Functional identification of specialized diterpene synthases from Chamaecyparis obtusa and C. obtusa var. formosana to illustrate the putative evolution of diterpene synthases in Cupressaceae.

Chamaecyparis obtusa and C. obtusa var. formosana of the Cupressaceae family are well known for their fragrance and excellent physical properties. To investigate the biosynthesis of unique diterpenoid compounds, diterpene synthase genes for specialized metabolite synthesis were cloned from C. obtusa and C. obtusa var. formosana. Using an Escherichia coli co-expression system, eight diterpene synthases (diTPSs) were characterized. CoCPS and CovfCPS are class II monofunctional (+)-copalyl diphosphate synthases [(+)-CPSs]. Class I monofunctional CoLS and CovfLS convert (+)-copalyl diphosphate [(+)-CPP] to levopimaradiene, CoBRS, CovfBRS1, and CovfBRS3 convert (+)-CPP to (-)-beyerene, and CovfSDS converts (+)-CPP to (-)-sandaracopimaradiene. These enzymes are all monofunctional diterpene syntheses in Cupressaceae family of gymnosperm, and differ from those in Pinaceae. The discovery of the enzyme responsible for the biosynthesis of tetracyclic diterpene (-)-beyerene was characterized for the first time. Diterpene synthases with different catalytic functions exist in closely related species within the Cupressaceae family, indicating that this group of monofunctional diterpene synthases is particularly prone to the evolution of new functions and development of species-specific specialized diterpenoid constituents.

Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments.

Cryptomeria fortunei has become one of the main timber afforestation species in subtropical high-altitude areas of China due to its fast growth, good material quality, and strong adaptability, showing broad application prospects. Quantitative real-time PCR (qRT-PCR) is the most accurate and widely used gene expression evaluation technique, and selecting appropriate reference genes (RGs) is essential for normalizing qRT-PCR results. However, suitable RGs for gene expression normalization in C. fortunei have not been reported. Here, we tested the expression stability for 12 RGs in C. fortunei under various experimental conditions (simulated abiotic stresses (cold, heat, drought, and salinity) and hormone treatments (methyl jasmonate, abscisic acid, salicylic acid, and gibberellin) and in different tissues (stems, tender needles, needles, cones, and seeds) using four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper). Then, geometric mean rankings from these algorithms and the RefFinder program were used to comprehensively evaluate RG stability. The results indicated CYP, actin, UBC, and 18S as good choices for studying C. fortunei gene expression. qRT-PCR analysis of the expression patterns of three target genes (CAT and MAPK1/6) further verified that the selected RGs were suitable for gene expression normalization. This study provides an important basis for C. fortunei gene expression standardization and quantification.

Metasequoia glyptostroboides potentiates anticancer effect against cervical cancer via intrinsic apoptosis pathway.

This study was undertaken to investigate the anticancer effects of organic extracts derived from the floral cones of Metasequoia glyptostroboides. Dried powder of M. glyptostroboides floral cones was subjected to methanol extraction, and the resulting extract was further partitioned by liquid-liquid extraction using the organic solvents n-hexane, dichloromethane (DME), chloroform, and ethyl acetate in addition to deionized water. HeLa cervical and COS-7 cells were used as a cancer cell model and normal cell control, respectively. The anticancer effect was evaluated by using the Cell Counting Kit-8 assay. The viability of COS-7 cells was found to be 12-fold higher than that of the HeLa cells under the administration of 50 µg/ml of the DME extract. Further, the sub-G1 population was determined by FACS analysis. The number of cells at the sub-G1 phase, which indicates apoptotic cells, was increased approximately fourfold upon treatment with the DME and CE extracts compared with that in the negative control. Furthermore, RT-qPCR and western blotting were used to quantitate the relative RNA and protein levels of the cell death pathway components, respectively. Our results suggest that the extracts of M. glyptostroboides floral cones, especially the DME extract, which possesses several anticancer components, as determined by GC-MS analysis, could a potential natural anticancer agent.

Differential Gene Profiling of the Heartwood Formation Process in Taiwania cryptomerioides Hayata Xylem Tissues.

