Greater mesophyll conductance and leaf photosynthesis in the field through modified cell wall porosity and thickness via AtCGR3 expression in tobacco.

Mesophyll conductance (gm) describes the ease with which CO2 passes from the sub-stomatal cavities of the leaf to the primary carboxylase of photosynthesis, Rubisco. Increasing gm is suggested as a means to engineer increases in photosynthesis by increasing [CO2] at Rubisco, inhibiting oxygenation and accelerating carboxylation. Here, tobacco was transgenically up-regulated with Arabidopsis Cotton Golgi-related 3 (CGR3), a gene controlling methylesterification of pectin, as a strategy to increase CO2 diffusion across the cell wall and thereby increase gm. Across three independent events in tobacco strongly expressing AtCGR3, mesophyll cell wall thickness was decreased by 7%-13%, wall porosity increased by 75% and gm measured by carbon isotope discrimination increased by 28%. Importantly, field-grown plants showed an average 8% increase in leaf photosynthetic CO2 uptake. Up-regulating CGR3 provides a new strategy for increasing gm in dicotyledonous crops, leading to higher CO2 assimilation and a potential means to sustainable crop yield improvement.

The response of mesophyll conductance to short-term CO2 variation is related to stomatal conductance.

The response of mesophyll conductance (gm) to CO2 plays a key role in photosynthesis and ecosystem carbon cycles under climate change. Despite numerous studies, there is still debate about how gm responds to short-term CO2 variations. Here we used multiple methods and looked at the relationship between stomatal conductance to CO2 (gsc) and gm to address this aspect. We measured chlorophyll fluorescence parameters and online carbon isotope discrimination (Δ) at different CO2 mole fractions in sunflower (Helianthus annuus L.), cowpea (Vigna unguiculata L.), and wheat (Triticum aestivum L.) leaves. The variable J and Δ based methods showed that gm decreased with an increase in CO2 mole fraction, and so did stomatal conductance. There were linear relationships between gm and gsc across CO2 mole fractions. gm obtained from A-Ci curve fitting method was higher than that from the variable J method and was not representative of gm under the growth CO2 concentration. gm could be estimated by empirical models analogous to the Ball-Berry model and the USO model for stomatal conductance. Our results suggest that gm and gsc respond in a coordinated manner to short-term variations in CO2, providing new insight into the role of gm in photosynthesis modelling.

Differential growth rate, water use efficiency and climate sensitivity between males and females of Ilex aquifolium in north-western Spain.

BACKGROUND AND AIMS: Dioecious plant species, i.e., those in which male and female functions are housed in different individuals, are particularly vulnerable to global environmental changes. For long-lived plant species, such as trees, long-term studies are imperative to understand how growth patterns and their sensitivity to climate variability differentially affect the sexes. METHODS: Here, we explore long-term intersexual differences in wood traits, namely radial growth rates, water use efficiency quantified as stable carbon isotope abundance of wood cellulose, and their climate sensitivity in Ilex aquifolium trees growing in a natural population in NW Spain. KEY RESULTS: We found that sex differences in secondary growth rates were variable over time, with males outperforming females in both radial growth rates and water use efficiency in recent decades. Summer water stress significantly reduced the growth of female trees in the following growing season, while the growth of male trees was primarily favoured by cloudy and rainy conditions the previous fall and winter combined with low cloud cover and warm conditions in summer. Sex-dependent lagged correlations between radial growth and water availability were found, with a strong association between tree growth and cumulative water availability in females at 30 months and in males at 10 months. CONCLUSIONS: Overall, our results point to greater vulnerability of female tress to increasing drought, which could lead to sex-ratio biases threatening population viability in the future.

Genome-wide association identifies candidate genes for drought tolerance in coast redwood and giant sequoia.

