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Trees regenerating in the understory respond to increased availability of light caused by gap formation by undergoing a range of morphological and physiological adjustments. These adjustments include the production of thick, sun-type leaves containing thicker mesophyll and longer palisade cells than in shade-type leaves. We asked whether in the shade-regenerating tree Acer pseudoplatanus, the increase in leaf thickness and expansion of leaf tissues are possible also in leaves that had been fully formed prior to the increase in irradiance, a response reported so far only for a handful of species. We acclimated potted seedlings to eight levels (from 1 to 100%) of solar irradiance and, in late summer, transferred a subset of them to full sunlight. Within 30 days, the shaded leaves increased leaf mass per area and became thicker mostly due to elongation of palisade cells, except for the most shaded individuals which suffered irreversible photo-oxidative damage. This anatomical acclimation was accompanied by partial degradation of chlorophyll and a transient decline in photosynthetic efficiency of PSII (Fv/FM). These effects were related to the degree of pre-shading. The Fv/FM recovered substantially within the re-acclimation period. However, leaves of transferred plants were shed significantly earlier in the fall, indicating that the acclimation was not fully effective. These results show that A. pseudoplatanus is one of the few known species in which mature leaves may re-acclimate anatomically to increased irradiance. This may be a potentially important mechanism enhancing utilization of gaps created during the growing season.

Trees regenerating in the understory respond to increased availability of light caused by gap formation by undergoing a range of morphological and physiological adjustments. These adjustments include the production of thick, sun-type leaves containing thicker mesophyll and longer palisade cells than in shade-type leaves. We asked whether in the shade-regenerating tree Acer pseudoplatanus, the increase in leaf thickness and expansion of leaf tissues are possible also in leaves that are already fully formed, a response reported so far only for a handful of species. We acclimated potted seedlings to eight levels (from 1 to 100%) of solar irradiance and, in late summer, transferred a subset of them to full sunlight. Within 30 days, the pre-shaded leaves increased leaf mass per area and became thicker mostly due to the elongation of palisade cells, except for the most shaded individuals which suffered irreversible photo-oxidative damage. This anatomical acclimation was accompanied by a transient decline in photosynthetic efficiency of PSII (Fv/FM), the magnitude of which was related to the degree of pre-shading. The Fv/FM recovered substantially within the re-acclimation period. However, leaves of transferred plants were shed earlier in the fall, indicating that the acclimation was not fully effective. These results show that A. pseudoplatanus is one of the few known species in which mature leaves may re-acclimate anatomically to increased irradiance. This may be an important mechanism enhancing utilization of gaps created during the growing season.

Background and Aims The reliance on external support by lianas has been hypothesized to imply a reduction in the biomass cost of stem construction and root anchorage, and an increased investment in leaves, relative to self-supporting plants. These evolutionary trade-offs have not been adequately tested in an ontogenetic context and on the whole-plant scale. Moreover, the hypothesis may be extended to other potentially limiting resources, such as nitrogen (N.) Methods Plants belonging to five con-familiar pairs of temperate liana/shrub species were cultivated in 120 L barrels and sequentially harvested over up to three growing seasons. To account for the ontogenetic drift, organ biomass and nitrogen fractions were adjusted for plant biomass and N pool, respectively. Key Results Lianas invested, on average, relatively less biomass in the root fraction in comparison with shrubs. This was offset by only insignificant increases in leaf or stem investment. Even though liana stems and roots showed higher N concentration in comparison with shrubs, plant N distribution was mostly driven by, and largely matched, the pattern of biomass distribution. Lianas also showed a greater relative growth rate than shrubs. The differences between the growth forms became apparent only when ontogenetic drift was controlled for. These results were confirmed regardless of whether reproductive biomass was included in the analysis. Conclusions Our results suggest that temperate lianas, in spite of their diverse, species-specific resource distribution patterns, preferentially allocate resources to above-ground organs at the expense of roots. By identifying this trade-off and demonstrating the lack of a general trend for reduction in stem investment in lianas, we significantly modify the prevailing view of liana allocation strategies and evolutionary advantages. Such a resource distribution pattern, along with the cheap unit leaf area and stem unit length construction, situates lianas as a group close to the fast acquisition/rapid growth end of the life strategy spectrum.

