Long-term water stress leads to acclimation of drought sensitivity of photosynthetic capacity in xeric but not riparian Eucalyptus species.

BACKGROUND AND AIMS: Experimental drought is well documented to induce a decline in photosynthetic capacity. However, if given time to acclimate to low water availability, the photosynthetic responses of plants to low soil moisture content may differ from those found in short-term experiments. This study aims to test whether plants acclimate to long-term water stress by modifying the functional relationships between photosynthetic traits and water stress, and whether species of contrasting habitat differ in their degree of acclimation. METHODS: Three Eucalyptus taxa from xeric and riparian habitats were compared with regard to their gas exchange responses under short- and long-term drought. Photosynthetic parameters were measured after 2 and 4 months of watering treatments, namely field capacity or partial drought. At 4 months, all plants were watered to field capacity, then watering was stopped. Further measurements were made during the subsequent 'drying-down', continuing until stomata were closed. KEY RESULTS: Two months of partial drought consistently reduced assimilation rate, stomatal sensitivity parameters (g1), apparent maximum Rubisco activity (V'(cmax)) and maximum electron transport rate (J'(max)). Eucalyptus occidentalis from the xeric habitat showed the smallest decline in V'(cmax) and J'(max); however, after 4 months, V'(cmax) and J'(max) had recovered. Species differed in their degree of V'(cmax) acclimation. Eucalyptus occidentalis showed significant acclimation of the pre-dawn leaf water potential at which the V'(cmax) and 'true' V(cmax) (accounting for mesophyll conductance) declined most steeply during drying-down. CONCLUSIONS: The findings indicate carbon loss under prolonged drought could be over-estimated without accounting for acclimation. In particular, (1) species from contrasting habitats differed in the magnitude of V'(cmax) reduction in short-term drought; (2) long-term drought allowed the possibility of acclimation, such that V'(cmax) reduction was mitigated; (3) xeric species showed a greater degree of V'(cmax) acclimation; and (4) photosynthetic acclimation involves hydraulic adjustments to reduce water loss while maintaining photosynthesis.

Genetic variation in circadian regulation of nocturnal stomatal conductance enhances carbon assimilation and growth.

Circadian resonance, whereby a plant's endogenous rhythms are tuned to match environmental cues, has been repeatedly shown to be adaptive, although the underlying mechanisms remain elusive. Concomitantly, the adaptive value of nocturnal transpiration in C3 plants remains unknown because it occurs without carbon assimilation. These seemingly unrelated processes are interconnected because circadian regulation drives temporal patterns in nocturnal stomatal conductance, with maximum values occurring immediately before dawn for many species. We grew individuals of six Eucalyptus camaldulensis genotypes in naturally lit glasshouses and measured sunset, predawn and midday leaf gas exchange and whole-plant biomass production. We tested whether sunrise anticipation by the circadian clock and subsequent increases in genotype predawn stomatal conductance led to rapid stomatal opening upon illumination, ultimately affecting genotype differences in carbon assimilation and growth. We observed faster stomatal responses to light inputs at sunrise in genotypes with higher predawn stomatal conductance. Moreover, early morning and midday stomatal conductance and carbon assimilation, leaf area and total plant biomass were all positively correlated with predawn stomatal conductance across genotypes. Our results lead to the novel hypothesis that genotypic variation in the circadian-regulated capacity to anticipate sunrise could be an important factor underlying intraspecific variation in tree growth.

Concerted transcription of auxin and carbohydrate homeostasis-related genes underlies improved adventitious rooting of microcuttings derived from far-red treated Eucalyptus globulus Labill mother plants.

