Steven J. Crafts-Brandner

Relationship between the Heat Tolerance of Photosynthesis and the Thermal Stability of Rubisco Activase in Plants from Contrasting Thermal Environments

Inhibition of net photosynthesis (Pn) by moderate heat stress has been attributed to an inability of Rubisco activase to maintain Rubisco in an active form. To examine this proposal, the temperature response of Pn, Rubisco activation, chlorophyll fluorescence, and the activities of Rubisco and Rubisco activase were examined in species from contrasting environments. The temperature optimum of Rubisco activation was 10°C higher in the creosote bush (Larrea tridentata) compared with the Antarctic hairgrass (Deschampsia antarctica), resembling the temperature response of Pn. Pn increased markedly with increasing internal CO2 concentration in Antarctic hairgrass and creosote bush plants subjected to moderate heat stress even under nonphotorespiratory conditions. Nonphotochemical quenching of chlorophyll fluorescence, the effective quantum yield of photochemical energy conversion (ΔF/Fm′) and the maximum yield of PSII (Fv/Fm) were more sensitive to temperature in Antarctic hairgrass and two other species endemic to cold regions (i.e. Lysipomia pumila and spinach [Spinacea oleracea]) compared with creosote bush and three species (i.e. jojoba [Simmondsia chinensis], tobacco [Nicotiana tabacum], and cotton [Gossypium hirsutum]) from warm regions. The temperature response of activity and the rate of catalytic inactivation of Rubisco from creosote bush and Antarctic hairgrass were similar, whereas the optimum for ATP hydrolysis and Rubisco activation by recombinant creosote bush, cotton, and tobacco activase was 8°C to 10°C higher than for Antarctic hairgrass and spinach activase. These results support a role for activase in limiting photosynthesis at high temperature.

Sensitivity of photosynthesis in a C4 plant, maize, to heat stress

Our objective was to determine the sensitivity of components of the photosynthetic apparatus of maize (Zea mays), a C4 plant, to high temperature stress. Net photosynthesis (Pn) was inhibited at leaf temperatures above 38°C, and the inhibition was much more severe when the temperature was increased rapidly rather than gradually. Transpiration rate increased progressively with leaf temperature, indicating that inhibition was not associated with stomatal closure. Nonphotochemical fluorescence quenching (qN) increased at leaf temperatures above 30°C, indicating increased thylakoid energization even at temperatures that did not inhibit Pn. Compared with CO2 assimilation, the maximum quantum yield of photosystem II (F v/F m) was relatively insensitive to leaf temperatures up to 45°C. The activation state of phosphoenolpyruvate carboxylase decreased marginally at leaf temperatures above 40°C, and the activity of pyruvate phosphate dikinase was insensitive to temperature up to 45°C. The activation state of Rubisco decreased at temperatures exceeding 32.5°C, with nearly complete inactivation at 45°C. Levels of 3-phosphoglyceric acid and ribulose-1,5-bisphosphate decreased and increased, respectively, as leaf temperature increased, consistent with the decrease in Rubisco activation. When leaf temperature was increased gradually, Rubisco activation acclimated in a similar manner as Pn, and acclimation was associated with the expression of a new activase polypeptide. Rates of Pn calculated solely from the kinetics of Rubisco were remarkably similar to measured rates if the calculation included adjustment for temperature effects on Rubisco activation. We conclude that inactivation of Rubisco was the primary constraint on the rate of Pn of maize leaves as leaf temperature increased above 30°C.

