Planta ,1990)182:492-500
P l a n t a 9 Springer-Verlag1990
Factors influencing the capacity for photosynthetic carbon assimilation in barley leaves at low temperatures Carlos A. Labate* and Richard C. Leegood Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield, Sheffield SI0 2TN, UK Received 24 January; accepted 21 May 1990
Abstract. The aim of this work was to examine the effect upon photosynthetic capacity of short-term exposure (up to 10 h) to low temperatures (5 ~ C) of darkened leaves of barley (Hordeum vulgare L.) plants. The carbohydrate content, metabolite status and the photosynthetic rate of leaves were measured at low temperature, high light and higher than ambient CO2. Under these conditions we could detect whether previous exposure of leaves to low temperature overcame the limitation by phosphate which occurs in leaves of plants not previously exposed to low temperatures. The rates of CO2 assimilation measured at 8~ C differed by as much as twofold, depending upon the pretreatment. (i) Leaves from plants which had previously been darkened for 24 h had a low content of carbohydrate, had the lowest CO2-assimilation rates at low temperature, and photosynthesis was limited by carbohydrate, as shown by a large stimulation of photosynthesis by feeding glucose. (ii) Leaves from plants which had previously been illuminated for 24 h and which contained large carbohydrate reserves showed an accumulation of phosphorylated intermediates and higher CO2-assimilation rates at low temperature, but nevertheless remained limited by phosphate. (iii) Maximum rates of CO2 assimilation at low temperature were observed in leaves which had intermediate reserves of carbohydrate or in leaves which were rich in carbohydrate and which were also fed phosphate. It is suggested that carbohydrate reserves potentiate the system for the achievement of high rates of photosynthesis at low temperatures by accumulation of photosynthetic intermediates such as hexose phosphates, but that this potential cannot be realised if, at the same time, carbohydrate accumulation is itself leading to feedback inhibition of photosynthesis. *Present address: Escola Superior de Agricultura "Luiz de Queiroz", Departamento de Gen+tica, Universidada de S~o Paulo, Piracicaba, C.P. 83, CEP 13400, Brazil Abbreviations: A = rate of photosynthetic CO2 assimilation; Chl = chlorophyll; hexose-P = hexose phosphate(s); PFD = photon flux density; PGA=glycerate-3-phosphate; Pi=inorganic phosphate; RuBP = ribulose-1,5-bisphosphate; triose-P = triose phosphate(s)
Key words: Carbon assimilation, p h o t o s y n t h e t i c - Hordeum (leaf, carbon assimilation) - Photosynthesis - Temperature, low (carbon assimilation)
Introduction Abrupt transfer of leaves of C3 plants to low temperatures can result in a limitation of the rate of photosynthetic carbon assimilation by phosphate. This occurs when leaves are illuminated at high photon flux densities (PFDs) and CO2 concentrations which are at, or above, ambient (reviewed by Leegood 1985, 1989). This limitation occurs because sucrose synthesis runs too slowly at low temperatures. The restriction of sucrose synthesis, and hence recycling of inorganic phosphate (Pi) to resynthesis ATP in the chloroplast, might occur in one or all o f a number of ways. The observation that this limitation by the rate of sucrose synthesis at low temperatures can be ameliorated by feeding Pi (Leegood and Furbank 1986; Labate and Leegood 1988) indicates that more phosphate (either as Pi or as esterified phosphate) is required for photosynthetic metabolism at lower than at higher temperatures. Sucrose synthesis could be curtailed (i) by the amount of free Pi in the cytosol [and hence diminished export of triose phosphate (triose-P) from the chloroplast via the phosphate translocator]; this could occur either because the cytosolic Pi optimum for export o f carbon from the chloroplast increases (Leegood and Walker 1983), or because sequestration of cytosolic Pi into accumulated phosphorylated intermediates occurs [leaves illuminated at lower temperatures tend to accumulate phosphorylated intermediates (Leegood 1985; Stitt and Grosse 1988; Labate et al. 1990), which may be needed to compensate for decreases in enzyme affinity and velocity at low temperatures]; (ii) by limiting amounts of phosphorylated intermediates, if insufficient Pi was available in the cytosol to allow for the accumulation of phosphate esters at low temperatures; or (iii) by temperature-dependent changes in the
C.A. Labate and R.C. Leegood: Photosynthesis at low temperatures properties of, for example, the cytosolic fructose bisphosphatase or sucrose-phosphate synthase (Stitt and Grosse 1988). Acclimation of photosynthetic carbon assimilation to lower temperatures is readily observed in the longterm, e.g. by increased Oz sensitivity of photosynthesis after growth at low temperatures (e.g. Cornic and Louason 1980; Sage and Sharkey 1987) and, after 2 d at low temperatures, by a substantial increase in photosynthetic rates in barley leaves (Sicher et al. 1988). However, exposures of even a few hours to low temperatures in the dark can lead to the accumulation of phosphorylated intermediates (Labate and Leegood 1989), significantly increased rates of CO2 assimilation, and relief of symptoms of phosphate deficiency such as O2-insensitivity (Labate and Leegood 1988). O f course, such short-term acclimation to low temperature will not be solely dependent upon increasing the metabolically-active phosphate pools. Limitation by Pi will only be important in saturating light and high CO2, and other changes in the regulation of photosynthesis will be necessary in limiting light. Many parallel processes will also occur over different periods of time. The aim of this study was to investigate how altering the a m o u n t of carbohydrate and phosphorylated metabolites in leaves, by prolonged light or dark pretreatment of barley plants (Labate and Leegood 1989), affected short-term acclimation to low temperature of the photosynthetic rate, measured in saturating light and high CO2 so as to promote a limitation by phosphate.
