CASP microdomain formation requires cross cell wall stabilization of domains and non-cell autonomous action of LOTR1.

Efficient uptake of nutrients in both animal and plant cells requires tissue-spanning diffusion barriers separating inner tissues from the outer lumen/soil. However, we poorly understand how such contiguous three-dimensional superstructures are formed in plants. Here, we show that correct establishment of the plant Casparian Strip (CS) network relies on local neighbor communication. We show that positioning of Casparian Strip membrane domains (CSDs) is tightly coordinated between neighbors in wild-type and that restriction of domain formation involves the putative extracellular protease LOTR1. Impaired domain restriction in lotr1 leads to fully functional CSDs at ectopic positions, forming 'half strips'. LOTR1 action in the endodermis requires its expression in the stele. LOTR1 endodermal expression cannot complement, while cortex expression causes a dominant-negative phenotype. Our findings establish LOTR1 as a crucial player in CSD positioning acting in a directional, non-cell-autonomous manner to restrict and coordinate CS positioning.

REPRISAL: mapping lignification dynamics using chemistry, data segmentation, and ratiometric analysis.

This article describes a methodology for detailed mapping of the lignification capacity of plant cell walls that we have called "REPRISAL" for REPorter Ratiometrics Integrating Segmentation for Analyzing Lignification. REPRISAL consists of the combination of three separate approaches. In the first approach, H*, G*, and S* monolignol chemical reporters, corresponding to p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, are used to label the growing lignin polymer in a fluorescent triple labeling strategy based on the sequential use of three main bioorthogonal chemical reactions. In the second step, an automatic parametric and/or artificial intelligence segmentation algorithm is developed that assigns fluorescent image pixels to three distinct cell wall zones corresponding to cell corners, compound middle lamella and secondary cell walls. The last step corresponds to the exploitation of a ratiometric approach enabling statistical analyses of differences in monolignol reporter distribution (ratiometric method [RM] 1) and proportions (RM 2) within the different cell wall zones. We first describe the use of this methodology to map developmentally related changes in the lignification capacity of wild-type Arabidopsis (Arabidopsis thaliana) interfascicular fiber cells. We then apply REPRISAL to analyze the Arabidopsis peroxidase (PRX) mutant prx64 and provide further evidence for the implication of the AtPRX64 protein in floral stem lignification. In addition, we also demonstrate the general applicability of REPRISAL by using it to map lignification capacity in poplar (Populus tremula × Populus alba), flax (Linum usitatissimum), and maize (Zea mays). Finally, we show that the methodology can be used to map the incorporation of a fucose reporter into noncellulosic cell wall polymers.

A regulatory network driving shoot lignification in rapidly growing bamboo.

Woody bamboo is environmentally friendly, abundant, and an alternative to conventional timber. Degree of lignification and lignin content and deposition affect timber properties. However, the lignification regulatory network in monocots is poorly understood. To elucidate the regulatory mechanism of lignification in moso bamboo (Phyllostachys edulis), we conducted integrated analyses using transcriptome, small RNA, and degradome sequencing followed by experimental verification. The lignification degree and lignin content increased with increased bamboo shoot height, whereas phenylalanine ammonia-lyase and Laccase activities first increased and then decreased with shoot growth. Moreover, we identified 11,504 differentially expressed genes (DEGs) in different portions of the 13th internodes of different height shoots; most DEGs associated with cell wall and lignin biosynthesis were upregulated, whereas some DEGs related to cell growth were downregulated. We identified a total of 1,502 miRNAs, of which 687 were differentially expressed. Additionally, in silico and degradome analyses indicated that 5,756 genes were targeted by 691 miRNAs. We constructed a regulatory network of lignification, including 11 miRNAs, 22 transcription factors, and 36 enzyme genes, in moso bamboo. Furthermore, PeLAC20 overexpression increased lignin content in transgenic Arabidopsis (Arabidopsis thaliana) plants. Finally, we proposed a reliable miRNA-mediated "MYB-PeLAC20" module for lignin monomer polymerization. Our findings provide definite insights into the genetic regulation of bamboo lignification. In addition to providing a platform for understanding related mechanisms in other monocots, these insights could be used to develop strategies to improve bamboo timber properties.

Drought stress triggers proteomic changes involving lignin, flavonoids and fatty acids in tea plants.