Economically important plant species, such as Eucalyptus globulus, are often rooting recalcitrant. We have previously shown that far-red light enrichment applied to E. globulus donor-plants improved microcutting rooting competence and increased rooting zone/shoot carbohydrate ratio. To better understand this developmental response, the relative expression profiles of genes involved in auxin signaling (ARF6, ARF8, AGO1), biosynthesis (YUC3) and transport (AUX1, PIN1, PIN2); sucrose cleavage (SUS1, CWINV1), transport (SUC5), hexose phosphorylation (HXK1, FLN1) and starch biosynthesis (SS3) were quantified during adventitious rooting of E. globulus microcuttings derived from donor plants exposed to far-red or white light. Expression of auxin transport-related genes increased in the first days of root induction. Far-red enrichment of donor plants induced ARF6, ARF8 and AGO1 in microcuttings. The first two gene products could activate GH3 and other rooting related genes, whereas AGO1 deregulation of the repressor ARF17 may relief rooting inhibition. Increased sink strength at the basal stem with sucrose unloading in root tissue mediated by SUC and subsequent hydrolysis by SUS1 were also supported by gene expression profile. Fructose phosphorylation and starch biosynthesis could also contribute to proper carbon allocation at the site of rooting, as evidenced by increased expression of related genes. These data are in good agreement with increased contents of hexoses and starch at the cutting base severed from far-red exposed donor plants. To sum up, pathways integrating auxin and carbohydrate metabolism were activated in microcuttings derived from donor plants exposed to far red light enrichment, thereby improving rooting response in E. globulus.

Diurnal and seasonal variation in light and dark respiration in field-grown Eucalyptus pauciflora.

Respiration from vegetation is a substantial part of the global carbon cycle and the responses of plant respiration to daily and seasonal fluctuations in temperature and light must be incorporated in models of terrestrial respiration to accurately predict these CO2 fluxes. We investigated how leaf respiration (R) responded to changes in leaf temperature (T(leaf)) and irradiance in field-grown saplings of an evergreen tree (Eucalyptus pauciflora Sieb. ex Spreng). Seasonal shifts in the thermal sensitivity of leaf R in the dark (R(dark)) and in the light (R(light)) were assessed by allowing T(leaf) to vary over the day in field-grown leaves over a year. The Q10 of R (i.e., the relative increase in R for a 10 °C increase in T(leaf)) was similar for R(light) and R(dark) and had a value of ∼ 2.5; there was little seasonal change in the Q10 of either R(light) or R(dark), indicating that we may be able to use similar functions to model short-term temperature responses of R in the dark and in the light. Overall, rates of R(light) were lower than those of R(dark), and the ratio of R(light)/R(dark) tended to increase with rising T(leaf), such that light suppression of R was reduced at high T(leaf) values, in contrast to earlier work with this species. Our results suggest we cannot assume that R(light)/R(dark) decreases with increasing T(leaf) on daily timescales, and highlights the need for a better mechanistic understanding of what regulates light suppression of R in leaves.

Structural adjustments in resprouting trees drive differences in post-fire transpiration.

Following disturbance many woody species are capable of resprouting new foliage, resulting in a reduced leaf-to-sapwood area ratio and altered canopy structure. We hypothesized that such changes would promote adjustments in leaf physiology, resulting in higher rates of transpiration per unit leaf area, consistent with the mechanistic framework proposed by Whitehead et al. (Whitehead D, Jarvis PG, Waring RH (1984) Stomatal conductance, transpiration and resistance to water uptake in a Pinus sylvestris spacing experiment. Can J For Res 14:692-700). We tested this in Eucalyptus obliqua L'Hér following a wildfire by comparing trees with unburnt canopies with trees that had been subject to 100% canopy scorch and were recovering their leaf area via resprouting. In resprouting trees, foliage was distributed along the trunk and on lateral branches, resulting in shorter hydraulic path lengths. We evaluated measurements of whole-tree transpiration and structural and physiological traits expected to drive any changes in transpiration. We used these structural and physiological measurements to parameterize the Whitehead et al. equation, and found that the expected ratio of transpiration per unit leaf area between resprouting and unburnt trees was 3.41. This is similar to the observed ratio of transpiration per unit leaf area, measured from sapflow observations, which was 2.89 (i.e., resprouting trees had 188% higher transpiration per unit leaf area). Foliage at low heights (<2 m) was found to be significantly different to foliage in the tree crown (14-18 m) in a number of traits, including higher specific leaf area, midday leaf water potential and higher rates of stomatal conductance and photosynthesis. We conclude that these post-fire adjustments in resprouting trees help to drive increased stomatal conductance and hydraulic efficiency, promoting the rapid return of tree-scale transpiration towards pre-disturbance levels. These transient patterns in canopy transpiration have important implications for modelling stand-level water fluxes in forests capable of resprouting, which is frequently done on the basis of the leaf area index.