In some lianas the use of trellis modifies within-plant biomass allocation and stimulates growth, however in other species trellis use may negatively affect growth indicating a cost associated with climbing. In order to improve access to light, lianas use other plants as climbing trellises. Whereas in shaded lianas climbing a trellis may enhance biomass gain by increasing leaf-level irradiance, we suspected that trellis use may also stimulate growth by triggering modifications in whole-plant allocation of biomass and nitrogen. We evaluated responses to trellis in three temperate lianas: Hedera helix, Celastrus orbiculatus and Wisteria floribunda. Lianas were grown outdoors in 120-L barrels and with ample space, with and without trellises. Biomass and nitrogen accumulation, distribution patterns and total stem length were determined after three and four seasons. Responses were adjusted for individual plant biomass. Liana responses to trellises were species-specific. In W. floribunda trellis use enhanced growth, consistently with the increased leaf mass fraction and leaf area ratio while biomass distribution to roots was reduced. Biomass distribution to stems and total stem length were increased by trellis use. In contrast, growth and biomass distribution in C. orbiculatus were not altered by trellis use. In H. helix growth was reduced in plants climbing on trellises in comparison with plants creeping on the ground, but biomass distribution pattern was not altered. Moreover, supported H. helix plants accumulated less nitrogen than did unsupported plants, suggesting a less efficient uptake of this nutrient. The ability to plastically modify growth and allocation patterns in response to trellis found in some lianas may constitute an important aspect of their ecological strategy. In other species, however, the switch to climbing may incur physiological costs resulting in lower growth.

Climate change has likely altered high-latitude forests globally, but direct evidence remains rare. Here we show that throughout a ≈1000-km transect in Scots pine (Pinus sylvestris L.) forests in Sweden, mature trees in ≈2015 had longer needles with shorter lifetimes than did trees in ≈1915. These century-scale shifts in needle traits were detected by sampling needles at 74 sites from 2012 to 2017 along the same transect where needle traits had been assessed at 57 sites in 1914–1915. Climate warming of ≈1 °C all along the transect in the past century has driven this temporal shift in foliage traits known to be physiologically critical to growth and carbon cycling processes. These century-scale changes in Scandinavian Scots pine forests represent a fingerprint of climate change on a fundamental biological element, the leaf, with repercussions for productivity and sensitivity to future climate, which are likely to be mirrored by similar changes for evergreen conifers across the boreal biome. (Less)

Occupancy of cold habitats by evergreen species requires structural modification of photosynthetic organs for stress resistance and longevity. Such modifications have been described at interspecific level, while intraspecific variation has been underexplored. To identify structural and anatomical traits that may be adaptive in cold environments, we studied intraspecific variability of needles of Scots pine (Pinus sylvestris L.), a wide-ranging tree, along a 1,900 km temperate-boreal transect in Europe. Needles from 20 sites representing mean minimum winter temperatures between -4.0 and -19.9°C and mean annual temperatures between 8.3 and -1.7°C were sampled for measurements of leaf mass per area (LMA, g/m2), leaf density (LD, g/cm3) and 30 other morpho-anatomical traits. Needles from cold sites lived longer, were shorter, showed higher LMA and LD, had narrower and more collapse-resistant tracheids, thicker epidermal cells with thicker cell walls and wider resin ducts occupying larger fraction of needle volume in comparison to needles from warmer sites. Along the steep climatic gradient, needles presented a coordinated phenotypic spectrum of external and internal traits that are largely interpretable in functional, adaptive terms. This intraspecific pattern of covarying traits provides insight into the adaptive syndrome associated with stress tolerance and extended needle longevity under cold conditions of high latitudes. A plain language summary is available for this article. (Less)