Drought stress triggers a series of physiological and biochemical changes in tea plants. It is well known that flavonoids, lignin and long-chain fatty acids play important roles in drought resistance. However, changes in proteins related to these three metabolic pathways in tea plants under drought stress have not been reported. We analysed the proteomic profiles of tea plants by tandem mass tag and liquid chromatography-tandem mass spectrometry. A total of 4789 proteins were identified, of which 11 and 100 showed up- and downregulation, respectively. The proteins related to the biosynthesis of lignin, flavonoids and long-chain fatty acids, including phenylalanine ammonia lyase, cinnamoyl-CoA reductase, peroxidase, chalcone synthase, flavanone 3-hydroxylase, flavonol synthase, acetyl-CoA carboxylase 1,3-ketoacyl-CoA synthase 6 and 3-ketoacyl-CoA reductase 1, were downregulated. However, the contents of soluble proteins, malondialdehyde, total phenols, lignin and flavonoids in the tea plants increased. These results showed that tea plants might improve drought resistance by inhibiting the accumulation of synthases related to lignin, flavonoids and long-chain fatty acids. The proteomic spectrum of tea plants provides a scientific basis for studying the pathways related to lignin, flavonoid and long-chain fatty acid metabolism in response to drought stress.

The effect of altered lignin composition on mechanical properties of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) deficient poplars.

MAIN CONCLUSION: CAD-deficient poplars enabled studying the influence of altered lignin composition on mechanical properties. Severe alterations in lignin composition did not influence the mechanical properties. Wood represents a hierarchical fiber-composite material with excellent mechanical properties. Despite its wide use and versatility, its mechanical behavior has not been entirely understood. It has especially been challenging to unravel the mechanical function of the cell wall matrix. Lignin engineering has been a useful tool to increase the knowledge on the mechanical function of lignin as it allows for modifications of lignin content and composition and the subsequent studying of the mechanical properties of these transgenics. Hereby, in most cases, both lignin composition and content are altered and the specific influence of lignin composition has hardly been revealed. Here, we have performed a comprehensive micromechanical, structural, and spectroscopic analysis on xylem strips of transgenic poplar plants, which are downregulated for cinnamyl alcohol dehydrogenase (CAD) by a hairpin-RNA-mediated silencing approach. All parameters were evaluated on the same samples. Raman microscopy revealed that the lignin of the hpCAD poplars was significantly enriched in aldehydes and reduced in the (relative) amount of G-units. FTIR spectra indicated pronounced changes in lignin composition, whereas lignin content was not significantly changed between WT and the hpCAD poplars. Microfibril angles were in the range of 18°-24° and were not significantly different between WT and transgenics. No significant changes were observed in mechanical properties, such as tensile stiffness, ultimate stress, and yield stress. The specific findings on hpCAD poplar allowed studying the specific influence of lignin composition on mechanics. It can be concluded that the changes in lignin composition in hpCAD poplars did not affect the micromechanical tensile properties.

Effects of lignins as diet components on the physiological activities of a lower termite, Coptotermes formosanus Shiraki.

We investigated the effects of lignins as diet components on the physiological activities of a lower termite, Coptotermes formosanus Shiraki. Artificial diets composed of polysaccharides with and without purified lignins (milled-wood lignins) from Japanese cedar (softwood), Japanese beech (hardwood), and rice (grass), were fed to C. formosanus workers. The survival and body mass of the workers as well as the presence of three symbiotic protists in the hindguts of the workers were then periodically examined. The survival rates of workers fed on diets containing lignins were, regardless of the lignocellulose diet sources, significantly higher than those of workers fed on only polysaccharides. In addition, it was clearly observed that all the tested lignins have positive effects on the maintenance of two major protists in the hindguts of C. formosanus workers, i.e., Pseudotrichonympha grassii and Holomastigotoides hartmanni. Overall, our data suggest that the presence of lignin is crucial to maintaining the physiological activities of C. formosanus workers during their lignocellulose decomposition. Our data also suggested that some components, possibly minerals and/or non-structural carbohydrates, in grass lignocellulose negatively affect the survival of C. formosanus workers as well as the present rate of the symbiotic protists in their hindguts.

Leucaena leucocephala (Lam.) de Wit., "subabul" stem lignin: Isolation, structural characterization and thermal properties.

