Showing papers in "Tree Physiology in 2017"

PtoMYB170 positively regulates lignin deposition during wood formation in poplar and confers drought tolerance in transgenic Arabidopsis.

Abstract: Wood formation is a complex developmental process under multi-level transcriptional control executed by a large set of transcription factors. However, only limited members have been characterized to be key regulators of lignin biosynthesis in poplar. Here we report the conserved and unique functions of PtoMYB170, a transcription factor identified from Populus tomentosa (Chinese white poplar), in lignin deposition and drought tolerance in comparison with its duplicate paralog PtoMYB216. PtoMYB170 is preferentially expressed in young leaves and xylem tissues. Overexpression of PtoMYB170 in transgenic poplar plants resulted in stronger lignification and more thickened secondary wall in xylem compared with wild-type plants, whereas the CRISPR/Cas9-generated mutation of PtoMYB170 weakened lignin deposition, thereby leading to a more flexible and collapsed xylem phenotype. Transient expression experiments demonstrated that PtoMYB170 specifically activated the expression of lignin biosynthetic genes, consistent with the function of PtoMYB216. However, GUS staining assays revealed that PtoMYB170 was specifically expressed in guard cells of transgenic Arabidopsis while PtoMYB216 was not. Heterologous expression of PtoMYB170 in Arabidopsis enhanced stomatal closure in the dark and resulted in drought tolerance of the transgenic plants through reduced water loss, indicating a diversified role from PtoMYB216. These results revealed the PtoMYB170-dependent positive transcriptional regulation on lignin deposition in poplar and its coordinated function in enhancing drought tolerance by promoting dark-induced stomatal closure.

Long-term effects of drought on tree-ring growth and carbon isotope variability in Scots pine in a dry environment.

Abstract: Drought frequency is increasing in many parts of the world and may enhance tree decline and mortality. The underlying physiological mechanisms are poorly understood, however, particularly regarding chronic effects of long-term drought and the response to increasing temperature and vapor pressure deficit (VPD). We combined analyses of radial growth and stable carbon isotope ratios (δ13C) in tree rings in a mature Scots pine (Pinus sylvestris L.) forest over the 20th century to elucidate causes of tree mortality in one of the driest parts of the European Alps (Pfynwald, Switzerland). We further compared trees that have recently died with living trees in a 10-year irrigation experiment, where annual precipitation was doubled. We found a sustained growth increase and immediate depletion of δ13C values for irrigated trees, indicating higher stomatal conductance and thus indeed demonstrating that water is a key limiting factor for growth. Growth of the now-dead trees started declining in the mid-1980s, when both mean temperature and VPD increased strongly. But growth of these trees was reduced to some extent already several decades earlier, while intrinsic water-use efficiency derived from δ13C values was higher. This indicates a more conservative water-use strategy compared with surviving trees, possibly at the cost of low carbon uptake and long-term reduction of the needle mass. We observed reduced climatic sensitivity of raw tree-ring δ13C for the now-dead in contrast to surviving trees, indicating impaired stomatal regulation, although this difference between the tree groups was smaller after detrending the data. Higher autocorrelation and a lower inter-annual δ13C variability of the now-dead trees further indicates a strong dependence on (low) carbon reserves. We conclude that the recent increase in atmospheric moisture demand in combination with insufficient soil water supply was the main trigger for mortality of those trees that were weakened by long-term reduced carbon uptake.

Exogenous glutathione enhances cadmium accumulation and alleviates its toxicity in Populus × canescens.

Abstract: Glutathione (GSH) plays an important role in cadmium (Cd) tolerance in woody plants, but the underlying mechanisms remain largely unknown. To elucidate the physiological and transcriptional regulation mechanisms of GSH-mediated Cd tolerance in woody plants, we exposed Populus × canescens (Ait.) Smith saplings to either 0 or 75 μM Cd together with one of three external GSH levels. Glutathione treatments include buthionine sulfoximine (BSO, an inhibitor of GSH biosynthesis), no external GSH and exogenous GSH. External GSH resulted in higher Cd2+ uptake rate in the roots, greater Cd amount in poplars, lower Cd-induced H2O2 levels in the roots, and higher contents of endogenous GSH in Cd-treated roots and leaves. Furthermore, external GSH led to upregulated transcript levels of several genes including zinc/iron regulated transporter related protein 6.2 (ZIP6.2) and natural resistance-associated macrophage protein 1.3 (NRAMP1.3), which probably take part in Cd uptake, glutathione synthetase 2 (GS2) implicated in Cd detoxification, metal tolerance protein 1 (MTP1) and ATP-binding cassette transporter C3 (ABCC3) involved in Cd vacuolar accumulation in the roots, γ-glutamylcysteine synthetase (ECS) and phytochelatin synthetase family protein 1 (PCS1) involved in Cd detoxification, and oligopeptide transporter 7 (OPT7) probably implicated in Cd detoxification in the leaves of Cd-exposed P. × canescens. In contrast, BSO often displayed the opposite effects on Cd-triggered physiological and transcriptional regulation responses in poplars. These results suggest that exogenous GSH can enhance Cd accumulation and alleviate its toxicity in poplars. This is probably attributed to external-GSH-induced higher net Cd2+ influx in the roots, greater Cd accumulation in aerial parts, stronger scavenging of reactive oxygen species, and transcriptional overexpression of several genes involved in Cd uptake, detoxification and accumulation.

A multi-proxy assessment of dieback causes in a Mediterranean oak species.