Taiwania (Taiwania cryptomerioides) is an important tree species in Taiwan because of the excellent properties of its wood and fascinating color qualities of its heartwood (HW), as well as the bioactive compounds therein. However, limited information is available as to the HW formation of this species. The objective of this research is to analyze the differentially expressed genes (DEGs) during the HW formation process from specific Taiwania xylem tissues, and to obtain genes that might be closely associated with this process. The results indicated that our analyses have captured DEGs representative to the HW formation process of Taiwania. DEGs related to the terpenoid biosynthesis pathway were all up-regulated in the transition zone (TZ) to support the biosynthesis and accumulation of terpenoids. Many DEGs related to lignin biosynthesis, and two DEGs related to pinoresinol reductase (PrR)/pinoresinol lariciresinol reductase (PLR), were up-regulated in TZ. These DEGs together are likely involved in providing the precursors for the subsequent lignan biosynthesis. Several transcription factor-, nuclease-, and protease-encoding DEGs were also highly expressed in TZ, and these DEGs might be involved in the regulation of secondary metabolite biosynthesis and the autolysis of the cellular components of ray parenchyma cells in TZ. These results provide further insights into the process of HW formation in Taiwania.

Phylogenetically distant group of terpene synthases participates in cadinene and cedrane-type sesquiterpenes accumulation in Taiwania cryptomerioides.

Along with the species evolution, plants have evolved ways to produce a different collection of terpenoids to accommodate its biotic and abiotic environment, and terpene synthase (TPS) is one of the major contributors to various terpene compounds. The timber of a monotypic and relictual conifer species of Cupressace, Taiwania cryptomerioides, has excellent durability, and one of the essential factors for Taiwania to resist decay and insect pests is sesquiterpene. Compared to other conifers, Taiwania has much higher abundance of cadinene-type sesquiterpenes, and the presence of cedrene-type sesquiterpenes. To understand sesquiterpene biosynthesis in Taiwania, we functionally characterized 10 T. cryptomerioides TPSs (TcTPSs) in vivo or in planta, which could catalyze sesquiterpene formation and potentially are involved in biosynthesis of diverse sesquiterpenoids in Taiwania. The distant phylogenetic relationship and the intron loss event of TcTPSs correlate to the differentiation of chemical profile Taiwania compared to other conifers. Furthermore, we identified TcTPS3 and TcTPS12 as δ-cadinene synthase, and TcTPS6 as cedrol synthase, which demonstrates the important contributions of dynamic evolution in TPSs to the chemical diversity in plants. Combining with functional characterization and comparison of catalytic residues, we conclude at least three catalytic routes for sesquiterpene biosynthesis in this species, and the skeleton diversity has been expended in T. cryptomeriodes.

Biochemical characterization of diterpene synthases of Taiwania cryptomerioides expands the known functional space of specialized diterpene metabolism in gymnosperms.

Taiwania cryptomerioides is a monotypic gymnosperm species, valued for the high decay resistance of its wood. This durability has been attributed to the abundance of terpenoids, especially the major diterpenoid metabolite ferruginol, with antifungal and antitermite activity. Specialized diterpenoid metabolism in gymnosperms primarily recruits bifunctional class-I/II diterpene synthases (diTPSs), whereas monofunctional class-II and class-I enzymes operate in angiosperms. In this study, we identified a previously unrecognized group of monofunctional diTPSs in T. cryptomerioides, which suggests a distinct evolutionary divergence of the diTPS family in this species. Specifically, five monofunctional diTPS functions not previously observed in gymnosperms were characterized, including monofunctional class-II enzymes forming labda-13-en-8-ol diphosphate (LPP, TcCPS2) and (+)-copalyl diphosphate (CPP, TcCPS4), and three class-I diTPSs producing biformene (TcKSL1), levopimaradiene (TcKSL3) and phyllocladanol (TcKSL5), respectively. Methyl jasmonate (MeJA) elicited the accumulation of levopimaradiene and the corresponding biosynthetic diTPS genes, TcCPS4 and TcKSL3, is consistent with a possible role in plant defense. Furthermore, TcCPS4 and TcKSL3 are likely to contribute to abietatriene biosynthesis via levopimaradiene as an intermediate in ferruginol biosynthesis in Taiwania. In conclusion, this study provides deeper insight into the functional landscape and molecular evolution of specialized diterpenoid metabolism in gymnosperms as a basis to better understand the role of these metabolites in tree chemical defense.

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).

Thinning Effects on Biomass and Carbon Stock for Young Taiwania Plantations.