EXOGENOUS METHYL JASMONATE INDUCES VOLATILE EMISSIONS IN COTTON PLANTS

We investigated the effect of exogenous methyl jasmonate (MeJA) on the emission of herbivore-induced volatiles; these volatile chemicals can signal natural enemies of the herbivore to the damaged plant. Exogenous treatment of cotton cv. Deltapine 5415 plants with MeJA induced the emission of the same volatile compounds as observed for herbivore-damaged plants. Cotton plants treated with MeJA emitted elevated levels of the terpenes (E)-β-ocimene, linalool, (3E)-4,8-dimethyl-1,3,7-nonatriene, (E,E)-α-farnesene, (E),-β-farnesene, and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene compared to untreated controls. Other induced components included (Z)-3-hexenyl acetate, methyl salicylate, and indole. Methyl jasmonate treatment did not cause the release of any of the stored terpenes such as α-pinene, β-pinene, α-humulene, and (E)-β-caryophyllene. In contrast, these compounds were emitted in relatively large amounts from cotton due to physical disruption of glands by the herbivores. The timing of volatile release from plants treated with MeJA or herbivores followed a diurnal pattern, with maximal volatile release during the middle of the photoperiod. Similar to herbivore-treated plants, MeJA treatment led to the systemic induction of (Z)-3-hexenyl acetate, (E)-β-ocimene, linalool, (3E)-4,8-dimethyl-1,3,7-nonatriene, (E,E)-α-farnesene, (E)-β-farnesene, and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene. Our results indicate that treatment of cotton with MeJA can directly and systemically induce the emission of volatiles that may serve as odor cues in the host-search behavior of natural enemies.

Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase/oxygenase activation state

Abstract. Experiments were conducted to determine the relative contributions of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) activation state vis-à-vis Rubisco activase and metabolite levels to the inhibition of cotton (Gossypium hirsutum L.) photosynthesis by heat stress. Exposure of leaf tissue in the light to temperatures of 40 or 45 °C decreased the activation state of Rubisco to levels that were 65 or 10%, respectively, of the 28 °C control. Ribulose-1,5-bisphosphate (RuBP) levels increased in heat-stressed leaves, whereas the 3-phosphoglyceric acid pool was depleted. Heat stress did not affect Rubisco per se, as full activity could be restored by incubation with CO2 and Mg2+. Inhibition and recovery of Rubisco activation state and carbon dioxide exchange rate (CER) were closely related under moderate heat stress (up to 42.5 °C). Moderate heat stress had negligible effect on Fv/Fm, the maximal quantum yield of photosystem II. In contrast, severe heat stress (45 °C) caused significant and irreversible damage to Rubisco activation, CER, and Fv/Fm. The rate of Rubisco activation after alleviating moderate heat stress was comparable to that of controls, indicating rapid reversibility of the process. However, moderate heat stress decreased both the rate and final extent of CER activation during dark-to-light transition. Treatment of cotton leaves with methyl viologen or an oxygen-enriched atmosphere reduced the effect of heat stress on Rubisco inactivation. Both treatments also reduced tissue RuBP levels, indicating that the amount of RuBP present during heat stress may influence the degree of Rubisco inactivation. Under both photorespiratory and non-photorespiratory conditions, the inhibition of the CER during heat stress could be completely reversed by increasing the internal partial pressure of CO2 (Ci). However, the inhibition of the CER by nigericin, a K+ ionophore, was not reversible when the Ci was increased at ambient or high temperature. Our results indicate that inhibition of photosynthesis by moderate heat stress is not caused by inhibition of the capacity for RuBP regeneration. We conclude that heat stress inhibits Rubisco activation via a rapid and direct effect on Rubisco activase, possibly by perturbing Rubisco activase subunit interactions with each other or with Rubisco.

VOLATILE EMISSIONS TRIGGERED BY MULTIPLE HERBIVORE DAMAGE: BEET ARMYWORM AND WHITEFLY FEEDING ON COTTON PLANTS

Plants are commonly attacked by more than one species of herbivore, potentially causing the induction of multiple, and possibly competing, plant defense systems. In the present paper, we determined the interaction between feeding by the phloem feeder silverleaf whitefly (SWF), Bemisia tabaci Gennadius (B-biotype = B. argentifolii Bellows and Perring), and the leaf-chewing beet armyworm (BAW), Spodoptera exigua Hübner, with regard to the induction of volatile compounds from cotton plants. Compared to undamaged control plants, infestation with SWF did not induce volatile emissions or affect the number and density of pigment glands that store volatile and nonvolatile terpenoid compounds, whereas infestation by BAW strongly induced plant volatile emission. When challenged by the two insect herbivores simultaneously, volatile emission was significantly less than for plants infested with only BAW. Our results suggest that tritrophic level interactions between cotton, BAW, and natural enemies of BAW, that are known to be mediated by plant volatile emissions, may be perturbed by simultaneous infestation by SWF. Possible mechanisms by which the presence of whiteflies may attenuate volatile emissions from caterpillar-damaged cotton plants are discussed.