Lignin is the second most abundant renewable biopolymer on earth after cellulose. It is being used in many industrial applications due to its abundance. In the present study, lignin was isolated from the stems of Leucaena leucocephala (Lam.) de Wit., a high biomass yielding plant using acidic dioxane under N2 atmosphere. Structural characterization of isolated dioxane lignin (DL) was performed by analytical techniques: UV, FT-IR, ¹H NMR and ¹³C NMR. Their monolignol content was determined by nitrobenzene oxidation followed by HPLC-MS/MS analysis. The data was compared with commercial alkali lignin (AL). The results showed that DL is of hardwood guaiacyl-syringyl (GS) type, whereas AL is softwood type with more guaiacyl units and trace amounts of p-hydroxyphenyl units (H). Thermogravimetric analysis (TGA) of DL showed two stage thermal degradation profile similar to AL. The DTGmax for DL and AL were found in the second major loss event of second stage of TGA at 424°C and 404°C, respectively. Differential scanning calorimetry (DSC) study exhibited the glass transition temperatures (Tg) at 132°C and 122°C for DL and AL, respectively. The results from thermal stability studies suggest that dioxane lignin isolated from the "miracle tree" (subabul) can be exploited in various thermoplastic industrial applications.

Leveraging Genetic-Background Effects in Saccharomyces cerevisiae To Improve Lignocellulosic Hydrolysate Tolerance.

UNLABELLED: A major obstacle to sustainable lignocellulosic biofuel production is microbe inhibition by the combinatorial stresses in pretreated plant hydrolysate. Chemical biomass pretreatment releases a suite of toxins that interact with other stressors, including high osmolarity and temperature, which together can have poorly understood synergistic effects on cells. Improving tolerance in industrial strains has been hindered, in part because the mechanisms of tolerance reported in the literature often fail to recapitulate in other strain backgrounds. Here, we explored and then exploited variations in stress tolerance, toxin-induced transcriptomic responses, and fitness effects of gene overexpression in different Saccharomyces cerevisiae (yeast) strains to identify genes and processes linked to tolerance of hydrolysate stressors. Using six different S. cerevisiae strains that together maximized phenotypic and genetic diversity, first we explored transcriptomic differences between resistant and sensitive strains to identify common and strain-specific responses. This comparative analysis implicated primary cellular targets of hydrolysate toxins, secondary effects of defective defense strategies, and mechanisms of tolerance. Dissecting the responses to individual hydrolysate components across strains pointed to synergistic interactions between osmolarity, pH, hydrolysate toxins, and nutrient composition. By characterizing the effects of high-copy gene overexpression in three different strains, we revealed the breadth of the background-specific effects of gene fitness contributions in synthetic hydrolysate. Our approach identified new genes for engineering improved stress tolerance in diverse strains while illuminating the effects of genetic background on molecular mechanisms. IMPORTANCE: Recent studies on natural variation within Saccharomyces cerevisiae have uncovered substantial phenotypic diversity. Here, we took advantage of this diversity, using it as a tool to infer the effects of combinatorial stress found in lignocellulosic hydrolysate. By comparing sensitive and tolerant strains, we implicated primary cellular targets of hydrolysate toxins and elucidated the physiological states of cells when exposed to this stress. We also explored the strain-specific effects of gene overexpression to further identify strain-specific responses to hydrolysate stresses and to identify genes that improve hydrolysate tolerance independent of strain background. This study underscores the importance of studying multiple strains to understand the effects of hydrolysate stress and provides a method to find genes that improve tolerance across strain backgrounds.

An ethylene response-related factor, GbERF1-like, from Gossypium barbadense improves resistance to Verticillium dahliae via activating lignin synthesis.