Recent climatic changes have resulted in an increased frequency and prolonged periods of drought and strained water resources affecting plant production. We explored the possibility of reducing irrigation in a container nursery and studied the growth response of seedlings of economically important forest trees: broadleaf deciduous angiosperms Fagus sylvatica, Quercus petraea and evergreen conifers Abies alba and Pinus sylvestris. We also studied markers of water stress including modifications of biomass allocation, leaf anatomy, proline accumulation and expression of selected genes. Growth of the broadleaved deciduous species was more sensitive to the reduced water supply than that of conifers. Remarkably, growth of the shade tolerant Abies was not affected. Adjustment of biomass allocations was strongest in P. sylvestris, with a remarkable increase in allocation to roots. In response to water deficit both deciduous species accumulated proline in leaves and produced leaves with shorter palisade cells, reduced vascular tissues and smaller conduit diameters, but not conifers. Relative transcript abundance of a gene encoding a Zn-finger protein in Q. petraea and a gene encoding a pore calcium channel protein 1 in A. alba increased as water deficit increased. These findings suggest that in container nursery, the genetic selection can be initiated by water deficit. Our study shows major differences between functional groups in response to irrigation, with seedlings of evergreen conifers having higher tolerance than the deciduous species. This suggests that major water savings could be achieved by adjusting irrigation regime to functional group or species requirements.

The correlations of phenotypic traits with environmental drivers suggest that variability of these traits is a result of natural selection, especially if such trait correlations are based on genetic variability. We hypothesized that in situ correlations of structural needle traits of Scots pine (Pinus sylvestris L) with minimal winter temperature (Tmin) reported previously from a temperate/boreal transect would be conserved when plants are cultivated under common conditions. We tested this hypothesis by analyzing needles from two common gardens located in the temperate zone, one including adult trees and the other juvenile seedlings. The majority of adult needle traits for which correlations with Tmin were found in the field turned out to be under environmental influence. In contrast, the majority of traits studied in juvenile needles were correlated with the original Tmin suggesting the role of past natural selection in shaping their variability. Juvenile needles thus appeared to be inherently less plastic than adult needles, perhaps reflecting the stronger selective pressure acting during juvenile, as compared with adult, ontogenetic stage. Genetically based cold-climate adaptation in either juvenile or adult needles, or both, involved an increase in leaf mass per area and leaf density, decrease in needle length, reduction in the amount of xylem and phloem, increase in thickness of epidermis, decrease in tracheid diameter and increase in tracheid density, and increase in diameter and volume fraction of resin ducts. We also show that at least some traits, such as transverse xylem and phloem areas and number of fibers, scale with needle length, suggesting that climate-related trait variation may also be mediated by changes in needle length. Moreover, slopes of these allometric relationships may themselves be plastically modified. The phenotypic syndrome typical of needles from cold environments may thus be under environmental, genetic and allometric control.

In evergreen plants, old leaves may contribute photosynthate to initiation of shoot growth in the spring. They might also function as storage sites for carbohydrates and nitrogen (N). We hence hypothesized that whole-plant allocation of carbohydrates and N to storage in stems and roots may be lower in evergreen than in deciduous species. We selected three species pairs consisting of an evergreen and a related deciduous species: Mahonia aquifolium (Pursh) Nutt. and Berberis vulgaris L. (Berberidaceae), Prunus laurocerasus L. and Prunus serotina Ehrh. (Rosaceae), and Viburnum rhytidophyllum Hemsl. and Viburnum lantana L. (Adoxaceae). Seedlings were grown outdoors in pots and harvested on two dates during the growing season for the determination of biomass, carbohydrate and N allocation ratios. Plant size-adjusted pools of nonstructural carbohydrates in stems and roots were lower in the evergreen species of Berberidaceae and Adoxaceae, and the slope of the carbohydrate pool vs plant biomass relationship was lower in the evergreen species of Rosaceae compared with the respective deciduous species, consistent with the leading hypothesis. Pools of N in stems and roots, however, did not vary with leaf habit. In all species, foliage contained more than half of the plant’s nonstructural carbohydrate pool and, in late summer, also more than half of the plant’s N pool, suggesting that in juvenile individuals of evergreen species, leaves may be a major storage site. Additionally, we hypothesized that concentration of defensive phenolic compounds in leaves should be higher in evergreen than in deciduous species, because the lower carbohydrate pool in stems and roots of the former restricts their capacity for regrowth following herbivory and also because of the need to protect their longer-living foliage. Our results did not support this hypothesis, suggesting that evergreen plants may rely predominantly on structural defenses. In summary, our study indicates that leaf habit has consequences for storage economics at the whole-plant level, with evergreen shrub species storing less carbohydrates (but not N) per unit plant biomass than deciduous species.