Abstract: Drought stress causes forest dieback that is often explained by two interrelated mechanisms, namely hydraulic failure and carbon starvation. However, it is still unclear which functional and structural alterations, related to these mechanisms, predispose to dieback. Here we apply a multi-proxy approach for the characterization of tree structure (radial growth, wood anatomy) and functioning (δ13C, δ18O and non-structural carbohydrates (NSCs)) in tree rings before and after drought-induced dieback. We aim to discriminate which is the main mechanism and to assess which variables can act as early-warning proxies of drought-triggered damage. The study was tailored in southern Italy in two forests (i.e., San Paolo (SP) and Oriolo (OR)) where declining and non-declining trees of a ring-porous tree species (Quercus frainetto Ten.) showing anisohydric behavior coexist. Both stands showed growth decline in response to warm and dry spring conditions, although the onset of dieback was shifted between them (2002 in SP and 2009 in OR). Declining trees displayed a sharp growth drop after this onset with reductions of 49% and 44% at SP and OR sites, respectively. Further, contrary to what we expected, declining trees showed a lower intrinsic water-use efficiency compared with non-declining trees after the dieback onset (with reductions of 9.7% and 5.6% at sites SP and OR, respectively), due to enhanced water loss through transpiration, as indicated by the lower δ18O values. This was more noticeable at the most drought-affected SP stand. Sapwood NSCs did not differ between declining and non-declining trees, indicating no carbon starvation in affected trees. Thus, the characterized structural and functional alterations partially support the hydraulic failure mechanism of dieback. Finally, we show that growth data are reliable early-warning proxies of drought-triggered dieback.

Evolutionary trade-offs between drought resistance mechanisms across a precipitation gradient in a seasonally dry tropical oak (Quercus oleoides).

Abstract: In seasonally dry tropical forest regions, drought avoidance during the dry season coupled with high assimilation rates in the wet season is hypothesized to be an advantageous strategy for forest trees in regions with severe and long dry seasons In contrast, where dry seasons are milder, drought tolerance coupled with a conservative resource-use strategy is expected to maximize carbon assimilation throughout the year Tests of this hypothesis, particularly at the intraspecific level, have been seldom conducted In this study, we tested the extent to which drought resistance mechanisms and rates of carbon assimilation have evolved under climates with varying dry season length and severity within Quercus oleoidesCham and Schlect, a tropical dry forest species that is widely distributed in Central America For this purpose, we conducted a greenhouse experiment where seedlings originating from five populations that vary in rainfall patterns were grown under different watering treatments Our results revealed that populations from xeric climates with more severe dry seasons exhibited large mesophyllous leaves (with high specific leaf area, SLA), and leaf abscission in response to drought, consistent with a drought-avoidance strategy In contrast, populations from more mesic climates with less severe dry seasons had small and thick sclerophyllous leaves with low SLA and reduced water potential at the turgor loss point (πtlp), consistent with a drought-tolerance strategy Mesic populations also showed high plasticity in πtlp in response to water availability, indicating that osmotic adjustment to drought is an important component of this strategy However, populations with mesophyllous leaves did not have higher maximum carbon assimilation rates under well-watered conditions Furthermore, SLA was negatively associated with mass-based photosynthetic rates, contrary to expectations of the leaf economics spectrum, indicating that drought-resistance strategies are not necessarily tightly coupled with resource-use strategies Overall, our study demonstrates the importance of considering intraspecific variation in analyses of the vulnerability of tropical trees to climate change

Organic and inorganic nitrogen uptake by 21 dominant tree species in temperate and tropical forests.

Abstract: Evidence shows that many tree species can take up organic nitrogen (N) in the form of free amino acids from soils, but few studies have been conducted to compare organic and inorganic N uptake patterns in temperate and tropical tree species in relation to mycorrhizal status and successional state. We labeled intact tree roots by brief 15N exposures using field hydroponic experiments in a temperate forest and a tropical forest in China. A total of 21 dominant tree species were investigated, 8 in the temperate forest and 13 in the tropical forest. All investigated tree species showed highest uptake rates for NH4+ (ammonium), followed by glycine and NO3- (nitrate). Uptake of NH4+ by temperate trees averaged 12.8 μg N g-1 dry weight (d.w.) root h-1, while those by tropical trees averaged 6.8 μg N g-1 d.w. root h-1. Glycine uptake rates averaged 3.1 μg N g-1 d.w. root h-1 for temperate trees and 2.4 μg N g-1 d.w. root h-1 for tropical trees. NO3- uptake was the lowest (averaging 0.8 μg N g-1 d.w. root h-1 for temperate trees and 1.2 μg N g-1 d.w. root h-1 for tropical trees). Uptake of NH4+ accounted for 76% of the total uptake of all three N forms in the temperate forest and 64% in the tropical forest. Temperate tree species had similar glycine uptake rates as tropical trees, with the contribution being slightly lower (20% in the temperate forest and 23% in the tropical forest). All tree species investigated in the temperate forest were ectomycorrhizal and all species but one in the tropical forest were arbuscular mycorrhizal (AM). Ectomycorrhizal trees showed significantly higher NH4+ and lower NO3- uptake rates than AM trees. Mycorrhizal colonization rates significantly affected uptake rates and contributions of NO3- or NH4+, but depended on forest types. We conclude that tree species in both temperate and tropical forests preferred to take up NH4+, with organic N as the second most important N source. These findings suggest that temperate and tropical forests demonstrate similar N uptake patterns although they differ in physiology of trees and soil biogeochemical processes.

Continuous excitation chlorophyll fluorescence parameters: a review for practitioners.

Abstract: This review introduces, defines and critically reviews a number of chlorophyll fluorescence parameters with specific reference to those derived from continuous excitation chlorophyll fluorescence. A number of common issues and criticisms are addressed. The parameters fluorescence origin (F0) and the performance indices (PI) are discussed as examples. This review attempts to unify definitions for the wide range of parameters available for measuring plant vitality, facilitating their calculation and use.