Material and methods Plants. Barley (Hordeum vulgare L. cv. Sonja; Nickersons R.P.B., High Wycombe, Bucks, UK) was grown as described previously (Labate and Leegood 1988) in a greenhouse, under a maximum quantum flux density of 500-600 gmol quanta-m 2.s-1 at midday. Air temperature was kept at an average of 30~ C during the day and 18~ C at night. Gas-exchange measurements. Although in many leaves the maximum photosynthetic capacity can be measured in saturating (approx. 5%) CO2 in the 'leaf-disc O2 electrode' (Delieu and Walker 1981) this is not the case for barley, in which photosynthetic rates are considerably lower at these very high COz concentrations (data not shown). Maximum rates of photosynthetic CO2 uptake by barley primary leaves were therefore estimated in an infra-red gasanalysis system using a high CO2 concentration of 860 ~tbar, in 20% O2 and at a PFD of 1000 ~tmol.m- 2. s- 1. Water was circulated through an aluminium chamber (Doncaster et al. 1989) to give effective temperature control to 8_ 1~ C. The leaf temperature was monitored by a thermocouple attached to the underside of the leaf. Sections of barley leaves were mounted side-by-side in the chamber and fed with either water or 50 mM K2HPO4 (pH 7). The windows of the chamber were Parafilm discs held in place by O-rings. The branches of a bifurcated fibre optic (from a KL 1500 projector; Schott, Mainz, FRG) were placed at an angle of 45~ to illuminate the leaf from above. Metabolite measurements. The illuminated leaves were freezeclamped in situ (Doncaster et al. 1989) or darkened leaf samples were immersed in liquid N2. Leaf samples were then ground in a pestle and mortar, pre-cooled with liquid N2, and killed and extracted in HC104 as described previously (Leegood and Furbank
493 1986). Following neutralisation, sufficient charcoal was added to render the extract completely colourless. The extract was centrifuged at 2000 .g for 2 min. The supernatant was either immediately used or stored in liquid N2 for the determination of metabolites by the methods of Lowry and Passonneau (1972) in a Hitachi (Tokyo, Japan) 557 dual-wavelength spectrophotometer (340400 nm). Ribulose-l,5-bisphosphate (RuBP) and glycerate-3-phosphate (PGA) were measured as described by Doncaster et al. (1989). Total carbohydrate (starch, sucrose and hexose) was measured as described previously (Labate and Leegood 1989).
Chlorophyll. Pellets remaining from the HC104 extraction were washed with water and homogenised in 2 ml water. Following overnight extraction in 80% (v/v) acetone at room temperature, phaeophytin was measured by the method of Vernon (1960).
Results Changes in photosynthetic rates and metabolites during exposure to low temperature. The carbohydrate content of the leaves of intact barley plants was manipulated by preillumination, or darkening, of the leaves for a period of 24 h (Labate and Leegood 1989). Both sets of plants were then darkened for varying periods of time at 25 ~ C or at 5~ C. We then measured hexose phosphate (hexose-P) and the carbohydrate content of the leaves in the dark, before illuminating them and allowing them to reach steady-state photosynthesis at 8~ C. Steadystate photosynthesis was measured under conditions o f high P F D (1000gmol q u a n t a . m - Z . s -1) and higher than ambient CO2 (860 labar). This promotes a limitation of photosynthesis by phosphate in the leaves o f plants which have not previously been exposed to low temperatures (Labate and Leegood 1988). In this way we could follow the acclimation process (in respect o f overcoming the limitation by phoSphate) as an increase in the rate of CO2 assimilation. The leaves were then freeze-clamped in the light for the determination of metabolites. In parallel experiments, phosphate was supplied to similar leaves during illumination and metabolites measured. The following observations were made: (i) In plants which had previously been illuminated for 24 h there was a large accumulation of carbohydrate (Table 1). There was a gradual decrease in the COz-aSsimilation rate measured at 8~ C in those plants which were kept in the dark at 25 ~ C (Table 1 ; first column). In the plants which were kept in the dark at 5~ C there was an increase in the assimilation rate after 3 h compared with the control plants, followed by a decrease in the photosynthetic rate after 10 h in darkness (Table 1 ; third column). In plants which had been previously been darkened for 24 h the carbohydrate content was very low (Table 2). There was little change in the rate of carbon assimilation in the plants kept at 25 ~ C (it should be noted that the rates o f CO2 assimilation were comparable with those o f leaves of plants illuminated for 24 h and then darkened for 3 h or 10 h at 25 ~ C). In the plants kept at 5~ C, the assimilation rates were the same after 3 h and after 10 h as in the plants kept at 25 ~ C. There was, therefore, no evidence that the plants exposed to a long
494
C.A. Labate and R.C. Leegood: Photosynthesis at low temperatures
TaMe 1. Effect of temperature pretreatment on steady-state CO2-assimilation rate and metabolite contents of illuminated barley leaves. Plants were kept in the light for 24 h at 25~ C, and then at 25~ C, or at 5~ C in the dark for a further period of 0, 3 or 10 h. After this pretreatment, leaves were detached. One sample was extracted for measurement of hexose-P and carbohydrate while another was placed in a chamber for gas exchange and allowed to reach steady-state photosynthesis at 8~ C in the presence (" Fed Pi") or absence ("Fed HzO") of 50 mM K2HPO 4 fed through the transpiration stream. Contents of total carbohydrate are shown in gmol hexose equivalents.(mg Chl) -1. Photosynthesis was measured at a CO 2 concentration of 860 labar and a PFD of 1000 gmol.m 2.s-1. Data are means • SE of three experiments Parameter