A model system to study the lignification process in Eucalyptus globulus.

Recalcitrance of plant biomass is closely related to the presence of the phenolic heteropolymer lignin in secondary cell walls, which has a negative effect on forage digestibility, biomass-to-biofuels conversion and chemical pulping. The genus Eucalyptus is the main source of wood for pulp and paper industry. However, when compared to model plants such as Arabidopsis thaliana and poplar, relatively little is known about lignin biosynthesis in Eucalyptus and only a few genes were functionally characterized. An efficient, fast and inexpensive in vitro system was developed to study lignification in Eucalyptus globulus and to evaluate the potential role of candidate genes in this biological process. Seedlings were grown in four different conditions, in the presence or absence of light and with or without sucrose in the growth medium, and several aspects of lignin metabolism were evaluated. Our results showed that light and, to a lesser extent, sucrose induced lignin biosynthesis, which was followed by changes in S/G ratio, lignin oligomers accumulation and gene expression. In addition, higher total peroxidase activity and differential isoperoxidase profile were observed when seedlings were grown in the presence of light and sucrose. Peptide sequencing allowed the identification of differentially expressed peroxidases, which can be considered potential candidate class III peroxidases involved in lignin polymerization in E. globulus.

Elevated [CO2] does not ameliorate the negative effects of elevated temperature on drought-induced mortality in Eucalyptus radiata seedlings.

It has been reported that elevated temperature accelerates the time-to-mortality in plants exposed to prolonged drought, while elevated [CO(2)] acts as a mitigating factor because it can reduce stomatal conductance and thereby reduce water loss. We examined the interactive effects of elevated [CO(2)] and temperature on the inter-dependent carbon and hydraulic characteristics associated with drought-induced mortality in Eucalyptus radiata seedlings grown in two [CO(2)] (400 and 640 µL L(-1)) and two temperature (ambient and ambient +4 °C) treatments. Seedlings were exposed to two controlled drying and rewatering cycles, and then water was withheld until plants died. The extent of xylem cavitation was assessed as loss of stem hydraulic conductivity. Elevated temperature triggered more rapid mortality than ambient temperature through hydraulic failure, and was associated with larger water use, increased drought sensitivities of gas exchange traits and earlier occurrence of xylem cavitation. Elevated [CO(2)] had a negligible effect on seedling response to drought, and did not ameliorate the negative effects of elevated temperature on drought. Our findings suggest that elevated temperature and consequent higher vapour pressure deficit, but not elevated [CO(2)], may be the primary contributors to drought-induced seedling mortality under future climates.

Carbon dynamics of eucalypt seedlings exposed to progressive drought in elevated [CO2] and elevated temperature.

Elevated [CO2] and temperature may alter the drought responses of tree seedling growth, photosynthesis, respiration and total non-structural carbohydrate (TNC) status depending on drought intensity and duration. Few studies have addressed these important climatic interactions or their consequences. We grew Eucalyptus globulus Labill. seedlings in two [CO2] concentrations (400 and 640 µl l(-1)) and two temperatures (28/17 and 32/21 °C) (day/night) in a sun-lit glasshouse, and grew them in well-watered conditions or exposed them to two drought treatments having undergone different previous water conditions (i.e., rewatered drought and sustained drought). Progressive drought in both drought treatments led to similar limitations in growth, photosynthesis and respiration, but reductions in TNC concentration were not observed. Elevated [CO2] ameliorated the impact of the drought during the moderate drought phase (i.e., Day 63 to Day 79) by increasing photosynthesis and enhancing leaf and whole-plant TNC content. In contrast, elevated temperature exacerbated the impact of the drought during the moderate drought phase by reducing photosynthesis, increasing leaf respiration and decreasing whole-plant TNC content. Extreme drought (i.e., Day 79 to Day 103) eliminated [CO2] and temperature effects on plant growth, photosynthesis and respiration. The combined effects of elevated [CO2] and elevated temperature on moderate drought stressed seedlings were reduced with progressive drought, with no sustained effects on growth despite greater whole-plant TNC content.

