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Twelve-oxo-phytodienoic acid (OPDA), the cyclopentenone precursor of jasmonic acid (JA), is required for the wounding response of plants. OPDA is derived from plastid-localized α-linolenic acid (α-LeA; 18:3) via the octadecanoid pathway, and is further exported from plastids to the cytosol for JA biosynthesis. However, the mechanism of OPDA transport from plastids has yet to be elucidated. In the current study, a plastid inner envelope-localized protein, designated 12-oxo-Phtyodienoic Acid Transporter 1 (OPDAT1), was identified and shown to potentially be involved in OPDA export from plastids, in Populus trichocarpa. Torr. OPDAT1 is expressed predominantly in young leaves of P. trichocarpa. Functional expression of OPDAT1 in yeast cells revealed that OPDAT1 is involved in OPDA transport. Loss-of-function of OPDAT1 in poplar resulted in increased accumulation of OPDA in the extracted plastids and a reduction in JA concentration, whereas an OPDAT1-overexpressing line showed a reverse tendency in OPDA accumulation and JA biosynthesis. OPDAT1 transcripts were rapidly induced by mechanical wounding of leaves, and an opdat1 mutant transgenic plant displayed increased susceptibility to spider mite (Tetranychus urticae) infestation. Collectively, these data suggest that OPDAT1 is an inner envelope transporter for OPDA, and this has potential implications for JA biosynthesis in poplar under environmental stresses.

In trees, wood formation needs carbon import from the photosynthetic source tissues. Sugar transporters play important roles in carbohydrate transport into wood-forming cells. Sugars will eventually be exported transporters (SWEETs) play essential roles in many physiological processes. However, the roles of this family in the growth and development of woody plants have not been systematically investigated. In this study, 27 SWEET genes were identified in the Populus trichocarpa genome. These SWEET genes were classified into four clades based on their phylogenetic relationships, gene structures, conserved motifs and chromosomal locations. Representative SWEET members from each clade were selected for further studies. The PagSWEETs of Populus alba × Populus glandulosa were localized to plasma membrane, vacuolar, endoplasmic reticulum or Golgi. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that PagSWEETs have distinct expression patterns in various tissues, and PagSWEET5, 7, 10b, 10c, 15b, 17a and 17c exhibited high expression levels in stems. PagSWEET7 is localized to the cytoplasmic membrane and specifically expressed in the phloem as detected by histochemical GUS ($\beta $ - glucuronidase) assays. Xylem production and xylem sugar content were greater in developing wood of PagSWEET7 overexpression than wild-type lines. Collectively, these results provide valuable information for further investigating functions of PagSWEET genes, and identify PagSWEET7 as a candidate gene for using biotechnology to modify the wood formation in poplar.

The multisubunit Elongator complex plays key roles in transcription by interacting with RNA polymerase II and chromatin modeling. Kti proteins have been identified as the auxiliary protein for the Elongator complex. However, our knowledge of Kti proteins in woody plants remains limited. In this study, in total 16 KTI gene homologs were identified in Populus trichocarpa. Among them, the two KTI12 candidates were named PtKTI12A and PtKTI12B. Although PtKTI12A and PtKTI12B were largely different in gene expression level and tissue specificity, both genes were induced by heat and drought stresses. PtKTI12A and PtKTI12B RNAi transgenic poplar plants showed reduced levels of modified nucleosides, in particular 5-carbamoylmethyluridine and 5-methoxycarbonylmethyl-2-thiouridine. Meanwhile, their tolerance to drought was improved when subjected to withdrawal of watering. Also, the protein products of PtKTI12A and PtKTI12B had similar subcellular localization and predicted tertiary structure. The results suggest that Kti12 proteins are involved in tRNA wobble uridine modification, stress response and drought stress tolerance in hybrid poplar.

The remobilization and resorption of plant nutrients is considered as a crucial aspect of the seasonal senescence of plant organs. In leaves, the mechanisms responsible for the relocation of valuable compounds are well understood while the related processes in roots are still being debated. Some research indicates that remobilization in roots occurs, while other studies have not found evidence of this process. Considering that the total biomass of fine roots is equal to or greater than that of leaves, clarifying the conflicting reports and ambiguities may provide critical information on the circulation of chemical elements in forest ecosystems. This study provides new information concerning the basis for remobilization processes in roots by combining physiological data with gene expression and protein levels. We suggest that, as in leaves, molecular mechanisms involved in nitrogen (N) resorption are also activated in senescent roots. An analysis of N concentration indicated that N levels decreased during the senescence of both organs. The decrease was associated with an increase in the expression of a glutamine synthetase (GS) gene and a concomitant elevation in the amount of GS—one of the most important enzymes in N metabolism. In addition, significant accumulation of carbohydrates was observed in fine roots, which may represent an adaptation to unfavorable weather conditions that would allow remobilization to occur rather than a rapid death in response to ground frost or cold. Our results provide new insights into the senescence of plant organs and clarify contentious topics related to the remobilization process in fine roots

As one of the major groups of small post-translationally modified peptides, the CLV3/EMBRYO SURROUNDING REGION-RELATED (CLE)-like (CLEL) peptide family has been reported to regulate root growth, lateral root development and plant gravitropic responses in Arabidopsis thaliana. In this study, we identified 12 CLEL genes in Populus trichocarpa and performed a comprehensive bioinformatics analysis on these genes. Among them, five P. trichocarpa CLELs (PtrCLELs) were revised with new gene models. All of these PtrCLEL proteins were structurally similar to the A. thaliana CLELs (AtCLELs), including an N-terminal signal peptide, a conserved C-terminal 13-amino-acid CLEL motif and a variable intermediate region. In silico and quantitative real-time PCR analyses showed that PtrCLELs were widely expressed in various tissues, including roots, leaves, buds and stems. Exogenous application of chemically synthesized PtrCLEL peptides resulted in wavy or curly roots and reduced lateral root formation in A. thaliana. Moreover, germinating Populus deltoides seedlings on a growth medium containing these peptides caused the roots to thicken and to form abnormal lateral roots, in many cases in clusters. Anatomical and histological changes in thickened roots were further investigated by treating Populus 717 cuttings with the PtrCLEL10 peptide. We observed that root thickening was mainly due to an increased number of cells in the epidermis, hypodermis and cortex. The results of our study suggested that PtrCLEL and AtCLEL genes encode proteins with similar protein structures, sequences of peptide motif and peptide activities on developing roots. The activities of PtrCLEL peptides in root development were species-dependent.

Both anthocyanins and lignins are essential secondary metabolites in plant growth and development. Their biosynthesis is metabolically interconnected and diverges in the central metabolite 4-coumaroyl CoA of the phenylpropanoid pathway. Considerable progress has been made in understanding transcriptional regulation of genes involved in lignin and anthocyanin synthesis pathways, but the concerted regulation of these pathways is not yet fully understood. Here, we functionally characterized PtrMYB120, a R2R3-MYB transcription factor from Populus trichocarpa. Overexpression of PtrMYB120 in a hybrid poplar (i.e., 35S::PtrMYB120) was associated with increased anthocyanin (i.e., cyanidin 3-O-glucoside) accumulation and upregulation of anthocyanin biosynthetic genes. However, transgenic poplars with dominant suppression of PtrMYB120 function achieved by fusing the ERF-associated amphiphilic repression motif to PtrMYB120 (i.e., 35S::PtrMYB120-SRDX) had a dramatic decrease in not only anthocyanin but also Klason lignin content with downregulation of both anthocyanin and lignin biosynthetic genes. Indeed, 35S::PtrMYB120-SRDX poplars had irregularly shaped xylem vessels with reduced S-lignin content in stems, which was proportionally related to the level of the introduced PtrMYB120-SRDX gene. Furthermore, protoplast-based transcriptional activation assay using the PtrMYB120-GR system suggested that PtrMYB120 directly regulates genes involved in both anthocyanin and lignin biosynthesis, including chalcone synthase and ferulate-5 hydroxylase. Interestingly, the saccharification efficiency of line #6 of 35S::PtrMYB120-SRDX poplars, which had slightly reduced lignin content with a normal growth phenotype, was dramatically enhanced (>45%) by NaOH treatment. Taken together, our results suggest that PtrMYB120 functions as a positive regulator of both anthocyanin and lignin biosynthetic pathways and can be targeted to enhance saccharification efficiency in woody perennials.

