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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)

Intraspecific variability of anatomical leaf traits is usually determined on the basis of median sections and interpreted as adaptive. We hypothesized that anatomical traits may relate to the position within the leaf, reflecting functional coordination e.g., between transport and gas exchange capacities and between supporting tissue and the supported leaf weight. Anatomical variation may thus be partly controlled by leaf size. We examined variability of transverse anatomical traits and stomata at different positions along the leaf axis using Scots pine (Pinus sylvestris L.) needles collected from three geographical locations with contrasting climate (Poland, Southern and Northern Sweden) and differing in length. Transverse xylem and phloem area, and the number of tracheids at a given cross-section scaled positively with length of the distal needle part and the number of distal stomata. Similarly, the number of vascular sclerenchyma fibers in a cross-section scaled with the length and volume of the distal needle part. Allometric slopes were less steep in the longer, southern-sites needles. Additionally, our study revealed an increased sclerification of epidermis towards the needle apex, the occurrence of widest tracheids in central or subapical regions, a reduced number of resin ducts in basal and apical parts, as well as overall basipetal decreases in duct diameter, the number of stomatal files and stomatal density. These results show that allometry accounts for significant amount of intraspecific variability in xylem, phloem and fibers, and that environmental adaptation involves also modification of the within-needle allometric relationships. The allometric component of variability, together with the nonlinear distribution of certain traits along needle length, should be considered when designing comparative studies of foliar phenotypes and scaling up from transverse sections to entire needles.

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.

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.

SummaryFlowers and floral parts have seldom been used successfully as explants to initiate tissue culture. Flower buds of Mammillaria albicoma, M. carmenae, and M. schiedeana (Cactaceae) were cultured on solid Murashige and Skoog medium containing 0.1 mg l−1 -naphthaleneacetic acid (NAA) and 5.0 mg l−1 6-benzylaminopurine. The formation of vegetative buds (areoles) and the development of shoots were observed in all three species. Both direct and indirect mechanisms of shoot morphogenesis were observed. Direct shoot morphogenesis from the perianth (especially from the axils of the perianth segments) in M. albicoma and M. carmenae was confirmed by microscopic examination. Shoots that formed on explants were isolated and used to establish proliferating cultures. Finally, proliferated shoots were rooted in vitro on MS medium containing 0.01 mg l−1 NAA and acclimated to ex vitro conditions. M. carmenae shoots were also rooted non-aseptically in horticultural substrate. This is the first report of the complete ...

In several genera of Cactaceae flowers are produced by a specialized, nonphotosynthetic portion of the stem known as the cephalium. The objective of this study is to test if cephalial tissue can be induced to revert to vegetative growth under in vitro conditions. Segments excised from the surface of Melocactus matanzanus cephalium and placed on Murashige and Skoog medium supplemented with 0.1 mg l−1 1-napthaleneacetic acid (NAA) and 1.0 or 5.0 mg l−1 6-benzyloaminopurine (BAP) regularly produced vegetative shoots. Shoots were also obtained when kinetin was used instead of BAP, although the frequency of their formation was then lower. One of the reverted shoots was successfully established as a rooted plant ex vitro.

We tested the organogenetic capacity of floral buds of Mammillaria albicoma Boed. (Cactaceae). Buds were incubated on solid MS medium supplemented with 0.1 mg l−1 α-naphthaleneacetic acid (NAA) and 5.0 mg l−1 6-benzylaminopurine (BA). Callus growth was observed from the cut explant base and from within the perianth. These calli during subsequent subcultures to the same medium gave rise to adventitious shoots. Shoots formed also directly from the perianth, as confirmed by observations in the light microscope and scanning electron microscope (SEM). On transfer to a fresh medium, the shoots produced proliferating cultures. This is the first report of regeneration of cactus shoots from floral explants.