Plants illuminated for 24 h and then maintained in darkness Time in the dark
Temperature in the dark
(h)
25~ C
5~ C
Treatment during gas-exchange measurement Fed H20
Fed Pi
Fed H20
Fed Pi
Oh A (gmol.m-2.s -1) Carbohydrate in dark (gmol hexose-(mg Chl)-1) Metabolites (nmol-(mg Chl)- 1) Hexose-P in dark Hexose-P in light PGA in light Triose-P in light PGA/triose-P in light RuBP in light Z esterified phosphate in light
7.9•
0.7
14.6-+
1
266 _+ 22 456 _+ 44 240 -+ 13 55 _+ 1 4.8_+ 1 179 -+ 20 1109 • 100
8.4•
0.7
613 _+ 53 259 _+ 32 70 +_ 4 3.8_+ 1 163 _+ 5 1268 _+101
3h A (gmol.m-Z.s 1)
6.0-1-0.2
Carbohydrate in dark (I.tmolhexose. (mg Chl)- 1) Metabolites (nmol-(mg Chl)- 1) Hexose-P in dark Hexose-P in light PGA in light Triose-P in light PGA/triose-P in light RuBP in light X esterified phosphate in light
8.8_+
0.4
8.8_+ 0.4
15.1 -I- 1
15.9-+ 1
182 + 5 384 -+16 361 -+ 56 42 _+ 1 8.6_+ 1 105 +11 997 _+95
334 _+12 460 +36 302 -+ 16 65 -+ 4 4.7_+ 0.5 188 _+20 1203 _+96
346 _+ 20 278 _+ 66 79 + 18 3.5_+ 1 116 -+ 16 925 _+136
10.3__+ 0.5
709 -I-12 339 _+31 139 _+13 2.4+ 0.i 186 _+ 3 1559 _+82
10h A (gmol. m - 2. s- 1)
5.0_+ 0.7
Carbohydrate in dark (gmol hexose.(mg Chl)-X)
7.9+ 3
Metabolites (nmol.(mg Chl)-x) Hexose-P in dark Hexose-P- in light PGA in light Triose-P in light PGA/triose-P in light RuBP in light Z esterfied phosphate in light
d a r k p r e t r e a t m e n t h a d s u b s e q u e n t l y acclimated to low temperature. (ii) I n leaves f r o m p l a n t s which h a d previously been i l l u m i n a t e d for 24 h (Table 1) there was a g r a d u a l fall in the a m o u n t o f hexose-P d u r i n g d a r k n e s s at 2 5 ~ (Stitt et al. 1985; L a b a t e a n d L e e g o o d 1989). I n those p l a n t s which were m a i n t a i n e d at 5~ C, there was first a rise in hexose-P c o n t e n t after 3 h, followed by a fall,
85 _+20 326 _+32 358 _+17 34 _+ 5 10.9__+ 2 76 +22 870 _+98
7.4_+
1
6.0_+ 0.3
9.2_+
0.2
13.7_+ 1
504 _+ 67 369 -+ 13 88 _+ 16 4.4_+ 0.6 141 + 25 1243 _+166
278 467 422 66 7 131 1217
_+35 ___34 -+28 _+15 + 1 __10 _+97
629 _ 15 420 -+ 37 97 • 10 4.4_+ 0.5 173 _+ 30 1492 +122
a n d hexose-P r e m a i n e d very m u c h higher in the lowt e m p e r a t u r e t r e a t m e n t (see also L a b a t e a n d Leegood 1989). The s u m o f esterified p h o s p h a t e present in the phosph o r y l a t e d intermediates in i l l u m i n a t e d leaves was also calculated (these intermediates w o u l d be largely present in the chloroplasts a n d cytoplasm). Table 1 shows t h a t in leaves f r o m plants which h a d been kept for 24 h in
C.A. Labate and R.C. Leegood: Photosynthesis at low temperatures
495
Table 2. Effect of temperature pretreatment on steady-state CO2-assimilation rate and metabolite contents of illuminated barley leaves. Plants were kept in the dark for 24 h at 25~ C, and then at 25~ C, or at 5~ C in the dark for a further period of 0, 3 or 10 h. After this pretreatment, leaves were detached. One sample was extracted for measurement of hexose-P and carbohydrate while another placed in the chamber for gas exchange and allowed to reach steady-state photosynthesis at 8~ C in the presence (" Fed Pi") or absence (" Fed H20") of 50 mM K2HPO4 fed through the transpiration stream. Contents of total carbohydrate are shown in gmol hexose equivalents. (mg Chl)-1. Photosynthesis was measured at a CO2 concentration of 860 labar and a PFD of 1000 gmol.m - 2 . s - 1. Data are means• of three experiments Parameter
Plants darkened for 24 h and then maintained in darkness Time in the dark (h)
Temperature in the dark 25~ C
5~ C
Treatment during gas-exchange measurement Fed H20
Fed Pi
Fed HzO
Fed Pi
Oh A (gmol.m-Z.s -1)
5.6•
0.7
Carbohydrate in dark (gmol hexose. (mg Chl)-1)
0.8•
0.4
Metabolites (nmol. (mg Chl)- 1) Hexose-P in dark Hexose-P- in light PGA in light Triose-P in light PGA/triose-P in light RuBP in light X ester• phosphate in light
57 • 5 179 • 46 178 • 50 23 • 10 10.3• 4 76 • 23 532 •
8.4•
0.5
345 • 40 200 • 45 46 • 18 5.2• 1 120 • 16 831 •
3h A (gmol.m-2.s -1)
5.8•
Metabolites (nmol.(mg Chl)-1) Hexose-P in dark
0.5
65 • 7 290 • 40 237 • 56 43 • 4 5.5• 1 87 • 21 744 •
Hexose-P- in light PGA in light Triose-P in light PGA/triose-P in light RuBP in light X ester• phosphate in light
7.0•
1
275 • 47 167 • 14 44 • 8 4.0• 0.4 84 • 14 654 • 97
5.8•
0.3
124 • 17 237 • 27 189 • 51 3O • 6 6.1• 1 71 • 12 598 •
7.1_+ 1
312 • 212 • 53 • 4.8• 1 80 • 737 • 84
1Oh A (gmol. m - z. s- t) Metabolites (nmol. (mg Chl)- x) Hexose-P in dark Hexose-P- in light PGA in light Triose-P in light PGA/triose-P in light RuBP in light X ester• phosphate in light
the light a n d t h e n d a r k e n e d , the s u m o f ester• phosp h a t e in the light in leaves o f p l a n t s which h a d been kept at 5 ~ was c o n s i d e r a b l y greater t h a n in those w h i c h h a d b e e n kept at 2 5 ~ for b o t h 3 h a n d 10 h darkness. This change was p a r t l y caused b y higher c o n tents o f hexose-P, triose-P a n d R u B P a n d was a c c o m p a nied by lower P G A / t r i o s e - P ratios. T h e decrease in this ratio indicates a lessening, after exposure to low temperature, o f the restriction b y p h o s p h a t e availability o n the g e n e r a t i o n o f A T P a n d N A D P H necessary to reduce P G A to triose-P (Heber et al. 1986).