Forests play an important role as carbon sinks by sequestrating carbon through photosynthesis. Thinning treatments have large impacts on carbon storage, in addition to strengthening quality and quantity of plantations. This study analyzed the effects of different thinning treatments on carbon stocks in both individual trees and stands of Taiwania (Taiwania cryptomerioides) plantations. Repeated field measurements and allometric equations were used to calculate total C storage and sequestration rates of live trees. The results of this study showed that the total carbon stock of stands with thinning treatments was less than that of the non-thinned stands. The non-thinned 23-year old stands had an estimated carbon stock of 96.8 Mg C ha-1, which is higher than the carbon stock found in either medium- (84.1 Mg C ha-1) or heavily-thinned (74.7 Mg C ha-1) treatment plots of the same age. If the objective of Taiwania plantations was to store large amounts of carbon in the young growth stage, without regard to the initial rate of storage, a better option is no-thinning. However, the medium thinned forests seem to be more promising for carbon sequestration than the no-thinned forests if a longer period is considered.

Latitudinal patterns of leaf N, P stoichiometry and nutrient resorption of Metasequoia glyptostroboides along the eastern coastline of China.

Latitudinal patterns of leaf stoichiometry and nutrient resorption were not consistent among published studies, likely due to confounding effects from taxonomy (e.g., plant distribution and community composition), and environment, which is also influenced by altitude and longitude. Thus, the latitudinal patterns and environmental mechanism could be best revealed by testing a given species along a latitude gradient with similar altitude and longitude. We determined nitrogen (N) and phosphorus (P) concentrations of green (leaf) and senesced leaves (litter) from eight Metasequoia glyptostroboides forests along the eastern coastline of China, with similar altitude and longitude. Leaf N, P concentrations increased along latitude, mainly driven by mean annual temperature (MAT), mean annual precipitation (MAP), annual evaporation (AE), aridity index (AI), and annual total solar radiation (ATSR); While leaf N:P ratio was stable with no latitudinal pattern. Nitrogen resorption efficiency (NRE) increased along latitude, and was also mainly influenced by MAT, MAP, AE, and AI. Phosphorus resorption efficiency (PRE) first increased and then decreased with latitude, which was impacted by soil available P. These results indicated that only climate (such as heat, water, and light) controlled the shift in leaf stoichiometry and NRE, while soil nutrient was likely responsible for the shift in PRE along eastern China. Our findings also suggested that leaf N, P stoichiometry and NRE displayed similar latitudinal patterns at regional scale when studied for a given species (this study) or multi-species (previous studies).

Plant Species Rather Than Climate Greatly Alters the Temporal Pattern of Litter Chemical Composition During Long-Term Decomposition.

A feedback between decomposition and litter chemical composition occurs with decomposition altering composition that in turn influences the decomposition rate. Elucidating the temporal pattern of chemical composition is vital to understand this feedback, but the effects of plant species and climate on chemical changes remain poorly understood, especially over multiple years. In a 10-year decomposition experiment with litter of four species (Acer saccharum, Drypetes glauca, Pinus resinosa, and Thuja plicata) from four sites that range from the arctic to tropics, we determined the abundance of 11 litter chemical constituents that were grouped into waxes, carbohydrates, lignin/tannins, and proteins/peptides using advanced (13)C solid-state NMR techniques. Decomposition generally led to an enrichment of waxes and a depletion of carbohydrates, whereas the changes of other chemical constituents were inconsistent. Inconsistent convergence in chemical compositions during decomposition was observed among different litter species across a range of site conditions, whereas one litter species converged under different climate conditions. Our data clearly demonstrate that plant species rather than climate greatly alters the temporal pattern of litter chemical composition, suggesting the decomposition-chemistry feedback varies among different plant species.

Physiology and proteomics research on the leaves of ancient Platycladus orientalis (L.) during winter.