A stable leaf temperature provides plants with a suitable microenvironment for photosynthesis. With global warming, extreme temperatures have become more frequent and severe; therefore, it is increasingly important to understand the fine regulation of leaf temperature under heat stress. In this study, five poplar species (Populus tomentosa, Populus simonii, Populus euphratica, Populus deltoides and Populus trichocarpa) that live in different native environments were used to analyze leaf temperature regulation. Leaf temperatures were more stable in Populus simonii and Populus euphratica (adapted to water-deficient regions) under elevated ambient temperature. Although transpiration contributes strongly to leaf cooling in poplar, the thicker epidermis and mesophyll and lower absorbance of Populus simonii and Populus euphratica leaves also help reduce leaf temperature, since their leaves absorb less radiation. Co-expression network and association analysis of a natural population of P. simonii indicated that PsiMYB60.2, PsiMYB61.2 and PsiMYB61.1 play dominant roles in coordinating leaf temperature, stomatal conductance and transpiration rate in response to heat stress. Individuals with CT-GT-GT genotypes of these three candidate genes have significantly higher water-use efficiency, and balance leaf temperature cooling with photosynthetic efficiency. Therefore, our findings have clarified the genetic basis of leaf cooling among poplar species and laid the foundation for molecular breeding of thermostable, water-conserving poplar varieties.

As a member of the CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION-related (CLE) peptide family, tracheary element differentiation inhibitory factor (TDIF) plays crucial roles in vascular meristem maintenance by promoting cell proliferation and inhibiting xylem cell differentiation. In Populus trichocarpa, six TDIF-encoding genes are all expressed in vascular tissues, and in Arabidopsis PtTDIFpro:GUS lines, the expression driven by PtTDIF promoters were predominantly detected in stem vascular bundles, initiating leaves and leaf veins. Although exogenous application of two poplar TDIF peptides did not evidently affect the shoot growth in vitro, overexpression of PtTDIF genes in hybrid poplar severely retarded the internodal elongation by upregulating the expression of GA2ox and GA20ox genes and thus decreasing the level of endogenous gibberellins (GAs), which phenotypic defect could be rescued by exogenously applied GA3. In addition, TDIF overexpression unexpectedly induced a more complex venation pattern in poplar leaves, which was underpinned by the elevated expression of WOX4 and WOX13 genes. Upon TDIF treatment, the DR5:GUS poplar leaves revealed a higher GUS activity and in TDIF-overexpressing leaves, the transcript abundances of several PIN-FORMED (PIN) genes, especially that of PIN1, were increased, which implied an integration of TDIF and auxin in mediating this process. Collectively, data of this work presented novel activities of TDIF involved in internode elongation and leaf vein formation, thus revealing the divergent functions of TDIF in perennial tree species from those in annual herbaceous Arabidopsis.

In plants, R2R3 MYB transcription factors (TFs) consist of one large gene family and are involved in the regulation of many developmental processes and various stresses. However, the functions of most of MYB TFs in woody plants remain unknown. Here, PtrMYB94, an R2R3 MYB TF from Populus trichocarpa, is characterized to be involved in the regulation of drought responses and abscisic acid (ABA) signaling. PtrMYB94 encodes a nuclear-localized R2R3 MYB TF. RT-PCR results showed that the PtrMYB94 transcripts were relatively abundant in leaves and stems, and were induced rapidly in response to dehydration stress. Overexpression of PtrMYB94 improved plant drought responses, suggesting that this MYB TF may functionally regulate poplar adaptability to drought stress. Furthermore, the analysis of transcriptional expression and PtrMYB94 promoter: GUS activity showed that PtrMYB94 responded to ABA induction. PtrMYB94-overexpressing plants exhibited the inhibition of seed germination compared with the wild-type (WT) control under ABA exposure condition. The ABA content was evidently increased in the PtrMYB94-overexpressing plants relative to the WT plants. In addition, transcript levels of several ABA- and drought-responsive genes, such as ABA1 and DREB2B, were up-regulated. Taken together, our results suggest that PtrMYB94 is involved in an ABA-dependent drought stress regulation in Populus.

Anthocyanins are a group of colorful and bioactive natural pigments with important physiological and ecological functions in plants. We found an MYB transcription factor (PtrMYB119) from Populus trichocarpa that positively regulates anthocyanin production when expressed under the control of the CaMV 35S promoter in transgenic Arabidopsis Amino acid sequence analysis revealed that PtrMYB119 is highly homologous to Arabidopsis PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1), a well-known transcriptional activator of anthocyanin biosynthesis. Independently produced transgenic poplars overexpressing PtrMYB119 or PtrMYB120 (a paralogous gene to PtrMYB119) (i.e., 35S::PtrMYB119 and 35S::PtrMYB120, respectively) showed elevated accumulation of anthocyanins in the whole plants, including leaf, stem and even root tissues. Using a reverse-phase high-performance liquid chromatography, we confirmed that the majority of the accumulated anthocyanin in our transgenic poplar is cyanidin-3-O-glucoside. Gene expression analyses revealed that most of the genes involved in the anthocyanin biosynthetic pathway were highly upregulated in 35S::PtrMYB119 poplars compared with the nontransformed control poplar. Among these genes, expression of PtrCHS1 (Chalcone Synthase1) and PtrANS2 (Anthocyanin Synthase2), which catalyze the initial and last steps of anthocyanin biosynthesis, respectively, was upregulated by up to 350-fold. Subsequent transient activation assays confirmed that PtrMYB119 activated the transcription of both PtrCHS1 and PtrANS2 Interestingly, expression of MYB182, a repressor of both anthocyanin and proanthocyanidin (PA) biosynthesis, was largely suppressed in 35S::PtrMYB119 poplars, while expression of MYB134, an activator of PA biosynthesis, was not changed significantly. More interestingly, high-level accumulation of anthocyanins in 35S::PtrMYB119 poplars did not have an adverse effect on plant growth. Taken together, our results demonstrate that PtrMYB119 and PtrMYB120 function as transcriptional activators of anthocyanin accumulation in both Arabidopsis and poplar.

Cellulose synthase A genes (CesAs) are responsible for cellulose biosynthesis in plant cell walls. In this study, functions of secondary wall cellulose synthases PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B were characterized during wood formation in Populus trichocarpa (Torr. & Gray). CesA RNAi knockdown transgenic plants exhibited stunted growth, narrow leaves, early necrosis, reduced stature, collapsed vessels, thinner fiber cell walls and extended fiber lumen diameters. In the RNAi knockdown transgenics, stems exhibited reduced mechanical strength, with reduced modulus of rupture (MOR) and modulus of elasticity (MOE). The reduced mechanical strength may be due to thinner fiber cell walls. Vessels in the xylem of the transgenics were collapsed, indicating that water transport in xylem may be affected and thus causing early necrosis in leaves. A dramatic decrease in cellulose content was observed in the RNAi knockdown transgenics. Compared with wildtype, the cellulose content was significantly decreased in the PtrCesA4, PtrCesA7 and PtrCesA8 RNAi knockdown transgenics. As a result, lignin and xylem contents were proportionally increased. The wood composition changes were confirmed by solid-state NMR, two-dimensional solution-state NMR and sum-frequency-generation vibration (SFG) analyses. Both solid-state nuclear magnetic resonance (NMR) and SFG analyses demonstrated that knockdown of PtrCesAs did not affect cellulose crystallinity index. Our results provided the evidence for the involvement of PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B in secondary cell wall formation in wood and demonstrated the pleiotropic effects of their perturbations on wood formation.

