Photosynthetic response to moderate heat stress for a temperate and boreal populus species

The objective of my study was to elucidate the mechanism that constrains the photosynthesis capacity under moderate heat stress condition using two Populus species. Populus species adapted to contrasting thermal environments were used. Populus deltoides and P. balsamifera seedlings were grown in large pots (46 L) in a greenhouse at 25/17 oC (day/night) and natural lighting. Toward the end of the growing season, the photoperiod was prolonged by 5 h using two 400 W metal halide lamps. Gas-exchange data (under photorespiratory and non-photorespiratory conditions), including the response of CO2 assimilation (A) to internal leaf CO2 concentration (Ci), and ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) activation states were determined at 27, 36, and 40 oC for both species. To determine the minimum temperature at which a major phase shift occurred for thylakoid membranes, the minimal chlorophyll a fluorescence yield (F0) was measured as temperature increased for dark-acclimated leaf discs. A comparative characterization was performed on the molecular and kinetic differences of recombinant Rubisco activase from P. deltoides and P. balsamifera. The activase genes from both species were cloned, and the recombinant proteins were produced in transgenic E. coli and tested for their ATPase activity under different temperatures. The data indicated a significant difference between P. deltoids and P. balsamifera in their response of A to high temperature. As the leaf temperature increased above 27 oC, a decline in A occurred for both species under photorespiratory and non-photorespiratory conditions, however this decline was greater for P. balsamifera compared to P. deltoides, indicating a greater sensitivity of A to moderate high temperature for P. balsamifera compared to the response of A for P. deltoides. Exposure to 36 oC reduced A for P. balsamifera by 25% but only 11% for P. deltoides relative to A at 27 oC. When the temperature was raised to 40 oC, A was reduced 45% for P. balsamifera and 24% for P. deltoides. The analyses of A/Ci curves indicated that maximal electron transport limitation to A (Jmax) increased by about 38.5 % for P. deltoides and 21.7% for P. balsamifera when the temperature increased from 27 °C to 36 °C. Also, under non-photorespiratory conditions and high CO2 (1500 µL L-1) a stimulation of A occurred at temperatures as high as 40 oC for both species, and the values of F0 did not increase until a temperature of 48 oC was reached for both species. Taken together, these data indicate that problems with electron transport could not be constraining A at temperatures between 27 and 40 oC for these species and were not responsible for their differences in the response of A to moderately high temperatures. The A/Ci analyses also showed that there was a highly significant reduction in carboxylation efficiency (CE) as the temperature increased from 27 to 36 oC for both species, suggesting that Rubisco deactivation was occurring at these temperatures. Indeed, as leaf temperature was increased from 27 to 40 oC, a significant reduction in the activation state of the enzyme occurred for both species, but the greater reduction occurred for P. balsamifera Rubisco than for P. deltoides Rubisco at 36 and 40 oC. The problem of maintaining Rubisco activation state at high temperature has been linked to the heat-labile nature of Rubisco activase. Therefore, the genes for the â-isoform (short form) of Rubisco activase from both species were cloned. The full-length sequences of the cDNAs for the two species showed that the open reading frame (ORF) of the gene is 1323 bp (1149 bp without the cTP) in P. balsamifera and 1320 bp (1146 bp without the cTP) in P. deltoides. The mature protein for P. balsamifera is composed of 382 amino acids and that for P. deltoides is composed of 381 with a predicted protein size of 42.5 kDa and 94.2% sequence identity for the enzyme from both species.

Recombinant Rubisco activase protein without the chloroplast transit peptide was derived from the cDNA cloned from each species. The ATPase activity for the activases from both species increased as temperature increased from 10 up to 27 °C without any significant species differences. However, from 30 to 50 °C, the ATP hydrolysis activity of P. balsamifera activase was significantly lower than that for P. deltoides activase at each assay temperature. The optimum temperatures for ATPase activity coincided rather well with the optimum temperatures for A for both speceis (30°C for P. balsamifera activase and 35°C for P. deltoides activase).

Overall, the results of my study indicated that the reduction in A at moderately high temperatures was most likely caused by inactivation of Rubisco and not by electron transport limitation of A. The problem of maintaining Rubisco activation at high temperature in Populus species appears to be related to the thermal instability of the Rubisco activase as judged by ATPase activity.