Validation of reference genes from Eucalyptus spp. under different stress conditions.

BACKGROUND: The genus Eucalyptus consists of approximately 600 species and subspecies and has a physiological plasticity that allows some species to propagate in different regions of the world. Eucalyptus is a major source of cellulose for paper manufacturing, and its cultivation is limited by weather conditions, particularly water stress and low temperatures. Gene expression studies using quantitative reverse transcription polymerase chain reaction (qPCR) require reference genes, which must have stable expression to facilitate the comparison of the results from analyses using different species, tissues, and treatments. Such studies have been limited in eucalyptus. RESULTS: Eucalyptus globulus Labill, Eucalyptus urograndis (hybrid from Eucalyptus urophylla S.T. Blake X Eucalyptus grandis Hill ex-Maiden) and E. uroglobulus (hybrid from E. urograndis X E. globulus) were subjected to different treatments, including water deficiency and stress recovery, low temperatures, presence or absence of light, and their respective controls. Except for treatment with light, which examined the seedling hypocotyl or apical portion of the stem, the expression analyses were conducted in the apical and basal parts of the stem. To select the best pair of genes, the bioinformatics tools GeNorm and NormFinder were compared. Comprehensive analyses that did not differentiate between species, treatments, or tissue types, showed that IDH (isocitrate dehydrogenase), SAND (SAND protein), ACT (actin), and A-Tub (α-tubulin) genes were the most stable. IDH was the most stable gene in all of the treatments. CONCLUSION: Comparing these results with those of other studies on eucalyptus, we concluded that five genes are stable in different species and experimental conditions: IDH, SAND, ACT, A-Tub, and UBQ (ubiquitin). It is usually recommended a minimum of two reference genes is expression analysis; therefore, we propose that IDH and two others genes among the five identified genes in this study should be used as reference genes for a wide range of conditions in eucalyptus.

Transpiration alters the contribution of autotrophic and heterotrophic components of soil CO2 efflux.

• An unbiased partitioning of autotrophic and heterotrophic components of soil CO(2) efflux is important to estimate forest carbon budgets and soil carbon sequestration. The contribution of autotrophic sources to soil CO(2) efflux (F(A)) may be underestimated during the daytime as a result of internal transport of CO(2) produced by root respiration through the transpiration stream. • Here, we tested the hypothesis that carbon isotope composition of soil CO(2) efflux (δ(FS)) in a Eucalyptus plantation grown on a C(4) soil is enriched during the daytime, which will indicate a decrease in F(A) during the periods of high transpiration. • Mean δ(FS) of soil CO(2) efflux decreased to -25.7‰ during the night and increased to -24.7‰ between 11:00 and 15:00 h when the xylem sap flux density was at its maximum. • Our results indicate a decrease in the contribution of root respiration to soil CO(2) efflux during the day that may be interpreted as a departure of root-produced CO(2) in the transpiration stream, leading to a 17% underestimation of autotrophic contribution to soil CO(2) efflux on a daily timescale.

Light inhibition of leaf respiration in field-grown Eucalyptus saligna in whole-tree chambers under elevated atmospheric CO2 and summer drought.

We investigated whether the degree of light inhibition of leaf respiration (R) differs among large Eucalyptus saligna grown in whole-tree chambers and exposed to present and future atmospheric [CO(2) ] and summer drought. Associated with month-to-month changes in temperature were concomitant changes in R in the light (R(light) ) and darkness (R(dark) ), with both processes being more temperature dependent in well-watered trees than under drought. Overall rates of R(light) and R(dark) were not significantly affected by [CO(2) ]. By contrast, overall rates of R(dark) (averaged across both [CO(2) ]) were ca. 25% lower under drought than in well-watered trees. During summer, the degree of light inhibition of leaf R was greater in droughted (ca. 80% inhibition) than well-watered trees (ca. 50% inhibition). Notwithstanding these treatment differences, an overall positive relationship was observed between R(light) and R(dark) when data from all months/treatments were combined (R(2) = 0.8). Variations in R(light) were also positively correlated with rates of Rubisco activity and nitrogen concentration. Light inhibition resulted in a marked decrease in the proportion of light-saturated photosynthesis respired (i.e. reduced R/A(sat) ). Collectively, these results highlight the need to account for light inhibition when assessing impacts of global change drivers on the carbon economy of tree canopies.

