The ontogeny of disparity in Cupressaceae seed cones.

Ontogenetic shape change has long been recognized to be important in generating patterns of morphological diversity and may be especially important in plant reproductive structures. We explore how seed cone disparity in Cupressaceae changes over ontogeny by comparing pollination-stage and mature cones. We sampled cones at pollen and seed release and measured cone scales using basic morphometric shape variables. We used multivariate statistical methods, particularly hypervolume overlap calculations, to measure morphospace occupation and disparity. Cone scales at both pollination and maturity exhibit substantial variability, although the disparity is greater at maturity. Mature cone scales are also more clustered in trait space, showing less overlap with other taxa than at pollination. These patterns reflect two growth strategies that generate closed cones over maturation, either through thin laminar scales or relatively thick, peltate scales, resulting in two distinct regions of morphospace occupation. Disparity patterns in Cupressaceae seed cones change over ontogeny, reflecting shifting functional demands that require specific patterns of cone scale growth. The evolution of Cupressaceae reproductive disparity therefore represents selection for trajectories of ontogenetic shape change, a phenomenon that should be widespread across seed plants.

Chemical Composition and Cytotoxic Activity of the Fractionated Trunk Bark Essential Oil from Tetraclinis articulata (Vahl) Mast. Growing in Tunisia.

The aim of the present research was to determine the chemical composition and the cytotoxic effects of Tetraclinis articulata trunk bark essential oil (HEE) obtained by steam distillation and five fractions obtained by normal phase silica chromatographic separation. Chemical analysis allowed the identification of 54 known compounds. Relatively high amounts of oxygenated sesquiterpenes (44.4-70.2%) were detected, mainly consisting of caryophyllene oxide (13.1-26.6%), carotol (9.2-21.2%),14-hydroxy-9-epi-(E)-caryophyllene (3.2-15.5%) and humulene epoxide II (2.6-7.2%). The cytotoxic activity against human mammary carcinoma cell lines (MDA-MB-231) and colorectal carcinoma cell lines (SW620) of the essential oil and its fractions were assessed. All the samples displayed moderate to weak activity compared to 5-fluorouracil. The colorectal carcinoma cell line was relatively more sensitive to the essential oil and its fractions compared to the breast cancer cell line, showing IC50 values from 25.7 to 96.5 µg/mL. In addition, the essential oil and its fraction E.2 revealed a cytotoxic activity against colorectal carcinoma cell line, with IC50 values lower than 30 µg/mL. This is the first report on the chemical composition and cytotoxic activity of the trunk bark essential oil of T. articulata.

Revisiting the relative growth rate hypothesis for gymnosperm and angiosperm species co-occurrence.

PREMISE OF THE STUDY: It is unclear to what extent the co-occurrence of angiosperm and gymnosperm species in some marginal ecosystems is explained by reduced growth in angiosperms due to carbon (C) limitation and by high stress tolerance in gymnosperms associated with lack of vessels and resource conservation. METHODS: We examined growth patterns and traits associated with C balance in four evergreen angiosperm species (including one vesselless species, Drimys winteri) and three gymnosperm tree species of a cold-temperate rainforest in southern Chile. We measured the mean basal area increment for the first 50 (BAI50 ) and the last 10 years (BAI10 ), wood density, leaf lifespan, and nonstructural carbohydrate (NSC) concentrations in different organs. KEY RESULTS: BAI50 was 6-fold higher in angiosperms than in gymnosperms and ca. 4-fold higher in Drimys than in the fastest-growing gymnosperm. BAI10 and aboveground NSC concentrations were significantly higher and leaf lifespan lower in angiosperms than in gymnosperms; these differences, however, were largely driven by the slow growth and low NSC concentrations of the Cupressaceae species (Pilgerodendron uviferum), while the two Podocarpaceae had BAI10 and NSC concentrations similar to angiosperms. In angiosperms, NSC and starch concentrations were generally higher in species with lower BAI10 , indicating no severe C limitation. CONCLUSIONS: The co-occurrence of angiosperms and gymnosperms in cold-temperate rainforests of southern Chile is not explained by growth disadvantages and C limitation in angiosperms. Long leaf longevity, but not lack of vessels, appeared to favor resource conservation and C balance in some gymnosperms (Podocarpaceae).

