Plant Physiology Division, Department of Scientific and Industrial Research, Palmerston North, New Zealand. ABSTRACT. Patterns of radiocarbon exchange ...
Plant Physiol. (1973) 52, 180-182
Short Communication
Plants under Climatic Stress V. CHILLING AND LIGHT EFFECTS ON RADIOCARBON EXCHANGE BETWEEN PHOTOSYNTHETIC INTERMEDIATES OF SORGHUM Received for publication February 13, 1973 I. R. BROOKING AND A. 0. TAYLOR
Plant Physiology Division, Department of Scientific and Industrial Research, Palmerston North, New Zealand ABSTRACT
Patterns of radiocarbon exchange between photosynthetic intermediates of the chilling sensitive Sorghum bicolor were modified by exposure to a combined environmental stress of low temperature (10 C) and moderate light levels (170 w m-2, visible). Pulse chase experiments with"CO2 showed that this stress initially slowed the release of photosynthetically absorbed radiocarbon from malate. Further exposure caused an increased proportion of the radiocarbon to accumulate in aspartate. This trend continued, so that after 30 hours, some 80% of absorbed radiocarbon remained in aspartate after 1 minute of chasing and subsequent release of carbon into the C3 cycle was very slow. In Sorghum, chilling combined with light seemed to cause a restriction in an early step of the C pathway before ultrastructural changes could be detected in the mesophyll chloroplasts.
Chilling temperatures combined with high levels of light have been shown (6, 8) to cause a progressive reduction in the photosynthetic capacity of the leaves of several plants of tropical and subtropical origin. Chloroplasts, which were most exposed to light during such environmental stress, underwent marked and quite rapid ultrastructural changes before finally bursting and contributing to cell and eventual leaf necrosis (9). Many C4 photosynthetic pathway species (3) seemed to be particularly chilling-sensitive, and in these plants chilling under high light rapidly altered the level of amino acids formed from intermediates of the C4 pathway (10). This led us to suggest that some time- and temperature-dependent blockages were developing in the interconversion of C4 pathway intermediates and possibly in the flow of other intermediates to and from the sites of C4 photosynthesis. In the present communication, we have looked at the effects of chilling and light on rates of radiocarbon exchange between photosynthetic intermediates of the chilling-sensitive C4 species, Sorghum bicolor. The data presented here show that increasing periods of this combined environmental stress cause a proportionate increase in the photosynthetic labeling of aspartate in this plant. This apparent restriction in carbon exchange between early C4 pathway intermediates seems to precede any marked changes in chloroplast ultrastructure.
MATERIALS AND METHODS
Plants of Sorghum bicolor hybrid NK145 were grown in controlled environment cabinets (8) at a constant 25 C with 12-hr photoperiods of 170 w m-2. They were shifted to similar cabinets maintained at 10 C for low temperature treatment at the commencement of a photoperiod. Light intensities were maintained at 170 w m-2 during this chilling or were reduced to 50 w m-2 by neutral screens. Mid-portions of attached most recently mature leaves were pulse-labeled with radiocarbon in a half liter Perspex chamber (8) maintained within the larger environment cabinet. CO.-free air was drawn over the enclosed leaf for approximately 1 min, then CO, levels returned to ambient by the addition of 200 ,uc of "4CO2. After 20-sec photosynthesis, leaves were moved into the radiocarbon-free air of the main cabinet. A sequence of 14 mm 4 discs was punched from the main lamina of these leaves into methanol-chloroform-2 M formic acid (12:5:3) maintained at -70 C. CO2 supply was not limiting during this period of time, since leaves maintained continuously at 25 C used less than one-fifth of the available CO2 during the 20-sec pulse, while those at 10 C fixed less (see "Results"). Leaf discs were ground first in the killing medium, then reextracted with 50% aqueous methanol, and the combined supernatants were separated into phases by adding water and chloroform. Aliquots of the aqueous layer were separated on thin layers of MN 300 cellulose after first being passed through Sephadex G25. Two-dimensional chromatography in watersaturated phenol followed in the second direction with a propyl acetate based solvent (1), or a combination of thin layer electrophoresis followed by chromatography (7) were both used. Developed plates were then autoradiographed and radioactive areas removed after coating the plates with collodion (2).