Ancient trees have an important value in humanities and history, and also have an important scientific value in the investigation of the decline and senescence mechanisms. Thus, we conducted an environmental stress study using ancient trees. To evaluate age-dependent changes in physiology and the leaf proteome, we assessed the low-temperature stress responses of 20 ± 5-, 500 ± 100- and 1200 ± 200-year-old Platycladus orientalis (L.) samples obtained outdoors during winter. Several physiological parameters were evaluated. Leaf proteomes were obtained using two-dimensional electrophoresis gels, and 77 protein spots were identified successfully using MALDI TOF/TOF MS/MS. The majority of the identified protein species were classified into functional categories including defense/stress-related, energy and carbohydrate metabolism, photosynthesis, and hormone-related functions. A general reduction in the abundance of protein species was observed as the age of the studied trees increased; reduction in photosynthesis and defense/stress-related categories were particularly apparent in the leaves of ancient trees. However, the number of protein species with functions in energy and carbohydrate metabolism increased with age. An increase in the abundance of lipid metabolism and hormone-related protein species was a primary characteristic of the leaves of ancient trees under low-temperature stress during winter. These results improve our understanding of the biochemical mechanisms of stress responses in ancient trees. BIOLOGICAL SIGNIFICANCE: Low temperature is the most common meteorological challenge in the study area. For evergreen plants, low-temperature stress has a great impact on the leaves of ancient P. orientalis. Thus, we conducted an environmental stress study using ancient trees. Recently, various studies were carried out in ancient trees. However, no information is available on the molecular mechanisms of defense to low-temperature stress in ancient trees. Therefore, this original study comprises the following differential proteomic analysis of ancient P. orientalis: (1) age-dependent changes in the physiology and leaf proteome are evaluated under low-temperature stress to (2) understand the differences in metabolic responses between ancient and adult trees under low-temperature stress during winter. This analysis will provide an understanding of the complex physiological changes that occur in ancient trees. The results suggest that certain identified proteins can be used as markers of low-temperature stress in ancient P. orientalis.

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.

Effect of forest structural change on carbon storage in a coastal Metasequoia glyptostroboides stand.

Forest structural change affects the forest's growth and the carbon storage. Two treatments, thinning (30% thinning intensity) and underplanting plus thinning, are being implemented in a coastal Metasequoia glyptostroboides forest shelterbelt in Eastern China. The vegetation carbon storage significantly increased in the underplanted and thinned treatments compared with that in the unthinned treatment (P < 0.05). The soil and litterfall carbon storage in the underplanted treatment were significantly higher than those in the unthinned treatment (P < 0.05). The total forest ecosystem carbon storage in the underplanted and thinned treatments increased by 35.3% and 26.3%, respectively, compared with that in the unthinned treatment, an increase that mainly came from the growth of vegetation aboveground. Total ecosystem carbon storage showed no significant difference between the underplanted and thinned treatments (P > 0.05). The soil light fraction organic carbon (LFOC) was significantly higher at the 0-15 cm soil layer in the thinned and underplanted stands compared with that in the unthinned stand (P < 0.05). The soil respiration of the underplanted treatment was significantly higher than that of the unthinned treatment only in July (P < 0.05). This study concludes that 30% thinning and underplanting after thinning could be more favorable to carbon sequestration for M. glyptostroboides plantations in the coastal areas of Eastern China.

Plant-mediated synthesis of platinum nanoparticles and its bioreductive mechanism.

Pt nanoparticles (PtNPs) were biologically synthesized by reducing Na2PtCl4 with Cacumen Platycladi Extract (CPE). The effects of reaction temperature, initial Pt(II) concentration, and CPE percentage on Pt(II) conversion and the size distribution of the PtNPs were studied. The results showed that the Pt(II) conversion rate reached 95.9% and that PtNPs measuring 2.4±0.8nm were obtained under the following conditions: reaction temperature, 90°C; CPE percentage, 70%; initial Pt(II) concentration, 0.5mM; reaction time, 25h. In addition, the bioreduction of Pt(II) was attributed to reducing sugars and flavonoids rather than proteins. The elucidation of bioreductive mechanism of Pt(II) ions was achieved by investigating the changes that occurred in the reducing sugar, flavonoid and protein concentrations in the plant extract, leading to a good insight into the formation mechanism of such biosynthesized PtNPs.

The development of seasonal tree water deficit in Callitris intratropica.

The tropical conifer Callitris intratropica (Cupressaceae) produces clear annual growth rings, and has been shown to be potentially useful for understanding past climate variability in northern Australia. As climate patterns in this region become less predictable, an understanding of plant responses to different weather patterns is of importance. In this paper, we examine tree water relations using a parameter here called tree water deficit (ΔD), determined from de-trended stem size variability in densely grown ('grove') and isolated trees. This parameter provides an integrated measure of the trees' response to water supply and demand under constantly changing environmental conditions. The work, conducted over 12 months, found that daily variation in tree water deficit was determined mainly by soil water availability, but temperature and relative humidity contributed more to the variability over some periods. Isolated and grove trees exhibited quite distinct patterns of ΔD development during the year, but particularly during the transition between the dry and wet seasons. The results of this work suggest that the dendrochronological interpretation of tree rings in the context of strongly seasonal water availability should incorporate an understanding of the development of seasonal drought in isolated trees compared with trees experiencing strong intra-specific competition. Different responses based on the ecological situations of the trees will affect their patterns of stem growth, and ultimately the climatic information that is incorporated in ring width variability.

