Different photorespiratory mechanisms in conifer leaves, where peroxisomes have intrinsically low catalase activity.

Photorespiration is an essential metabolic mechanism associated with photosynthesis; however, little is known about the photorespiratory pathway of conifer gymnosperms. Metabolite analyses of the leaves of 27 tree species showed that the mean glycerate content in conifer leaves was lower than that in angiosperm leaves. We performed experiments where [13 C]-serine was fed to detached shoots of a conifer (Cryptomeria japonica), via the transpiration stream, and compared the labeling patterns of photorespiratory metabolites with those of an angiosperm tree (Populus nigra), because glycerate is produced from serine via hydroxypyruvate in peroxisomes. In P. nigra, hydroxypyruvate, glycerate and glycine were labeled with 13 C, whereas in C. japonica, glycolate and a non-canonical photorespiratory metabolite, formate, were also labeled, suggesting that an H2 O2 -mediated non-enzymatic decarboxylation (NED) reaction occurs in C. japonica. We analyzed changes in the metabolite contents of leaves kept in the dark and leaves exposed to illuminated photorespiration-promoting conditions: a positive relationship between formate and serine levels in C. japonica implied that the active C1 -metabolism pathway synthesizes serine from formate. Leaf gas exchange analyses revealed that CO2 produced through NED was recaptured by chloroplasts. Database analysis of the peroxisomal targeting signal motifs of an H2 O2 -scavenging enzyme, catalase, derived from various species, including nine coniferous species, as well as analyses of peroxisomal fractions isolated from C. japonica and P. nigra leaves indicated that conifer peroxisomes had less catalase activity. These results suggest that NED and the subsequent C1 metabolism are involved in the photorespiratory pathway of conifer leaves, where peroxisomes have intrinsically low catalase activity.

Dehydroquinate dehydratase/shikimate dehydrogenases involved in gallate biosynthesis of the aluminum-tolerant tree species Eucalyptus camaldulensis.

MAIN CONCLUSION: Eucalyptus camaldulensis EcDQD/SDH2 and 3 combine gallate formation, dehydroquinate dehydratase, and shikimate dehydrogenase activities. They are candidates for providing the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B. The tree species Eucalyptus camaldulensis shows exceptionally high tolerance against aluminum, a widespread toxic metal in acidic soils. In the roots of E. camaldulensis, aluminum is detoxified via the complexation with oenothein B, a hydrolyzable tannin. In our approach to elucidate the biosynthesis of oenothein B, we here report on the identification of E. camaldulensis enzymes that catalyze the formation of gallate, which is the phenolic constituent of hydrolyzable tannins. By systematical screening of E. camaldulensis dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDHs), we found two enzymes, EcDQD/SDH2 and 3, catalyzing the NADP+-dependent oxidation of 3-dehydroshikimate to produce gallate. Based on extensive in vitro assays using recombinant EcDQD/SDH2 and 3 enzymes, we present for the first time a detailed characterization of the enzymatic gallate formation activity, including the cofactor preferences, pH optima, and kinetic constants. Sequence analyses and structure modeling suggest the gallate formation activity of EcDQD/SDHs is based on the reorientation of 3-dehydroshikimate in the catalytic center, which facilitates the proton abstraction from the C5 position. Additionally, EcDQD/SDH2 and 3 maintain DQD and SDH activities, resulting in a 3-dehydroshikimate supply for gallate formation. In E. camaldulensis, EcDQD/SDH2 and 3 are co-expressed with UGT84A25a/b and UGT84A26a/b involved in hydrolyzable tannin biosynthesis. We further identified EcDQD/SDH1 as a "classical" bifunctional plant shikimate pathway enzyme and EcDQD/SDH4a/b as functional quinate dehydrogenases of the NAD+/NADH-dependent clade. Our data indicate that in E. camaldulensis the enzymes EcDQD/SDH2 and 3 provide the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.

Increased phytotoxic O3 dose accelerates autumn senescence in an O3-sensitive beech forest even under the present-level O3.

Ground-level ozone (O3) concentrations are expected to increase over the 21(st) century, especially in East Asia. However, the impact of O3 has not been directly assessed at the forest level in this region. We performed O3 flux-based risk assessments of carbon sequestration capacity in an old cool temperate deciduous forest, consisting of O3-sensitive Japanese beech (Fagus crenata), and in a warm temperate deciduous and evergreen forest dominated by O3-tolerant Konara oak (Quercus serrata) based on long-term CO2 flux observations. On the basis of a practical approach for a continuous estimation of canopy-level stomatal conductance (Gs), higher phytotoxic ozone dose above a threshold of 0 uptake (POD0) with higher Gs was observed in the beech forest than that in the oak forest. Light-saturated gross primary production, as a measure of carbon sequestration capacity of forest ecosystem, declined earlier in the late growth season with increasing POD0, suggesting an earlier autumn senescence, especially in the O3-sensitive beech forest, but not in the O3-tolerant oak forest.

In situ CO2 efflux from leaf litter layer showed large temporal variation induced by rapid wetting and drying cycle.

We performed continuous and manual in situ measurements of CO2 efflux from the leaf litter layer (R(LL)) and water content of the leaf litter layer (LWC) in conjunction with measurements of soil respiration (RS) and soil water content (SWC) in a temperate forest; our objectives were to evaluate the response of R(LL) to rainfall events and to assess temporal variation in its contribution to R(S). We measured R(LL) in a treatment area from which all potential sources of CO2 except for the leaf litter layer were removed. Capacitance sensors were used to measure LWC. R(LL) increased immediately after wetting of the leaf litter layer; peak R(LL) values were observed during or one day after rainfall events and were up to 8.6-fold larger than R(LL) prior to rainfall. R(LL) declined to pre-wetting levels within 2-4 day after rainfall events and corresponded to decreasing LWC, indicating that annual R(LL) is strongly influenced by precipitation. Temporal variation in the observed contribution of R(LL) to RS varied from nearly zero to 51%. Continuous in situ measurements of LWC and CO2 efflux from leaf litter only, combined with measurements of RS, can provide robust data to clarify the response of R(LL) to rainfall events and its contribution to total R(S).

Seasonal ozone uptake by a warm-temperate mixed deciduous and evergreen broadleaf forest in western Japan estimated by the Penman-Monteith approach combined with a photosynthesis-dependent stomatal model.

Canopy-level stomatal conductance over a warm-temperate mixed deciduous and evergreen broadleaf forest in Japan was estimated by the Penman-Monteith approach, as compensated by a semi-empirical photosynthesis-dependent stomatal model, where photosynthesis, relative humidity, and CO2 concentration were assumed to regulate stomatal conductance. This approach, using eddy covariance data and routine meteorological observations at a flux tower site, permits the continuous estimation of canopy-level O3 uptake, even when the Penman-Monteith approach is unavailable (i.e. in case of direct evaporation from soil or wet leaves). Distortion was observed between the AOT40 exposure index and O3 uptake through stomata, as AOT40 peaked in April, but with O3 uptake occurring in July. Thus, leaf pre-maturation in the predominant deciduous broadleaf tree species (Quercus serrata) might suppress O3 uptake in springtime, even when the highest O3 concentrations were observed.