The Arabidopsis vacuolar H(+)-pyrophosphatase (AVP1) has been well studied and subsequently employed to improve salt and/or drought resistance in herbaceous plants. However, the exact function of H(+)-pyrophosphatase in woody plants still remains unknown. In this work, we cloned a homolog of type I H(+)-pyrophosphatase gene, designated as PtVP1.1, from Populus trichocarpa, and investigated its function in both Arabidopsis and poplar. The deduced translation product PtVP1.1 shares 89.74% identity with AVP1. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR analyses revealed a ubiquitous expression pattern of PtVP1.1 in various tissues, including roots, stems, leaves and shoot tips. Heterologous expression of PtVP1.1 rescued the retarded-root-growth phenotype of avp1, an Arabidopsis knock out mutant of AVP1, on low carbohydrate medium. Overexpression of PtVP1.1 in poplar (P. davidiana × P. bolleana) led to more vigorous growth of transgenic plants in the presence of 150 mM NaCl. Microsomal membrane vesicles derived from PtVP1.1 transgenic plants exhibited higher H(+)-pyrophosphatase hydrolytic activity than those from wild type (WT). Further studies indicated that the improved salt tolerance was associated with a decreased Na(+) and increased K(+) accumulation in the leaves of transgenic plants. Na(+) efflux and H(+) influx in the roots of transgenic plants were also significantly higher than those in the WT plants. All these results suggest that PtVP1.1 is a functional counterpart of AVP1 and can be genetically engineered for salt tolerance improvement in trees.

The C-repeat binding factors (CBFs), also termed dehydration-responsive element-binding protein 1 (DREB1) family members, play crucial roles in the acquisition of stress tolerance, but in trees, the underlying mechanisms of stress tolerance remain elusive. To gain insight into these mechanisms, we isolated five CBF1 orthologs from four poplar sections (Populus spp.) and assessed their expression under drought, cold, heat and salt stress conditions. Globally induced expression in response to cold suggested a correlation between poplar CBF1 expression and the acquisition of cold tolerance. Responses that varied between sections may reflect section-specific stress tolerance mechanisms, suggesting an effect of ecological context on the development of CBF1-mediated stress tolerance in poplar. We then used a genome-wide search strategy in Populus trichocarpa to predict 2263 putative CBF target genes; the identified genes participate in multiple biological processes and pathways. Almost all of the putative target genes contained multiple cis-acting elements that mediate responses to various environmental and endogenous signals, consistent with an important role of CBF1s in an integrated cold regulatory network. Finally, analysis of an association population of 528 individuals of Populus simonii identified six single-nucleotide polymorphisms (false discovery rate Q < 0.10) significantly (P < 0.005) associated with malondialdehyde production and electrolyte leakage, suggesting the potential importance of PsCBF1 in the regulation of some membrane-related functions. Our findings provide new insights into the function of PsCBF1 and shed light on the CBF-mediated regulatory network in poplar.

Knowledge of genetic variation in response of Populus to heavy metals like cadmium (Cd) is an important step in understanding the underlying mechanisms of tolerance. In this study, a pseudo-backcross pedigree of Populus trichocarpa and Populus deltoides was characterized for Cd exposure. The pedigree showed significant variation for Cd tolerance thus enabling the identification of relatively tolerant and susceptible genotypes for intensive characterization. A total of 16 QTLs at logarithm of odds (LOD) ratio > 2.5, were found to be associated with total dry weight, its components, and root volume. Four major QTLs for total dry weight were mapped to different linkage groups in control (LG III) and Cd conditions (LG XVI) and had opposite allelic effects on Cd tolerance, suggesting that these genomic regions were differentially controlled. The phenotypic variation explained by Cd QTL for all traits under study varied from 5.9% to 11.6% and averaged 8.2% across all QTL. Leaf Cd contents also showed significant variation suggesting the phytoextraction potential of Populus genotypes, though heritability of this trait was low (0.22). A whole-genome microarray study was conducted by using two genotypes with extreme responses for Cd tolerance in the above study and differentially expressed genes were identified. Candidatemore » genes including CAD2 (CADMIUM SENSITIVE 2), HMA5 (HEAVY METAL ATPase5), ATGTST1 (Arabidopsis thaliana Glutathione S-Transferase1), ATGPX6 (Glutathione peroxidase 6), and ATMRP 14 (Arabidopsis thaliana Multidrug Resistance associated Protein 14) were identified from QTL intervals and microarray study. Functional characterization of these candidate genes could enhance phytoremediation capabilities of Populus.« less

Poplars are one of the woody plants that are very sensitive to water stress, which may reduce the productivity of fast- growing plantations. Poplars can exhibit several drought tolerance strategies that may impact productivity differently. Trees from two improved hybrids, Populus balsamifera × Populus trichocarpa Torr. & Gray (clone B × T) and P. balsamifera × Populus maximowiczii A. Henry (clone B × M), having P. balsamifera L. as a parent and trees from native and unimproved P. balsamifera were subjected to a 1-month drying cycle in a growth chamber and then rewatered. The unimproved and native B clone maintained higher stomatal conductance (gs) than the hybrids, and high photosynthetic activity and transpiration, even when soil water content was nearly zero. As a result, both instantaneous water use efficiency (WUE i) and leaf carbon isotope composition (δ 13 C) indicated that this clone was less affected by drought than both hybrids at maximal drought stress. However, this clone shed its leaves when the drought threshold was exceeded, which implied a greater loss of productivity. The B × M hybrid showed a relatively conservative response to water stress, with the greatest decrease in transpiring versus absorbing surface (total leaf area to root biomass ratio). This clone was also the only one to develop new leaves after rewatering, and its total biomass production was not significantly decreased by drought. Among the two hybrids, clone B × T was the most vigorous, with the greatest transpiration (Ei) and net CO2 assimilation (A) rates, allowing for high biomass production. However, it had a more risky strategy under drought conditions by keeping its stomata open and high Ei rates under moderate drought, resulting in a lower recovery rate after rewatering. The opposite drought response strategies of the two hybrids were reflected by clone B × T having lower WUEi values than clone B × M at maximal drought, with a very low Ψmin value of −3.2 MPa, despite closed stomata and stopped photosynthetic activity. Positive linear relationships between A and gs for the three hybrids indicated strong stomatal control of photosynthesis. Moreover, the three poplar clones showed anisohydric behaviour for stomatal control and their use under long-term drought should be of interest, especially the B × M clone.

We studied the influence of nitrogen (N) on hydraulic traits and aquaporin (AQP) expression in the stem xylem of hybrid poplar saplings (Populus trichocarpa (Torr. & Gray) × deltoides Bartr. ex Marsh clone H11-11). Plants were grown in a controlled environment and were kept well watered throughout the experiments. Hydraulic measurements were done on basal and distal stem segments of plants receiving high N fertilization (high N plants) versus plants receiving only adequate N fertilization (adequate N plants). High N plants grew faster and exhibited more leaf area than adequate N controls. These morphological differences were paralleled by wider vessels and higher specific conductivities (K S ) in high N plants. However, stems of high N plants were more vulnerable to xylem cavitation, at least in one of two experiments, and showed lower wood densities than stems of adequate N plants. Leaf area was strongly correlated with cross-sectional xylem area in both plant groups. Since higher K S in high N plants was accompanied by concomitant increases in leaf area, leaf-specific conductivities were similar in both plant groups. Influences of N on hydraulic traits were paralleled by changes in AQP expression. Seven AQPs were upregulated in the stem xylem of high N plants, five of which have been identified recently as water transporters. The enhanced growth of secondary xylem of high N plants has been shown to result from both increased cambial activity as well as increased cell size. We suggest that some of these water-transporting AQPs could play a role in xylogenesis, facilitating the influx of water into the zone of differentiating and maturing cells in secondary xylem, including expanding vessels.

International audience; The objective of this study was to evaluate the environmental, temporal and genetic stability of the relationships between growth and a selection of tree architectural, leaf and phenological traits (selection based on the conclusions of previous studies carried out on the same experimental trial). Therefore, the growth of two hybrid families, Populus deltoides ‘S9-2’ • Populus nigra ‘Ghoy’ (D • N family, 180 F1) and P. deltoides ‘S9-2’ • Populus trichocarpa ‘V24’ (D • T family, 182 F1), was investigated during a 3-year period at two sites, i.e., in northern Italy and central France. At the end of the second growing season, all trees were coppiced and the resprouts were thinned to a single stem. At the end of each growing season, stem circumference and height were measured for all F1 hybrids. The number of sylleptic branches, individual leaf area (LA) and petiole length of the largest leaf along the main stem, production of new leaves, bud flush and bud set were estimated for a selection of genotypes (31 F1) per family at each site during the course of the 3-year experiment. The D • T family was clearly the most productive family and displayed the highest heterosis values. However, there appeared to be a compromise between good growth at a given site and stability between the two different sites, both at family and at genotype levels. Particularly, the less performing trees were stable between Italy and France. Among the studied growth components, the number of sylleptic branches and individual LA of the largest leaf along the main stem were the best growth predictors, irrespective of site and family. Growth strategies in terms of leaf development differed between the two families. Hence, leaf production rate was strongly associated with growth of the D • N family only. These results have important consequences for the use of the studied traits as selection criteria in breeding programmes.