[Environmental responses of four urban tree species transpiration in northern China].

By using thermal dissipation probes (TDP), this paper monitored the sap flow of four tree species (Cedrus deodara, Zelkova schneideriana, Euonymus bungeanus, and Metasequoia glyptostroboides) at the Laodong Park in Dalian City from June to August 2008, and the soil moisture content and micrometeorological variables were mehsured simultaneously. Due to the absence of water-stress in the habitat, the sap flow of all sampled trees had no significant correlation with soil moisture content (R2 < 0.050, P > 0.211, n=1296). The correlation coefficient between solar radiation and sap flow reached 0.624-0.773 (P = 0.00, n=1296) despite the existing hysteresis. Solar radiation had major effect (R2 > 0.700, P < 0.05) during early morning (5:00-8:00) and late afternoon (18:00-20:00) when undergoing dramatic changes. As the main factor determining nighttime sap flow (R2 > 0.660, P < 0.05, n=1872), vapor pressure deficit (VPD) had a correlation coefficient as high as 0.650-0.823 (P = 0.00, n=1296) with the sap flow in whole-day scale. Meanwhile, the models constructed on the basis of VPD were able to explain 90% of daily sap flow change (P = 0.00). The correlation coefficient between sap flow and wind speed was relatively smaller than the previous two (R2 < 0.380, P = 0.00, n=1296), though showing significant correlation in affecting sap flow. Observations also detected the saturation phenomenon of sap flow to the environmental demands.

Analyses of delta(13)C and delta(18)O in tree rings of Callitris columellaris provide evidence of a change in stomatal control of photosynthesis in response to regional changes in climate.

We examined relationships between stable isotopes of carbon (delta(13)C) and oxygen (delta(18)O) in tree rings of Callitris columellaris F. Muell in the semi-arid Pilbara region of north-western Australia. To test the hypothesis that stomatal control of photosynthesis decreases during drier periods, we developed delta(13)C and delta(18)O chronologies spanning 1919-1999, and used a permutation regression approach to relate a 21-year running correlation between delta(13)C and delta(18)O to rainfall and temperature at Marble Bar and our study site. The relationship between delta(13)C and delta(18)O switched from being always negative before 1955 to being consistently positive after 1976, suggesting an increase in stomatal control of photosynthesis in recent decades. Changes in the delta(13)C-delta(18)O relationship reflected changes in rainfall, which has increased in the region by 30% since 1976. The correlation between delta(13)C and delta(18)O was positively related to the 21-year running mean of normalized rainfall anomalies at both the study site (P = 0.045, Adj. r(2) = 0.47) and Marble Bar (P = 0.046, Adj. r(2) = 0.48). In addition, the delta(13)C-delta(18)O correlation was negatively related (P = 0.047, Adj. r(2) = 0.61) to temperatures at Marble Bar. Our interpretation of the role of changes in climate affecting the relationship between tree-ring delta(13)C and delta(18)O is supported by evidence from the isotope composition of foliage samples: foliar delta(13)C and delta(18)O were negatively correlated with log stomatal conductance (delta(13)C, r = -0.41; delta(18)O, r = -0.42), whereas the correlation between foliar delta(13)C and delta(18)O was positive (r = 0.63, P = 0.027) after the summer wet period. Our data indicate that stomatal control of photosynthesis in Callitris adjusts to region-wide changes in climate and that, in a warmer and drier world, trees might adapt by increasing non-stomatal control of photosynthesis.

Coordinating leaf functional traits with branch hydraulic conductivity: resource substitution and implications for carbon gain.