Responses of Intrinsic Water-use Efficiency and Tree Growth to Climate Change in Semi-Arid Areas of North China.

Tree-level intrinsic water-use efficiency (iWUE) is derived from the tree-ring 13C isotope composition (δ13C) and is an important indicator of the adaptability for trees to climate change. However, there is still uncertainty regarding the relationship between long-term forest ecosystem carbon sequestration capacity and iWUE. To determine whether elevated atmospheric CO2 concentration (Ca) increase iWUE and tree growth (basal area increment, BAI), dendrochronological methods and stable isotope analyses were used to examine annual changes in the tree-ring width and carbon isotope composition (δ13C) of Platycladus orientalis in northern China. The iWUE derived from δ13C has increased significantly (p < 0.01). Long-term iWUE trend was largely and positively driven by the elevated atmospheric CO2 concentration and temperature. We observed a general increase in averaged BAI, which had significant positive correlation with iWUE (R2 = 0.3186, p < 0.01). Increases in iWUE indeed translated into enhanced P. orientalis growth in semi-arid areas of northern China. Elevated atmospheric CO2 concentration significantly (p < 0.01) stimulated P. orientalis biomass accumulation when Ca was less than approximately 320 ppm in the early phase; however, this effect was not pronounced when Ca exceeded 320 ppm.

Tree growth response of Fokienia hodginsii to recent climate warming and drought in southwest China.

To date, few attempts have been made to assess the influence of climate change on forest ecosystems and on the relationship between tree growth and climate in humid areas of low latitudes. In this paper, we studied the response of tree growth and forest ecosystem to climate change by using Fokienia hodginsii tree-ring cores from the northern Yunnan-Guizhou Plateau, southwest of China. Tree growth correlates the highest (r = -0.64, p < 0.01) with mean temperature (July-September), but the coefficients were changing with time as revealed by a moving correlation analysis. Tree growth is significantly (p < 0.05) and positively correlated with January-April mean temperature from AD 1961-1987, while correlations with precipitation are insignificant. In contrast, from 1988 to 2014, tree growth correlated negatively with mean temperature of previous summer and positively with precipitation of previous August-September. This indicated that the limiting factors for tree growth have changed under different climate conditions. The meteorological data suggested that from 1961 to 1987 it was cold and wet in the study area and radial growth is limited by winter and spring temperatures. This restriction is weaker if the climate is appropriate in general. However, from 1988 to 2014, the combined effects of recent warming and decreasing precipitation have led to an increasing response of tree-ring width to drought. In addition, a large proportion of mature F. hodginsii mortality occurred from 2007 to 2013, which corresponds with a drastic reduction of radial growth (narrowest in recent 100 years). The recent drought, induced by decreasing precipitation and increasing temperature, may have passed the threshold which F. hodginsii could tolerate, causing tree growth reduction, tree growth-climate relationship change, as well as catastrophic tree mortality. All these changes may lead to further responses of the local ecosystem to climate change which should be highly regarded.

Development and evaluation of height diameter at breast models for native Chinese Metasequoia.

Accurate tree height and diameter at breast height (dbh) are important input variables for growth and yield models. A total of 5503 Chinese Metasequoia trees were used in this study. We studied 53 fitted models, of which 7 were linear models and 46 were non-linear models. These models were divided into two groups of single models and multivariate models according to the number of independent variables. The results show that the allometry equation of tree height which has diameter at breast height as independent variable can better reflect the change of tree height; in addition the prediction accuracy of the multivariate composite models is higher than that of the single variable models. Although tree age is not the most important variable in the study of the relationship between tree height and dbh, the consideration of tree age when choosing models and parameters in model selection can make the prediction of tree height more accurate. The amount of data is also an important parameter what can improve the reliability of models. Other variables such as tree height, main dbh and altitude, etc can also affect models. In this study, the method of developing the recommended models for predicting the tree height of native Metasequoias aged 50-485 years is statistically reliable and can be used for reference in predicting the growth and production of mature native Metasequoia.