5.0•
0.3
80 • 5 206 • 26 203 • 51 35 • 9 6.4• 2 66 • 7 576 •
5.2•
0.5
245 • 56 253 • 48 46 • 10 5.8• 1 81 • 6 706 •
5.6•
1
96 • 372 • 295 • 40 • 7.8• 90 • 887 •
36 25 53 7 2 25
6.5•
1
506 • 54 280 • 76 60 • 22 5.3• 1 98 • 15 1042 •
(iii) I n the leaves f r o m p l a n t s which h a d previously b e e n d a r k e n e d for 24 h (Table 2), there was a small rise in the c o n t e n t o f hexose-P in the d a r k b o t h at 5~ C a n d at 25 ~ C. C o n t e n t s o f total p h o s p h o r y l a t e d i n t e r m e d i a t e s in i l l u m i n a t e d leaves were m u c h lower t h a n i n the leaves o f those p l a n t s which h a d b e e n kept in the light for 24 h. A l t h o u g h there was a n increase after 3 h f u r t h e r darkness, followed by a decrease after 10 h at 25 ~ C, there was a n increase in total ester• phosphate in the light b e t w e e n 3 h a n d 10 h in p l a n t s which h a d b e e n kept at 5~ C. After 10 h, total ester• p h o s p h a t e in
496
C.A. Labate and R.C. Leegood: Photosynthesis at low temperatures the PGA/triose-P ratio at low temperatures (in the absence of Pi feeding) in illuminated leaves of plants which had been predarkened for 24 h, then kept in the dark at 5 ~ C, and a p o o r stimulation of photosynthesis by short-term feeding of Pi. These observations indicate that leaves which have spent a long period in the dark and which are low in carbohydrate may shift from a situation at low temperature where photosynthesis is phosphate-limited to one in which photosynthesis is limited by some other factor. We tested the possibility that photosynthesis was limited by the carbohydrate status o f the leaves by feeding glucose to leaves of plants which had been darkened at 2 5 ~ for 24 h. The assimilation rate increased from 5.6 _ 0.5 to 10.6 _ 1 lamol 9m - 2. s - 1 (mean_+ SE of three leaves), when the leaves were illuminated at 8~ C and fed 25 m M glucose, showing that the assimilation rate was being lowered by some factor related to the availability of carbohydrate. Figure I shows that feeding glucose to barley leaves also shortened induction considerably. When Pi was fed to the leaves fed glucose there was a small, but more or less immediate, increase in the assimilation rate (compare Table 2). On the other hand, when Pi was fed to the control leaves there was initially no response, but after 20-30 min the assimilation started to rise gradually and continued to do so over the next 1-2 h until it reached a rate comparable to that observed in the leaves fed glucose and Pi (Fig. 1). In leaves preilluminated for 6 h or 24 h there were only slight stimulations of photosynthesis after feeding glucose (less than 10%; data not shown). Table 3 shows the effect of glucose feeding on metabolite pools in leaves which had been darkened and transferred to sorbitol or glucose at 5 ~ for 6 h. Glucose feeding led to a doubling of the pool of hexose-P in the dark, similar to the observations made by Stitt et al. (1984), and led to an increase in the glucose-6-phosphate/fructose-6-phosphate ratio, indicating an accumulation of these compounds in the cytosol (Gerhardt et al. 1987). However, there were relatively small adjustments in the metabolite pools in the illuminated leaves compared with the control leaves incubated on sorbitol, except for a large fall in the content of PGA. This fall
the light was higher at 5 ~ but PGA/triose-P ratios were also slightly higher in plants which had been kept at 5 ~ C (Table 2; column 3) than in plants kept at 25 ~ C (Table 2; column 1).
Influence of feeding Pi. Tables 1 and 2 also show how short-term Pi feeding (i.e. eliciting a response within a b o u t 10 min of the c o m m e n c e m e n t of Pi feeding) affected rates of CO2 assimilation and metabolite pools in these leaves. In plants which had been illuminated for 24 h and maintained at 25 ~ C, the decrease over 10 h in the CO2-assimilation rate measured at 8 ~ C was largely reversed by feeding Pi. In the plants which had been kept in the dark at 5~ C, Pi feeding led to substantial further increases in the rate of carbon assimilation so that the rate after b o t h 3 h and 10 h darkness was above that of the zero-time treatment. The ability of Pi to stimulate the CO2-assimilation rate rose (from 11% to 54%) during the course of incubation at 5 ~ in the dark. In m o s t cases (except after the treatment of plants for 3 h at 25 ~ C), feeding Pi led to a large increase in esterified phosphate (see also Stitt and Schreiber 1988). After 10 h (Table 1) there were increases in the amounts of hexose-P, triose-P and R u B P accompanying the increase in photosynthetic rate and very much lower P G A triose-P ratios for both pretreatments after 3 h and 10 h, indicating a lessening of the restriction by phosphate on the reduction of P G A to triose-P. In plants which had been darkened for 24 h, the short-term ability of Pi to stimulate the CO2-assimilation rate declined during the course of the incubation in the dark at 25 ~ C and at 5~ C. However, in these experiments we could distinguish two types of response to feeding Pi. Although an immediate response (within 10 min of feeding Pi through the transpiration stream) was usually observed, in leaves darkened for 24 h there was a much slower response to Pi feeding (see below) which took between 1 and 2 h.