Evergreen plants are an important component of many ecosystems of the world and occur in nu- merous evolutionary lineages. In this article we review phenotypic traits of evergreen woody angiosperms occurring in habitats that regularly experience frost. Leaf anatomical traits such as sclerenchymatic tissues or prominent cuticles ensure mechanical strength while often enhancing tolerance of water deficit. The low ratio of photosynthetic to nonphotosynthetic tissues as well as modified cell wall structure and nitrogen allocation patterns in evergreen leaves result in lower mass-based photosynthetic rate and photosynthetic nitrogen use efficiency in comparison with deciduous leaves. Their photosynthetic apparatus is adapted for the survival of frost in a down-regulated state with potential for photosynthetic activity in winter during periods of permissive temperatures. Leaf structure interacts with the mechanisms of frost survival. Stem xylem in evergreen plants tends to contain smaller diameter conduits incurring greater resistance to freeze/ thaw induced cavitation than in deciduous plants, although at the cost of reduced hydraulic efficiency. In contrast, no such differences in hydraulic conductivity have been documented at the leaf level. There is ev- idence for reduced structural plasticity of evergreen leaves in response to variability in irradiance, however photosynthetic downregulation occurs in mature leaves in response to self shading. Some evergreen species exhibit slow leaf development and "delayed greening", while in many species aging is also a very protracted process. Finally, evergreen leaves may participate in carbohydrate and, less obviously, in nitrogen storage for the support of spring shoot and foliage growth, although the importance of this function is under debate. In conclusion, the evergreen leaf habit is correlated with numerous structural and functional traits at the leaf and also at the stem level. These correlations may generate trade-offs that shape the ecological strategies of evergreen plants.

Xeromorphism is a set of structural traits facilitating plant functioning under the conditions of water deficit. While leaf xeromorphic traits have been widely described in a variety of species, much less is known about the ability of plants to acclimate to drought by plastically modifying their leaf structure. We hypothesized that such modification would recapitulate the xeromophic syndrome. We conducted a greenhouse experiment in which a genetically and phenotypically diverse population of barley (Hordeum vulgare L.) cultivars and hybrid lines were subjected to controlled drought followed by re-watering. Leaves produced after the onset of drought were sampled for determination of lamina length and 18 anatomical traits of adaxial epidermis and central transverse lamina sections. Across all studied genotypes, mean leaf length, width, and dimensions of all internal structures were reduced by drought, whereas the density of veins, stomata, and trichomes increased. However, since anatomical traits were commonly correlated with leaf length and following drought the leaves became shorter, we statistically controlled for the leaf size-related variation. Although leaf shortening explained much anatomical response to drought, several traits exhibited additional, leaf-length independent modification. For example, vein and stomatal density increased and xylem area and vessel diameter decreased to a greater extent than expected from leaf size reduction. These results confirm the occurrence of modest facultative xeromorphism in barley but also emphasize the need to use allometric analysis to uncouple the true plasticity of traits from changes attributable to altered plant and organ size.

Background As proposed by Darwin, climbers have been assumed to allocate a smaller fraction of biomass to support organs in comparison with self-supporting plants. They have also been hypothesized to possess a set of traits associated with fast growth, resource uptake and high productivity. Scope In this review, these hypotheses are evaluated by assembling and synthesizing published and unpublished data sets from across the globe concerning resource allocation, growth rates and traits of leaves, stems and roots of climbers and self-supporting species. Conclusions The majority of studies offer little support for the smaller allocation of biomass to stems or greater relative growth rates in climbers; however, these results are based on small sized (