Species and hybrids in the genus Populus have become the focus of investigation for use in biofuels production and their capacity to sequester carbon (C) in the environment. The identification of species resistant to marginal edaphic sites may be important in both of these endeavors. Plant growth, total dissolved organic carbon (TOC) and low molecular weight organic acid (OA) production, antioxidative enzyme activities and mineral content were assessed in Populus tremuloides L. and Populus trichocarpa Torr. & Gray seedlings under exposure to aluminum (Al). Both species were sensitive to Al, with significant reductions in shoot and root biomass at and above 50 microM Al. Exposure to Al induced 40-fold increases in TOC deposition in P. tremuloides and 100-fold increases in P. trichocarpa. In P. tremuloides, Al treatment induced root exudation of malic and citric acids, while Al increased exudation of citrate and oxalate in P. trichocarpa. Organic acids accounted for 20-64% of total C released upon Al exposure, with the proportion of OAs increasing in P. tremuloides and decreasing in P. trichocarpa. Dose-dependent responses of catalase and ascorbate peroxidase were observed in both root and leaf tissues, indicating that Al exposure induced oxidative stress in poplar. Treatment at and above 100 microM Al reduced the concentrations of calcium (Ca) and magnesium (Mg) in roots and leaves, whereas Al at or above 50 microM reduced root and leaf phosphorous (P) concentrations. The majority of Al taken up was retained in the root system. Even with the induction of OA exudation and accumulation, P. tremuloides and P. trichocarpa remained sensitive to Al, as evidenced by elevated antioxidative enzyme activities, which may reflect inhibition of Ca or P uptake and destabilization of cell homeostasis in these poplar species. Although plants exhibited reductions in growth and evidence of oxidative and nutritional stress, total C rhizodeposition rates for both species increased with increasing Al exposure concentration. Estimated C deposition rates of 16 mg C plant(-1) day(-1) were four-times larger than previously reported values for forest tree species, indicating that edaphic stress plays an important role in C flux to the rhizosphere.

Long-lived tree species are subject to attack by various pests and pathogens during their lifetime. This problem is exacerbated by climate change, which may increase the host range for pathogens and extend the period of infestation by pests. Plant defences may involve preformed barriers or induced resistance mechanisms based on recognition of the invader, complex signalling cascades, hormone signalling, activation of transcription factors and production of pathogenesis-related (PR) proteins with direct antimicrobial or anti-insect activity. Trees have evolved some unique defence mechanisms compared with wellstudied model plants, which are mostly herbaceous annuals. The genome sequence of Eucalyptus grandis W. Hill ex Maiden has recently become available and provides a resource to extend our understanding of defence in large woody perennials. This review synthesizes existing knowledge of defence mechanisms in model plants and tree species and features mechanisms that may be important for defence in Eucalyptus, such as anatomical variants and the role of chemicals and proteins. Based on the E. grandis genome sequence, we have identified putative PR proteins based on sequence identity to the previously described plant PR proteins. Putative orthologues for PR-1, PR-2, PR-4, PR-5, PR-6, PR-7, PR-8, PR-9, PR-10, PR-12, PR-14, PR-15 and PR-17 have been identified and compared with their orthologues in Populus trichocarpa Torr. & A. Gray ex Hook and Arabidopsis thaliana (L.) Heynh. The survey of PR genes in Eucalyptus provides a first step in identifying defence gene targets that may be employed for protection of the species in future. Genomic resources available for Eucalyptus are discussed and approaches for improving resistance in these hardwood trees, earmarked as a bioenergy source in future, are considered.

Young individuals of a single black cottonwood (Populus trichocarpa Torr. & Gray) clone were raised for three growing seasons in whole-tree chambers and exposed to either ambient or elevated atmospheric carbon dioxide concentration ([CO2]), with either a high or a low mineral nutrient supply, in a factorial experimental design. Nutrient availability had a larger effect on growth and dry matter partitioning than did [CO2]. Total biomass did not differ significantly with CO2 treatment when nutrient availability was low. However, elevated [CO2] increased whole-plant biomass by 47% in the high nutrient availability treatment. Carbon dioxide enrichment reduced leaf area ratio and specific leaf area significantly, but had no significant effect on mean leaf size or leaf mass ratio. Root mass ratio was significantly increased by elevated [CO2] at low, but not at high nutrient availability. A modified "demographic harvesting approach" made possible the retrospective estimation of stem and branch dry masses for different years. The relative growth rates of stem and branch were significantly enhanced by elevated [CO2] with high, but not with low nutrient availability. Canopy productivity index (CPI), i.e., the amount of stem and branch wood produced annually per unit leaf area, was raised 12% by elevated [CO2] when nutrient availability was high, but was reduced when nutrient availability was low, because of increased below ground allocation.

Cottonwoods occur in riparian areas where water table depth generally varies with the elevation of the adjacent river. Plant adaptation to the riparian zone requires the coordination of root elongation to maintain contact with the water table during the summer decline. We investigated the effects of rate of water decline on growth allocation and concentrations of endogenous gibberellins (GAs) in black cottonwood (Populus trichocarpa Torr. & A. Gray ex Hook.) saplings. Rhizopods were used to achieve water decline rates of 0, 2 and 4 cm day −1 . Root elongation approximately doubled in response to the 2 cm day −1 treatment, whereas leaf area was reduced. A water decline rate of 4 cm day −1 led to water stress, as evidenced by reduced growth, increased leaf diffusive resistance, decreased water potential, and leaf senescence and abscission. Endogenous GAs were extracted, purified and analyzed by gas chromatography-selected ion monitoring with internal [ 2 H 2 ]GA standards. Across the sampled plant organs, GAs were generally highest in shoot tips and sequentially lower in basal stems, root tips, leaves and upper roots; GAs were thus abundant in rapidly growing tissues. Of the GAs measured, GA 1 tended to predominate, followed sequentially by GA 3 , GA 8 , GA 19 , GA 20 , GA 29 and GA 4 . There was little relationship between GA concentration and growth allocation across the water table decline treatments, although GA 8 was consistently reduced in plants experiencing water table decline. Because GA 8 is the final gibberellin in the metabolic sequence, it might be useful for assessing historic patterns of GAs and growth rate. This study demonstrated changes in growth allocation in response to water table decline, but provided little evidence that endogenous GAs play a primary role in the regulation of root elongation in response to water table decline.

Fast-growing tree clones selected for biomass plantations are highly productive and therefore likely to use more water than the agricultural crops they replace. We report field measurements of transpiration through the summer of 1994 from two poplar clones, Beaupre (Populus trichocarpa Torr. & A. Gray x P. deltoides Bartr. ex Marsh.) and Dorschkamp (P. deltoides x P. nigra L.), grown as unirrigated short-rotation coppice in southern England. Stand transpiration was quantified by scaling up from sap flow measurements made with the heat balance method in a sample of stems. Leaf conductances, leaf area development, meteorological variables and soil water deficit were also measured to investigate the response of the trees to the environment. High rates of transpiration were found for Beaupre. In June, when soil water was plentiful, the mean (+/- SD) transpiration rate over an 18-day period was 5.0 +/- 1.8 mm day(-1), reaching a maximum of 7.9 mm day(-1). Transpiration rates from Dorschkamp were lower, as a result of its lower leaf area index. High total leaf conductances were measured for both Beaupre (0.34 +/- 0.17 mol m(-2) s(-1)) and Dorschkamp (0.39 +/- 0.16 mol m(-2) s(-1)). Leaf conductance declined slightly with increasing atmospheric vapor pressure deficit in both clones, but only in Beaupre did leaf conductance decrease as soil water deficit increased.