Drought is a major limitation for survival and growth in plants. With more frequent and severe drought episodes occurring due to climate change, it is imperative to understand the genomic and physiological basis of drought tolerance to be able to predict how species will respond in the future. In this study, univariate and multitrait multivariate genome-wide association study methods were used to identify candidate genes in two iconic and ecosystem-dominating species of the western USA, coast redwood and giant sequoia, using 10 drought-related physiological and anatomical traits and genome-wide sequence-capture single nucleotide polymorphisms. Population-level phenotypic variation was found in carbon isotope discrimination, osmotic pressure at full turgor, xylem hydraulic diameter, and total area of transporting fibers in both species. Our study identified new 78 new marker × trait associations in coast redwood and six in giant sequoia, with genes involved in a range of metabolic, stress, and signaling pathways, among other functions. This study contributes to a better understanding of the genomic basis of drought tolerance in long-generation conifers and helps guide current and future conservation efforts in the species.

Phenotypic variation in leaf photosynthetic traits, leaf area index, and carbon discrimination of field-grown wheat genotypes and their relationship with yield performance in Mediterranean environments.

MAIN CONCLUSION: Leaf water potential, gas exchange, and chlorophyll fluorescence exhibited significant differences among genotypes, high environmental effects, but low heritability. The highest-yielding and drought-tolerant genotypes presented superior harvest index and grain weight, compared to drought-susceptible ones. Physiological phenotyping can help identify useful traits related to crop performance under water-limited conditions. A set of fourteen bread wheat genotypes with contrasting grain yield (GY) was studied in eight Mediterranean environments in Chile, resulting from the combination of two sites (Cauquenes and Santa Rosa), two water conditions (rainfed-WL and irrigated-WW), and four growing seasons (2015-2018). The objectives were to (i) evaluate the phenotypic variation of leaf photosynthetic traits after heading (anthesis and grain filling) in different environments; (ii) analyze the relationship between GY and leaf photosynthetic traits and carbon isotope discrimination (Δ13C); and (iii) identify those traits that could have a greater impact in the determination of tolerant genotypes under field conditions. Agronomic traits exhibited significant genotypic differences and genotype × environment (GxE) interaction. The average GY under the WW condition at Santa Rosa was 9.2 Mg ha-1 (range 8.2-9.9 Mg ha-1) and under the WL condition at Cauquenes was 6.2 Mg ha-1 (range 3.7-8.3 Mg ha-1). The GY was closely related to the harvest index (HI) in 14 out of 16 environments, a trait exhibiting a relatively high heritability. In general terms, the leaf photosynthetic traits presented low GxE interaction, but high environmental effects and low heritability, except for the chlorophyll content. The relationships between GY and leaf photosynthetic traits were weaker when performed across genotypes in each environment, indicating low genotypic effects, and stronger when performed across environments for each genotype. The leaf area index and Δ13C also presented high environmental effects and low heritability, and their correlations with GY were influenced by environmental effects. The highest-yielding and drought-tolerant genotypes presented superior HI and grain weight, but no clear differences in leaf photosynthetic traits or Δ13C, compared to drought-susceptible ones. It seems that the phenotypic plasticity of agronomic and leaf photosynthetic traits is very important for crop adaptation to Mediterranean environments.

Domestication has reduced leaf water use efficiency associated with the anatomy of abaxial stomata in cotton.

Crop domestication for increasing growth rates and yields appears to have altered the features of adaxial and abaxial stomata, but its effect on leaf water use efficiency (WUE) have not been experimentally verified. In this study, we characterized stomatal anatomy and carbon isotope discrimination (δ13C) in 32 wild and 36 domesticated genotypes of cotton grown under agricultural field conditions. The results showed that domesticated genotypes possessed lower WUE, as indicated by low or more negative δ13C compared with wild genotypes. Higher theoretical maximum stomatal conductance (gsmax) after domestication was accounted for by more stomata rather than significantly enlarged stomata. Specifically, abaxial stomatal density was higher whilst there was no change in the adaxial density. The size of both adaxial and abaxial stomata was greater due to larger guard cells but without there being any increase in pore size. However, there was a negative relationship between δ13C and stomatal size across wild and domesticated genotypes, especially on the abaxial leaf surface, because bigger stomata resulted in a lower maximum stomatal response rate to fluctuating canopy light, resulting in increased water loss. Overall, our results indicate that cotton domestication has resulted in substantial variation in stomatal anatomy, and that WUE and drought tolerance can potentially be improved in future breeding by decreasing the size of abaxial stomata to produce a faster stomatal response and hence a reduction in unnecessary water loss.

