Restrictions to photosynthesis can limit plant growth at high temperature in a variety of ways. In addition to increasing photorespiration, moderately high temperatures (35-42 ∞ ∞ ∞ ∞ C) can cause direct injury to the photosynthetic apparatus. Both carbon metabolism and thylakoid reactions have been suggested as the primary site of injury at these temperatures. In the present study this issue was addressed by first characterizing leaf temperature dynamics in Pima cotton ( Gossypium barbadense ) grown under irrigation in the US desert south-west. It was found that cotton leaves repeatedly reached temperatures above 40 ∞ ∞ ∞ ∞ C and could fluctuate as much as 8 or 10 ∞ ∞ ∞ ∞ C in a matter of seconds. Laboratory studies revealed a maximum photosynthetic rate at 30-33 ∞ ∞ ∞ ∞ C that declined by 22% at 45 ∞ ∞ ∞ ∞ C. The majority of the inhibition persisted upon return to 30 ∞ ∞ ∞ ∞ C. The mechanism of this limitation was assessed by measuring the response of photosynthesis to CO 2 in the laboratory. The first time a cotton leaf (grown at 30 ∞ ∞ ∞ ∞ C) was exposed to 45 ∞ ∞ ∞ ∞ C, photosynthetic electron transport was stimulated (at high CO 2 ) because of an increased flux through the photorespiratory pathway. However, upon cooling back to 30 ∞ ∞ ∞ ∞ C, photosynthetic electron transport was inhibited and fell substantially below the level measured before the heat treatment. In the field, the response of assimilation ( A ) to various internal levels of CO 2 ( C i ) revealed that photosynthesis was limited by ribulose-1,5-bisphosphate (RuBP) regeneration at normal levels of CO 2 (presumably because of limitations in thylakoid reactions needed to support RuBP regeneration). There was no evidence of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) limitation at air levels of CO 2 and at no point on any of 30 A -C i curves measured on leaves at temperatures from 28 to 39 ∞ ∞ ∞ ∞ C was RuBP regeneration capacity measured to be in substantial excess of the capacity of Rubisco to use RuBP. It is therefore concluded that photosynthesis in field-grown Pima cotton leaves is functionally limited by photosynthetic electron transport and RuBP regeneration capacity, not Rubisco activity.

Photosynthesis is inhibited by high temperatures that plants are likely to experience under natural conditions. Both increased thylakoid membrane ionic conductance and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) deactivation have been suggested as the primary cause. The moderately heat-tolerant crop Pima S-6 cotton ( Gossypium barbadense ) was used to examine heat stressinduced inhibition of photosynthesis. Previous field-work indicated that moderate heat stress ( T = 35-45 ∞ ∞ ∞ ∞ C) is associated with very rapid leaf temperature changes. Therefore, a system was devised for rapidly heating intact, attached leaves to mimic natural field heat-stress conditions and monitored Rubisco activation, carbon-cycle metabolites, thylakoid ionic conductance, and photosystem I activity. As a proxy for NADPH and stromal redox status the activation state of NADP-malate dehydrogenase (NADP-MDH) was measured. In dark-adapted cotton leaves, heating caused an increase in thylakoid permeability at temperatures as low as 36 ∞ ∞ ∞ ∞ C. The increased permeability did not cause a decline in adenosine 5 ¢ ¢ ¢ ¢ -triphosphate (ATP) levels during steady-state or transient heating. Rapid heating caused a transient decline in ribulose 1,5-bisphosphate without a decrease in Rubisco activation. Sustained heating caused a decline in Rubisco activation and also oxidized the stroma as judged by NADP-MDH activation and this is hypothesized to result from increased cyclic photophosphorylation, explaining the maintenance of ATP content in the face of increased thylakoid membrane ion leakiness.

Transitory starch is formed in chloroplasts during the day and broken down at night. Transitory starch degradation could be regulated by light, circadian rhythms, or carbon balance. To test the role of these potential regulators, starch breakdown rates and metabolites were measured in bean (Phaseolus vulgaris) and Arabidopsis (Arabidopsis thaliana) plants. In continuous light, starch and maltose levels oscillated in a circadian manner. Under photorespiratory conditions, transitory starch breakdown occurred in the light faster than at night and glucose-6-P (G6P) was elevated. Nonaqueous fractionation showed that the increase in G6P occurred in the chloroplast. When Arabidopsis plants lacking the plastidic starch phosphorylase enzyme were placed under photorespiratory conditions, G6P levels remained constant, indicating that the increased chloroplastic G6P resulted from phosphorolytic starch degradation. Maltose was increased under photorespiratory conditions in both wild type and plants lacking starch phosphorylase, indicating that regulation of starch breakdown may occur at a point preceding the division of the hydrolytic and phosphorolytic pathways. When bean leaves were held in N 2 to suppress photosynthesis and Suc synthesis without increasing photorespiration, starch breakdown did not occur and maltose and G6P levels remained constant. The redox status of the chloroplasts was found to be oxidized under conditions favoring starch degradation.

High temperature inhibits photosynthesis by several mechanisms including reduction in Rubisco activity. While the initial reaction velocity of purified, fully carbamylated, inhibitor-free Rubisco increases with temperature in vitro, over time, the reaction velocity slowly declines (fallover) because of the enzymatic and non-enzymatic production of inhibitors from the substrate ribulose-1,5-bisphosphate. We tested whether fallover could contribute to the decline in Rubisco activity observed in leaf extracts at high temperature. Production of d-xylulose-1,5-bisphosphate (XuBP), an inhibitor of Rubisco, was greater at 35 and 45°C than at 25°C but fallover was less severe at 35 and 45°C than at 25°C, both in rate and extent under saturating CO2 and ambient O2. This apparent dichotomy is consistent with the catalytic site of Rubisco loosening at higher temperatures and releasing inhibitors more easily. The loosening of the catalytic site was supported by the observation that RuBP and XuBP were released from their complexes with uncarbamylated, Mg2+-free Rubisco faster at 35 and 45°C than at 25°C. We conclude that, although XuBP production increased relative to catalytic throughput at higher temperatures, this was more than compensated for by its faster release, resulting in less fallover inhibition at higher temperatures.