Identification of potential genes that contributed to the variation in the taxoid contents between two Taxus species (Taxus media and Taxus mairei)

Abstract: Taxol is currently a valuable anticancer drug; however, the accumulated mixture of taxoids can vary greatly among Taxus species. So far, there is very little genomic information for the genus Taxus, except for Taxus baccata. Transcriptome analysis is a powerful approach to explore the different regulatory mechanisms underlying the taxoid biosynthesis pathway in Taxus species. First, we quantified the variation in the taxoid contents between Taxus media and Taxus mairei. The contents of paclitaxel and 10-deacetylpaclitaxel in T. media are higher than that in T. mairei. Then, the transcriptome profiles of T. media and T. mairei were analyzed to investigate the altered expressions. A total of 20,704 significantly differentially expressed genes (DEGs), including 9865 unigenes predominantly expressed in T. media and 10,839 unigenes predominantly expressed in T. mairei, were identified. In total, 120 jasmonic acid-related DEGs were analyzed, suggesting variations in 'response to JA stimulus' and 'JA biosynthetic process' pathways between T. media and T. mairei. Furthermore, a number of genes related to the precursor supply, taxane skeleton formation and hydroxylation, and C13-side chain assembly were also identified. The differential expression of the candidate genes involved in taxoid biosynthetic pathways may explain the variation in the taxoid contents between T. media and T. mairei.

DNA methylation and small interference RNAs participate in the regulation of MADS-box genes involved in dormancy in sweet cherry (Prunus avium L.).

Abstract: Epigenetic modifications can yield information about connections between genotype, phenotype variation and environmental conditions. Bud dormancy release in temperate perennial fruit trees depends on internal and environmental signals such as cold accumulation and photoperiod. Previous investigations have noted the participation of epigenetic mechanisms in the control of this physiological process. We examined whether epigenetic modifications were modulated in MADS-box genes, potential candidates for the regulation of bud dormancy and flowering in sweet cherry (Prunus avium L.). We identified and cloned two MADS-box genes homologous to the already-characterized dormancy regulators DORMANCY-ASSOCIATED MADS-box (DAM3 and DAM5) from Prunus persica (L.) Batsch. Bisulfite sequencing of the identified genes (PavMADS1 and PavMADS2), Methylated DNA Immunoprecipitation and small RNA deep sequencing were performed to analyze the presence of DNA methylations that could be guided by non-coding RNAs in the floral buds exposed to differential chilling hours. The results obtained reveal an increase in the level of DNA methylation and abundance of matching small interference RNAs (siRNAs) in the promoter of PavMADS1 when the chilling requirement is complete. For the first intron and 5' UTR of PavMADS1, de novo DNA methylation could be associated with the increase in the abundance of 24-nt siRNA matching the promoter area. Also, in the second large intron of PavMADS1, maintenance DNA methylation in all cytosine contexts is associated with the presence of homologous siRNAs in that zone. For PavMADS2, only maintenance methylation was present in the CG context, and no matching siRNAs were detected. Silencing of PavMADS1 and PavMADS2 coincided with an increase in Flowering Locus T expression during dormancy. In conclusion, DNA methylations and siRNAs appear to be involved in the silencing of PavMADS1 during cold accumulation and dormancy release in sweet cherry.

Single-provenance mature conifers show higher non-structural carbohydrate storage and reduced growth in a drier location.

Abstract: Since growth is more sensitive to drought than photosynthesis, trees inhabiting dry regions are expected to exhibit higher carbohydrate storage and less growth than their conspecifics from more humid regions. However, the same pattern can be the result of different genotypes inhabiting contrasting humidity conditions. To test if reduced growth and high carbohydrate storage are environmentally driven by drought, we examined the growth and non-structural carbohydrate (NSC) concentrations in single-provenance stands of mature trees of Pinus contorta Douglas and Pinus ponderosa Douglas ex C. Lawson planted at contrasting humidity conditions (900 versus 300 mm of annual precipitation) in Patagonia, Chile. Individual tree growth was measured for each species and at each location as mean basal area increment of the last 10 years (BAI10), annual shoot elongation for the period 2011-14, and needle length for 2013 and 2014 cohorts. Additionally, needle, branch, stem sapwood and roots were collected from each sampled tree to determine soluble sugars, starch and total NSC concentrations. The two species showed lower mean BAI10 and 2013 needle length in the dry site; P. ponderosa also had lower annual shoot extension for 2011 and 2014, and lower 2014 needle length, in the dry than in the mesic site. By contrast, NSC concentrations of all woody tissues for both species were either similar or higher in the dry site when compared with the mesic site. Patterns of starch and sugars were substantially different: starch concentrations were similar between sites except for roots of P. ponderosa, which were higher in the dry site, while sugar concentrations of all woody tissues in both species were higher in the dry site. Overall, our study provides evidence that reduced growth along with carbon (C) accumulation is an environmentally driven response to drought. Furthermore, the significant accumulation of low-molecular weight sugars in the dry site is compatible with a prioritized C allocation for osmoregulation. However, since this accumulation did not come at the expense of reduced starch, it is unlikely that growth was limited by C supply in the dry site.

Genetic adaptation and phenotypic plasticity contribute to greater leaf hydraulic tolerance in response to drought in warmer climates.

Abstract: The ability of plants to maintain an intact water transport system in leaves under drought conditions is intimately linked to survival and can been be seen as adaptive in shaping species climatic limits. Large differences in leaf hydraulic vulnerability to drought are known among species from contrasting climates, yet whether this trait varies among populations within a single species and, furthermore, whether it is altered by changes in growth conditions, remain unclear. We examined intraspecific variation in both leaf water transport capacity (Kleaf) and leaf hydraulic vulnerability to drought (P50leaf) among eight populations of Corymbia calophylla (R. Br.) K.D. Hill & L.A.S. Johnson (Myrtaceae) from both cool and warm climatic regions grown reciprocally under two temperature treatments representing the cool and warm edge of the species distribution. Kleaf did not vary between cool and warm-climate populations, nor was it affected by variable growth temperature. In contrast, population origin and growth temperature independently altered P50leaf. Using data pooled across growth temperatures, cool-climate populations showed significantly higher leaf hydraulic vulnerability (P50leaf = -3.55 ± 0.18 MPa) than warm-climate populations (P50leaf = -3.78 ± 0.08 MPa). Across populations, P50leaf decreased as population home-climate temperature increased, but was unrelated to rainfall and aridity. For populations from both cool and warm climatic regions, P50leaf was lower under the warmer growth conditions. These results provide evidence of trait plasticity in leaf hydraulic vulnerability to drought in response to variable growth temperature. Furthermore, they suggest that climate, and in particular temperature, may be a strong selective force in shaping intraspecific variation in leaf hydraulic vulnerability to drought.