Poplar hybrids were grown with irrigation in a large-scale plantation to investigate the mechanisms underlying clonal differences in drought resistance. Beginning in spring 1992, Populus trichocarpa x P. deltoides (TD) and P. deltoides x P. nigra (DN) cuttings received 46, 76, or 137 cm year(-1) of irrigation to supplement the 18-20 cm of annual precipitation, and all trees received the same fertilization regime. Stem volume, assessed as the square of stem diameter at breast height times tree height (D(2)H), and water relations of the trees were studied from the end of their second growing season until the end of their fifth growing season. By the end of the second growing season, stem volume of Clone TD was 40-146% larger than that of Clone DN, but stem volume growth was independent of irrigation in excess of 46 cm year(-1) in both clones. During the third growing season, stem volume growth of both clones was limited by both the 46- and 76-cm irrigation treatments, so that by the end of the third growing season trees in the 46-cm irrigation treatment were only half the size of trees in the 137-cm irrigation treatment. These treatment differences were maintained through the fifth growing season. Although stem volumes of Clone TD trees in the 76- and 137-cm irrigation treatments were larger than the corresponding values for Clone DN trees at the end of the third growing season (1994), these clonal differences gradually decreased in subsequent years and were not detectable after 5 years, because stem volume relative growth rate of Clone DN was greater than that of Clone TD in all treatments. Although both clones exhibited similar predawn leaf water potentials, Clone DN typically maintained higher midday leaf water potentials, suggesting better stomatal control of water loss. Clonal and treatment differences in osmotic potential at full turgor were minimal and could not explain the clonal differences in drought resistance. Root density and root density to stem volume ratio increased more in response to moderate drought in Clone DN than in Clone TD, resulting in enhanced drought resistance (high stem volume growth rate under moderate drought conditions) and an increased capacity to withdraw water from the soil. We conclude that the greater drought resistance of Clone DN compared with Clone TD was the result of the maintenance of a more favorable water balance by stomatal regulation and greater carbon allocation to roots during the early stages of drought. However, the low root density to stem volume ratio in Clone DN growing in the 46-cm irrigation treatment suggests that severe water limitation restricted the preferential allocation of carbon to belowground tissues, so that both root and shoot growth were constrained by severe drought.

Cuttings of balsam spire hybrid poplar (Populus trichocarpa var. Hastata Henry x Populus balsamifera var. Michauxii (Dode) Farwell) were grown in sand culture and irrigated every 2 (W) or 10 (w) days with a solution containing either 3.0 (N) or 0.5 (n) mol nitrogen m(-3) for 90 days. Trees in the WN (control) and wn treatments had stable leaf nitrogen concentrations averaging 19.4 and 8.4 mg g(-1), respectively, over the course of the experiment. Trees in the Wn and wN treatments had a similar leaf nitrogen concentration, which increased from 12.0 to 15.8 mg g(-1) during the experiment. By the final harvest, mean stomatal conductances of trees in the wN and wn treatments were less than those of trees in the Wn and WN treatments (1.8 versus 4.6 mm s(-1)). Compared to the WN treatment, biomass at the final harvest was reduced by 61, 72 and 75% in the Wn, wN and wn treatments, respectively. At the final harvest, WN trees had a mean total leaf area of 4750 +/- 380 cm(2) tree(-1) and carried 164 +/- 8 leaves tree(-1) with a specific leaf area of 181 +/- 16 cm(2) g(-1), whereas Wn trees had a smaller mean total leaf area (1310 +/- 30 cm(2) tree(-1)), because of the production of fewer leaves (41 +/- 6) with a smaller specific leaf area (154 +/- 2 cm(2) g(-1)). A greater proportion of biomass was allocated to roots in Wn trees than in WN trees, but component nitrogen concentrations adjusted such that there was no Wn treatment effect on nitrogen allocation. Compared with WN trees, rates of photosynthesis and respiration per unit weight of tissue of Wn trees decreased by 28 and 31%, respectively, but the rate of photosynthesis per unit leaf nitrogen remained unaltered. The wN and Wn trees had similar leaf nitrogen concentrations; however, compared with the Wn treatment, the wN treatment decreased mean total leaf area (750 +/- 50 cm(2) tree(-1)), number of leaves per tree (29 +/- 2) and specific leaf area (140 +/- 6 cm(2) g(-1)), but increased the allocation of biomass and nitrogen to roots. Net photosynthetic rate per unit leaf nitrogen was 45% lower in the wN treatment than in the other treatments. Rates of net photosynthesis and respiration per unit weight of tissue were 48 and 33% less, respectively, in wN trees than in Wn trees.

Effects of mineral nutrition on susceptibility to cavitation were examined in four hybrid poplar clones. Two drought-sensitive and two drought-resistant hybrid clones of black cottonwood (Populus trichocarpa Torr. & Gray) and eastern cottonwood (P. deltoides Bartr.) were grown at three concentrations of nitrogen (N) applied factorially with two concentrations of phosphorus (P) in a greenhouse, and subjected to varying degrees of drought stress before measurement of cavitation and of anatomical features that might affect cavitation. Mean vessel pit pore diameters were 0.132 micro m at low P, and 0.074 micro m at high P, but no other significant effects of mineral nutrition on vessel dimensions were observed. Vessel diameter and specific conductivity were greater in the drought-resistant clones than in the drought-susceptible clones. Drought-resistant clones did not reach such low water potentials as drought-sensitive clones during the cavitation induction experiments, suggesting better stomatal and cuticular control of water loss. Scanning electron microscope observations showed less damage to pit membranes, also suggesting greater membrane strength in drought-resistant clones than in drought-sensitive clones. High concentrations of N increased cavitation, whereas high concentrations of P decreased cavitation as measured by both hydraulic flow apparatus and dye perfusion techniques. For one test, cavitation was 48% at high N and low P, but only 28% at high N and high P. We consider that N fertilization may make poplars more susceptible to cavitation on dry sites, but P fertilization may reduce this effect.

Ten years ago, it was announced that the Joint Genome Institute with funds provided by the Department of Energy, Office of Science, Biological and Environmental Research would sequence the black cottonwood (Populus trichocarpa Torr. & Gray) genome. This landmark decision was the culmination of work by the forest science community to develop Populus as a model system. Since its public release in late 2006, the availability of the Populus genome has spawned research in plant biology, morphology, genetics and ecology. Here we address how the tree physiologist has used this resource. More specifically, we revisit our earlier contention that the rewards of sequencing the Populus genome would depend on how quickly scientists working with woody perennials could adopt molecular approaches to investigate the mechanistic underpinnings of basic physiological processes. Several examples illustrate the integration of functional and comparative genomics into the forest sciences, especially in areas that target improved understanding of the developmental differences between woody perennials and herbaceous annuals (e.g., phase transitions). Sequencing the Populus genome and the availability of genetic and genomic resources has also been instrumental in identifying candidate genes that underlie physiological and morphological traits of interest. Genome-enabled research has advanced our understanding of how phenotype and genotype are related and provided insights into the genetic mechanisms whereby woody perennials adapt to environmental stress. In the future, we anticipate that low-cost, high-throughput sequencing will continue to facilitate research in tree physiology and enhance our understanding at scales of individual organisms and populations. A challenge remains, however, as to how genomic resources, including the Populus genome, can be used to understand ecosystem function. Although examples are limited, progress in this area is encouraging and will undoubtedly improve as future research targets the many unique aspects of Populus as a keystone species in terrestrial ecosystems.