An ethylene response-related factor, GbERF1-like, from Gossypium barbadense cv. '7124' involved in the defence response to Verticillium dahliae was characterized. GbERF1-like transcripts present ubiquitously in various tissues, with higher accumulation in flower organs. GbERF1-like was also responsive to defence-related phytohormones and V. dahliae infection. The downregulation of GbERF1-like increased the susceptibility of cotton plants to V. dahliae infection, while overexpression of this gene improved disease resistance in both cotton and Arabidopsis, coupled with activation of the pathogenesis-related proteins. Further analysis revealed that genes involved in lignin synthesis, such as PAL, C4H, C3H, HCT, CCoAOMT, CCR and F5H, showed higher expression levels in the overexpressing cotton and Arabidopsis lines and lower expression levels in the RNAi cotton lines. The expression levels of these genes increased obviously when the GbERF1-like-overexpressing plants were inoculated with V. dahliae. Meanwhile, significant differences in the content of whole lignin could be found in the stems of transgenic and wild-type plants after inoculation with V. dahliae, as revealed by metabolic and histochemical analysis. More lignin could be detected in GbERF1-like-overexpressing cotton and Arabidopsis but less in GbERF1-like-silencing cotton compared with wild-type plants. The ratio of S and G monomers in GbERF1-like-overexpressing cotton and Arabidopsis increased significantly after infection by V. dahliae. Moreover, our results showed that the promoters of GhHCT1 and AtPAL3 could be transactivated by GbERF1-like in vivo based on yeast one-hybrid assays and dual-luciferase reporter assays. Knockdown of GhHCT1 in GbERF1-like over-expressing cotton decreases resistance to V. dahliae. Collectively, our results suggest that GbERF1-like acts as a positive regulator in lignin synthesis and contributes substantially to resistance to V. dahliae in plants.

Evidence of the late lignification of the G-layer in Simarouba tension wood, to assist understanding how non-G-layer species produce tensile stress.

To recover verticality after disturbance, angiosperm trees produce 'tension wood' allowing them to bend actively. The driving force of the tension has been shown to take place in the G-layer, a specific unlignified layer of the cell wall observed in most temperate species. However, in tropical rain forests, the G-layer is often absent and the mechanism generating the forces to reorient trees remains unclear. A study was carried out on tilted seedlings, saplings and adult Simarouba amara Aubl. trees-a species known to not produce a G-layer. Microscopic observations were done on sections of normal and tension wood after staining or observed under UV light to assess the presence/absence of lignin. We showed that S. amara produces a cell-wall layer with all of the characteristics typical of G-layers, but that this G-layer can be observed only as a temporary stage of the cell-wall development because it is masked by a late lignification. Being thin and lignified, tension wood fibres cannot be distinguished from normal wood fibres in the mature wood of adult trees. These observations indicate that the mechanism generating the high tensile stress in tension wood is likely to be the same as that in species with a typical G-layer and also in species where the G-layer cannot be observed in mature cells.

[The development, differentiation and composition of flax fiber cells]. / Rozwój, róznicowanie sie i sklad komórek wlókna lnianego.

Having vascular origin, flax fiber belongs to the sclerenchyma (steroids) and its structure is limited to the cell wall. What determines fiber properties is its composition, which in practice means the composition of the secondary cell wall. It consists of four main polymers which constitute approximately 90% of the fiber: cellulose, hemicellulose, pectin, lignin, and a variety of secondary metabolites, proteins, waxes and inorganic compounds. The cell wall is a structure with a high complexity of both the composition and interactions of the particular elements between themselves. It is determined by differentiation and cell growth as well as environmental factors, biotic and abiotic stresses. The molecular background of these processes and mechanisms regulating the synthesis and rearrangement of secondary cell walls components are being intensively studied. In this work we described the latest news about the development, composition and metabolism of flax fiber cell wall components together with the molecular explanation of these processes.

Thermophysical properties of lignocellulose: a cell-scale study down to 41 K.

Thermal energy transport is of great importance in lignocellulose pyrolysis for biofuels. The thermophysical properties of lignocellulose significantly affect the overall properties of bio-composites and the related thermal transport. In this work, cell-scale lignocellulose (mono-layer plant cells) is prepared to characterize their thermal properties from room temperature down to ∼ 40 K. The thermal conductivities of cell-scale lignocellulose along different directions show a little anisotropy due to the cell structure anisotropy. It is found that with temperature going down, the volumetric specific heat of the lignocellulose shows a slower decreasing trend against temperature than microcrystalline cellulose, and its value is always higher than that of microcrystalline cellulose. The thermal conductivity of lignocellulose decreases with temperature from 243 K to 317 K due to increasing phonon-phonon scatterings. From 41 K to 243 K, the thermal conductivity rises with temperature and its change mainly depends on the heat capacity's change.