Estimating intra-seasonal photosynthetic discrimination and water use efficiency using δ13C of leaf sucrose in Scots pine.

Sucrose has a unique role in recording environmental and physiological signals during photosynthesis in its carbon isotope composition (δ13C) and transport of the signal to tree rings. Yet, instead of sucrose, total organic matter (TOM) or water-soluble carbohydrates (WSC) are typically analysed in studies that follow δ13C signals within trees. To study how the choice of organic material may bias the interpretation of δ13C records, we used mature field-grown Scots pine (Pinus sylvestris) to compare for the first time δ13C of different leaf carbon pools with δ13C of assimilates estimated by a chamber-Picarro system (δ13CA_Picarro), and a photosynthetic discrimination model (δ13CA_model). Compared with sucrose, the other tested carbon pools, such as TOM and WSC, poorly recorded the seasonal trends or absolute values of δ13CA_Picarro and δ13CA_model. Consequently, in comparison with the other carbon pools, sucrose δ13C was superior for reconstructing changes in intrinsic water use efficiency (iWUE), agreeing in both absolute values and intra-seasonal variations with iWUE estimated from gas exchange. Thus, deriving iWUE and environmental signals from δ13C of bulk organic matter can lead to misinterpretation. Our findings underscore the advantage of using sucrose δ13C to understand plant physiological responses in depth.

Agrivoltaics mitigate drought effects in winter wheat.

Climate change is expected to decrease water availability in many agricultural production areas around the globe. At the same time renewable energy concepts such as agrivoltaics (AV) are necessary to manage the energy transition. Several studies showed that evapotranspiration can be reduced in AV systems, resulting in increased water availability for crops. However, effects on crop performance and productivity remain unclear to date. Carbon-13 isotopic composition (δ13 C and discrimination against carbon-13) can be used as a proxy for the effects of water availability on plant performance, integrating crop responses over the entire growing season. The aim of this study was to assess these effects via carbon isotopic composition in grains, as well as grain yield of winter wheat in an AV system in southwest Germany. Crops were cultivated over four seasons from 2016-2020 in the AV system and on an unshaded adjacent reference (REF) site. Across all seasons, average grain yield did not significantly differ between AV and REF (4.7 vs 5.2 t ha-1 ), with higher interannual yield stability in the AV system. However, δ13 C as well as carbon-13 isotope discrimination differed significantly across the seasons by 1‰ (AV: -29.0‰ vs REF: -28.0‰ and AV: 21.6‰ vs REF: 20.6‰) between the AV system and the REF site. These drought mitigation effects as indicated by the results of this study will become crucial for the resilience of agricultural production in the near future when drought events will become significantly more frequent and severe.

Increased bundle-sheath leakiness of CO2 during photosynthetic induction shows a lack of coordination between the C4 and C3 cycles.

Use of a complete dynamic model of NADP-malic enzyme C4 photosynthesis indicated that, during transitions from dark or shade to high light, induction of the C4 pathway was more rapid than that of C3 , resulting in a predicted transient increase in bundle-sheath CO2 leakiness (ϕ). Previously, ϕ has been measured at steady state; here we developed a new method, coupling a tunable diode laser absorption spectroscope with a gas-exchange system to track ϕ in sorghum and maize through the nonsteady-state condition of photosynthetic induction. In both species, ϕ showed a transient increase to > 0.35 before declining to a steady state of 0.2 by 1500 s after illumination. Average ϕ was 60% higher than at steady state over the first 600 s of induction and 30% higher over the first 1500 s. The transient increase in ϕ, which was consistent with model prediction, indicated that capacity to assimilate CO2 into the C3 cycle in the bundle sheath failed to keep pace with the rate of dicarboxylate delivery by the C4 cycle. Because nonsteady-state light conditions are the norm in field canopies, the results suggest that ϕ in these major crops in the field is significantly higher and energy conversion efficiency lower than previous measured values under steady-state conditions.

Multienvironment QTL analysis delineates a major locus associated with homoeologous exchanges for water-use efficiency and seed yield in canola.