Populus trichocarpa Torr. and Grey x P. balsamifera L. TT32 lines were regenerated from calli that had been maintained under differing in vitro conditions for sixteen months. In the final months, calli were maintained with one of six concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D, 0.1, 0.2, 0.3, 0.4, 0.5 or 0.6 mg l(-1))and regenerated with 0.25, 0.50 or 1.0 mg l(-1) benzylaminopurine (BA). Regenerant lines were obtained from 15 of these 18 treatments. The spectrum of variation in several morphological, physiological and leaf gas exchange traits was evaluated in the primary regenerants in 1986, and in their secondary vegetative propagules in the two subsequent years, in relation to differences in the original culture conditions. The results indicate that somoclonal variation was induced largely as a result of prolonged culture in the presence of 2,4-D, but that the terminal maintenance and regeneration phases also induced changes in the regenerants. Qualitative differences among the regenerant lines were detected by the end of 1986. For most traits, these differences were statistically confirmed within the 3-year period. The treatment lines ultimately diverged sufficiently to produce lines showing general performance that was either above or below that of the original TT32 clone. An early visible indicator of this divergence was variation in leaf shape (leaf length/width ratio), which could be related to 2,4-D-BA interactions in the final stages of culture. Graphic illustration of the independent effects of either 2,4-D or BA on stem height and gas exchange parameters suggested an inverse relationship with BA concentration and a complex interaction with 2,4-D. Significant correlations were detected between gas exchange parameters and morphological characteristics representing leaf form and stem development. Overall, the results indicate the presence of somaclonal lines that offer potential for the selective improvement of growth using morphological and gas exchange parameters as screening tools.

Members of glycosyltransferase protein families GT8, GT43 and GT47 are implicated in the biosynthesis of xylan in the secondary cell walls of Arabidopsis. The Arabidopsis mutant irx8 has a 60% reduction in xylan. However, over-expression of an ortholog of Arabidopsis IRX8, poplar PoGT8D, in Arabidopsis irx8 mutant could not restore xylan synthesis. The functions of tree GT8D genes remain unclear. We identified two GT8 gene homologs, PtrGT8D1 and PtrGT8D2, in Populus trichocarpa. They are the only two GT8D members and are abundantly and specifically expressed in the differentiating xylem of P. trichocarpa. PtrGT8D1 transcript abundance was >7 times that of PtrGT8D2. To elucidate the genetic function of GT8D in P. trichocarpa, the expression of PtrGT8D1 and PtrGT8D2 was simultaneously knocked down through RNAi. Four transgenic lines had 85–94% reduction in transcripts of PtrGT8D1 and PtrGT8D2, resulting in 29–36% reduction in stem wood xylan content. Xylan reduction had essentially no effect on cellulose quantity but caused an 11–25% increase in lignin. These transgenics exhibit a brittle wood phenotype, accompanied by increased vessel diameter and thinner fiber cell walls in stem xylem. Stem modulus of elasticity and modulus of rupture were reduced by 17–29% and 16–23%, respectively, and were positively correlated with xylan content but negatively correlated with lignin quantity. These results suggest that PtrGT8Ds play key roles in xylan biosynthesis in wood. Xylan may be a more important factor than lignin affecting the stiffness and fracture strength of wood.

We located fully sequenced putative genes of the plasma membrane intrinsic proteins (PIPs) family in the Populus trichocarpa (Torr. Gray), genome. Of 23 gene candidates, we assigned eight genes to the PIP2 subfamily. All eight putative genes were expressed in vegetative tissues (roots, leaves, bark and wood), and all of them showed water channel activity after being expressed in Xenopus oocytes. Six of eight proteins were affected by mercury ions. No proteins were affected by the presence of nickel or tungsten ions, or by lowering the pH of bathing external solution from 7.4 to 6.5. The presence of copper ions caused seven of eight PIP2 proteins to increase their water transport capacity by as much as 50%. This systematic study of the PIP2 subfamily of proteins in P. trichocarpa provides a basic overview of their activity as water channels and will be a useful reference for future physiological studies of plant water relations that use P. trichocarpa as a model system.

The recent completion of a draft sequence of the poplar (Populus trichocarpa Torr. & Gray ex Brayshaw) genome has advanced forest tree genetics to an unprecedented level. A "parts list" for a forest tree has been produced, opening up new opportunities for dissecting the interworkings of tree growth and development. In the relatively near future we can anticipate additional reference genome sequences, including the much larger Pinus genome. One goal is to use this information to define the genomic attributes that affect the phenotypic performances of trees growing in various environments. A first step is the definition of ideotypes that constitute optimal tree and stand-level performance. Following this, the genome can be systematically searched for genetic elements and their allelic variants that affect the specified traits. Knowledge of these alleles and their effects will facilitate the development of efficient tree improvement programs through genome-guided breeding and genetic engineering and further our mechanistic understanding of trait variation. Improved mechanistic understanding of tree growth and development is needed to develop process models that will allow us to anticipate and manage change in forest ecosystems. Here we consider the development of an ideotype for loblolly pine (Pinus taeda L.) and discuss genomic approaches for studying the component traits that will enable advances in process model development and the genetic improvement of this important conifer.

At coastal sites, trees are exposed to marine aerosols that may cause foliar necrosis and shoot dieback, which can result in deformed crowns and contorted stems. A six-year study of leaf primordia in terminal buds of black cottonwood trees (Populus trichocarpa Torr. & Gray) on Heimaey Island off the south coast of Iceland was undertaken to elucidate the physiological events associated with salt-deposition-related bud failure. Leaf and bud lengths, dry mass, water content and chloride concentrations were monitored and related to four phenological stages: (1) bud set; (2) dormancy induction; (3) dormancy release; and (4) bud break. The trees set buds in July and shed their leaves by late September. Leaf primordia generally stopped growing by September 10 +/- 22 days and attained midwinter water content in late September. Leaf growth commenced in the terminal buds by March 2 +/- 16 days, but mean dates of bud swelling and bud break were April 29 +/- 19 and May 10 +/- 12 days. In summer and until November, chloride concentrations in leaf primordia were low, but increasing. Chloride concentrations remained stable from December to February, even though the dormant trees were exposed to large amounts of marine aerosols. In February and March, three events occurred more or less simultaneously: (1) leaf extension growth commenced; (2) chloride concentration surged in the leaf primordia; and (3) the leaf primordia began to hydrate. Following dormancy release, growth and hydration of leaf primordia were negatively related to chloride concentration in the leaf primordia, with inhibition of leaf growth, tissue hydration and chloride acquisition occurring at a chloride concentration threshold estimated at 7.3 mg Cl- g(-1) tissue water. Necrosis of leaf primordia was observed above 14 mg Cl- g(-1) tissue water. Growth and hydration of leaves at bud break in mid-May was explained by a three-parameter logistic model of chloride concentration in leaf primordia at the end of March. By mid-May, 90% of all buds remained non-necrotic, but only 56% the terminal buds had broken. Salt alone explained the observed growth suppression of leaf primordia in the buds and the resultant failure of terminal buds to break by mid-May.

Elucidation of the mechanisms of dehydration tolerance in poplar (Populus sp.) trees will permit development of biochemical and molecular indicators to identify dehydration-tolerant genotypes during genetic selection. The objectives of this study were to characterize the degree of phenotypic variation in osmotic potential (a determinant of dehydration tolerance), determine the relationship between osmotic potential at full turgor and relative growth rate, and identify quantitative trait loci (QTL) for osmotic potential in an advanced-generation, interspecific poplar pedigree established in contrasting environments. A three-generation, sib-mated black cottonwood (Populus trichocarpa Torr.Gray) and eastern cottonwood (P. deltoides Bartr.) segregating F(2) family (Family 331) was analyzed at a dry site east of the Cascade Mountain Range (Boardman, OR) and at a wet site west of the mountains (Clatskanie, OR). At the Boardman site, 2-year-old trees (59 clones) were either irrigated everyday (wet) or every other day (dry), whereas 3- and 4-year-old trees (58 clones) at the Clatskanie site were unirrigated. At the Boardman site, the typically narrow range of osmotic potentials exhibited by grandparents and parents was greatly expanded in the F(2) population, spanning from -1.38 to -2.35 MPa under wet conditions, with a similar range under dry conditions (-1.40 to -2.15 MPa). Clones that had osmotic potentialsor = -1.90 MPa generally displayed full maintenance of stem relative growth rates under dry conditions in contrast to clones with osmotic potentials that wereor = -1.60 MPa, in which stem relative growth rates were reduced by an average of 38% in the dry treatment relative to the wet treatment. Although osmotic adjustments of 0.13 to 0.36 MPa were observed in nine out of 59 clones, adjustment typically occurred from relatively high baseline osmotic potentials. The range in osmotic potential at the wetter Clatskanie site at age three was higher (-1.27 to -1.84 MPa) and was further expanded the following year (-1.14 to -1.94 MPa), which had a wetter spring than the previous year, followed by a typically dry July. Seven QTL for osmotic potential were identified that each explained7.5% of the variation in osmotic potential. Given that four clones (7%) had osmotic potentials of -2.00 MPa or less and that QTL for osmotic potential have been identified, we suggest that there are opportunities to extend the limit of dehydration tolerance in Populus.