Topochemical and transmission electron microscopic studies of bacterial decay in pine (Pinus sylvestris L.) harbour foundation piles.

Bacterial degradation of Pinus sylvestris harbour foundation piles was studied topochemically by scanning UV-microspectrophotometry. This analytical technique enables direct imaging of lignin distribution within individual cell wall layers. Additionally, light and transmission electron microscopy (TEM) was used to characterise structural changes of the cell walls. Various decay stages were found in the samples. TEM revealed that the bacterial degradation occurred mainly in the S2, leaving granular remnants in degraded wall portions with lower as well as higher electron density than the surrounding unmodified wall. In the initial stages, topochemical investigations revealed that lignin modification starts in the innermost parts of the secondary wall, most clearly observed in latewood tracheids. During advanced degradation, lignin modification occurs more or less severe in walls of all cell types. However, even in cell portions with intensive decay, the compound middle lamellae and ray tracheids were undegraded. The knowledge about lignin modification at initial stages of wood degradation by bacteria is of fundamental importance to provide more information on the process of cell wall decay.

Anatomy and lignification status of the Lophanthera lactescens Ducke plant grown in vitro.

This aim of this study was to evaluate the lignification in young stems of the Lophanthera lactescens Ducke plant grown in vitro L. lactescens (Malpighiaceae), a species endemic in the Brazilian Amazon that possesses both medicinal properties and could be used in the forest product industry. Plants grown in vitro condition in MS medium were analyzed using Infrared (IR) microspectroscopy in a diffuse reflectance mode, fluorescence microscopy. Moreover, histochemical tests such as the Wiesner and Maüle test were run to monitor the process of lignification in cell walls. The spectra of IR microscopy recorded using cross section tissue are representative of guaiacyl/syringyl lignin, based on the 1336 and 1246 cm-1 signal. Individuals presenting stem sprains, resulting from the marked development, produced gelatinous fibers with a clear cellulose layer. Initially, fluorescence microscopy demonstrated lignin deposition in the cell corner region having progressive deposition in the secondary wall of tracheary elements of the stem.

Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without suberin.

Casparian strips are ring-like cell-wall modifications in the root endodermis of vascular plants. Their presence generates a paracellular barrier, analogous to animal tight junctions, that is thought to be crucial for selective nutrient uptake, exclusion of pathogens, and many other processes. Despite their importance, the chemical nature of Casparian strips has remained a matter of debate, confounding further molecular analysis. Suberin, lignin, lignin-like polymers, or both, have been claimed to make up Casparian strips. Here we show that, in Arabidopsis, suberin is produced much too late to take part in Casparian strip formation. In addition, we have generated plants devoid of any detectable suberin, which still establish functional Casparian strips. In contrast, manipulating lignin biosynthesis abrogates Casparian strip formation. Finally, monolignol feeding and lignin-specific chemical analysis indicates the presence of archetypal lignin in Casparian strips. Our findings establish the chemical nature of the primary root-diffusion barrier in Arabidopsis and enable a mechanistic dissection of the formation of Casparian strips, which are an independent way of generating tight junctions in eukaryotes.

Anatomy and lignification status of the Lophanthera lactescens Ducke plant grown in vitro / Anatomia e status da lignificação de plantas de Lophanthera lactescens Ducke crescidas in vitro

This aim of this study was to evaluate the lignification in young stems of the Lophanthera lactescens Ducke plant grown in vitro L. lactescens (Malpighiaceae), a species endemic in the Brazilian Amazon that possesses both medicinal properties and could be used in the forest product industry. Plants grown in vitro condition in MS medium were analyzed using Infrared (IR) microspectroscopy in a diffuse reflectance mode, fluorescence microscopy. Moreover, histochemical tests such as the Wiesner and Maüle test were run to monitor the process of lignification in cell walls. The spectra of IR microscopy recorded using cross section tissue are representative of guaiacyl/syringyl lignin, based on the 1336 and 1246 cm-1 signal. Individuals presenting stem sprains, resulting from the marked development, produced gelatinous fibers with a clear cellulose layer. Initially, fluorescence microscopy demonstrated lignin deposition in the cell corner region having progressive deposition in the secondary wall of tracheary elements of the stem.