Lygus hesperus feeding and salivary gland extracts induce volatile emissions in plants.

Induction of plant volatiles by leaf-chewing caterpillars is well documented. However, there is much less information about volatile induction by insects with different feeding habits. We studied the induction of plant volatiles by a piercing–sucking insect, the western tarnished plant bug Lygus hesperus Knight. Adults of both genders and nymphs of Lygus induced the local emission of a blend of volatiles from both cotton and maize. Feeding by Lygus also induced the systemic emission of volatiles that was similar but less complex than the blend emitted at the site of feeding. Infestation by mated, mature adult females (>4 days old), but not by nymphs or mature males, caused detectable emission of α-pinene, myrcene, and (E)-β-caryophyllene, compounds that are stored in the glands of cotton tissue. This indicated that damage to glands in the petiole and leaf by the female ovipositor, rather than feeding, contributed significantly to the emission of these volatiles. Girdling the plant stem to disrupt phloem transport markedly decreased the movement of 14C-labeled photosynthetic products to the apex of the plant, and this treatment also markedly reduced the amount of systemically induced volatiles caused by Lygus feeding. Lygus salivary gland extracts were capable of inducing emission of the same volatile blend as measured for plants infested by feeding insects or treated with volicitin, an elicitor isolated from caterpillar regurgitant. The results indicate that L. hesperus is capable of inducing the emission of plant volatiles and that induction is caused by an elicitor that is contained in the insect salivary gland.

Changes in ribulosebisphosphate carboxylase/oxygenase and ribulose 5-phosphate kinase abundances and photosynthetic capacity during leaf senescence.

The abundances of ribulose-1,5-bisphosphate carboxylate/oxygenase (Rubisco) and ribulose-5-phosphate (Ru5P) kinase in field-grown soybean (Glycine max L. Merr.) leaves were quantified by a Western blot technique and related to changes in chlorophyll and photosynthetic capacity during senescence. Even though the leaf content of Rubisco was approximately 80-fold greater than that of Ru5P kinase, the decline in the levels of these two Calvin cycle enzymes occurred in parallel during the senescence of the leaves. Moreover, the decrease in the content of Rubisco was accompanied by parallel decreases of both the large and small subunits of this enzyme but not by an accumulation of altered large or small subunit isoforms. With increasing senescence, decreases in abundances of Rubisco, Ru5P kinase and chlorophyll were closely correlated with the decline in photosynthetic capacity; thus, the specific photosynthetic capacity when expressed per abundance of any of these parameters was rather constant despite an 8-fold decrease in photosynthetic capacity. These results suggest that during senescence of soybean leaves the chloroplast is subject to autolysis by mechanisms causing an approximately 80-fold greater rate of loss of Rubisco than Ru5P kinase.

Ribulose-1,5-bis-Phosphate Carboxylase/Oxygenase Degradation in Isolated Pea Chloroplasts Incubated in the Light or in the Dark

Summary Intact pea ( Pisum sativum L.) chloroplasts isolated mechanically were incubated in the dark or in the light. After incubation intact chloroplasts were reisolated on Percoll steps prior to analysis. A 37 kD polypeptide derived from the large subunit of ribulose-1,5-bis-phosphate carboxylase/oxygenase accumulated during incubation in darkness. Other degradation products (45, 42, 37, and 32 kD) were detected on immunoblots from organelles incubated in the light. The catabolism of Rubisco in the chloroplasts was affected by the composition of the incubation medium.