Physiological and transcriptional responses of Catalpa bungei to drought stress under sufficient- and deficient-nitrogen conditions.

Abstract: Many semi-arid ecosystems are simultaneously limited by soil water and nitrogen (N). We conducted a greenhouse experiment to address how N availability impacts drought-resistant traits of Catalpa bungei C. A. Mey at the physiological and molecular level. A factorial design was used, consisting of sufficient-N and deficient-N combined with moderate drought and well-watered conditions. Seedling biomass and major root parameters were significantly suppressed by drought under the deficient-N condition, whereas N application mitigated the inhibiting effects of drought on root growth, particularly that of fine roots with a diameter <0.2 mm. Intrinsic water-use efficiency was promoted by N addition under both water conditions, whereas stable carbon isotope compositions (δ13C) was promoted by N addition only under the well-watered condition. Nitrogen application positively impacted drought adaptive responses including osmotic adjustment and homeostasis of reactive oxygen species, the content of free proline, soluble sugar and superoxide dismutase activity: all were increased upon drought under sufficient-N conditions but not under deficient-N conditions. The extent of abscisic acid (ABA) inducement upon drought was elevated by N application. Furthermore, an N-dependent crosstalk between ABA, jasmonic acid and indole acetic acid at the biosynthesis level contributed to better drought acclimation. Moreover, the transcriptional level of most genes responsible for the ABA signal transduction pathway, and genes encoding the antioxidant enzymes and plasma membrane intrinsic proteins, are elevated upon drought only under sufficient-N addition. These observations confirmed at the molecular level that major adaptive responses to drought are dependent on sufficient N nutrition. Although N uptake was decreased under drought, N-use efficiency and transcription of most genes encoding N metabolism enzymes were elevated, demonstrating that active N metabolism positively contributed drought resistance and growth of C. bungei under sufficient-N conditions.

Relationship between fine-root exudation and respiration of two Quercus species in a Japanese temperate forest.

Abstract: Plants allocate a considerable amount of carbon (C) to fine roots as respiration and exudation. Fine-root exudation could stimulate microbial activity, which further contributes to soil heterotrophic respiration. Although both root respiration and exudation are important components of belowground C cycling, how they relate to each other is less well known. In this study, we aimed to explore this relationship on mature trees growing in the field. The measurements were performed on two canopy species, Quercus serrata Thunb. and Quercus glauca, in a warm temperate forest. The respiration and exudation rates of the same fine-root segment were measured in parallel with a syringe-basis incubation and a closed static chamber, respectively. We also measured root traits and ectomycorrhizal colonization ratio because these indexes commonly relate to root respiration and reflect root physiology. The microbial activity enhanced by root exudation was investigated by comparing the dissolved organic carbon (DOC) and microbial biomass carbon (MBC) between rhizosphere soils and bulk soils. Mean DOC concentration and MBC were ca two times higher in the rhizosphere soils and positively related to exudation rates, indicating that exudation further relates to the C dynamics in the soils. Flux rates of exudation and respiration were positively correlated with each other. Both root exudation and respiration rates positively related to ectomycorrhizal colonization and root tissue nitrogen, and therefore the relationship between the two fluxes may be attributed to fine-root activity. The flux rates of root respiration were 8.7 and 10.5 times as much as those of exudation on a root-length basis and a root-weight basis, respectively. In spite of the fact that flux rates of respiration and exudation varied enormously among the fine-root segments of the two Quercus species, exudation was in proportion to respiration. This result gives new insight into the fine-root C-allocation strategy and the belowground C dynamics.

New research perspectives from a novel approach to quantify tracheid wall thickness.

Abstract: The analysis of xylem cell anatomical features in dated tree rings provides insights into xylem functional responses and past growth conditions at intra-annual resolution. So far, special focus has been given to the lumen of the water-conducting cells, whereas the equally relevant cell wall thickness (CWT) has been less investigated due to methodological limitations. Here we present a novel approach to measure tracheid CWT in high-resolution images of wood cross-sections that is implemented within the specialized image-analysis tool 'ROXAS'. Compared with the traditional manual line measurements along a selection of few radial files, this novel image-analysis tool can: (i) measure CWT of all tracheids in a tree-ring cross-section, thus increasing the number of individual tracheid measurements by a factor of ~10-20; (ii) measure the tangential and radial walls separately; and (iii) laterally integrate the measurements in a customizable way from only the thinnest central part of the cell walls up to the thickest part of the tracheids at the corners. Cell wall thickness measurements performed with our novel approach and the traditional manual approach showed comparable accuracy for several image resolutions, with an optimal accuracy-efficiency balance at 100× magnification. The configurable settings intended to underscore different cell wall properties indeed changed the absolute levels and intra- and inter-annual patterns of CWT. This versatility, together with the high data production capacity, allows to tailor the measurements of CWT to the specific goal of each study, which opens new research perspectives, e.g., for investigating structure-function relationships, tree stress responses and carbon allocation patterns, and for reconstructing climate based on intra- and inter-annual variability of anatomical wood density.

Leaf surface traits and water storage retention affect photosynthetic responses to leaf surface wetness among wet tropical forest and semiarid savanna plants.

