and 2% (w/v) PVP-40 at pH 7.5. The homogenate was centrifuged at 5000 g for 5 min at 2°C and the supernatant was immediately used for assays. Measurement of H C O ; - / M g 2+-activated RuBPCase activity was performed at 30°C in a manner similar to that described previously./28/The activity of PEPCase was assayed in a reaction medium which consisted of 50 m M H e p e s - N a O H , 5 m M D T T , 10 m M MgC12, 0.1 m M EDTA, 5 m M PEP and l0 m M NaHI4CO~ (0.2 Ci/mol) at p H 8.0. The reaction was initiated with 0.1 ml enzyme extract and stopped after 3 min at 30°C with 0.1 ml of 6N HC1 saturated with D N P H . Kinetic determinations of PEPCase were made on extracts from leaves of trees that had been maintained for 30 days in the H T and L T cham-

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bers. Assays were performed in Reacti-Vials (Pierce Chemical Co., Rockford, Illinois) sealed with serum stoppers and screw caps. A CO2-free buffer solution was used and all reaction mixtures were flushed with N 2 for 3 rain before addition of the buffer and H14CO3. The incorporation of 14C into acid-stable products was then measured by liquid scintillation spectrometry.

Measurements of leaf water potential, chlorophyll, protein and proline Total water potentials of single leaves were estimated by the pressure chamber technique as described by KAtJF~ANN./~°~Proline was extracted from oven-dried leaf subsamples, and concentration was determined according to TROLL and LINDSLEY,(26) with modifications by SiNcn et al. 12°~Total chlorophyll was extracted and measured by the method ofARNon./l~ Soluble protein was estimated by the method of BRADFORO,/7/ using bovine serum albumin (fraction V) as standard. RESULTS

Table 1 shows CER, Cs, E, ~, Chl, protein, and proline concentrations of'Valencia' orange leaves measured after 30 days of growth under high temperature (32.2/21.1 °C) and low temperature (15.6/4.4°C) regimes. CER, C~ and E of attached leaves, which were determined during growth conditions under which trees had been maintained, were lower in the L T treatment as compared to the t t T treatment. Values of CER and Cs for the H T treatment were about twofold and transpiration rates were almost fourfold those of the L T treatment. In contrast, Os of leaves from H T treatment were about 0.5 M P a more negative than those from L T leaves (Table 1). Differences in proline concentration were obvious in leaves of trees exposed to two growth temperature regimes (Table 1). Leaves of trees grown in the H T chamber had 62% less proline content relative to that in leaves of trees of the L T chamber. Chlorophyll content per unit leaf weight increased with increase in growth temperatures. Plants grown for 30 days at L T contained about 78% Chl compared to plants grown at HT. Leaf protein of plants grown in HT, however, was about 89% of that from L T treatment. After 30 days of growth at high- or low-tem-

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Table 1. Leaf CO2 exchange rate (CER), stomatal conductance (C~), transpiration (E), water potential (~), proline, Chl, and soluble protein of'Valencia' orange determined after 30-day growth at high temperature (HT, 32.2/21. I°C) and low temperature (L 7", 15.6/4.4°C). Mean values ± standard deviations are presented

Treatment

CER * (/2tool/ m 2" sec)

C,* (cm/sec)

E* (mg/ m 2. sec)

0* (MPa)

Proline1" (mg/g dry wt)

Chl~" (mg/g fresh wt)

Protein ~" (mg/g fresh wt)

HT LT

4.64±0.83 2.25+_0.53

0.062_+0.009 0.034±0.003

18.71+-4.06 4.78±0.85

- 1.10_+0.12 -0.55+_0.12

5.6+_0.5 14.8+_0.1

2.14+_0.10 1.67±0.06

22.6_+0.9 25.5+_1.0

• N = 16-21 measurements. i N = 6 measurements. Table 2. Leaf C02 exchange rate (CER), stomatal conductance (C~), transpiration (E), and water potential ( tp ) of' Valencia' orange determined after 4, 24 and 96 hr of transfer of 30-day HT-treated trees to L T, or 30-day L T-treated trees to H T conditions. Mean values +-standard deviations are presented

ceR?