16 p., 1 mapa, tablas -- Postprint del artículo publicado en Diversity and Distributions. A Journal of Conservation Biogeopraphy. Versión revisada y corregida, [Aim] Scots pine is one of the dominant tree species in forest ecosystems of the temperate and boreal zones in Eurasia. Since the Tertiary, it has persisted also in the Mediterranean region, forming relict populations. In this study, we investigate range-wide genetic diversity of Scots pine, aiming to provide comprehensive information on the phylogeography and genetic resources of the species., [Location] Europe and Asia., [Methods] Thirteen paternally inherited chloroplast microsatellite (cpSSR) loci were used to investigate the genetic structure of 62 populations from Fennoscandia and 30 populations from the Mediterranean. We also surveyed variability of two maternally inherited mitochondrial DNA (mtDNA) regions and additionally included literature data from 156 populations (248 populations in total)., [Results] Scots pine retains an overall high genetic diversity in the chloroplast genome but, unexpectedly, the historically younger populations from Fennoscandia showed statistically higher mean intrapopulation diversity than the Mediterranean stands (0.807 and 0.750, respectively). The latter also contained a lower number of private cpSSR haplotypes. Population differentiation with respect to mtDNA was higher (GST = 0.628) than at cpSSR (GST = 0.015) indicating an efficient pollen-mediated gene flow among refugial populations of the species. One novel haplotype mtDNA was found, increasing the number of known mitotypes for the species from 4 to 5. The geographic distribution of mitotypes was structured into four groups corresponding to the main refugial areas of the species., [Main conclusions] Genetic impoverishment of the Mediterranean populations may enhance their vulnerability to future environmental changes. The spatial distribution of two mitotypes in Fennoscandia, featuring predominance of mitotype a in Norway and Sweden and mitotype b in Finland, gives strong support for dual colonization of that region from south-eastern (Finland) and south-western (Sweden) directions. These results thus provide new insights into both Holocene expansion of Scots pine and the present distribution of species' genetic resources., This study was supported by the National Science Centre (2011/02/A/NZ9/00108) and Institute of Dendrology, Polish Academy of Sciences.

Spectra of leaf traits in northern temperate forest canopies reflect major differences in leaf longevity between evergreen conifers and deciduous broadleaf angiosperms, as well as plastic modifications caused by within-crown shading. We investigated (1) whether long-lived conifer leaves exhibit similar intra-canopy plasticity as short-lived broadleaves, and (2) whether global interspecific relationships between photosynthesis, nitrogen, and leaf structure identified for sun leaves adequately describe leaves differentiated in response to light gradients. We studied structural and photosynthetic properties of intra-tree sun and shade foliage in adult trees of seven conifer and four broadleaf angiosperm species in a common garden in Poland. Shade leaves exhibited lower leaf mass-per-area (LMA) than sun leaves; however, the relative difference was smaller in conifers than in broadleaves. In broadleaves, LMA was correlated with lamina thickness and tissue density, while in conifers, it was correlated with thickness but not density. In broadleaves, but not in conifers, reduction of lamina thickness was correlated with a thinner palisade layer. The more conservative adjustment of conifer leaves could result from a combination of phylogenetic constraints, contrasting leaf anatomies and shoot geometries, but also from functional requirements of long-lived foliage. Mass-based nitrogen concentration (Nmass) was similar between sun and shade leaves, and was lower in conifers than in deciduous broadleaved species. Given this, the smaller LMA in shade corresponded with a lower area-based N concentration (Narea). In evergreen conifers, LMA and Narea were less powerful predictors of area-based photosynthetic rate (Amax(area)) in comparison with deciduous broadleaved angiosperms. Multiple regression for sun and shade leaves showed that, in each group, Amax(mass) was related to Nmass but not to LMA, whereas LMA became a significant codeterminant of Amax(mass) in analysis combining both groups. Thus, a fundamental mass-based relationship between photosynthesis, nitrogen, and leaf structure reported previously also exists in a dataset combining within-crown and across-functional type variation. Electronic supplementary material The online version of this article (doi:10.1007/s00442-012-2279-y) contains supplementary material, which is available to authorized users.