Abstract: While it is reasonable to predict that photosynthetic rates are inhibited while leaves are wet, leaf gas exchange measurements during wet conditions are challenging to obtain due to equipment limitations and the complexity of canopy-atmosphere interactions in forested environments. Thus, the objective of this study was to evaluate responses of seven tropical and three semiarid savanna plant species to simulated leaf wetness and test the hypotheses that (i) leaf wetness reduces photosynthetic rates (Anet), (ii) leaf traits explain different responses among species and (iii) leaves from wet environments are better adapted for wet leaf conditions than those from drier environments. The two sites were a tropical rainforest in northern Costa Rica with ~4200 mm annual rainfall and a savanna in central Texas with ~1100 mm. Gas exchange measurements were collected under dry and wet conditions on five sun-exposed leaf replicates from each species. Additional measurements included leaf wetness duration and stomatal density. We found that Anet responses varied greatly among species, but all plants maintained a baseline of activity under wet leaf conditions, suggesting that abaxial leaf Anet was a significant percentage of total leaf Anet for amphistomatous species. Among tropical species, Anet responses immediately after wetting ranged from -31% (Senna alata (L.) Roxb.) to +21% (Zamia skinneri Warsz. Ex. A. Dietr.), while all savanna species declined (up to -48%). After 10 min of drying, most species recovered Anet towards the observed status prior to wetting or surpassed it, with the exception of Quercus stellata Wangenh., a savanna species, which remained 13% below Anet dry. The combination of leaf wetness duration and leaf traits, such as stomatal density, trichomes or wax, most likely influenced Anet responses positively or negatively. There was also overlap between leaf traits and Anet responses of savanna and tropical plants. It is possible that these species converge on a relatively conservative response to wetness, each for divergent purposes (cooling, avoiding stomatal occlusion, or by several unique means of rapid drying). A better understanding of leaf wetness inhibiting photosynthesis is vital for accurate modeling of growth in forested environments; however, species adapted for wet environments may possess compensatory traits that mitigate these effects.

Magnesium-deficiency-induced alterations of gas exchange, major metabolites and key enzymes differ among roots, and lower and upper leaves of Citrus sinensis seedlings.

Abstract: Magnesium (Mg)-deficiency is a widespread problem adversely affecting the quality and yield of crops, including citrus. 'Xuegan' [Citrus sinensis (L.) Osbeck] seedlings were irrigated every other day with nutrient solution at an Mg concentration of 0 mM (Mg-deficiency) or 1 mM (Mg-sufficiency) for 16 weeks. Thereafter, biomass, leaf mass per area, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), pigments in the upper and lower leaves, Mg, gas exchange, organic acids, nonstructural carbohydrates, total soluble proteins, amino acids, phenolics and anthocyanins, and key enzymes related to organic acid, amino acid and phenolic metabolisms in the roots, and upper and lower leaves were assayed in order to test the hypothesis that Mg-deficiency-induced alterations of gas exchange, major metabolites and key enzymes may differ among the roots, and upper and lower leaves. Magnesium-deficiency affected the most measured parameters more in the lower than in the upper leaves except for the nonstructural carbohydrates, but the variation trends were similar between the two. Despite increased accumulation of nonstructural carbohydrates, the lower CO2 assimilation in the Mg-deficient leaves was not caused by the feedback inhibition mechanism via sugar accumulation. Both dark respiration and organic acid metabolism were elevated in the Mg-deficient lower leaves to 'consume' the excess carbohydrates, and inhibited in the Mg-deficient roots with less accumulation of nonstructural carbohydrates to keep the balance of net carbon. More total phenolics and fewer anthocyanins were accumulated in the Mg-deficient lower leaves, whereas the accumulation of both total phenolics and anthocyanins was reduced in the Mg-deficient roots. Interestingly, amino acid biosynthesis was repressed in the Mg-deficient roots and lower leaves, thus lowering the level of total free amino acids in these roots and leaves. To conclude, great differences existed in the Mg-deficiency-induced alterations of gas exchange, major metabolites and key enzymes among the roots, and upper and lower leaves.

Contrasting ecophysiological strategies related to drought: the case of a mixed stand of Scots pine (Pinus sylvestris) and a submediterranean oak (Quercus subpyrenaica).

Abstract: Submediterranean forests are considered an ecotone between Mediterranean and Eurosiberian ecosystems, and are very sensitive to global change. A decline of Scots pine (Pinus sylvestris L.) and a related expansion of oak species (Quercus spp.) have been reported in the Spanish Pre-Pyrenees. Although this has been associated with increasing drought stress, the underlying mechanisms are not fully understood, and suitable monitoring protocols are lacking. The aim of this study is to bring insight into the physiological mechanisms anticipating selective decline of the pines, with particular focus on carbon and water relations. For this purpose, we performed a sampling campaign covering two growing seasons in a mixed stand of P. sylvestris and Quercus subpyrenaica E.H del Villar. We sampled seasonally twig xylem and soil for water isotope composition (δ18O and δ2H), leaves for carbon isotope composition (δ13C) and stems to quantify non-structural carbohydrates (NSC) concentration, and measured water potential and leaf gas exchange. The first summer drought was severe for both species, reaching low predawn water potential (-2.2 MPa), very low stomatal conductance (12 ± 1.0 mmol m-2 s-1) and near-zero or even negative net photosynthesis, particularly in P. sylvestris (-0.6 ± 0.34 μmol m-2 s-1 in oaks, -1.3 ± 0.16 μmol m-2 s-1 in pines). Hence, the tighter stomatal control and more isohydric strategy of P. sylvestris resulted in larger limitations on carbon assimilation, and this was also reflected in carbon storage, showing twofold larger total NSC concentration in oaks than in pines (7.8 ± 2.4% and 4.0 ± 1.3%, respectively). We observed a faster recovery of predawn water potential after summer drought in Q. subpyrenaica than in P. sylvestris (-0.8 MPa and -1.1 MPa, respectively). As supported by the isotopic data, this was probably associated with a deeper and more reliable water supply in Q. subpyrenaica. In line with these short-term observations, we found a more pronounced negative effect of steadily increasing drought stress on long-term growth in pines compared with oaks. All these observations confer evidence of early warning of P. sylvestris decline and indicate the adaptive advantage of Q. subpyrenaica in the area.