Effects of elevated mean and extremely high temperatures on the physio-ecological characteristics of geographically distinctive populations of Cunninghamia lanceolata.

Conventional models for predicting species distribution under global warming scenarios often treat one species as a homogeneous whole. In the present study, we selected Cunninghamia lanceolata (C. lanceolata), a widely distributed species in China, to investigate the physio-ecological responses of five populations under different temperature regimes. The results demonstrate that increased mean temperatures induce increased growth performance among northern populations, which exhibited the greatest germination capacity and largest increase in the overlap between the growth curve and the monthly average temperature. However,tolerance of the southern population to extremely high temperatures was stronger than among the population from the northern region,shown by the best growth and the most stable photosynthetic system of the southern population under extremely high temperature. This result indicates that the growth advantage among northern populations due to increased mean temperatures may be weakened by lower tolerance to extremely high temperatures. This finding is antithetical to the predicted results. The theoretical coupling model constructed here illustrates that the difference in growth between populations at high and low latitudes and altitudes under global warming will decrease because of the frequent occurrence of extremely high temperatures.

[Effects of gel made by super absorbent polymers and urea on fine root growth and nitrogen use efficiency of Platycladus orientalis bareroot seedlings.] / 保水剂-å°¿ç´ å‡èƒ¶å¯¹ä¾§æŸè£¸æ ¹è‹—ç»†æ ¹ç”Ÿé•¿å’Œæ°®ç´ åˆ©ç”¨çŽ‡çš„å½±å“.

A pot experiment including five treatments, i.e., CK (neither fertilizer nor super absorbent polymers), U (urea alone), S [super absorbent polymers (SAP) alone], SUM (SAP mixed with urea) and SUG (gel made of SAP and urea) was conducted to evaluate their effects on fine root morphological characteristics, fine root absorption area, fine root nitrogen metabolism and nitrogen use efficiency of Platycladus orientalis bareroot seedlings. Results showed that compared with U treatment, the SUG treatment significantly increased the biomass, length, specific root length, surface area and volume of fine root. The total absorption area and active absorption area of fine root were also increased remarkably under the SUG treatment. The specific root length in the SUG treatment was increased by 34.7%, 37.9%, 41.1% and 12.4% compared with the treatments of CK, U, S and SUM, respectively. Compared with the U treatment, the activities of nitrate reductase, glutamine synthase, glutamate synthase and glutamate dehydrogenase in fine root of the SUG treatment was improved by 41.2%, 76.6%, 30.7% and 125.8%, respectively, while the ratio of GS to GDH decreased. Not only the ground diameter and plant height, but also the dry matter accumulation of aboveground and underground parts was remarkably enhanced under the SUG treatment. The nitrogen use efficiency was 40.7% in the SUG treatment, being 118.8% and 44.5% higher than the U and SUM treatments, respectively. Gel made of SAP and urea was able to significantly increase the nitrogen use efficiency, promote the growth and enhance the ability of drought resistance by improving the morphological characteristics, enhancing the absorption area and key enzymes activities of nitrogen metabolism in the fine root of P. orientalis bareroot seedlings.

Survival, growth and vulnerability to drought in fire refuges: implications for the persistence of a fire-sensitive conifer in northern Patagonia.