Physiological N response of field-grown maize hybrids (Zea mays L.) with divergent yield potential and grain protein concentration

Fertilizer N availability impacts photosynthesis and crop performance, although cause–effect relationships are not well established, especially for field-grown plants. Our objective was to determine the relationship between N supply and photosynthetic capacity estimated by leaf area index (LAI) and single leaf photosynthesis using genetically diverse field-grown maize (Zea mays L.) hybrids. We compared a high yield potential commercial hybrid (FR1064 x LH185) and an experimental hybrid (FR1064 x IHP) with low yield potential but exceptionally high grain protein concentration. Plant biomass and physiological traits were measured at tassel emergence (VT) and at the grain milk stage (R3) to assess the effects of N supply on photosynthetic source capacity and N uptake, and grain yield and grain N were measured at maturity. Grain yield of FR1064 x LH185 was much greater than FR1064 x IHP even though plant biomass and LAI were larger for FR1064 x IHP, and single leaf photosynthesis was similar for both hybrids. Although photosynthetic capacity was not related to hybrid differences in productivity, increasing N supply led to proportional increases in grain yield, plant biomass, LAI, photosynthesis, and Rubisco and PEP carboxylase activities for both hybrids. Thus, a positive relationship between photosynthetic capacity and yield was revealed by hybrid response to N supply, and the relationship was similar for hybrids with a marked difference in yield potential. For both hybrids the N response of single leaf CER and initial Rubisco activity was negative when expressed per unit of leaf N. In contrast, PEP carboxylase activity per unit leaf N increased in response to N availability, indicating that PEP carboxylase served as a reservoir for excess N accumulation in field-grown maize leaves. The correlation between CER and initial Rubisco activity was highly significant when expressed on a leaf area or a total leaf basis. The results suggest that regardless of genotypic yield potential, maize CER, and potentially grain yield, could be improved by increasing the partitioning of N into Rubisco.

Coordination of protein and mRNA abundances of stromal enzymes and mRNA abundances of the Clp protease subunits during senescence of Phaseolus vulgaris (L.) leaves

Our objective was to determine the coordination of transcript and/or protein abundances of stromal enzymes during leaf senescence. First trifolioliate leaves of Phaseolus vulgaris L. plants were sampled beginning at the time of full leaf expansion; at this same time, half of the plants were switched to a nutrient solution lacking N. Total RNA and soluble protein abundances decreased after full leaf expansion whereas chlorophyll abundance remained constant; N stress enhanced the decline in these traits. Abundances of ribulose-1,5-bisposphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39), Rubisco activase and phosphoribulokinase (Ru5P kinase; EC 2.7.1.19) decreased after full leaf expansion in a coordinated manner for both treatments. In contrast, adenosine diphosphate glucose (ADPGlc) pyrophosphorylase (EC 2.7.7.27) abundance was relatively constant during natural senescence but did decline similar to the other enzymes under N stress. Northern analyses indicated that transcript abundances for all enzymes declined markedly on a fresh-weight basis just after full leaf expansion. This rapid decline was particularly strong for the Rubisco small subunit (rbcS) transcript. The decline was enhanced by N stress for rbcS and Rubisco activase (rca), but not for Ru5P kinase (prk) and ADPGlc pyrophosphorylase (agp). Transcripts of the Clp protease subunits clpC and clpP declined in abundance just after full leaf expansion, similar to the other mRNA species. When Northern blots were analyzed using equal RNA loads, rbcS transcripts still declined markedly just after full leaf expansion whereas rca and clpC transcripts increased over time. The results indicated that senescence was initiated near the time of full leaf expansion, was accelerated by N stress, and was characterized by large decline in transcripts of stromal enzymes. The decreased mRNA abundances were in general associated with steadily declining stromal protein abundances, with ADPGlc pyrophosphorylase being the notable exception. Transcript analyses for the Clp subunits supported a recent report (Shanklin et al., 1995, Plant Cell 7: 1713–1722) indicating that the Clp protease subunits were constitutive throughout development and suggested that ClpC and ClpP do not function as a senescence-specific proteolytic system in Phaseolus.