RESULTS AND DISCUSSION Photosynthetic rates of Sorghum leaves prior to stress treatment were 20 to 22 X 10-' mg CO2/ cm' min at 25 C, corresponding to a radiocarbon uptake of 2.8 X 106 dpm/20 sec pulse in each leaf disc. Chilling progressively reduced this rate so that fixation in experiments shown as Figure 1, B, C, D, E, and F, fell to 42, 40, 7, 40, and 35%, respectively, of that at 25 C. Because of this, label in the major products, aspartate and malate, has been expressed as a percentage of the total radiocarbon isolated from the chromatograms. Other components not represented accounted for less than one-tenth of this total, while higher molecular weight compounds excluded by the Sephadex or centrifuging with the cell debris accounted 180
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PLANTS UNDER CLIMATIC STRESS. V
LOG TIME OF CHASE (MIN)
9
C)
-I
J
I_
0
I-
4 I-
0
0 0 *4*o
LOG
TIME
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CHASE (MIN)
FIG. 1. Effects of chilling and light on rates of exchange of radiocarbon between photosynthetic intermediates of Sorghum. Leaves developed at 25 C were pulsed (20 sec) and chased at 25 C (A) or chilled to 10 C and pulse chased after increasing periods of time at 170 w -m2 (B, C, D) or 50 w-mM2 (F). Leaves of some plants held at 10 C and 170 w-m2 for 30 hr were returned to 25 C for 15 min prior to pulse chasing (E). All 0 ; phosphate esters *----; alanine -0-; and sucrose labeling and chasing was done at 170 w- m2. Malate: *-. .; aspartate -
-
-A- -.
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BROOKING AND TAYLOR
for only a minor amount of the total carbon fixed by the leaf after even 10 min of chasing. Graphs shown are of individual experiments but were chosen as representative from several other runs. Phosphate esters were counted as a single pool, so no differentiation is made of any sequential labeling of the glycerate-3-P and the hexose phosphates. Immediate Effects of Low Temperature. Patterns of radiocarbon exchange between photosynthetic intermediates measured shortly after lowering the temperature of Sorghum (Fig. IB) were similar to those reported in corn at 8 C by Hofstra and Nelson (4). Radiocarbon was released more slowly from malate at 10 C than at 25 C (Fig. 1A), and there was a small lag before incorporation into sucrose commenced. The time taken for maximum radiocarbon to accumulate in the phosphate ester pool increased from some 10 to 50 sec; these times were measured from the start of the chase period. This delay in phosphate ester and subsequent sucrose labeling at 10 C could be due to a slow down in the presumed movement of malate (5) from mesophyll to bundle sheath chloroplasts. Unfortunately, the data were not extensive enough to permit quantitative evaluation of the effect of temperature on the kinetics of this postulated step in the C4-pathway. Time-dependent Effects of Low Temperature and Light. When low temperatures were maintained for longer periods under high light (170 w m-2) further changes in labeling patterns developed (Fig. 1, C and D). After only 6.5 hr at 10 C, the proportion of radiocarbon initially transferred into aspartic acid was higher and label was not lost so rapidly into subsequent photosynthetic intermediates. After 30 hr, this effect was very marked, with nearly 80% of the assimilated radiocarbon remaining in aspartate after 1 min chasing. This pattern became more pronounced, the longer the period of low temperature, high light stress. For example, 54 and 78 hr of stress treatment caused 60 and 80% of assimilated radiocarbon to be retained in aspartate after 10 min of chasing. Irregularities were often seen in graphs of the data obtained from more seriously injured leaves (Fig. ID). The smaller the amount of radiocarbon absorbed by individual discs taken from a leaf, the greater was the proportion of this radiocarbon seen in aspartate in these discs. Enhanced labeling of aspartate seems to be symptomatic of the damage process. Chilling Sorghum under high light may cause some serious restriction in the conversion of oxaloacetate to malate so that carbon entering the C, cycle (3) is trapped in aspartate. In support of this, we noted previously (10) that the level of free aspartate in Sorghiumn more than doubled during the 2nd day of stress treatment, while alanine levels fell sharply. This fall in alanine could have arisen from a reduced supply of 3carbon intermediates in later parts of the C4 pathway (3). One might also predict that malate would also be depleted under the same conditions, and yet we reported (10) that total malate levels were reduced only slowly by this stress. The pool of photosynthetic malate is thought to be quite small (5), however, and malate pools within the leaf may be rather rigidly delimited. In spite of this presumed blockage in the C4 photosynthetic pathway, little carbon from '4CO2 seems to appear in sucrose via C3 cycle activity without its passing through the C4 cycle.