Fire severity and extent are expected to increase in many regions worldwide due to climate change. Therefore, it is crucial to assess the relative importance of deterministic vs. stochastic factors producing remnant vegetation to understand their function in the persistence of fire-sensitive plants. Vegetation remnants (areas within the landscape that have not burned for a considerable amount of time) may occur stochastically or in more predictable locations (fire refuges) where physical conditions decrease fire severity. Our aim was to determine if remnant forests of the fire-sensitive conifer Austrocedrus chilensis are associated with biophysical attributes that allow persistence in a fire-prone Patagonian landscape. We conducted a multi-scale approach, determining attributes of forest remnants and their surroundings (matrices) through remote sensing and field-based biophysical and functional characteristics, and quantifying how tree survival probability relates to microsite conditions. Trees within remnants displayed abundant fire scars, were twofold older and had threefold larger growth rates than matrix trees. Remnants were associated with high rocky cover and elevated topographical positions. Tree survival increased in hilltops, eastern aspects, and with sparse vegetation. Trees within remnants experienced severe reductions in growth during droughts. Our results suggest that A. chilensis remnants are mainly the result of refuges, where environmental conditions increase fire survival, but also increase susceptibility to drought. A trade-off between fire survival and drought vulnerability may imply that under increasing drought and fire severity, locations that in the past have served as refuges may reduce their ability to allow the persistence of fire-sensitive taxa.

Tree Rings Show Recent High Summer-Autumn Precipitation in Northwest Australia Is Unprecedented within the Last Two Centuries.

An understanding of past hydroclimatic variability is critical to resolving the significance of recent recorded trends in Australian precipitation and informing climate models. Our aim was to reconstruct past hydroclimatic variability in semi-arid northwest Australia to provide a longer context within which to examine a recent period of unusually high summer-autumn precipitation. We developed a 210-year ring-width chronology from Callitris columellaris, which was highly correlated with summer-autumn (Dec-May) precipitation (r = 0.81; 1910-2011; p < 0.0001) and autumn (Mar-May) self-calibrating Palmer drought severity index (scPDSI, r = 0.73; 1910-2011; p < 0.0001) across semi-arid northwest Australia. A linear regression model was used to reconstruct precipitation and explained 66% of the variance in observed summer-autumn precipitation. Our reconstruction reveals inter-annual to multi-decadal scale variation in hydroclimate of the region during the last 210 years, typically showing periods of below average precipitation extending from one to three decades and periods of above average precipitation, which were often less than a decade. Our results demonstrate that the last two decades (1995-2012) have been unusually wet (average summer-autumn precipitation of 310 mm) compared to the previous two centuries (average summer-autumn precipitation of 229 mm), coinciding with both an anomalously high frequency and intensity of tropical cyclones in northwest Australia and the dominance of the positive phase of the Southern Annular Mode.

Community Structure and Survival of Tertiary Relict Thuja sutchuenensis (Cupressaceae) in the Subtropical Daba Mountains, Southwestern China.

A rare coniferous Tertiary relict tree species, Thuja sutchuenensis Franch, has survived in the Daba Mountains of southwestern China. It was almost eliminated by logging during the past century. We measured size and age structures and interpreted regeneration dynamics of stands of the species in a variety of topographic contexts and community associations. Forest communities containing T. sutchuenensis were of three types: (1) the Thuja community dominated by T. sutchuenensis, growing on cliffs; (2) the Thuja-Quercus-Cyclobalanopsis community dominated by T. sutchuenensis, Quercus engleriana and Cyclobalanopsis oxyodon, along with Fagus engleriana and Carpinus fargesiana, on steep slopes; (3) the Thuja-Tsuga-Quercus community dominated by T. sutchuenensis, Tsuga chinensis, and Quercus spinosa, on crest ridges. The established seedlings/saplings were found in limestone crevices, on scarred cliff-faces, cliff-edges, fallen logs, canopy gaps and forest margins. The radial growth rate was 0.5-1.1 mm per year. Its growth forms were distorted. It had strong sprouting ability after disturbances. The T. sutchuenensis population thrives on cliffs where there is little competition from other species because of harsh conditions and rockslide disturbances. It is shade-intolerant but stress-tolerant. Its regeneration has depended on natural disturbances.