Are gas exchange responses to resource limitation and defoliation linked to source:sink relationships?

Productivity of trees can be affected by limitations in resources such as water and nutrients, and herbivory. However, there is little understanding of their interactive effects on carbon uptake and growth. We hypothesized that: (1) in the absence of defoliation, photosynthetic rate and leaf respiration would be governed by limiting resource(s) and their impact on sink limitation; (2) photosynthetic responses to defoliation would be a consequence of changing source:sink relationships and increased availability of limiting resources; and (3) photosynthesis and leaf respiration would be adjusted in response to limiting resources and defoliation so that growth could be maintained. We tested these hypotheses by examining how leaf photosynthetic processes, respiration, carbohydrate concentrations and growth rates of Eucalyptus globulus were influenced by high or low water and nitrogen (N) availability, and/or defoliation. Photosynthesis of saplings grown with low water was primarily sink limited, whereas photosynthetic responses of saplings grown with low N were suggestive of source limitation. Defoliation resulted in source limitation. Net photosynthetic responses to defoliation were linked to the degree of resource availability, with the largest responses measured in treatments where saplings were ultimately source rather than sink limited. There was good evidence of acclimation to stress, enabling higher rates of C uptake than might otherwise have occurred.

Effect of gamma and e-beam radiation on the essential oils of Thymus vulgaris thymoliferum, Eucalyptus radiata, and Lavandula angustifolia.

The microbiological contamination of raw plant materials is common and may be adequately reduced by radiation processing. This study evaluated the effects of gamma- and e-beam ionizing radiations (25 kGy) on three plants used as food or as medicinal products (Thymus vulgaris L., Eucalyptus radiata D.C., and Lavandula angustifolia Mill.) as well as their effects on extracted or commercial essential oils and pure standard samples. Comparison between irradiated and nonirradiated samples was performed by GC/FID and GC/MS. At the studied doses, gamma and e-beam ionizing radiation did not induce any detectable qualitative or quantitative significant changes in the contents and yields of essential oils immediately after ionizing radiation of plants or commercial essential oils and standards. As the maximum dose tested (25 kGy) is a sterilizing dose (much higher than doses used for decontamination of vegetable drugs), it is likely that even decontamination with lower doses will not modify yields or composition of essential oils of these three plants.

Leaf respiration of snow gum in the light and dark. Interactions between temperature and irradiance.

We investigated the effect of temperature and irradiance on leaf respiration (R, non-photorespiratory mitochondrial CO(2) release) of snow gum (Eucalyptus pauciflora Sieb. ex Spreng). Seedlings were hydroponically grown under constant 20 degrees C, controlled-environment conditions. Measurements of R (using the Laisk method) and photosynthesis (at 37 Pa CO(2)) were made at several irradiances (0-2,000 micromol photons m(-2) s(-1)) and temperatures (6 degrees C-30 degrees C). At 15 degrees C to 30 degrees C, substantial inhibition of R occurred at 12 micromol photons m(-2) s(-1), with maximum inhibition occurring at 100 to 200 micromol photons m(-2) s(-1). Higher irradiance had little additional effect on R at these moderate temperatures. The irradiance necessary to maximally inhibit R at 6 degrees C to 10 degrees C was lower than that at 15 degrees C to 30 degrees C. Moreover, although R was inhibited by low irradiance at 6 degrees C to 10 degrees C, it recovered with progressive increases in irradiance. The temperature sensitivity of R was greater in darkness than under bright light. At 30 degrees C and high irradiance, light-inhibited rates of R represented 2% of gross CO(2) uptake (v(c)), whereas photorespiratory CO(2) release was approximately 20% of v(c). If light had not inhibited leaf respiration at 30 degrees C and high irradiance, R would have represented 11% of v(c). Variations in light inhibition of R can therefore have a substantial impact on the proportion of photosynthesis that is respired. We conclude that the rate of R in the light is highly variable, being dependent on irradiance and temperature.