We studied relationships among branch hydraulic conductivity, xylem embolism, stomatal conductance (gs), foliar nitrogen (N) concentration and specific leaf area (SLA) of seven tree species growing at four temperate woodland sites spanning a 464-1350 mm rainfall gradient. Specifically, we examined the question: are gs and foliar N concentration coordinated with branch hydraulic conductivity and, if so, what are the implications for carbon assimilation? Area-based, light-saturated photosynthetic rate (Aa) was uniquely and positively correlated with gs and foliar N concentration. Multiple regression analyses showed that, when variability in SLA was controlled for, the (positive) partial slope for each predictor remained significant. In contrast, there was a negative correlation between gs and foliar N concentration such that, for any given Aa, leaves with a high gs allocated less N to foliage than leaves with a low gs. Foliar N concentration was negatively correlated with branch hydraulic conductivity, whereas gs was positively correlated with branch hydraulic conductivity. These relationships were also significant when variability in leaf area to sapwood area ratio, gs and SLA were controlled for in a multiple regression, suggesting that the relationships were unique and independent of other confounding factors. Trees with low water transport capacity were able to support a high Aa by increasing investment in foliar N. Resource substitution occurred such that there was a trade-off between gs and foliar N in relation to branch hydraulic conductivity. High Aa could be sustained through either a high branch hydraulic conductivity and hence high gs and a low allocation to foliar N, or the effect of a low branch hydraulic conductivity and hence low gs could be offset by a high allocation to foliar N. The results are discussed in relation to mechanisms for minimizing the negative effects of limited water availability on carbon gain.

Identification of proteins that interact with a TcMago-TcY14 heterodimer complex in Taiwania crytomerioides.

The Mago-Y14 heterodimer, which is highly conserved among eukaryotes, is the core component of the exon-exon junction complex (EJC) and regulates oogenesis, embryogenesis and germ-line sex determination in Drosophila and the nematode Caenorhabditis elegans. To further survey these proteins in plants and to identify plant developmental processes with which the Mago-Y14 heterodimer is associated, we used the TcMago-TcY14 heterodimer as the bait protein in a yeast two-hybrid screen and searched for its binding partners in a cDNA library from a 3-year-old Taiwania (Taiwania cryptomerioides Hayata) seedling. We found three clones, an unknown protein (Tc61), a pectin methylesterase-like protein (Tc62) and a TPR (tetratricopeptide repeat)-like protein (Tc72). The Tc61 protein interacted strongly with the TcMago-TcY14 heterodimer, and its transcript was expressed in stems and leaves of 3-year-old Taiwania seedlings and in microsporangiate and ovulate cones. The pectin methylesterase-like protein was expressed abundantly in the roots of 3-year-old Taiwania seedlings and in pollinated ovulate cones, indicating that binding with the TcMago-TcY14 heterodimer may be involved in root development and growth of pollen tubes during pollination. The Tc72 protein encoded a complete TPR-like protein that is highly conserved among plants, with 10 alpha-helices and three conserved TPR motifs containing five consensus residues necessary for stable inter-helix packing. A tissue expression assay and light/dark treatments showed that the Tc72 gene was expressed abundantly in the leaves of seedlings and was sensitive to light and dark, as are the Tcmago and TcY14 genes, implying that the TcMago-TcY14 heterodimer interaction with Tc72 may be related to chloroplast protein transport.

Molecular identification and characterization of Tcmago and TcY14 in Taiwania (Taiwania cryptomerioides).

Mago nashi (Mago) and Y14 proteins are highly conserved among eukaryotes, and not only play important roles in oogenesis, embryogenesis and germ-line determination during animal development, but also participate in mRNA localization and splicing in cell growth. We identified mago (Tcmago) and Y14 (TcY14) homologues derived from expressed sequence tags of Taiwania cryptomerioides Hayata seedlings. Tcmago and TcY14 contain coding regions encoding 149 and 216 amino acids, respectively. Multiple amino acid sequence alignments as well as secondary and tertiary structure all predicted that TcMago and TcY14 possessed similar protein structures to the crystal structures of Drosophila melanogaster and human Mago and Y14 proteins. We demonstrated by yeast two-hybrid analysis and a GST pull-down assay that TcMago and TcY14 interacted in vivo and in vitro, confirming structural predictions of their interaction. TcMago and TcY14 were predominately localized in the nucleus. Whole mount in situ hybridization and immunolocalization showed that Tcmago and TcY14 were both detected in root hairs at the levels of transcription and protein expression. Overexpression of Tcmago in transgenic tobacco plants resulted in longer roots and a more complex root system. TcMago and TcY14 may have cellular functions similar to the Mago and Y14 proteins in animals and may be involved in root development in plants.