Effects of high-severity fire drove the population collapse of the subalpine Tasmanian endemic conifer Athrotaxis cupressoides.

Athrotaxis cupressoides is a slow-growing and long-lived conifer that occurs in the subalpine temperate forests of Tasmania, a continental island to the south of Australia. In 1960-1961, human-ignited wildfires occurred during an extremely dry summer that killed many A. cupressoides stands on the high plateau in the center of Tasmania. That fire year, coupled with subsequent regeneration failure, caused a loss of ca. 10% of the geographic extent of this endemic Tasmanian forest type. To provide historical context for these large-scale fire events, we (i) collected dendroecological, floristic, and structural data, (ii) documented the postfire survival and regeneration of A. cupressoides and co-occurring understory species, and (iii) assessed postfire understory plant community composition and flammability. We found that fire frequency did not vary following the arrival of European settlers, and that A. cupressoides populations were able to persist under a regime of low-to-mid severity fires prior to the 1960 fires. Our data indicate that the 1960 fires were (i) of greater severity than previous fires, (ii) herbivory by native marsupials may limit seedling survival in both burned and unburned A. cupressoides stands, and (iii) the loss of A. cupressoides populations is largely irreversible given the relatively high fuel loads of postfire vegetation communities that are dominated by resprouting shrubs. We suggest that the feedback between regeneration failure and increased flammability will be further exacerbated by a warmer and drier climate causing A. cupressoides to contract to the most fire-proof landscape settings.

First occurrence of Platycladus from the upper Miocene of Southwest China and its phytogeographic implications.

Platycladus Spach is native to Central China, but its natural occurrences are very difficult to establish. According to molecular phylogenetic data, this genus might have originated since the Oligocene, but no fossil record has been reported. Here, we describe eight foliage branches from the upper Miocene in western Yunnan, Southwest China as a new species, P. yunnanensis sp. nov., which is characterized by foliage branches spread in flattened sprays, and leaves decussate, imbricate, scale-like and dimorphic. The leaves are amphistomatic, and the stomata are elliptical or oblong, haplocheilic, and monocyclic type. Based on a detailed comparison with the extant genera of Cupressaceae sensu lato, our fossils are classified into the genus Platycladus. The occurrence of P. yunnanensis sp. nov. indicates that this genus had a more southernly natural distribution in the late Miocene than at present. Molecular phylogeny and fossil records support a pre-Oligocene common ancestor for the genera Platycladus, Microbiota and Calocedrus. The separation of the three taxa was most likely caused by the arid belt across Central China during the Oligocene. In addition, the cooling down of the global temperature and the strengthening of Asian monsoon since the Miocene will further promote the migration of these genera.

Impacts of elevated ozone on growth and photosynthesis of Metasequoia glyptostroboides Hu et Cheng.

One-year-old Metasequoia glyptostroboides seedlings were exposed to non-filtered ambient air (NF) and elevated ozone (E-O3, NF+60 ppb) in open-top chambers for two years. E-O3 accelerated leaf senescence, as indicated by significant decreases in photosynthetic pigment contents with the elongation of O3 exposure. E-O3 significantly affected gas exchange and carboxylation, inducing reductions in light-saturated photosynthesis (Asat), the maximum activity of Rubisco (Vc,max) and the maximum electron transport rate (Jmax). Chl a/b, Vc,max/Jmax and stomatal limitation (l) were not affected. Stomatal conductance (gs) was significantly decreased by E-O3 in the first year, but remained unchanged in the second year. It can be inferred that the decrease in Asat by E-O3 was mainly attributed to the changes in non-stomatal factors. After two years' exposure, E-O3 caused significant decreases in canopy photosynthesis and leaf mass per area, and a significant increase in the number of branches, but induced slight, not significant decreases in growth and biomass. Therefore, it can be concluded that the carbon accumulation of the species M. glyptostroboides could be negatively affected after long-term exposure to high O3 concentration.