Influence of feeding glucose and Pi to leaves depleted of carbohydrate. The data in Table 2 show an increase in I
I
r
I
I
I
r
I
t
r
I
i
t
r
T" Pi
10
l
glucose
+
E
E
//,
m tO
5
Pi
P l a n t a 9 Springer-Verlag1990
Factors influencing the capacity for photosynthetic carbon assimilation in barley leaves at low temperatures Carlos A. Labate* and Richard C. Leegood Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield, Sheffield SI0 2TN, UK Received 24 January; accepted 21 May 1990
Abstract. The aim of this work was to examine the effect upon photosynthetic capacity of short-term exposure (up to 10 h) to low temperatures (5 ~ C) of darkened leaves of barley (Hordeum vulgare L.) plants. The carbohydrate content, metabolite status and the photosynthetic rate of leaves were measured at low temperature, high light and higher than ambient CO2. Under these conditions we could detect whether previous exposure of leaves to low temperature overcame the limitation by phosphate which occurs in leaves of plants not previously exposed to low temperatures. The rates of CO2 assimilation measured at 8~ C differed by as much as twofold, depending upon the pretreatment. (i) Leaves from plants which had previously been darkened for 24 h had a low content of carbohydrate, had the lowest CO2-assimilation rates at low temperature, and photosynthesis was limited by carbohydrate, as shown by a large stimulation of photosynthesis by feeding glucose. (ii) Leaves from plants which had previously been illuminated for 24 h and which contained large carbohydrate reserves showed an accumulation of phosphorylated intermediates and higher CO2-assimilation rates at low temperature, but nevertheless remained limited by phosphate. (iii) Maximum rates of CO2 assimilation at low temperature were observed in leaves which had intermediate reserves of carbohydrate or in leaves which were rich in carbohydrate and which were also fed phosphate. It is suggested that carbohydrate reserves potentiate the system for the achievement of high rates of photosynthesis at low temperatures by accumulation of photosynthetic intermediates such as hexose phosphates, but that this potential cannot be realised if, at the same time, carbohydrate accumulation is itself leading to feedback inhibition of photosynthesis. *Present address: Escola Superior de Agricultura "Luiz de Queiroz", Departamento de Gen+tica, Universidada de S~o Paulo, Piracicaba, C.P. 83, CEP 13400, Brazil Abbreviations: A = rate of photosynthetic CO2 assimilation; Chl = chlorophyll; hexose-P = hexose phosphate(s); PFD = photon flux density; PGA=glycerate-3-phosphate; Pi=inorganic phosphate; RuBP = ribulose-1,5-bisphosphate; triose-P = triose phosphate(s)
Key words: Carbon assimilation, p h o t o s y n t h e t i c - Hordeum (leaf, carbon assimilation) - Photosynthesis - Temperature, low (carbon assimilation)
Introduction Abrupt transfer of leaves of C3 plants to low temperatures can result in a limitation of the rate of photosynthetic carbon assimilation by phosphate. This occurs when leaves are illuminated at high photon flux densities (PFDs) and CO2 concentrations which are at, or above, ambient (reviewed by Leegood 1985, 1989). This limitation occurs because sucrose synthesis runs too slowly at low temperatures. The restriction of sucrose synthesis, and hence recycling of inorganic phosphate (Pi) to resynthesis ATP in the chloroplast, might occur in one or all o f a number of ways. The observation that this limitation by the rate of sucrose synthesis at low temperatures can be ameliorated by feeding Pi (Leegood and Furbank 1986; Labate and Leegood 1988) indicates that more phosphate (either as Pi or as esterified phosphate) is required for photosynthetic metabolism at lower than at higher temperatures. Sucrose synthesis could be curtailed (i) by the amount of free Pi in the cytosol [and hence diminished export of triose phosphate (triose-P) from the chloroplast via the phosphate translocator]; this could occur either because the cytosolic Pi optimum for export o f carbon from the chloroplast increases (Leegood and Walker 1983), or because sequestration of cytosolic Pi into accumulated phosphorylated intermediates occurs [leaves illuminated at lower temperatures tend to accumulate phosphorylated intermediates (Leegood 1985; Stitt and Grosse 1988; Labate et al. 1990), which may be needed to compensate for decreases in enzyme affinity and velocity at low temperatures]; (ii) by limiting amounts of phosphorylated intermediates, if insufficient Pi was available in the cytosol to allow for the accumulation of phosphate esters at low temperatures; or (iii) by temperature-dependent changes in the
C.A. Labate and R.C. Leegood: Photosynthesis at low temperatures properties of, for example, the cytosolic fructose bisphosphatase or sucrose-phosphate synthase (Stitt and Grosse 1988). Acclimation of photosynthetic carbon assimilation to lower temperatures is readily observed in the longterm, e.g. by increased Oz sensitivity of photosynthesis after growth at low temperatures (e.g. Cornic and Louason 1980; Sage and Sharkey 1987) and, after 2 d at low temperatures, by a substantial increase in photosynthetic rates in barley leaves (Sicher et al. 1988). However, exposures of even a few hours to low temperatures in the dark can lead to the accumulation of phosphorylated intermediates (Labate and Leegood 1989), significantly increased rates of CO2 assimilation, and relief of symptoms of phosphate deficiency such as O2-insensitivity (Labate and Leegood 1988). O f course, such short-term acclimation to low temperature will not be solely dependent upon increasing the metabolically-active phosphate pools. Limitation by Pi will only be important in saturating light and high CO2, and other changes in the regulation of photosynthesis will be necessary in limiting light. Many parallel processes will also occur over different periods of time. The aim of this study was to investigate how altering the a m o u n t of carbohydrate and phosphorylated metabolites in leaves, by prolonged light or dark pretreatment of barley plants (Labate and Leegood 1989), affected short-term acclimation to low temperature of the photosynthetic rate, measured in saturating light and high CO2 so as to promote a limitation by phosphate.