Esta pesquisa teve como objetivo avaliar a lignificação em caules jovens de plantas de Lophanthera lactescens Ducke crescidas in vitro. L. lactescens (Malpighiaceae) é uma espécie endêmica da Amazônia Brasileira que, além de propriedades medicinais, apresenta potencial para a indústria de produtos florestais. As plantas foram crescidas in vitro, usando-se meio nutritivo MS. As análises foram realizadas por microespectrometria no infravermelho, no modo reflectância difusa. Microscopia com fluorescência e testes histoquímicos de Wiesner e Maüle foram realizados para o acompanhamento do processo de lignificação. Os espectros registrados a partir da microscopia no infravermelho mostraram sinais em 1336 e 1246 cm-1, característicos de ligninas siringílica e guaiacílica. Indivíduos que apresentaram entorses no caule, em decorrência do acentuado desenvolvimento, produziram fibras gelatinosas, com a camada de celulose evidente. A microscopia com fluorescência evidenciou deposição de lignina inicialmente nos ângulos das células, com depósito progressivo ocorrendo na parede secundária dos elementos traqueais do caule.

Effects of drying-induced fiber hornification on enzymatic saccharification of lignocelluloses.

This study investigated the effect of fiber hornification during drying on lignocellulosic substrate enzymatic saccharification. Two chemically pretreated wood substrates and one commercial bleached kraft hardwood pulp were used. Heat drying at 105 and 150°C and air drying at 50% RH and 23.8°C for different durations were applied to produce substrate with various degrees of hornification. It was found that substrate enzymatic digestibilities (SEDs) of hornified substrates made from the same never-dried sample correlate very well to an easily measurable parameter, water retention value (WRV), and can be fitted by a Boltzmann function. The hornification-produced SED reduction at a given degree of hornification as the percentage of the total SED reduction when the substrate is completely hornified depends on two parameters. The first is WRV¯, which is primarily a function of the effective enzyme molecule size, and Δ, which is related to the substrate pore size distribution shape. The low values of SED(CH), SED of a completely hornified substrate, obtained from curve fittings for the three sets of samples studied, suggest that enzyme accessibility to cellulose is mainly through the pores in the cell wall rather than substrate external surface. The SEDs of hornified substrates were found to correlate to Simons' staining measurements well. A new parameter was proposed to better correlate enzyme accessibility to cellulose using the two-color Simons' staining technique.

Carbon isotope discrimination in western hemlock and its relationship to mineral nutrition and growth.

Western hemlock (Tsuga heterophylla [Raf.] Sarg.) is a major component of temperate rainforests in coastal British Columbia. Forest fertilization can enhance the growth of forest trees, but results are inconsistent for western hemlock. We investigated the relationship between delta13C (foliage and stemwood), growth response and tree nutritional status in this species. To establish a sampling protocol for stemwood, we first assessed spot-to-spot variation around and along the bole, which exceeded 1 per thousand. We utilized the reaction wood (high lignin content) and adjacent normal wood in two curved western hemlock stems to evaluate whether this variation was related to wood composition. There was a consistent 3.43 per thousand difference between lignin and holocellulose, but the isotopic mass balance of whole wood was conserved and, therefore, did not vary with lignin content. Therefore, extraction of cellulose or holocellulose prior to analysis can introduce (not remove) bias. In a detailed study of a third stem, circumferential and longitudinal variation in delta13C was associated with spiral grain indicating limited physiological mixing of isotopic signatures originating from the crown. Wood was subsequently pooled from four cardinal positions around each stem. Eight even-aged western hemlock stands were selected and fertilized with different combinations of nitrogen (N), phosphorous (P) and a blend of S, K, Mg, Zn and Cu. Fertilization was effective in increasing foliar N, P, K and S depending on treatment. At the end of the first growing season after fertilization, the effect of treatments on foliar delta13C was nearly significant (P = 0.054), but did not persist into a second year. Effects on tree-ring delta13C were more obvious and persisted for about 3 years, averaging approximately 0.2-0.4 per thousand over this period, depending on treatment. Combinations of N, P and blend had the greatest effect, consistent with relative increases in basal area increment. Effects of fertilizer additions on delta13C, though clear, were superimposed on larger site and annual weather-related patterns in delta13C. Large tree-to-tree variation in delta13C was positively correlated with basal area increment, both before and after treatment imposition, suggesting that high water-use efficiencies are associated with greater growth.