Inoculation with arbuscular mycorrhizal fungi and addition of composted olive-mill waste enhance plant establishment and soil properties in the regeneration of a heavy metal-polluted environment.

A greenhouse experiment was carried out in order to investigate the effects of arbuscular mycorrhizal (AM) fungi inoculation and the use of composted olive waste (COW) in the establishment of Tetraclinis articulata and soil properties in a heavy metal-polluted soil. The treatments assayed were as follows: AM + 0% COW, AM + 1% COW, and AM + 3% COW. The higher doses of COW in combination with AM fungi increased shoot and root biomass production of T. articulata by 96 and 60%, respectively. These treatments trended to improve the soil properties evaluated, highlighting the C compounds and N as well as the microbiological activities. In relation to the metal translocation in T. articulata, doses of COW applied decreased the Cr, Ni, and Pb contents in shoot, as well as Cr and As in root, although the most of them reached low levels and far from phytotoxic. The COW amendment aided Glomus mosseae-inoculated T. articulata plants to thrive in contaminated soil, mainly through an improvement in both nutrients uptake, mainly P and soil microbial function. In addition, the combined use of AM fungi plus COW could be a feasible strategy to be incorporated in phytoremediation programs because it promotes soil properties, a better performance of plants for supporting the stress in heavy metal-contaminated soils derived from the mining process, and also can be a good way for olive-mill waste disposal.

Interspecific differences in whole-plant respiration vs. biomass scaling relationships: a case study using evergreen conifer and angiosperm tree seedlings.

PREMISE OF THE STUDY: Empirical studies and theory indicate that respiration rates (R) of small plants scale nearly isometrically with both leaf biomass (ML) and total plant biomass (MT). These predictions are based on angiosperm species and apply only across a small range of body mass. Whether these relationships hold true for different plants, such as conifers, remains unclear. METHODS: We tested these predictions using the whole-plant maintenance respiration rates and the biomass allocation patterns of the seedlings of two conifer tree species and two angiosperm tree species. Model Type II regression protocols were used to compare the scaling exponents (α) and normalization constants (ß) across all four species and within each of the four species. KEY RESULTS: The data show that the scaling exponents varied among the four species and that all differed significantly from isometry. For conifers, scaling exponents for R vs. MT, and R and ML were numerically smaller than those of the broadleaved angiosperm species. However, across the entire data set, R scaled isometrically with ML and with MT as predicted by the West, Brown, and Enquist (WBE) theory. We also observed higher respiration rates for small conifer seedlings compared to comparably sized angiosperm seedlings. CONCLUSIONS: Our data add credence to the view that the R vs. M scaling relationship differs among species, and that in general, the numerical values of this interspecific scaling relationship will depend on the species pooled in the analysis and on the range of body sizes within the data set.

Old-growth Platycladus orientalis as a resource for reproductive capacity and genetic diversity.

AIMS: Platycladus orientalis (Cupressaceae) is an old-growth tree species which distributed in the imperial parks and ancient temples in Beijing, China. We aim to (1) examine the genetic diversity and reproductive traits of old-growth and young populations of P. orientalis to ascertain whether the older populations contain a higher genetic diversity, more private alleles and a higher reproductive output compared with younger populations; (2) determine the relationships between the age of the population and the genetic diversity and reproductive traits; and (3) determine whether the imperial parks and ancient temples played an important role in maintaining the reproductive capacity and genetic diversity of Platycladus orientalis. METHODS: Samples from seven young (younger than 100 yrs.) and nine old-growth (older than 300 yrs.) artificial populations were collected. For comparison, three young and two old-growth natural populations were also sampled. Nine microsatellite loci were used to analyze genetic diversity parameters. These parameters were calculated using FSTAT version 2.9.3 and GenAlex v 6.41. IMPORTANT FINDINGS: The old-growth artificial populations of P. orientalis have significantly higher genetic diversity than younger artificial populations and similar levels to those in extant natural populations. The imperial parks and ancient temples, which have protected these old-growth trees for centuries, have played an important role in maintaining the genetic diversity and reproductive capacity of this tree species.

