Transgenic tobacco with suppressed zeaxanthin formation is susceptible to stress-induced photoinhibition.

Tobacco (Nicotiana tabacum cv. Xanthi) transformed with the antisense construct of tobacco violaxanthin de-epoxidase was analyzed for responses in growth chambers to both short and long-term stress treatments. Following a short-term (2 or 3 h) high-light treatment, antisense plants had a greater reduction in F(v)/F(m) relative to wild-type, indicating a greater susceptibility to photoinhibition. The responses of antisense plants to long-term stress were examined in two separate experiments, one with high light alone and the other wherein high light and water stress were combined. In the light-stress experiment, plants were grown at 1300 mumol photons m(-2) s(-1) under a 12 h photoperiod. In the light and water-stress experiment, plants were grown under moderately high light of 900 mumol photons m(-2) s(-1), under a 16 h photoperiod, in combination with water stress. Both conditions caused formation of high antheraxanthin and zeaxanthin levels in wild-type plants but not in antisense plants. In both cases, antisense plants showed significant reductions in F(v)/F(m) and total leaf-pigment content relative to wild-type. The data demonstrate a critical photoprotective function of the xanthophyll cycle-dependent energy dissipation in tobacco exposed suddenly to high amounts of excess light over extended times.

Suppression of zeaxanthin formation does not reduce photosynthesis and growth of transgenic tobacco under field conditions.

Tobacco (Nicotiana tabacum cv. Xanthi) transformed with an antisense cDNA construct of violaxanthin de-epoxidase (VDE) was examined for the effects of suppressed xanthophyll-cycle activity on photoinhibition, photosynthesis and growth under field conditions. De-epoxidation of violaxanthin and non-photochemical quenching were highly inhibited in antisense plants relative to vector-control and wild-type plants. However, no differences were observed between antisense and control plants in photosynthetic CO(2) uptake and maximum photochemical yield [(F(m)-F(o))/F(m)] measured at predawn or in actual photochemical yield [(F(m)'-F(s))/F(m)'] measured at midday. Moreover, growth rates of the plants were the same, as were the leaf area ratio, plant height and leaf number. Similarly, antisense plants did not exhibit greater susceptibility to photoinhibition than controls under field conditions. In contrast, when chloroplast protein (D1) synthesis was inhibited by lincomycin, antisense plants were more vulnerable to photoinhibition than wild-type plants. These results indicate that photoprotection under field conditions is not strictly dependent on the levels of the de-epoxidized xanthophylls, antheraxanthin and zeaxanthin.

Proteolytic footprinting titrations for estimating ligand-binding constants and detecting pathways of conformational switching of calmodulin.

To dissect the chemical basis for interactions controlling regulatory properties of macromolecular assemblies, it is essential to explore experimentally the linkage between ligand binding, conformational change, and subunit assembly. There are many advantages to using techniques that will probe the occupancy of individual binding sites or monitor conformational responses of individual residues, as described here. Proteolytic footprinting titrations may be used to infer binding free energies for ligands interacting with multiple sites or domains and to detect otherwise unrecognized "silent" interdomain interactions. Microgram quantities of pure protein are required, which is low relative to the hundreds of milligrams needed for comparable discontinuous equilibirum titrations monitored by NMR. By running comparative studies with several proteases, it is easy to determine whether resulting titration curves are consistent, independent of the protease used and therefore representative of the structural response of the protein to ligand binding or other differences in solution conditions (pH, salt, temperature). The results from multiple techniques (e.g., NMR, fluorescence, and footprinting) applied to aliquots from the same discontinuous titration may be compared easily to test for consistency. Classic methods for determining thermodynamic and kinetic properties of calcium binding to calmodulin include filter binding and equilibrium or flow dialysis (employing the isotope 45Ca), spectroscopic studies of stopped-flow fluorescence, calorimetry, and direct ion titrations. A cautionary note is that many different sets of microscopic data would be consistent with a single set of macroscopic constants determined by classic methods. This was well illustrated in Fig. 9. Thus, while it is important to compare results with those obtained by classic binding methods, they are, by definition, incapable of resolving the microscopic constants of interest. Thus, there is only one "direction" for comparison. Quantitative proteolytic footprinting titrations applied to studying calmodulin provided the first direct quantitative estimate of negative interactions between domains. Although studies of site-knockout mutants had suggested interactions between domains, this approach gave the first evidence for the pathway of anticooperative interactions between domains by showing that helix B responds structurally to calcium binding to sites III and IV in the C-domain. Despite two decades of study of calmodulin and the application of limited proteolysis studies to the apo and fully saturated forms, this finding emerged only when titration studies were undertaken as described. This highlights the general observation that while the behavior of the intermediate states in a cooperative switch are the key elements of the transition mechanism, they are the most difficult to observe. The unexpected finding that the isolated domains are nearly equivalent in their calcium-binding properties (Fig. 23 B) leaves us with many of the questions we had at the start: How does the sum of two nearly equivalent domains result in a molecule that switches sequentially rather than simultaneously? But it underscores why it is not yet possible to understand similar proteins by sequence gazing alone.