As part of fir reintroduction program in the Karkonosze Mountains (S. Poland) a study was undertaken to investigate growth and ecophysiological performance of young firs (Abies alba) under varying degrees of natural shade. Three-year-old fir seedlings were planted in five forest stands dominated by Picea abies, Fagus sylvatica, Betula pendula, Pinus sylvestris or Larix decidua. Over four growing seasons, annual apical increments became progressively greater under the light permeable canopy of Larix, but remained stunned under the deep shade of Picea, while intermediate irradiance at other sites resulted in intermediate growth responses. Similarly, apical dominance ratio was positively influenced by irradiance. These two contrasting sites were subsequently used for ecophysiological comparisons. Fir seedlings under the Larix canopy had consistently higher photosynthetic rates throughout the day compared to those under Picea, but in both stands light adapted quantum yield of PSII remained mostly above 0.8 indicating lack of photoinhibition. Quantum yield declined transiently only during sunflecks, which were more frequent under Larix. Light dependence of apparent electron transport rates in both sites was linear showing lack of light saturation, while slope differences between sites were only slight. In contrast, depression of PSII quantum yield by light was less pronounced under Larix, showing an acclimation to higher irradiance. Fir needles produced under Larix were thicker and contained more chlorophyll, carotenoid and nitrogen on area basis (but, except for nitrogen, not on dry mass basis) suggesting that structural modification is a major component of acclimation to light. On anatomical levels, needles produced under Larix had the traits typical for sun-acclimated ones, while those under Picea for shade-acclimated. The magnitude of modifications was, however, less than that reported in literature for sun and shade needles. Young seedlings were able to acclimate to higher irradiance under Larix without showing light stress symptoms. This suggests that success of stand regeneration may be enhanced by adjusting light penetration through the overstorey. Fir seedlings found best growth conditions under the relatively light permeable crowns of light-demanding tree species (Larix decidua, Pinus sylvestris) suggesting that stands dominated by these trees are more suitable for transformation using silver fir seedlings than those with shade-tolerant tree overstoreys.

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.

To evaluate the acclimative ability of current-year and previous-year needles of a shade tolerant conifer Taxus baccata L. to contrasting irradiance conditions, seedlings were raised under 27% solar irradiance and at 3 years of age they were transferred to an experimental garden and grown for one season under full irradiance (HL), 18% irradiance (ML) or 5% irradiance (LL). Whereas previous year needles did not change anatomically, current year needles in HL were thicker and had a thicker palisade and spongy mesophyll, and greater leaf mass per area than ML or LL needles. LL needles had greater nitrogen concentration than HL needles irrespective of age but only previous year LL needles also had an increased N per area content, thanks to their lack of reduction in LMA. Adjustment of chlorophyll and carotenoid content occurred in both needle age classes with LL and ML needles having much higher concentrations but, in current year needles, only slightly higher per area content than HL needles. Chlorophyll a/b ratio was not affected by age or irradiance. These modifications had no significant effect on photosynthetic capacities, which did not significantly differ between the age classes in HL or LL treatment and between treatments. On the other hand, high growth irradiance resulted in a greater photochemical yield, photochemical quenching, apparent electron transport rate and inducible non-photochemical quenching in needles formed in the current season. In previous year needles, however, only inducible NPQ was enhanced by high irradiance with other parameters remaining identical among treatments. To test sensitivity to photoinhibition, at the end of the summer plants from the three irradiance levels were transferred to a HL situation and F (v)/F (M) was determined over the following 18 days. Sensitivity to photoinhibition was negatively related to growth irradiance and previous year needles were less photoinhibited than current year needles. Thus, differences in acclimation ability between needle age classes were most pronounced at the level of anatomy and light reactions of photosynthesis, both of which showed almost no plasticity in previous year needles but were considerably modified by irradiance in current year needles.

In continuous light, the Crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perrier has a circadian rhythm of gas exchange with peaks occurring during the subjective night. The rhythm of gas exchange is coupled to a weak, reverse phased rhythm of quantum yield of photosystem II (Phi (PSII)). To test if the rhythm of Phi (PSII) persists in the absence of stomatal control, leaves were coated with a thin layer of translucent silicone grease which prevented CO2 and H2O exchange. In spite of this treatment, the rhythm of Phi (PSII) occurred with close to normal phase timing and with a much larger amplitude than in uncoated leaves. The mechanism underlying the Phi (PSII) rhythm in coated leaves can be explained by a circadian activity of phosphoenolpyruvate carboxylase (PEPC). At peaks of PEPC activity, the small amount of CO2 contained in the coated leaf could have become depleted, preventing the carboxylase activity of Rubisco and causing decreases in electron transport rates (observed as deep troughs of Phi (PSII) at 23-h in LL and at ca. 24-h intervals afterwards). Peaks of Phi (PSII) would be caused by a downregulation of PEPC leading to improved supply of CO2 to Rubisco. Substrate limitation of photochemistry at 23 h (trough of Phi (PSII)) was also suggested by the weak response of ETR in coated leaves to stepwise light enhancement. These results show that photosynthetic rhythmicity in K. daigremontiana is independent of stomatal regulation and may originate in the mesophyll.