Xylem development in trees is affected by dynamic mechanical stresses imposed on stems by wind. To assess clonal differences in response to mechanical perturbation (MP), we subjected seven greenhouse-grown F1 hybrids of Populus trichocarpa Torr. and A. Gray. x P. deltoides Bartr. ex Marsh. to a standard MP treatment consisting of 20 manually imposed stem flexures per day for 70-90 days. Effects of MP on aboveground biomass, hydraulic conductivity (k(h)), specific conductivity (k(s)), flexural stiffness (EI), modulus of elasticity (MOE) and modulus of rupture (MOR) were determined. Treatment increased stem radial growth and decreased height growth, leaf area and total aboveground biomass. It also significantly decreased k(s), MOE and MOR, but significantly increased EI and wood specific gravity in most clones. Mechanical perturbation caused greater stem rigidity, without having a significant effect on whole-stem k(h) or percent loss of conductivity due to embolism. Maximum k(h) was positively correlated with EI in both control (r(2) = 0.54, P < 0.0001) and MP-treated (r(2) = 0.61, P < 0.0001) plants, and k(s) and MOE were positively correlated with percent vessel lumen area (r(2) = 0.45, P < 0.0001 and r(2) = 0.28, P = 0.002, respectively). Thus, contrary to our expectation of a trade-off between conductivity and wood strength, there may be an opportunity to select clones for woody biomass production that are superior in both mechanical strength and hydraulic conductivity, as is the triploid Clone 19-61.

We compared the physiological and morphological responses of rooted cuttings of Populus trichocarpa Torr. & Gray and P. trichocarpa x P. deltoides Bartr. ex Marsh. grown in either near-ambient solar ultraviolet-B (UV-B; 280-320 nm) radiation (cellulose diacetate film) or subambient UV-B radiation (polyester film) for one growing season. Midday biologically effective UV-B radiation was 120.6 and 1.6 mJ m(-2) s(-1) under the cellulose diacetate and polyester films, respectively. Gas exchange, leaf chlorophyll, light harvesting efficiency of photosystem II, and foliar UV-B radiation-absorbing compounds (i.e., flavonoid derivatives) were measured in expanding (leaf plastochron index (LPI) 5), nearly expanded (LPI 10), and fully expanded mature (LPI 15) leaves of intact plants of plastochron index 30 to 35. Plants were then harvested and height, diameter, biomass allocation and leaf anatomical attributes determined. Net photosynthesis, transpiration, and stomatal conductance were significantly greater in mature leaves exposed to subambient UV-B radiation than in mature leaves exposed to near-ambient UV-B radiation. Concentrations of UV-B radiation-absorbing compounds (measured as absorbance of methanol-extracts at 300 nm) were significantly greater in mature leaves exposed to near-ambient UV-B radiation than in mature leaves exposed to subambient UV-B radiation. The UV-B radiation treatments had no effects on chlorophyll content or intrinsic light harvesting efficiency of photosystem II. Height, diameter, and biomass were not significantly affected by UV-B radiation regime in either clone. Leaf anatomical development was unaffected by UV-B radiation treatment in P. trichocarpa x P. deltoides. For P. trichocarpa, leaf anatomical development was complete by LPI 10 in the near-ambient UV-B radiation treatment, but continued through to LPI 15 in the subambient UV-B radiation treatment. Mature leaves of P. trichocarpa were thicker in the subambient UV-B radiation treatment than in the near-ambient UV-B radiation treament as a result of greater development of palisade parenchyma tissue. We conclude that exposure to near-ambient UV-B radiation for one growing season caused shifts in carbon allocation from leaf development to other pools, probably including but not limited to, UV-B absorbing compounds. This reallocation curtailed leaf development and reduced photosynthetic capacity of the plants compared with those in the subambient UV-B radiation treatment and may affect growth over longer periods of exposure.

Effects of N and K nutrition on drought and cavitation resistance were examined in six greenhouse-grown poplar clones: Populus trichocarpa (Torr.Gray) and its hybrids with P. deltoides Bartr. and P. euramericana (Dole) Guinier, before and after preconditioning to water stress. Both tendency to cavitate and water-use efficiency (WUE) increased when N supply was increased, whereas K supply had little impact on cavitation. Mean xylem vessel diameters increased from 36.6mgr;m at low-N supply to 45.2mgr;m at high-N supply. Drought-hardy clones, which were relatively resistant to cavitation, had the smallest mean vessel diameters. Vulnerability to cavitation had a weakly positive relationship with vessel diameter, and a negative correlation with transpiration. Drought hardening offered no protection against cavitation in a subsequent drought. Under drought conditions, increasing N supply increased leaf loss and decreased water potentials, whereas increasing K supply decreased leaf loss. Drought-resistant clones exhibited similar WUE to drought-susceptible clones, but had smaller, more numerous stomata and greater leaf retention under drought conditions.

Experiments were conducted during the growing seasons of 1993-1995 to determine whether exposure to ozone (O 3 ) affected the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in hybrid poplar, Populus maximowizii A. Henry × trichocarpa Torr. & A. Gray, Clone 245. As the canopy aged, the concentration of Rubisco decreased at a more rapid rate in lower leaves of hyrid poplar ramets subjected to chronic O 3 exposure in open-top chambers than in comparable foliage of plants grown in charcoal-filtered air. There was no difference in rate of synthesis of Rubisco between treatments, suggesting that loss of this protein in O 3 -treated leaves was caused by an accelerated rate of proteolysis. In foliage higher in the canopy, both concentration and rate of synthesis of Rubisco were stimulated by O 3 for a brief period when the leaves were young. Quantification of mRNA for the small (rbcS) and large (rbcL) transcripts of Rubisco did not reveal changes that were likely to reflect altered synthesis of Rubisco as a prime response to O 3 . Analyses of Rubisco concentration and rate of Rubisco synthesis in foliage connected by vascular traces within the canopy indicated that loss of Rubisco in older leaves was associated with an increase in this protein in younger leaves higher in the canopy. These data support the notion that accelerated senescence may provide some compensatory benefit to the plant. In 1995, the rate of synthesis of Rubisco was almost always higher in O 3 -treated foliage than in nontreated foliage, even when the concentration of Rubisco was adversely affected by the O 3 treatment. Because accelerated foliar abscission in response to O 3 was minimal in 1995 compared to other years, we speculate that, when abscission is delayed, Rubisco synthesis and concentration become uncoupled.

Leaf osmotic potential at full turgor (leaf osmotic potential) and the major solutes that contribute to osmotic potential were characterized in five hybrid poplar clones of Populus trichocarpa Torr. & Gray x P. deltoides Bartr. (TD) and P. deltoides x P. nigra L. (DN), growing under field conditions at two sites in eastern Washington and Oregon, USA. Trees were drip irrigated with 46, 76 or 137 cm of supplemental irrigation during each growing season. Trees at Wallula, WA, which were in their third growing season in 1994, were sampled twice a year for two years (1994 and 1995), and trees at Boardman, OR, which were in their second growing season in 1994, were sampled once a year or three years (1994-1996). At Wallula, the TD and DN clones exhibited lower predawn leaf water potentials in the 46-cm treatment than in the 137-cm treatment (-1.2 versus -0.7 MPa) during a hot, dry period in July 1994. Clone TD had a lower leaf osmotic potential than Clone DN (-1.67 versus -1.56 MPa) during the same period and the difference was also evident in 1995 (-1.81 versus -1.72 MPa) when trees were in their fourth growing season. There was also a significant treatment effect on leaf osmotic potential in Clone TD, with trees in the 46-cm treatment having lower leaf osmotic potential than trees in the 137-cm treatment in July 1994. At Boardman, leaf osmotic potential was generally high with no treatment differences during the 1994-96 samplings. The TD clones had significantly lower leaf osmotic potential than the DN clones in 1994 (-1.44 versus -1.36 MPa) and 1996 (-1.72 versus -1.54 MPa), but there was no difference between clones in 1995 (-1.40 versus -1.43 MPa). In 1995, at Wallula, osmotic adjustment in Clone TD was largely accounted for by an increase in sucrose, which constituted 70% of total organic solutes. Although the total concentration of free primary amino acids in this clone was 28% higher in trees in the 46-cm treatment than in trees in the 137-cm treatment, amino acids constituted only a small fraction of the total solute pool. Sixty-two percent of total solutes were inorganic ions in Clone TD compared to 52% in Clone DN, and potassium was the main ion constituting about 30% of total solutes and 50% of total ions. However, the clonal difference in leaf osmotic potential was not fully accounted for by the difference in solute concentration. Osmotic potential at full turgor declined over the growing season and with age. We conclude that, because the extent of osmotic adjustment exhibited by these clones was small, other drought resistance mechanisms contributed to the clonal differences in field performance.