Material and methods Plants. Barley (Hordeum vulgare L. cv. Sonja; Nickersons R.P.B., High Wycombe, Bucks, UK) was grown as described previously (Labate and Leegood 1988) in a greenhouse, under a maximum quantum flux density of 500-600 gmol quanta-m 2.s-1 at midday. Air temperature was kept at an average of 30~ C during the day and 18~ C at night. Gas-exchange measurements. Although in many leaves the maximum photosynthetic capacity can be measured in saturating (approx. 5%) CO2 in the 'leaf-disc O2 electrode' (Delieu and Walker 1981) this is not the case for barley, in which photosynthetic rates are considerably lower at these very high COz concentrations (data not shown). Maximum rates of photosynthetic CO2 uptake by barley primary leaves were therefore estimated in an infra-red gasanalysis system using a high CO2 concentration of 860 ~tbar, in 20% O2 and at a PFD of 1000 ~tmol.m- 2. s- 1. Water was circulated through an aluminium chamber (Doncaster et al. 1989) to give effective temperature control to 8_ 1~ C. The leaf temperature was monitored by a thermocouple attached to the underside of the leaf. Sections of barley leaves were mounted side-by-side in the chamber and fed with either water or 50 mM K2HPO4 (pH 7). The windows of the chamber were Parafilm discs held in place by O-rings. The branches of a bifurcated fibre optic (from a KL 1500 projector; Schott, Mainz, FRG) were placed at an angle of 45~ to illuminate the leaf from above. Metabolite measurements. The illuminated leaves were freezeclamped in situ (Doncaster et al. 1989) or darkened leaf samples were immersed in liquid N2. Leaf samples were then ground in a pestle and mortar, pre-cooled with liquid N2, and killed and extracted in HC104 as described previously (Leegood and Furbank
493 1986). Following neutralisation, sufficient charcoal was added to render the extract completely colourless. The extract was centrifuged at 2000 .g for 2 min. The supernatant was either immediately used or stored in liquid N2 for the determination of metabolites by the methods of Lowry and Passonneau (1972) in a Hitachi (Tokyo, Japan) 557 dual-wavelength spectrophotometer (340400 nm). Ribulose-l,5-bisphosphate (RuBP) and glycerate-3-phosphate (PGA) were measured as described by Doncaster et al. (1989). Total carbohydrate (starch, sucrose and hexose) was measured as described previously (Labate and Leegood 1989).
Chlorophyll. Pellets remaining from the HC104 extraction were washed with water and homogenised in 2 ml water. Following overnight extraction in 80% (v/v) acetone at room temperature, phaeophytin was measured by the method of Vernon (1960).
Results Changes in photosynthetic rates and metabolites during exposure to low temperature. The carbohydrate content of the leaves of intact barley plants was manipulated by preillumination, or darkening, of the leaves for a period of 24 h (Labate and Leegood 1989). Both sets of plants were then darkened for varying periods of time at 25 ~ C or at 5~ C. We then measured hexose phosphate (hexose-P) and the carbohydrate content of the leaves in the dark, before illuminating them and allowing them to reach steady-state photosynthesis at 8~ C. Steadystate photosynthesis was measured under conditions o f high P F D (1000gmol q u a n t a . m - Z . s -1) and higher than ambient CO2 (860 labar). This promotes a limitation of photosynthesis by phosphate in the leaves o f plants which have not previously been exposed to low temperatures (Labate and Leegood 1988). In this way we could follow the acclimation process (in respect o f overcoming the limitation by phoSphate) as an increase in the rate of CO2 assimilation. The leaves were then freeze-clamped in the light for the determination of metabolites. In parallel experiments, phosphate was supplied to similar leaves during illumination and metabolites measured. The following observations were made: (i) In plants which had previously been illuminated for 24 h there was a large accumulation of carbohydrate (Table 1). There was a gradual decrease in the COz-aSsimilation rate measured at 8~ C in those plants which were kept in the dark at 25 ~ C (Table 1 ; first column). In the plants which were kept in the dark at 5~ C there was an increase in the assimilation rate after 3 h compared with the control plants, followed by a decrease in the photosynthetic rate after 10 h in darkness (Table 1 ; third column). In plants which had been previously been darkened for 24 h the carbohydrate content was very low (Table 2). There was little change in the rate of carbon assimilation in the plants kept at 25 ~ C (it should be noted that the rates o f CO2 assimilation were comparable with those o f leaves of plants illuminated for 24 h and then darkened for 3 h or 10 h at 25 ~ C). In the plants kept at 5~ C, the assimilation rates were the same after 3 h and after 10 h as in the plants kept at 25 ~ C. There was, therefore, no evidence that the plants exposed to a long
494
C.A. Labate and R.C. Leegood: Photosynthesis at low temperatures
TaMe 1. Effect of temperature pretreatment on steady-state CO2-assimilation rate and metabolite contents of illuminated barley leaves. Plants were kept in the light for 24 h at 25~ C, and then at 25~ C, or at 5~ C in the dark for a further period of 0, 3 or 10 h. After this pretreatment, leaves were detached. One sample was extracted for measurement of hexose-P and carbohydrate while another was placed in a chamber for gas exchange and allowed to reach steady-state photosynthesis at 8~ C in the presence (" Fed Pi") or absence ("Fed HzO") of 50 mM K2HPO 4 fed through the transpiration stream. Contents of total carbohydrate are shown in gmol hexose equivalents.(mg Chl) -1. Photosynthesis was measured at a CO 2 concentration of 860 labar and a PFD of 1000 gmol.m 2.s-1. Data are means • SE of three experiments Parameter