C?

e?

O+

Treatment

(/~mol/m 2" sec)

(cm/sec)

(mg/m 2. sec)

(MPa)

HT (30 d)/LT (04 hr) HT (30 d)/LT (24 hr) HT (30 d)/LT (96 hr)

3.52+_0.78 1.97 +_0.47 2.68 +_0.80

0.055+_0.010 0.037 +_0.008 0.045 +_0.005

7.64_+ 1.19 4.47 +_ 1.75 6.92 +_ 1.57

(*) - 0.72_+ 0.04 - 0.47 +_0.05

LT (30 d)/HT (04 hr) LT (30 d)/HT (24 hr) LT (30 d)/HT (96 hr)

0.55+_0.93 2.90 _+0.88 4.51 _ + 0 . 7 2

0.035+_0.007 0.070 +- 0.012 0.095±0.018

10.53_+ 1.95 19.97 ± 3.78 21.88±4.45

(*) - 1.06 _+0.05 - 1.30+-0.11

(*) Not determined. ~ N = 9 16 measurements. ,+ N = 6 measurements.

p e r a t u r e e n v i r o n m e n t , trees in the H T were transferred to the L T c h a m b e r , and trees previously kept at the L T were transferred to the H T c h a m b e r . L e a f gas exchanges were again determ i n e d at diIi~rent periods following the transfer ( T a b l e 2). W h e n the 30-day H T - t r e a t e d trees were transtizrred to the L T c h a m b e r , decreases in CER, (~ a n d E occurred 4 hr after transfer. After 24 hr, CER, Cs, E, and ~9 were c o m p a r a b l e to 30d a y L T - t r e a t e d trees ( T a b l e 1). Similar events occurred for trees that h a d been m a i n t a i n e d for 30 days at L T a n d then transt~rred to the H T t r e a t m e n t . For these L T - t r e a t e d trees, there were increases in CER, C~ a n d E, and decreases in with increasing time in the H T e n v i r o n m e n t (Table 2). T a b l e 3 shows activities of R u B P C a s e and P E P C a s e in leaf extracts from trees that had been m a i n t a i n e d at H T or L T for 30 days and

were transferred thereafter to the thermally-contrasting growth c h a m b e r for 24 and 96 hr. Since total Chl c o n c e n t r a t i o n in leaf tissues of the 30d a y L T t r e a t m e n t was only a b o u t 78% that of the 30-day H T treatment, unit leaf fresh weight a p p e a r s to provide a more realistic basis for comparison of enzyme activities. F o r i n t b r m a t i o n a l purposes, however, enzyme activities on a Chl and protein basis were also included (Table 3). Therefore, on a leaf fresh weight basis, activities of R u B P C a s e and P E P C a s e in leaves of orange trees m a i n t a i n e d for 30 days at H T were 91 and 49 ° / , respectively, of those from trees kept for the same length of time u n d e r LT. W h e n the 30-day H T - t r e a t e d trees were transferred to L T conditions, activities of R u B P C a s e decreased a b o u t 18% 96 hr after the transfer. P E P C a s e activity, however, increased a b o u t 31 °/o after 96 hr in the L T c h a m b e r . Switching trees from the

T E M P E R A T U R E EFFECTS ON C I T R U S PHOTOSYNTHESIS

283

Table 3. Activities of RuBPCase and PEPCase in "Valencia' orange trees grown at H T or L T for 30 days, and 24 and 96 hr after transferring to a temperature-contrasting growth chamber. Mean values ± standard deviations represent triplicate determinations from the combined sampling pool of fully developed top leaves randomly selectedfrom trees of each treatment RuBPCase activity

PEPCase activity

(#mol CO2/hr) mg Chl mg protein

(/.tmol CO2/hr ) mg Chl mg protein

Treatment

g fresh wt

HT (30 d) HT (30d)/LT (24hr) HT(30d)/LT(96hr)