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If a plant was returned to 25 C after an intermediate period of stress (30 hr), its photosynthetic rate recovered within 15 min to almost one-half of that seen previously at 25 C. When labeling patterns were investigated after this period at 25 C (Fig. 1E), a higher proportion of radiocarbon than usual (Fig. 1A) was still incorporated into aspartate, and this carbon was released somewhat more slowly than in nonstressed plants. Nevertheless, this short period at the warmer temperature has enabled the plant to recover much of its normal pattern of photosynthetic carbon metabolism. If Sorghum plants were kept for extended periods at low temperatures while under shaded conditions (50 w m-2), patterns of radiocarbon flow were altered only marginally after 55 hr (Fig. 1F) from those seen after 10 min at 10 C. Proportionate labeling of the phosphate ester pool was increased, however, and the lag in sucrose labeling became more pronounced. Correlation with Chloroplast Ultrastructural Changes. We have described previously (9), the rate and type of ultrastructural change which mesophyll chloroplasts of Sorghum undergo when exposed to the same stress conditions as used in this present labeling work. No chloroplast ultrastructural changes were detected after 6 hr of chilling at high light. After 30 hr of stress, only about one-third of chloroplasts in cells of the upper mesophyll were badly swollen, others less exposed to light in the same cells had just commenced thylakoid contraction, whereas the majority of mesophyll chloroplasts showed only a reduction in starch grain size. Yet we have shown that patterns of photosynthetic radiocarbon exchange, which presumably integrate the functioning of the whole mesophyll, have been significantly disrupted by this time. Changes in the rate of flow of radiocarbon between photosynthetic intermediates were in fact commencing after only 6.5 hr of stress. It seems that the phenomena which lead to a proportionate increase in aspartate labeling precede any visible changes in chloroplast structure. LITERATURE CITED
1. BIELESKI, R. L. AND R. E. YOu NG. 1963. Extraction and separation of plhSphate esters from plant tisstues. Anal. Biochem. 6: 54-68. 2. BIELESKI, R. L. AN-D N. A. Tu-NER. 1966. Separation and estimation of amino acids in crIi(I plant extracts bs- tliin layer electroplhoresis andl chromatography. Anal. Biochem. 17: 278-293. 3. H.ATCI, -M. D. AN-D C. R. SL;ACK. 1970. Photosynthetic COx-fixation patlisrNvys. Annu. Rev. Plant Physiol. 21: 141-162. 4. HOFSTRA, G. AN-D C. D. Nci.so-\. 1969. The translocation of photosynt-hetically assimilated 14C in corn. Can. J. Bot. 47: 1435-1442. 5. OSNION-D, C. B. 1971. AIetalholite transport in C4 photosynthesis. .AXst. J. Biol. Sci. 24: 159-163. 6. RONALEY, J. A. AN-D A. 0. TAYLOR. 1972. Plants under climatic stress. IV. Effects of C02 and 02 on plsotosysntesis un(ler- higlh liglht, low temperature stress. New Phytol. 71: 477-481. 7. SCHuRAi\N-N, P. 1969. Separation of phosphate esters airl algal extracts byv thIin layer electroplhoresis and chromatography. J. Chromatog. 39: 507-
509. 8. TAYLOR, A. 0. AND J. A. ROWlEY. 1971. Plants uinder climatic stress. I. Low temperature, high liglht effects on pliotosyntlhesis. Plant Physiol. 47: 713718. 9. TAYLOR, A. 0. AN-D A. S. CRAIG. 1971. Plants undler climatic stress. II. Low temperature, high light effects on chloroplast ultrastructure. Plant Physiol. 47: 719-725. 10. TAYLOR, A. O., N. Ml. JEPSENZ, A'N-D J. T. CHRISTELLER. 1972. Plants uinder climatic stress. III. Low temperatuire, higlh light effects on photosynthetic products. Plant Physiol. 49: 798-802.