Semidwarf barley cultivars have been created in the past decades featuring shortened straw length. The semidwarfness trait is desirable because plants with reduced height are more resistant to lodging and, as a consequence, have an improved harvest index. Accordingly, both breeding and molecular mapping experiments were undertaken to enhance and evaluate the performance of semidwarf barley lines. To study the morpho-anatomical effects of the denso locus twenty barley lines developed by single seed descent technique were analysed. We measured several macro and microscopic traits of aboveground organs. We detected reduction in leaf size, which at tillering stage was also reflected by smaller sizes of some, but not all, categories of epidermal cells. Lower cell division frequencies were deduced for cell files with unchanged cell lengths. Denso leaf blades were thinner and had smaller vascular bundles and narrower tracheal elements compared to the wild type. On the other hand, a lack of effect was noted e.g. for sizes of stomata and size of spike. Denso gene thus exerted a multilevel dwarfing effect on cells, tissues and organs although not all organs and cells were affected to the same extent, and cell size or division frequency were modified depending on cell type.

Seasonal variability of maximum quantum yield of PSII photochemistry (Fv/Fm) was studied in needles of Taxus baccata seedlings acclimated to full light (HL, 100% solar irradiance), medium light (ML, 18% irradiance) or low light (LL, 5% irradiance). In HL plants, Fv/Fm was below 0.8 (i.e. state of photoinhibition) throughout the whole experimental period from November to May, with the greatest decline in January and February (when Fv/Fm value reached 0.37). In ML seedlings, significant declines of Fv/Fm occurred in January (with the lowest level at 0.666), whereas the decline in LL seedlings (down to 0.750) was not significant. Full recovery of Fv/Fm in HL seedlings was delayed until the end of May, in contrast to ML and LL seedlings. Fv/Fm was significantly correlated with daily mean (Tmean), maximal (Tmax) and minimal (Tmin) temperature and Tmin was consistently the best predictor of Fv/Fm in HL and ML needles. Temperature averages obtained over 3 or 5 days prior to measurement were better predictors of Fv/Fm than 1- or 30-day averages. Thus our results indicate a strong light-dependent seasonal photoinhibition in needles of T. baccata as well as suggest a coupling of Fv/Fm to cumulative temperature from several preceding days. The dependence of sustained winter photoinhibition on light level to which the plants are acclimated was further demonstrated when plants from the three light environments were exposed to full daylight over single days in December, February and April and Fv/Fm was followed throughout the day to determine residual sensitivity of electron transport to ambient irradiance. In February, the treatment revealed a considerable midday increase in photoinhibition in ML plants, much less in HL (already downregulated) and none in LL plants. This suggested a greater capacity for photosynthetic utilization of electrons in LL plants and a readiness for rapid induction of photoinhibition in ML plants. Further differences between plants acclimated to contrasting light regimes were revealed during springtime de-acclimation, when short term regeneration dynamics of Fv/Fm and the relaxation of nonphotochemical quenching (NPQ) indicated a stronger persistent thermal mechanism for energy dissipation in HL plants. The ability of Taxus baccata to sustain winter photoinhibition from autumn until late spring can be beneficial for protection against an excessive light occurring together with frosts but may also restrict photosynthetic carbon gain by this shade-tolerant species when growing in well illuminated sites.

Leaves developing in different irradiances undergo structural and functional acclimation, although the extent of trait plasticity is species specific. We tested the hypothesis that irradiance-induced plasticity of photosynthetic and anatomical traits is lower in highly shade-tolerant species than in moderately shade-tolerant species. Seedlings of two evergreen conifers, shade-tolerant Abies alba Mill. and moderately shade-tolerant Picea abies Karst., and two deciduous angiosperm species, highly shade-tolerant Fagus sylvatica L. and moderately shade-tolerant Acer pseudoplatanus L., were grown in deep shade (LL, 5% of full irradiance) or in full solar irradiance (HL) during 2003 and 2004. Steady state responses of quantum yield of PSII (Phi(PSII)), apparent electron transport rate (ETR), nonphotochemical quenching (NPQ) and photochemical quenching (qP) were generally modified by the light environment, with slower declines in Phi(PSII) and qP and greater maximal ETR and NPQ values in HL plants in at least one season; however, no link between quantitative measures of plasticity of these traits and shade tolerance was found. Plasticity of nine anatomical traits (including palisade cell length, which was reduced in LL) showed no relationship with shade tolerance, but was less in conifers than in deciduous trees, suggesting that leaf life span may be a significant correlate of plasticity. When LL-acclimated plants were exposed to HL conditions, the degree and duration of photoinhibition (measured as a decline in maximum quantum yield) was greatest in F. sylvatica, much lower in P. abies and A. alba, and lowest in A. pseudoplatanus. Thus, as with the other traits studied, vulnerability to photoinhibition showed no relationship with shade tolerance.