672.6-t-28.9 652.4-t-24.5 548.3± 17.0

312.5_+ 10.6 284.3+11.8 242.9+ 8.9

28.8_+1.3 27.8±0.8 24.7_+hl

102.1 ±4.3 106.3±5.2 148.6±6.1

47.5-t-2.3 48.3_+2.4 65.9_+2.8

4.4±0.2 4.6±0.3 6.4±0.3

L T (30d) LT (30 d)/HT (24 hr) LT (30d)/HT (96hr)

742.7+24.6 743.6±20.8 670.9±23.6

440.7_+18.8 396.4± 14.2 326.7±16.4

29.9±1.2 30.8_+1.5 27.5+_1.3

210.4±9.7 215.6±8.4 212.6±7.6

123.1±6.4 124.2±5.3 103.5±4.5

8.7±0.4 8.9_+0.5 8.7_+0.4

30-day L T t r e a t m e n t to the H T c h a m b e r for 96 hr resulted in a b o u t 10% r e d u c t i o n of R u B P C a s e activity. P E P C a s e activity was relatively not affected even 96 hr after transfer to the H T c h a m b e r ( T a b l e 3). T h e effects of various b i c a r b o n a t e concentrations on the activity of P E P C a s e extracted from leaves of 30-day H T - and L T - t r e a t e d trees are shown in Fig. 1. H y p e r b o l i c curves, characteristic of enzymes which follow the M i c h a e l i ~ M e n t e n kinetics, were observed. S a t u r a t i o n with HCO:~ occurred at a b o u t 5 m M for the H T treatm e n t enzyme a n d 10 m M for the L T t r e a t m e n t enzyme, with activities always lower for the H T g r o w t h regime. P E P C a s e of both t e m p e r a t u r e treatments, however, were kinetically similar (Fig. 1, inset), having the a p p a r e n t Km ( H C O 3 ) of 0.61 m M . T h e Vmax (HCO:~) were 105 a n d 212 ~tmol/g fresh wt" hr for the H T and L T treatment, respectively. T h e effect of PEP concentration on PEPCase is shown in Fig. 2. As before, P E P C a s e from L T treated trees exhibited the highest reaction rate at all substrate concentrations. In spite of the a p p a r e n t h y p e r b o l i c characteristics of the rate curves in Fig. 2, the d a t a did not follow the M i c h a e l i s - M e n t e n kinetics. T h e Vmax (PEP) values, which were derived from d o u b l e reciprocal plots of the velocity vs PEP concentration, were 110 and 225 # m o l / g ti~esh w t - hr for the H T a n d L T t r e a t m e n t , respectively. Consequently, the Hill plot <~9~ was used to d e t e r m i n e the Km (PEP) (Fig. 2, inset), which was 0.15 m M for both H T - a n d L T - t r e a t m e n t enzymes.

g fresh wt

F

0 15-6/4,4°c 032. .o 200

o

150

II(3 < ., (n

100

X O m ~g

~, s0 II1 Q,

-2

' RCO~

-1

0

1

S' CONCENTRATION

2 1/S '

3

4

1 k0

5

%

1 5'

(raM)

FIG. |. PEPCasc activity as a tlanction of HCO:, concentration in extracts from leaves of 'Valencia' orange grown for 30 days under high-temperature (HT, 32.2/21.1°C) and low-temperature (LT, 15.6/4.4°C) regimes. Data points are averages of three determinations±standard deviation. Inset shows a double reciprocal plot of the same data, ,giving a Km (HCO:~) of 0.61 mM for the enzyme of both treatments. DISCUSSION

T h e factors controlling the t e m p e r a t u r e response of photosynthetic c a r b o n assimilation in

284

J.c.v.

0

vu

and G. YELENOSKY

T I~

15-6/4-4°C

~200

i

~

150

100

50

it f

l 0

-1,5 i

o

j

-0.5 o., ~Oi,

0.510g o:.%,,

.....