Dynamics of stem water uptake among isohydric and anisohydric species experiencing a severe drought

Abstract: Predicting the impact of drought on forest ecosystem processes requires an understanding of trees' species-specific responses to drought, especially in the Eastern USA, where species composition is highly dynamic due to historical changes in land use and fire regime. Here, we adapted a framework that classifies trees' water-use strategy along the spectrum of isohydric to anisohydric behavior to determine the responses of three canopy-dominant species to drought. We used a collection of leaf-level gas exchange, tree-level sap flux and stand-level eddy covariance data collected in south-central Indiana from 2011 to 2013, which included an unusually severe drought in the summer of 2012. Our goal was to assess how patterns in the radial profile of sap flux and reliance on hydraulic capacitance differed among species of contrasting water-use strategies. In isohydric species, which included sugar maple (Acer saccharum Marsh.) and tulip poplar (Liriodendron tulipifera L.), we found that the sap flux in the outer xylem experienced dramatic declines during drought, but sap flux at inner xylem was buffered from reductions in water availability. In contrast, for anisohydric oak species (Quercus alba L. and Quercus rubra L.), we observed relatively smaller variations in sap flux during drought in both inner and outer xylem, and higher nighttime refilling when compared with isohydric species. This reliance on nocturnal refilling, which occurred coincident with a decoupling between leaf- and tree-level water-use dynamics, suggests that anisohydric species may benefit from a reliance on hydraulic capacitance to mitigate the risk of hydraulic failure associated with maintaining high transpiration rates during drought. In the case of both isohydric and anisohydric species, our work demonstrates that failure to account for shifts in the radial profile of sap flux during drought could introduce substantial bias in estimates of tree water use during both drought and non-drought periods.

The WRKY transcription factors PtrWRKY18 and PtrWRKY35 promote Melampsora resistance in Populus.

Abstract: WRKY transcription factors play important roles in response to diverse environmental stresses, but exact functions of these proteins in poplar defense are still largely unknown. In a previous study, we have shown that poplar WRKY89 is induced by salicylic acid (SA) treatment and plays an important role in resistance against fungi in transgenic poplars. Here, we determined an increase in transcript levels of Group IIa WRKY members in transgenic poplars overexpressing WRKY89 using quantitative real-time polymerase chain reaction analysis. Yeast one-hybrid assay showed that PtrWRKY18 and PtrWRKY35 were potential target genes of WRKY89. Furthermore, we demonstrated that PtrWRKY18 and PtrWRKY35 were localized in the nucleus, and exhibited no transcription activation activity. Constitutive overexpression of PtrWRKY18 and PtrWRKY35 in poplars activated pathogenesis-related genes, and increased resistance to the biotrophic pathogen Melampsora. The results also provided support for the involvement of SA-mediated signaling in Melampsora resistance.

Drought effects on root and needle terpenoid content of a coastal and an interior Douglas fir provenance.

Abstract: Douglas fir (Pseudotsuga menziesii) is a conifer species that stores large amounts of terpenoids, mainly monoterpenoids in resin ducts of various tissues. The effects of drought on stored leaf terpenoid concentrations in trees are scarcely studied and published data are partially controversial, since reduced, unaffected or elevated terpenoid contents due to drought have been reported. Even less is known on the effect of drought on root terpenoids. In the present work, we investigated the effect of reduced water availability on the terpenoid content in roots and needles of Douglas fir seedlings. Two contrasting Douglas fir provenances were studied: an interior provenance (var. glauca) with assumed higher drought resistance, and a coastal provenance (var. menziesii) with assumed lower drought resistance. We tested the hypothesis that both provenances show specific patterns of stored terpenoids and that the patterns will change in response to drought in both, needles and roots. We further expected stronger changes in the less drought tolerant coastal provenance. For this purpose, we performed an experiment under controlled conditions, in which the trees were exposed to moderate and severe drought stress. According to our expectations, the study revealed clear provenance-specific terpenoid patterns in needles. However, such patterns were not detected in the roots. Drought slightly increased the needle terpenoid contents of the coastal but not of the interior provenance. We also observed increased terpenoid abundance mainly in roots of the moderately stressed coastal provenance. Overall, from the observed provenance-specific reactions with increased terpenoid levels in trees of the coastal origin in response to drought, we conclude on functions of terpenoids for abiotic stress tolerance that might be fulfilled by other, constitutively expressed mechanisms in drought-adapted interior provenances.

Elucidation of the polyamine biosynthesis pathway during Brazilian pine (Araucaria angustifolia) seed development.

Abstract: Polyamines (PAs), such as spermidine and spermine, as well as amino acids that are substrates for their biosynthesis, are known to be essential for plant development. However, little is known about the gene expression and metabolic switches associated with the ornithine/arginine and PA biosynthetic pathway during seed development in conifers. To understand these metabolic switches, the enzyme activity of arginine decarboxylase and ornithine decarboxylase, as well as the contents of PAs and amino acids were evaluated in three Araucaria angustifolia (Bertol. Kuntze) seed developmental stages in combination with expression profile analyses of genes associated with the ornithine/arginine and PA biosynthetic pathway. Twelve genes were selected for further analysis and it was shown that the expression profiles of AaADC and AaSAMDC were up-regulated during zygotic embryo development. Polyamines and amino acids were found to accumulate differently in embryos and megagametophytes, and the transition from the globular to the cotyledonary stage was marked by an increase in free and conjugated spermidine and spermine contents. Putrescine is made from arginine, which was present at low content at the late embryogenesis stage, when high content of citrulline was observed. Differences in amino acids, PAs and gene expression profiles of biosynthetic genes at specific seed stages and at each seed transition stage were investigated, providing insights into molecular and physiological aspects of conifer embryogenesis for use in future both basic and applied studies.