Temporal and spatial characteristics of male cone development in Metasequoia glyptostroboides Hu et Cheng.

Metasequoia glyptostroboides, a famous relic species of conifer that survived in China, has been successfully planted in large numbers across the world. However, limited information on male cone development in the species is available. In this study, we observed the morphological and anatomical changes that occur during male cone development in M. glyptostroboides using semi-thin sections and scanning electron microscopy. The male cones were borne oppositely on one-year-old twigs that were mainly located around the outer and sunlit parts of crown. Male cones were initiated from early September and shed pollen in the following February. Each cone consisted of spirally arranged microsporophylls subtended by decussate sterile scales, and each microsporophyll commonly consisted of three microsporangia and a phylloclade. The microsporangial wall was composed of an epidermis, endothecium, and tapetum. In mid-February, the endothecium and tapetum layers disintegrated, and in the epidermal layer the cell walls were thickened with inner protrusions. Subsequently, dehiscence of the microsporangia occurred through rupturing of the microsporangial wall along the dehiscence line. These results suggest that the structure, morphology, architecture and arrangement of male cones of M. glyptostroboides are mainly associated with the production, protection and dispersal of pollen for optimization of wind pollination.

[Measurement accuracy of granier calibration based on transpiration of Platycladus orientalis].

In order to understand the accuracy of Granier' s thermal dissipation method in measuring tree water consumption, a comparative study was made from May to June, 2010. The sap flow density of potted Platycladus orientalis was measured with thermal dissipation probe, which was compared with the whole-plant gravimetric measurement. There were significant linear relationships (R2 > 0.825) between the sap flow velocity in both north and south directions of P. orientalis measured by thermal dissipation probe and the transpiration rate measured by gravimetric method. The average daily sap flux in the north and south directions of P. orientalis were 10.6% and 15.1% lower than the daily average transpiration of P. orientalis, respectively, but the differences were not significant. Therefore, Granier's method had high reliability in the measurement of P. orientalis transpiration at daily scale, though the large temperature fluctuation between day and night could result in a lower daily sap flux than daily transpiration.

Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) Using real-time PCR.

Platycladus orientalis is a tree species that is highly resistant, widely adaptable, and long-lived, with lifespans of even thousands of years. To explore the mechanisms underlying these characteristics, gene expressions have been investigated at the transcriptome level by RNA-seq combined with a digital gene expression (DGE) technique. So, it is crucial to have a reliable set of reference genes to normalize the expressions of genes in P. orientalis under various conditions using the most accurate and sensitive method of quantitative real-time PCR (qRT-PCR). In this study, we selected 10 reference gene candidates from transcriptome data of P. orientalis, and examined their expression profiles by qRT-PCR using 29 different samples of P. orientalis, which were collected from plants of different ages, different tissues, and plants subjected to different treatments including cold, heat, salinity, polyethylene glycol (PEG), and abscisic acid (ABA). Three analytical software packages (geNorm, Bestkeeper, and NormFinder) were used to assess the stability of gene expression. The results showed that ubiquitin-conjugating enzyme E2 (UBC) and alpha-tubulin (aTUB) were the optimum pair of reference genes at all developmental stages and under all stress conditions. ACT7 was the most stable gene across different tissues and cold-treated samples, while UBQ was the most stably expressed reference gene for NaCl- and ABA-treated samples. In parallel, aTUB and UBC were used singly or in combination as reference genes to examine the expression levels of NAC (a homolog of AtNAC2) in plants subjected to various treatments with qRT-PCR. The results further proved the reliability of the two selected reference genes. Our study will benefit future research on the expression of genes in response to stress/senescence in P. orientalis and other members of the Cupressaceae.