1.5 ,i,

i i i 1 2 "5"10 PEP CONCENTRATION (raM)

Fro. 2. PEPCase activity as a function of PEP concentration in extracts from leaves of 'Valencia' orange grown for 30 days under high-temperature (HT, 32.2/21. I°C) and low-temperature (LT, 15.6/4.4°C) regimes. Data points are averages of three determinations_+ standard deviation. Inset shows a Hill plot of the same data, giving a Km (PEP) of 0.15 mM for the enzyme of both treatments. green leaf tissues are not well understood. T h e capacity for photosynthesis in attached leaves strongly depends on the temperatures under which plants have been grown and is an integrated result of specific temperature effects on various photosynthetic component reactions. (3~ Stomata, which control the resistance to the diffusive transfer of water vapor and CO2 between the leaf and ambient air, would exert a strong influence on both the rate and the temperature dependence of photosynthesis. Changes in the temperature dependence of stomatal conductance as a function of growth temperature appear to control the shifts in temperature dependence of CO2 uptake of m a n y plants, m/ It has been reported that stomata of well-watered plants in m a n y species tend to remain open as the temperature is increased over a wide range. (2i T h e internal plant water status and the VPD between

the leaf and surrounding air are particularly important in stomatal response to temperature. From the C+ and E data presented in Table 1, the computed VPD values of the two temperature treatments were 18.7 and 40.6 mb tbr the 30-day LT- and HT-treated trees, respectively. Thus, in the absence of water stress and high vapor pressure gradients, stomata of 'Valencia' orange leaves tend to open in response to increased growth temperature. Data from this study as well as others (2'4'1~'2~'31) suggest that low temperature effects on photosynthesis involve changes in stomatal and nonstomata! characteristics. The primary change consists of reducing the physical diffusion of gases into and out of the leaf. This is likely due to lower stomatal conductance as a result of partial closure of the stomata (Table 1). Also, increases in CER and E observed shortly after transfer of trees from the L T - g r o w t h regime to H T conditions would generally reflect an increase in stomatal conductance rather than an acclimatory change in photosynthetic characteristics (Tables 1 and 2). Nonstomatal characteristics, however, appeared to change over a wide temperature range later in the period and would require sufficient time following transfer of the trees to the contrasting growth temperature regime. (~'~/ There are reports on the effects of different growth temperatures which can result in different photosynthetic capacities in terms of a m o u n t and/or activity of the catalytic proteins. (3'5'6'a'23'25'31/ Enzyme activity m a y also impose a limitation on the photosynthetic rate of plants grown at different temperatures./8'25~ There is evidence that the a m o u n t of chloroplast protein tends to fall with increasing growth temperature./3'~5/ In 'Valencia' orange, there were decreases in leaf soluble protein of the HT-treated trees (Table 1). Also, the proline in leaf tissues of liT-treated trees was only 38~o that of LT-treated trees. Free proline levels, which are associated with citrus frost hardiness, increase in citrus during chilling growth temperature regimes./32/ It is the most a b u n d a n t amino acid found in the tracheal sap of orange trees throughout the entire year, and is especially high in concentration during the winter season./33/ W h e t h e r proline contents are high enough to help protect citrus against frost injury is still an enigma at the present time.

TEMPERATURE EFFECTS ON CITRUS PHOTOSYNTHESIS Activities of both RuBPCase and PEPCase, when assayed in vitro at 30°C, were higher in the LT- than in HT-treated trees (Table 3). PEPCase is an important enzyme protein in higher plants functioning in malate synthesis. In C4 plants, malate and aspartate are intermediates of photosynthesis while in C3 plants, malate is an end product of the carbon reduction cycle. (24/ The twofold increase in activity of PEPCase in 'Valencia' orange grown at a LT regime is of particular interest (Table 3). From the data shown in Table 3, the RuBPCase/PEPCase ratio was 6.6 for the H T treatment and 3.5 for the LT treatment. Transfer of 30-day HT-treated trees to a LT chamber for 4 days decreased the RuBPCase/ PEPCase ratio to that of the 30-day LT-treated trees. In contrast, the RuBPCase/PEPCase ratio was relatively unaffected 4 days after transfer of the 30-day LT-treated trees to H T growth chamber (Table 3). RuBPCase activities of 30day LT-treated trees decreased about 10% after 4-day incubation in the H T chamber. Unexpectedly, RuBPCase activities declined to even lower levels when 30-day HT-treated trees were transferred to the LT chamber. PEPCase activity of 30-day HT-treated trees, however, increased about 46°J£~ 4 days after the transfer to a LT chamber. In contrast, PEPCase activity of trees maintained for 30 days at LT had not changed much 4 days after transfer to H T chamber. Decreased activities of RuBPCase in high as compared to low growth temperature have been reported in other plant species./3'25/ In Brassica napus, the activity of PEPCase increased considerably when plants were subjected to continuous cold treatment at 2°C for 8 days. (23) Since increase in temperature increases enzyme activity, less RuBPCase and PEPCase protein may be required at high growth temperatures, resulting in a saving in synthesis and of maintenance energy costs. (17/ Such changes would be particularly important for RuBPCase, which represents a large fraction of the total leaf protein in higher plants. Orange trees grown under a LT regime for 30 days contained less Chl than a similar group of trees grown for the same period at H T (Table 1). The development of chlorotic leaves is a common symptom in plants subjected to stress. In sorghum, a C4 photosynthetic category, chlorotic