Drought and reproductive effort interact to control growth of a temperate broadleaved tree species (Fagus sylvatica)

Abstract: Interannual variation in radial growth is influenced by a range of physiological processes, including variation in annual reproductive effort, although the importance of reproductive allocation has rarely been quantified. In this study, we use long stand-level records of annual seed production, radial growth (tree ring width) and meteorological conditions to analyse the relative importance of summer drought and reproductive effort in controlling the growth of Fagus sylvatica L., a typical masting species. We show that both summer drought and reproductive effort (masting) influenced growth. Importantly, the effects of summer drought and masting were interactive, with the greatest reductions in growth found in years when high reproductive effort (i.e., mast years) coincided with summer drought. Conversely, mast years that coincided with non-drought summers were associated with little reduction in radial growth, as were drought years that did not coincide with mast years. The results show that the strength of an inferred trade-off between growth and reproduction in this species (the cost of reproduction) is dependent on environmental stress, with a stronger trade-off in years with more stressful growing conditions. These results have widespread implications for understanding interannual variability in growth, and observed relationships between growth and climate.

Different hydraulic traits of woody plants from tropical forests with contrasting soil water availability.

Abstract: In southwestern China, tropical karst forests (KF) and non-karst rain forests (NKF) have different species composition and forest structure owing to contrasting soil water availability, but with a few species that occur in both forests. Plant hydraulic traits are important for understanding the species' distribution patterns in these two forest types, but related studies are rare. In this study, we investigated hydraulic conductivity, vulnerability to drought-induced cavitation and wood anatomy of 23 abundant and typical woody species from a KF and a neighboring NKF, as well as two Bauhinia liana species common to both forests. We found that the KF species tended to have higher sapwood density, smaller vessel diameter, lower specific hydraulic conductivity (ks) and leaf to sapwood area ratio, and were more resistant to cavitation than NKF species. Across the 23 species distinctly occurring in either KF or NKF, there was a significant tradeoff between hydraulic efficiency and safety, which might be an underlying mechanism for distributions of these species across the two forests. Interestingly, by possessing rather large and long vessels, the two Bauhinia liana species had extremely high ks but were also high resistance to cavitation (escaping hydraulic tradeoff). This might be partially due to their distinctly dimorphic vessels, but contribute to their wide occurrence in both forests.

A steady-state stomatal model of balanced leaf gas exchange, hydraulics and maximal source–sink flux

Abstract: Trees must simultaneously balance their CO2 uptake rate via stomata, photosynthesis, the transport rate of sugars and rate of sugar utilization in sinks while maintaining a favourable water and carbon balance. We demonstrate using a numerical model that it is possible to understand stomatal functioning from the viewpoint of maximizing the simultaneous photosynthetic production, phloem transport and sink sugar utilization rate under the limitation that the transpiration-driven hydrostatic pressure gradient sets for those processes. A key feature in our model is that non-stomatal limitations to photosynthesis increase with decreasing leaf water potential and/or increasing leaf sugar concentration and are thus coupled to stomatal conductance. Maximizing the photosynthetic production rate using a numerical steady-state model leads to stomatal behaviour that is able to reproduce the well-known trends of stomatal behaviour in response to, e.g., light, vapour concentration difference, ambient CO2 concentration, soil water status, sink strength and xylem and phloem hydraulic conductance. We show that our results for stomatal behaviour are very similar to the solutions given by the earlier models of stomatal conductance derived solely from gas exchange considerations. Our modelling results also demonstrate how the 'marginal cost of water' in the unified stomatal conductance model and the optimal stomatal model could be related to plant structural and physiological traits, most importantly, the soil-to-leaf hydraulic conductance and soil moisture.

Populus simonii × Populus nigra WRKY70 is involved in salt stress and leaf blight disease responses.

Abstract: WRKY transcription factors (TFs) are important regulators in the complex stress response signaling networks in plants, but the detailed mechanisms underlying these regulatory networks have not been fully characterized. In the present study, we identified a Group III WRKY gene (PsnWRKY70, Potri.016G137900) from Populussimonii × Populusnigra and explored its function under salt and pathogen stresses. The promoter sequence that is located 2471-bp upstream from the start codon (SC) of PsnWRKY70 contained many stress-responsive cis-elements. Yeast one-hybrid assay suggested the upstream regulators, PsnWRKY70, PsnNAM (Potri.009G141600), PsnMYB (Potri.006G000800) and PsnGT1 (Potri.010G055000), probably modulate the expression of the PsnWRKY70 gene by specifically binding to the W-box or GT1GMSCAM4 (GT1) element. Yeast two-hybrid assay and transcriptome analysis revealed that HP1 (Potri.004G092100), RRM (Potri.008G146700), Ulp1 (Potri.002G105700) and some mitogen-activated protein kinase cascade members probably interact with PsnWRKY70 TF to response to salt stress. Compared with non-transgenic (NT) plants, PsnWRKY70-overexpressing (OEX) plants exhibited improved leaf blight disease resistance, while PsnWRKY70-repressing (REX) plants displayed enhanced salt stress tolerance. PsnWRKY70, PsnNAM, PsnMYB and PsnGT1 exhibited similar expression patterns in NT under salt and leaf blight disease stresses. The differentially expressed genes (DEGs) from NT vs OEX1 and the DEGs from NT vs REX1 exhibited considerable diversification. Most of the DEGs between NT and OEX1 were involved in aromatic amino acid biosynthesis, secondary metabolism, programmed cell death, peroxisomes and disease resistance. Most of the DEGs between NT and REX1 were related to desiccation response, urea transmembrane transport, abscisic acid response, calcium ion transport and hydrogen peroxide transmembrane transport. Our findings not only revealed the salt stress response signal transduction pathway of PsnWRKY70, but also provided direct evidence for the opposite biological functions of PsnWRKY70 TF in response to salt stress and leaf blight disease in P. simonii × P. nigra.

Isohydric species are not necessarily more carbon limited than anisohydric species during drought.