Plants illuminated for 24 h and then maintained in darkness Time in the dark
Temperature in the dark
(h)
25~ C
5~ C
Treatment during gas-exchange measurement Fed H20
Fed Pi
Fed H20
Fed Pi
Oh A (gmol.m-2.s -1) Carbohydrate in dark (gmol hexose-(mg Chl)-1) Metabolites (nmol-(mg Chl)- 1) Hexose-P in dark Hexose-P in light PGA in light Triose-P in light PGA/triose-P in light RuBP in light Z esterified phosphate in light
7.9•
0.7
14.6-+
1
266 _+ 22 456 _+ 44 240 -+ 13 55 _+ 1 4.8_+ 1 179 -+ 20 1109 • 100
8.4•
0.7
613 _+ 53 259 _+ 32 70 +_ 4 3.8_+ 1 163 _+ 5 1268 _+101
3h A (gmol.m-Z.s 1)
6.0-1-0.2
Carbohydrate in dark (I.tmolhexose. (mg Chl)- 1) Metabolites (nmol-(mg Chl)- 1) Hexose-P in dark Hexose-P in light PGA in light Triose-P in light PGA/triose-P in light RuBP in light X esterified phosphate in light
8.8_+
0.4
8.8_+ 0.4
15.1 -I- 1
15.9-+ 1
182 + 5 384 -+16 361 -+ 56 42 _+ 1 8.6_+ 1 105 +11 997 _+95
334 _+12 460 +36 302 -+ 16 65 -+ 4 4.7_+ 0.5 188 _+20 1203 _+96
346 _+ 20 278 _+ 66 79 + 18 3.5_+ 1 116 -+ 16 925 _+136
10.3__+ 0.5
709 -I-12 339 _+31 139 _+13 2.4+ 0.i 186 _+ 3 1559 _+82
10h A (gmol. m - 2. s- 1)
5.0_+ 0.7
Carbohydrate in dark (gmol hexose.(mg Chl)-X)
7.9+ 3
Metabolites (nmol.(mg Chl)-x) Hexose-P in dark Hexose-P- in light PGA in light Triose-P in light PGA/triose-P in light RuBP in light Z esterfied phosphate in light
d a r k p r e t r e a t m e n t h a d s u b s e q u e n t l y acclimated to low temperature. (ii) I n leaves f r o m p l a n t s which h a d previously been i l l u m i n a t e d for 24 h (Table 1) there was a g r a d u a l fall in the a m o u n t o f hexose-P d u r i n g d a r k n e s s at 2 5 ~ (Stitt et al. 1985; L a b a t e a n d L e e g o o d 1989). I n those p l a n t s which were m a i n t a i n e d at 5~ C, there was first a rise in hexose-P c o n t e n t after 3 h, followed by a fall,
85 _+20 326 _+32 358 _+17 34 _+ 5 10.9__+ 2 76 +22 870 _+98
7.4_+
1
6.0_+ 0.3
9.2_+
0.2
13.7_+ 1
504 _+ 67 369 -+ 13 88 _+ 16 4.4_+ 0.6 141 + 25 1243 _+166
278 467 422 66 7 131 1217
_+35 ___34 -+28 _+15 + 1 __10 _+97
629 _ 15 420 -+ 37 97 • 10 4.4_+ 0.5 173 _+ 30 1492 +122
a n d hexose-P r e m a i n e d very m u c h higher in the lowt e m p e r a t u r e t r e a t m e n t (see also L a b a t e a n d Leegood 1989). The s u m o f esterified p h o s p h a t e present in the phosph o r y l a t e d intermediates in i l l u m i n a t e d leaves was also calculated (these intermediates w o u l d be largely present in the chloroplasts a n d cytoplasm). Table 1 shows t h a t in leaves f r o m plants which h a d been kept for 24 h in
C.A. Labate and R.C. Leegood: Photosynthesis at low temperatures
495
Table 2. Effect of temperature pretreatment on steady-state CO2-assimilation rate and metabolite contents of illuminated barley leaves. Plants were kept in the dark for 24 h at 25~ C, and then at 25~ C, or at 5~ C in the dark for a further period of 0, 3 or 10 h. After this pretreatment, leaves were detached. One sample was extracted for measurement of hexose-P and carbohydrate while another placed in the chamber for gas exchange and allowed to reach steady-state photosynthesis at 8~ C in the presence (" Fed Pi") or absence (" Fed H20") of 50 mM K2HPO4 fed through the transpiration stream. Contents of total carbohydrate are shown in gmol hexose equivalents. (mg Chl)-1. Photosynthesis was measured at a CO2 concentration of 860 labar and a PFD of 1000 gmol.m - 2 . s - 1. Data are means• of three experiments Parameter
Plants darkened for 24 h and then maintained in darkness Time in the dark (h)
Temperature in the dark 25~ C
5~ C
Treatment during gas-exchange measurement Fed H20
Fed Pi
Fed HzO
Fed Pi
Oh A (gmol.m-Z.s -1)
5.6•
0.7
Carbohydrate in dark (gmol hexose. (mg Chl)-1)
0.8•
0.4
Metabolites (nmol. (mg Chl)- 1) Hexose-P in dark Hexose-P- in light PGA in light Triose-P in light PGA/triose-P in light RuBP in light X ester• phosphate in light
57 • 5 179 • 46 178 • 50 23 • 10 10.3• 4 76 • 23 532 •
8.4•
0.5
345 • 40 200 • 45 46 • 18 5.2• 1 120 • 16 831 •
3h A (gmol.m-2.s -1)
5.8•
Metabolites (nmol.(mg Chl)-1) Hexose-P in dark
0.5
65 • 7 290 • 40 237 • 56 43 • 4 5.5• 1 87 • 21 744 •
Hexose-P- in light PGA in light Triose-P in light PGA/triose-P in light RuBP in light X ester• phosphate in light
7.0•
1
275 • 47 167 • 14 44 • 8 4.0• 0.4 84 • 14 654 • 97
5.8•
0.3
124 • 17 237 • 27 189 • 51 3O • 6 6.1• 1 71 • 12 598 •
7.1_+ 1
312 • 212 • 53 • 4.8• 1 80 • 737 • 84
1Oh A (gmol. m - z. s- t) Metabolites (nmol. (mg Chl)- x) Hexose-P in dark Hexose-P- in light PGA in light Triose-P in light PGA/triose-P in light RuBP in light X ester• phosphate in light
the light a n d t h e n d a r k e n e d , the s u m o f ester• phosp h a t e in the light in leaves o f p l a n t s which h a d been kept at 5 ~ was c o n s i d e r a b l y greater t h a n in those w h i c h h a d b e e n kept at 2 5 ~ for b o t h 3 h a n d 10 h darkness. This change was p a r t l y caused b y higher c o n tents o f hexose-P, triose-P a n d R u B P a n d was a c c o m p a nied by lower P G A / t r i o s e - P ratios. T h e decrease in this ratio indicates a lessening, after exposure to low temperature, o f the restriction b y p h o s p h a t e availability o n the g e n e r a t i o n o f A T P a n d N A D P H necessary to reduce P G A to triose-P (Heber et al. 1986).