In continuous light, leaves of the Crassulacean acid metabolism (CAM) plant Kalanchoedaigremonti- ana Hamet et Perrier exhibit a circadian rhythm of CO2 uptake, stomatal conductance and leaf-internal CO2 pressure. According to a current quantitative model of CAM, the pacemaking mechanism involves periodic turgor-related tension and relaxation of the tonoplast, which determines the direction of the net flux of malate between the vacuole and the cytoplasm. Cytoplasmic malate, in turn, through its inhibitory effect on phospho enolpyruvate carboxylase, controls the rate of CO2 up- take. According to this mechanism, when the accumu- lation of malate is disrupted by removing CO2 from the ambient air, the induction of a phase delay with respect to an unperturbed control plant is expected. First, using the mathematical model, such phase delays were ob- served in numerical simulations of three scenarios of CO2 removal: (i) starting at a trough of CO2 uptake, lasting for about half a cycle (ca. 12 h in vivo); (ii) with the identical starting phase, but lasting for 1.5 cycles (ca. 36 h); and (iii) starting while CO2 increases, lasting for half a cycle again. Applying the same protocols to leaves of K. daigremontiana in vivo did not induce the predicted phase shifts, i.e. after the end of the CO2 removal the perturbed rhythm adopted nearly the same phase as that of the control plant. Second, when leaves were exposed to a nitrogen atmosphere for three nights prior to onset of continuous light to prevent malate accumulation, a small, 4-h phase advance was observed instead of a de- lay, again contrary to the model-based expectations. Hence, vacuolar malic acid accumulation is ruled out as the central pacemaking process. This observation is in line with our earlier suggestion (T.P. Wyka, U. Luttge (2003) J Exp Bot 54:1471-1479) that in extended con- tinuous light, CO2 uptake switches gradually from a CAM-like to a C3-like mechanism, with oscillations of the two CO2 uptake systems being tightly coordinated. It appears that the circadian rhythm of gas exchange in this CAM plant emerges from one or several devices that are capable of generating temporal information in a robust manner, i.e. they are protected from even severe metabolic perturbations.

During the endogenous circadian rhythm of carbon dioxide uptake in continuous light by a Crassula cean acid metabolism plant, Kalanchoe daigremontiana, the two carboxylating enzymes, phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco), are active simultaneously, although, until now, only the role of PEPC in generating the rhythm has been acknowledged. According to the established model, the rhythm is primarily regulated at the PEPC activity level, modulated by periodic compartmentation of its inhibitor, malate, in the vacuole and controlled by tension/relaxation of the tonoplast. However, the circadian accumulation of malic acid (the main indicator of PEPC activity) dampened significantly within the first few periods without affecting the rhythm's amplitude. Moreover, the amount of malate accumulated during a free-running oscillation was several-fold lower than the amount expected if PEPC were the key carboxylating enzyme, based on a 1:1 stoichiometry of CO(2) and malate. Together with the observation that rates of CO(2) uptake under continuous light were higher than in darkness, the evidence shows that C(3) carboxylation greatly contributes to the generation of rhythmic CO(2) uptake in continuous light in this 'obligate' CAM plant. Because the shift from predominantly CAM to predominantly C(3) carboxylation is smooth and does not distort the trajectory of the rhythm, its control probably arises from a robust network of oscillators, perhaps also involving stomata.