Abstract: Isohydry (i.e., strong regulation of leaf water potential, Ψl) is commonly associated with strict stomatal regulation of transpiration under drought, which in turn is believed to minimize hydraulic risk at the expense of reduced carbon assimilation. Hence, the iso/anisohydric classification has been widely used to assess drought resistance and mortality mechanisms across species, with isohydric species being hypothetically more prone to carbon starvation and anisohydric species more vulnerable to hydraulic failure. These hypotheses and their underlying assumptions, however, have rarely been tested under controlled, experimental conditions. Our objective is to assess the physiological mechanisms underlying drought resistance differences between two co-occurring Mediterranean forest species with contrasting drought responses: Phillyrea latifolia L. (anisohydric and more resistant to drought) and Quercus ilex L. (isohydric and less drought resistant). A total of 100 large saplings (50 per species) were subjected to repeated drought treatments for a period of 3 years, after which Q. ilex showed 18% mortality whereas no mortality was detected in P. latifolia. Relatively isohydric behavior was confirmed for Q. ilex, but higher vulnerability to cavitation in this species implied that estimated embolism levels were similar across species (12-52% in Q. ilex vs ~30% in P. latifolia). We also found similar seasonal patterns of stomatal conductance and assimilation between species. If anything, the anisohydric P. latifolia tended to show lower assimilation rates than Q. ilex under extreme drought. Similar growth rates and carbon reserves dynamics in both species also suggests that P. latifolia was as carbon-constrained as Q. ilex. Increasing carbon reserves under extreme drought stress in both species, concurrent with Q. ilex mortality, suggests that mortality in our study was not triggered by carbon starvation. Our results warn against making direct connections between Ψl regulation, stomatal behavior and the mechanisms of drought-induced mortality in plants.

Isotope signals and anatomical features in tree rings suggest a role for hydraulic strategies in diffuse drought-induced die-back of Pinus nigra.

Abstract: The 2003 and 2012 summer seasons were among the warmest and driest of the last 200 years over southeastern Europe, and in particular in the Karst region (northeastern Italy). Starting from winter-spring 2013, several black pines (Pinus nigra J.F. Arnold) suffered crown die-back. Declining trees occurred nearby individuals with no signs of die-back, raising hypotheses about the occurrence of individual-specific hydraulic strategies underlying different responses to extreme drought. We investigated possible processes driving black pine decline by dendrochronological and wood anatomical measurements, coupled with analysis of tree-ring carbon (δ13C) and oxygen (δ18O) isotopic composition in healthy trees (H) and trees suffering die-back (D). Die-back trees showed higher growth rates than H trees at the beginning of the last century, but suffered important growth reduction following the dry summers in 2003 and 2012. After the 2012 drought, D trees produced tracheids with larger diameter and greater vulnerability to implosion than H ones. Healthy trees had significantly higher wood δ13C than D trees, reflecting higher water-use efficiency for the surviving trees, i.e., less water transpired per unit carbon gain, which could be related to lower stomatal conductance and a more conservative use of water. Relatively high δ18O for D trees indicates that they were strongly dependent on shallow water sources, or that they sustained higher transpiration rates than H trees. Our results suggest that H trees adopted a more conservative water-use strategy under drought stress compared with D trees. We speculate that this diversity might have a genotypic basis, but other possible explanations, like different rooting depth, cannot be ruled out.

Acclimation of light and dark respiration to experimental and seasonal warming are mediated by changes in leaf nitrogen in Eucalyptus globulus.

Abstract: Quantifying the adjustments of leaf respiration in response to seasonal temperature variation and climate warming is crucial because carbon loss from vegetation is a large but uncertain part of the global carbon cycle. We grew fast-growing Eucalyptus globulus Labill. trees exposed to +3 °C warming and elevated CO2 in 10-m tall whole-tree chambers and measured the temperature responses of leaf mitochondrial respiration, both in light (RLight) and in darkness (RDark), over a 20-40 °C temperature range and during two different seasons. RLight was assessed using the Laisk method. Respiration rates measured at a standard temperature (25 °C - R25) were higher in warm-grown trees and in the warm season, related to higher total leaf nitrogen (N) investment with higher temperatures (both experimental and seasonal), indicating that leaf N concentrations modulated the respiratory capacity to changes in temperature. Once differences in leaf N were accounted for, there were no differences in R25 but the Q10 (i.e., short-term temperature sensitivity) was higher in late summer compared with early spring. The variation in RLight between experimental treatments and seasons was positively correlated with carboxylation capacity and photorespiration. RLight was less responsive to short-term changes in temperature than RDark, as shown by a lower Q10 in RLight compared with RDark. The overall light inhibition of R was ∼40%. Our results highlight the dynamic nature of leaf respiration to temperature variation and that the responses of RLight do not simply mirror those of RDark. Therefore, it is important not to assume that RLight is the same as RDark in ecosystem models, as doing so may lead to large errors in predicting plant CO2 release and productivity.

Amphistomatic leaf surfaces independently regulate gas exchange in response to variations in evaporative demand.

Abstract: The occurrence of amphistomatic leaves (stomata on both surfaces) versus hypostomatic leaves (stomata limited to the lower or abaxial surface) has strong associations with environment. Amphistomy provides the advantage of higher conductance of CO2 for photosynthesis, however, unless the stomata on both leaf surfaces can be independently controlled in response to environmental cues, amphistomy may lead to inefficient gas exchange. While previous studies have found evidence that stomata can operate independently across and between surfaces of dorsiventral leaves, we investigate whether an independent stomatal response can be induced for isobilateral leaves by largely natural conditions. Here, we exposed surfaces of isobilateral, amphistomatic Eucalyptus globulus Labill. leaves to natural diurnal variation in differential evaporative demand, using leaf orientation to drive differences in irradiance and heat load on leaf surfaces. We identified preferential closure of stomata on the surface exposed to higher irradiation (and therefore evaporative demand) during the afternoon under natural conditions and similarly induced differential stomatal closure under experimental conditions in the laboratory. The differential response confirms that sufficient hydraulic isolation exists for independent stomatal response to occur between surfaces of amphistomatic, isobilateral leaves, and importantly, we show that natural conditions can induce surface-specific stomatal closure.