285

bands appeared following a series of low night temperatures, i2~) Under these thermal stress conditions, chlorosis may occur because either the synthesis of chlorophyll precursors is depressed or the rate ofphotodestruction of Chl and associated pigments occurs more rapidly than synthesis. (1:~'~7~ There was no indication in the present study that long-term temperature effects on PEPCase were due to changes in the kinetics (Km) of the enzyme (Figs 1 and 2), and this is also true for the Kms of RuBPCase extracted from LT- and HT-treated leaf tissues (Km (CO2) = 20.5/tM). The Krn values for HCO3 and PEP (Figs 1 and 2, insets) were the same for PEPCase extracted from LT- and HT-treated 'Valencia' orange, a C3 photosynthetic category. These Km values are in general consistent with values reported fbr PEPCase of other C3 plants. :!~'14'24) The lower Vmax values would indicate that decreases in PEPCase and RuBPCase activity in the HT-treated leaves may be partially due to lower enzyme concentration, although higher enzyme extractability from cold-treated leaf tissues cannot be ruled out. '1~'23! A comparison of the CER (Table 1) to RuBPCase activities (Table 3) was made, using the average value of 2.65 g leaf fresh weight per dm 2 leaf area for 1-year-old greenhouse-grown 'Valencia' orange, indicating that activities of the enzyme from trees grown at both temperature regimes are more than adequate to support the observed CO~ fixation rates of attached leaves. These enzyme activities (Table 3) were determined in vitro at 0.5 mM RuBP and 20 mM H C O 3 , the levels of which were likely much higher than those present in the leaf tissues. In a related study with 1-year-old 'Valencia' orange coming from the same group of trees used in this report, CER of attached leaves, when determined outdoors under natural irradiance, approached maximum levels of 7 /~mol/m 2" sec at about 600 /tmol (quanta)/m2.sec solar PPFD and 26°C air temperature. !29/ Even the activities of RuBPCase obtained from leaf extracts of both 30-day HTand LT-treated trees (Table 3) are still sufficient to maintain these optimum CER levels. Therefore, under our experimental temperature conditions, RuBPCase may not be considered a primary factor in mediating the observed temperature responses on CO 2 exchange in 'Valencia' orange.

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T h e large increases in PEPCase activity in the leaf tissues of L T - t r e a t e d trees that lead to decreases in the R u B P C a s e / P E P C a s e ratio (Table 3) suggest that some alteration in photosynthetic carbon metabolism might occur in citrus d u r i n g cold acclimation. O u r c u r r e n t working hypothesis is that, u n d e r low t e m p e r a t u r e cold h a r d e n i n g , an increase in aspartate metabolism may occur in ' V a l e n c i a ' orange leaves as a result of a twofold increase in PEPCase activity (Table 3). This would help explain a threefold increase in proline c o n t e n t as observed in leaves of orange trees subjected to a L T growth regime (Table 1).

Acknowledgements We would like to thank Doris Hawthorne for her technical assistance during various aspects of this research.

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