Canola varieties exhibit variation in drought avoidance and drought escape traits, reflecting adaptation to water-deficit environments. Our understanding of underlying genes and their interaction across environments in improving crop productivity is limited. A doubled haploid population was analysed to identify quantitative trait loci (QTL) associated with water-use efficiency (WUE) related traits. High WUE in the vegetative phase was associated with low seed yield. Based on the resequenced parental genome data, we developed sequence-capture-based markers and validated their linkage with carbon isotope discrimination (Δ13 C) in an F2 population. RNA sequencing was performed to determine the expression of candidate genes underlying Δ13 C QTL. QTL contributing to main and QTL × environment interaction effects for Δ13 C and yield were identified. One multiple-trait QTL for Δ13 C, days to flower, plant height, and seed yield was identified on chromosome A09. Interestingly, this QTL region overlapped with a homoeologous exchange (HE) event, suggesting its association with the multiple traits. Transcriptome analysis revealed 121 significantly differentially expressed genes underlying Δ13 C QTL on A09 and C09, including in HE regions. Sorting out the negative relationship between vegetative WUE and seed yield is a priority. Genetic and genomic resources and knowledge so developed could improve canola WUE and yield.

Lack of leaf carbonic anhydrase activity eliminates the C4 carbon-concentrating mechanism requiring direct diffusion of CO2 into bundle sheath cells.

Carbonic anhydrase (CA) performs the first enzymatic step of C4 photosynthesis by catalysing the reversible hydration of dissolved CO2 that diffuses into mesophyll cells from intercellular airspaces. This CA-catalysed reaction provides the bicarbonate used by phosphoenolpyruvate carboxylase to generate products that flow into the C4 carbon-concentrating mechanism (CCM). It was previously demonstrated that the Zea mays ca1ca2 double mutant lost 97% of leaf CA activity, but there was little difference in the growth phenotype under ambient CO2 partial pressures (pCO2 ). We hypothesise that since CAs are among the fastest enzymes, minimal activity from a third CA, CA8, can provide the inorganic carbon needed to drive C4 photosynthesis. We observed that removing CA8 from the maize ca1ca2 background resulted in plants that had 0.2% of wild-type leaf CA activity. These ca1ca2ca8 plants had reduced photosynthetic parameters and could only survive at elevated pCO2 . Photosynthetic and carbon isotope analysis combined with modelling of photosynthesis and carbon isotope discrimination was used to determine if ca1ca2ca8 plants had a functional C4 cycle or were relying on direct CO2 diffusion to ribulose 1,5-bisphosphate carboxylase/oxygenase within bundle sheath cells. The results suggest that leaf CA activity in ca1ca2ca8 plants was not sufficient to sustain the C4 CCM.

Mesophyll conductance is unaffected by expression of Arabidopsis PIP1 aquaporins in the plasmalemma of Nicotiana.

In plants with C3 photosynthesis, increasing the diffusion conductance for CO2 from the substomatal cavity to chloroplast stroma (mesophyll conductance) can improve the efficiencies of both CO2 assimilation and photosynthetic water use. In the diffusion pathway from substomatal cavity to chloroplast stroma, the plasmalemma and chloroplast envelope membranes impose a considerable barrier to CO2 diffusion, limiting photosynthetic efficiency. In an attempt to improve membrane permeability to CO2, and increase photosynthesis in tobacco, we generated transgenic lines in Nicotiana tabacum L. cv Petite Havana carrying either the Arabidopsis PIP1;2 (AtPIP1;2) or PIP1;4 (AtPIP1;4) gene driven by the constitutive dual 2x35S CMV promoter. From a collection of independent T0 transgenics, two T2 lines from each gene were characterized, with western blots confirming increased total aquaporin protein abundance in the AtPIP1;2 tobacco lines. Transient expression of AtPIP1;2-mGFP6 and AtPIP1;4-mGFP6 fusions in Nicotiana benthamiana identified that both AtPIP1;2 and AtPIP1;4 localize to the plasmalemma. Despite achieving ectopic production and correct localization, gas exchange measurements combined with carbon isotope discrimination measurements detected no increase in mesophyll conductance or CO2 assimilation rate in the tobacco lines expressing AtPIP. We discuss the complexities associated with trying to enhance gm through modified aquaporin activity.

isocalcR: An R package to streamline and standardize stable isotope calculations in ecological research.