To identify hybrid-specific differences in developmental response to mechanical perturbation (MP), we compared the effects of stem flexure on several morphological and mechanical properties of two Populus trichocarpa Torr. & A. Gray x P. deltoides Bartr. ex Marsh. hybrids, 47-174 and 11-11. In response to the MP treatment, both hybrids exhibited a significant increase in radial growth, especially in the direction of the MP (47-174, P = 0.0001; 11-11, P = 0.002), and a significant decrease in height to diameter growth ratio (P = 0.0001 for both hybrids), suggesting that MP-treated stems are more tapered than control stems. A direct consequence of the MP-induced increase in radial growth was a significant increase in flexural rigidity (EI, N mm(2)) in stems of both hybrids (47-174, P = 0.0001; 11-11, P = 0.009). Both control and MP-treated stems of Hybrid 47-174 had significantly greater height to diameter ratios and EI values than the corresponding stems of Hybrid 11-11 (11-11 stem ratios and EI values were 85 and 76%, respectively, of those of 47-174). In Hybrid 47-174, Young's modulus of elasticity (E, N mm(-2)), a measure of stem flexibility, for MP-treated stems was only 80% of the control value (P = 0.0034), whereas MP had no significant effect on E of stems of Hybrid 11-11 (P = 0.2720). Differences in flexure response between the hybrids suggest that Hybrid 47-174 can produce a stem that is more tolerant of wind-induced flexure by altering both stem allometry and material properties, whereas Hybrid 11-11 relies solely on changes in stem allometry for enhanced stability under MP conditions.

Variations in resistance to drought-induced xylem cavitation, xylem air-entry points, stomatal behavior, and hydraulic conductivity were measured in four populations of Populus trichocarpa Torr. & A. Gray collected along an east-west humidity and temperature gradient in Washington State, USA. Xylem air-entry points were less negative in trees from moist environments (-0.71 and -1.32 MPa in the Hoh and Nisqually populations, respectively) than in trees from dry environments (-1.55 and -1.67 MPa in the Palouse and Yakima populations, respectively). Xylem cavitation in response to experimental drought was consistent with air-injection measures of xylem air-entry points for a given population. Populations vulnerable to cavitation also exhibited higher stem specific hydraulic conductivities and limited stomatal control compared with resistant populations. Populations exhibiting vulnerability to cavitation and limited stomatal control desiccated more rapidly during drought compared with resistant populations. This study provides evidence of interpopulation variation in resistance to drought-induced xylem cavitation, stomatal behavior, and hydraulic conductivity within Populus trichocarpa.

Genetic variation in stomatal initiation and density, and epidermal cell size and number were examined in a hybrid pedigree of Populus trichocarpa T. & G. and P. deltoides Marsh in both ambient ([aCO2]) and elevated ([eCO2]) concentrations of CO2. We aimed to link anatomical traits with the underlying genetic map of F2 Family 331, composed of 350 markers across 19 linkage groups. Leaf stomatal and epidermal cell traits showed pronounced differences between the original parents. We considered the following traits in the F2 population: stomatal density (SD), stomatal index (SI), epidermal cell area (ECA) and the number of epidermal cells per leaf (ECN). In [eCO2], adaxial SD and SI were reduced in the F2 population, whereas ECA increased and ECN remained unchanged. In [aCO2], four putative quantitative trait loci (QTL) with logarithm of the odds ratio (LOD) scores greater than 2.9 were found for stomatal traits on linkage group B: adaxial SI (LOD scores of 5.4 and 5.2); abaxial SI (LOD score of 3.3); and SD (LOD score of 3.2). These results imply that QTL for SI and SD share linkage group B and are under genetic control. More moderate LOD scores (LOD scores >/= 2.5) suggest QTL for SI on linkage groups A and B and for SD on linkage groups B, D and X with a probable co-locating quantitative trait locus for SI and SD on linkage group D (position 46.3 cM). The QTL in both [aCO2] and [eCO2] for adaxial SD were co-located on linkage group X (LOD scores of 3.5 and 2.6, respectively) indicating a similar response across both treatments. Putative QTL were located on linkage group A (position 89.2 cM) for both leaf size and ECN in [aCO2] and for ECA at almost the same position. The data provide preliminary evidence that leaf stomatal and cell traits are amenable to QTL analysis.

Bark storage proteins (BSP) store nitrogen (N) translocated from senescing leaves in autumn, and supply reduced N for spring growth. Expression of bsp and BSP accumulation are associated with short day photoperiod. To determine if photoperiod-associated bsp expression varies among poplars native to different latitudes, Populus deltoides Bartr. clones originating from six latitudes were grown under natural conditions at a common location. Relative amounts of BSP mRNA in these clones were measured at 2-week intervals from August 7 to October 16. The date of maximum BSP mRNA accumulation was correlated with latitude of origin, and maximum accumulation of BSP mRNA occurred earlier in clones native to northern latitudes than in clones native to southern latitudes. This pattern of variation is consistent with photoperiodic responses of plants native to temperate climates. Genotypic variations in BSP accumulation, bark protein concentration and bark N concentration were compared among clones of six hybrid poplar (Populus trichocarpa Torr. and Gray × P. deltoides) full-sib families (three F 2 families, two F 1 families and one BC 1 family) after 6 weeks in a short day photoperiod and at midwinter. Significant differences in BSP accumulation occurred among clones within four of the six full-sib families after 6 weeks in a short day photoperiod and also at midwinter for outdoor-grown plants. Bark protein and bark N concentrations also varied significantly among clones within certain families. In general, the greatest variation was found in F 2 and BC 1 families. Within several families, relative BSP amounts were positively correlated with bark protein concentration and total bark N concentration. These results indicate a role of photoperiod in regulating bsp expression and demonstrate a genetic component underlying seasonal BSP accumulation. The results could have significance in selecting for clones with improved N storage capacity and N-use efficiency.

We studied first-order root development of field-grown plants of 44 F(1) Populus trichocarpa x P. deltoides hybrids as well as 15 clones of P. trichocarpa (T) and 20 clones of P. deltoides (D) during their first growing season. The cuttings were planted in the field on May 11-12, 1988 and destructively sampled in July, August and September. By September 12, hybrids had an average of 50 first-order roots per cutting (range = 32 to 85) with an average of 25% dead roots (range = 10 to 51%). The longest roots of hybrids averaged 107 cm (range = 58 to 182 cm) and originated at the base of the cutting. Roots originating at the side of the cutting averaged 54 cm in length (range = 12 to 96 cm). For most aboveground and root parameters measured, the values for hybrids exceeded those for both parent species clones. For all clones, aboveground weights were positively correlated with weights of collected roots (R(2) = 0.66 to 0.86). In contrast, aboveground weights were negatively (but poorly) correlated with root/shoot ratios (R(2) = 0.08 to 0.21). Roots differed allometrically depending on their location on the cutting. Total weight of roots originating along the sides of the cutting was not correlated with aboveground weight (R(2) = 0.001 to 0.27), whereas total weight of roots originating at the base of the cutting was correlated with aboveground weight (R(2) = 0.52 to 0.80). Basal roots dominated the first-order root systems of most clones in terms of maximum length and total mass, but not in total numbers of roots.