The xanthophyll cycle and acclimation of Pinus ponderosa and Malva neglecta to winter stress.

Seasonal differences in the efficiency of open PSII units (F v/F m), leaf pigment composition and xanthophyll cycle conversion (Z+A)/(V+A+Z), leaf adenylate status, and photosynthetic capacity were investigated in Pinus ponderosa (Ponderosa pine) and Malva neglecta. In P. ponderosa, acclimation to winter involved a lower photosynthetic capacity, higher carotenoid to chlorophyll ratio, persistent reductions in F v/F m corresponding to persistent retention of Z+A, and no change in foliar ATP/ADP ratios. In contrast, M. neglecta characterized in winter exhibited higher rates of photosynthesis than in summer with no change in carotenoid to chlorophyll ratio, while small nocturnally persistent reductions in F v/F m were observed exclusively on colder winter nights when nocturnal retention of Z+A, and high ATP/ADP ratios were also present. Upon removal of winter-stressed leaves or needles from the field to room temperature, a portion of F v/F m relaxed within 15 min of warming and recovery was completed within 5 h in M. neglecta but required 100 h in P. ponderosa. In M. neglecta, the entire recovery of F v/F m correlated with decreases in the foliar ATP/ADP ratio, while in P. ponderosa this ratio remained unchanged. Possible ATP-dependent forms of sustained (Z+A)-dependent energy dissipation are discussed including a nocturnally retained pH gradient on cold winter nights. The slow recovery in pine involved not only retention of Z+A, but apparently also a persistent engagement of Z+A for energy dissipation via an unidentified mechanism.

Enhanced Employment of the Xanthophyll Cycle and Thermal Energy Dissipation in Spinach Exposed to High Light and N Stress.

The involvement of the xanthophyll cycle in photoprotection of N-deficient spinach (Spinacia oleracea L. cv Nobel) was investigated. Spinach plants were fertilized with 14 mM nitrate (control, high N) versus 0.5 mM (low N) fertilizer, and grown under both high- and low-light conditions. Plants were characterized from measurements of photosynthetic oxygen exchange and chlorophyll fluorescence, as well as carotenoid and cholorophyll analysis. Compared with the high-N plants, the low-N plants showed a lower capacity for photosynthesis and a lower chlorophyll content, as well as a lower rate of photosystem II photosynthetic electron transport and a corresponding increase in thermal energy dissipation activity measured as nonphotochemical fluorescence quenching. The low-N plants displayed a greater fraction of the total xanthophyll cycle pool as zeaxanthin and antheraxanthin at midday, and an increase in the ratio of xanthophyll cycle pigments to total chlorophyll. These results indicate that under N limitation both the light-collecting system and the photosynthetic rate decrease. However, the increased dissipation of excess energy shows that there is excess light absorbed at midday. We conclude that spinach responds to N limitation by a combination of decreased light collection and increased thermal dissipation involving the xanthophyll cycle.

Calcium-induced interactions of calmodulin domains revealed by quantitative thrombin footprinting of Arg37 and Arg106.

Calcium-dependent conformational states of calmodulin (CaM) were probed by thrombin to determine quantitative differences in the susceptibility of two bonds: Arg37-Ser38 (R37-S38, near site I in the N-terminal domain) and Arg106-His107 (R106-H107, near site III in the C-terminal domain). Quantitative thrombin footprinting of a discontinuous equilibrium calcium titration of wild-type calmodulin showed that the R37-S38 bond of the apoprotein was cleaved at a barely detectable level while the R106-H107 bond was maximally susceptible. Calcium binding to sites III and IV monotonically protected R106-H107 from proteolysis; concomitantly, the susceptibility of R37-S38 increased. However, calcium binding to sites I and II protected R37-S38 from cleavage, yielding a peaked biphasic profile composed of equal and opposite transitions. Both bonds were fully protected when calmodulin was saturated with calcium. Susceptibility profiles resolved from the fractional abundance of primary cleavage products (peptides 1-37, 38-148, 1-106, 107-148) were interpreted as directly reflecting calcium-induced conformational changes in whole calmodulin; free energies of calcium binding and cooperativity were estimated. Secondary cleavage was never observed; both R37 and R106 were sites of thrombinolysis in whole calmodulin only. In studies of E140Q-CaM (having a mutation in site IV), the susceptibility of R37-S38 decreased monotonically. Thus, the biphasic character of cleavage of R37 in helix B was not intrinsic to that domain but depended on propagation of effects of calcium-induced changes in the C-terminal domain. The observed patterns of susceptibility indicated that partially saturated wild-type calmodulin adopts at least one intermediate conformation whose structure is determined by calcium-mediated interactions between the domains.