5.0•
0.3
80 • 5 206 • 26 203 • 51 35 • 9 6.4• 2 66 • 7 576 •
5.2•
0.5
245 • 56 253 • 48 46 • 10 5.8• 1 81 • 6 706 •
5.6•
1
96 • 372 • 295 • 40 • 7.8• 90 • 887 •
36 25 53 7 2 25
6.5•
1
506 • 54 280 • 76 60 • 22 5.3• 1 98 • 15 1042 •
(iii) I n the leaves f r o m p l a n t s which h a d previously b e e n d a r k e n e d for 24 h (Table 2), there was a small rise in the c o n t e n t o f hexose-P in the d a r k b o t h at 5~ C a n d at 25 ~ C. C o n t e n t s o f total p h o s p h o r y l a t e d i n t e r m e d i a t e s in i l l u m i n a t e d leaves were m u c h lower t h a n i n the leaves o f those p l a n t s which h a d b e e n kept in the light for 24 h. A l t h o u g h there was a n increase after 3 h f u r t h e r darkness, followed by a decrease after 10 h at 25 ~ C, there was a n increase in total ester• phosphate in the light b e t w e e n 3 h a n d 10 h in p l a n t s which h a d b e e n kept at 5~ C. After 10 h, total ester• p h o s p h a t e in
496
C.A. Labate and R.C. Leegood: Photosynthesis at low temperatures the PGA/triose-P ratio at low temperatures (in the absence of Pi feeding) in illuminated leaves of plants which had been predarkened for 24 h, then kept in the dark at 5 ~ C, and a p o o r stimulation of photosynthesis by short-term feeding of Pi. These observations indicate that leaves which have spent a long period in the dark and which are low in carbohydrate may shift from a situation at low temperature where photosynthesis is phosphate-limited to one in which photosynthesis is limited by some other factor. We tested the possibility that photosynthesis was limited by the carbohydrate status o f the leaves by feeding glucose to leaves of plants which had been darkened at 2 5 ~ for 24 h. The assimilation rate increased from 5.6 _ 0.5 to 10.6 _ 1 lamol 9m - 2. s - 1 (mean_+ SE of three leaves), when the leaves were illuminated at 8~ C and fed 25 m M glucose, showing that the assimilation rate was being lowered by some factor related to the availability of carbohydrate. Figure I shows that feeding glucose to barley leaves also shortened induction considerably. When Pi was fed to the leaves fed glucose there was a small, but more or less immediate, increase in the assimilation rate (compare Table 2). On the other hand, when Pi was fed to the control leaves there was initially no response, but after 20-30 min the assimilation started to rise gradually and continued to do so over the next 1-2 h until it reached a rate comparable to that observed in the leaves fed glucose and Pi (Fig. 1). In leaves preilluminated for 6 h or 24 h there were only slight stimulations of photosynthesis after feeding glucose (less than 10%; data not shown). Table 3 shows the effect of glucose feeding on metabolite pools in leaves which had been darkened and transferred to sorbitol or glucose at 5 ~ for 6 h. Glucose feeding led to a doubling of the pool of hexose-P in the dark, similar to the observations made by Stitt et al. (1984), and led to an increase in the glucose-6-phosphate/fructose-6-phosphate ratio, indicating an accumulation of these compounds in the cytosol (Gerhardt et al. 1987). However, there were relatively small adjustments in the metabolite pools in the illuminated leaves compared with the control leaves incubated on sorbitol, except for a large fall in the content of PGA. This fall
the light was higher at 5 ~ but PGA/triose-P ratios were also slightly higher in plants which had been kept at 5 ~ C (Table 2; column 3) than in plants kept at 25 ~ C (Table 2; column 1).
Influence of feeding Pi. Tables 1 and 2 also show how short-term Pi feeding (i.e. eliciting a response within a b o u t 10 min of the c o m m e n c e m e n t of Pi feeding) affected rates of CO2 assimilation and metabolite pools in these leaves. In plants which had been illuminated for 24 h and maintained at 25 ~ C, the decrease over 10 h in the CO2-assimilation rate measured at 8 ~ C was largely reversed by feeding Pi. In the plants which had been kept in the dark at 5~ C, Pi feeding led to substantial further increases in the rate of carbon assimilation so that the rate after b o t h 3 h and 10 h darkness was above that of the zero-time treatment. The ability of Pi to stimulate the CO2-assimilation rate rose (from 11% to 54%) during the course of incubation at 5 ~ in the dark. In m o s t cases (except after the treatment of plants for 3 h at 25 ~ C), feeding Pi led to a large increase in esterified phosphate (see also Stitt and Schreiber 1988). After 10 h (Table 1) there were increases in the amounts of hexose-P, triose-P and R u B P accompanying the increase in photosynthetic rate and very much lower P G A triose-P ratios for both pretreatments after 3 h and 10 h, indicating a lessening of the restriction by phosphate on the reduction of P G A to triose-P. In plants which had been darkened for 24 h, the short-term ability of Pi to stimulate the CO2-assimilation rate declined during the course of the incubation in the dark at 25 ~ C and at 5~ C. However, in these experiments we could distinguish two types of response to feeding Pi. Although an immediate response (within 10 min of feeding Pi through the transpiration stream) was usually observed, in leaves darkened for 24 h there was a much slower response to Pi feeding (see below) which took between 1 and 2 h.
Influence of feeding glucose and Pi to leaves depleted of carbohydrate. The data in Table 2 show an increase in I
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