The use of stable isotopes to characterize ecosystem dynamics and infer leaf gas exchange processes has become increasingly prevalent over the last few decades within the ecological community. While advancements in theory and our understanding of the physiological processes controlling isotopic signatures in plants has been well-documented, no standardized tool currently exists to facilitate the computation of common isotope-derived plant physiological indices. Here, we present isocalcR, an R package intended to facilitate the use of stable isotope data from plant tissues by providing an integrated collection of functions and recommended reference data. The isocalcR R package contains a suite of functions that compute leaf carbon isotope discrimination (∆13 C), leaf intercellular [CO2 ], the ratio of leaf intercellular to atmospheric [CO2 ], the difference between atmospheric and leaf intercellular [CO2 ], and intrinsic water use efficiency from carbon isotope signatures in leaf or wood tissue with minimal inputs from the user. isocalcR also implements and provides recommended input atmospheric [CO2 ] (ppm) and atmospheric δ13 CO2 (‰) data for the period 0-2021 C.E. A major goal of isocalcR is to provide a standardized, open-source tool to streamline the calculation of reproducible physiological indices from stable isotope signatures in plant tissues, incorporating the most up-to-date theory, while simultaneously eliminating potential errors associated with complex calculations. isocalcR can be used for any location globally as long as the user provides information regarding temperature and elevation to the main workhorse functions.

Changes in tree drought sensitivity provided early warning signals to the California drought and forest mortality event.

Climate warming in recent decades has negatively impacted forest health in the western United States. Here, we report on potential early warning signals (EWS) for drought-related mortality derived from measurements of tree-ring growth (ring width index; RWI) and carbon isotope discrimination (∆13 C), primarily focused on ponderosa pine (Pinus ponderosa). Sampling was conducted in the southern Sierra Nevada Mountains, near the epicenter of drought severity and mortality associated with the 2012-2015 California drought and concurrent outbreak of western pine beetle (Dendroctonus brevicomis). At this site, we found that widespread mortality was presaged by five decades of increasing sensitivity (i.e., increased explained variation) of both tree growth and ∆13 C to Palmer Drought Severity Index (PDSI). We hypothesized that increasing sensitivity of tree growth and ∆13 C to hydroclimate constitute EWS that indicate an increased likelihood of widespread forest mortality caused by direct and indirect effects of drought. We then tested these EWS in additional ponderosa pine-dominated forests that experienced varying mortality rates associated with the same California drought event. In general, drier sites showed increasing sensitivity of RWI to PDSI over the last century, as well as higher mortality following the California drought event compared to wetter sites. Two sites displayed evidence that thinning or fire events that reduced stand basal area effectively reversed the trend of increasing hydroclimate sensitivity. These comparisons indicate that reducing competition for soil water and/or decreasing bark beetle host tree density via forest management-particularly in drier regions-may buffer these forests against drought stress and associated mortality risk. EWS such as these could provide land managers more time to mitigate the extent or severity of forest mortality in advance of droughts. Substantial efforts at deploying additional dendrochronological research in concert with remote sensing and forest modeling will aid in forecasting of forest responses to continued climate warming.

Global decadal variability of plant carbon isotope discrimination and its link to gross primary production.

Carbon isotope discrimination (Δ13 C) in C3 woody plants is a key variable for the study of photosynthesis. Yet how Δ13 C varies at decadal scales, and across regions, and how it is related to gross primary production (GPP), are still incompletely understood. Here we address these questions by implementing a new Δ13 C modelling capability in the land-surface model JULES incorporating both photorespiratory and mesophyll-conductance fractionations. We test the ability of four leaf-internal CO2 concentration models embedded in JULES to reproduce leaf and tree-ring (TR) carbon isotopic data. We show that all the tested models tend to overestimate average Δ13 C values, and to underestimate interannual variability in Δ13 C. This is likely because they ignore the effects of soil water stress on stomatal behavior. Variations in post-photosynthetic isotopic fractionations across species, sites and years, may also partly explain the discrepancies between predicted and TR-derived Δ13 C values. Nonetheless, the "least-cost" (Prentice) model shows the lowest biases with the isotopic measurements, and lead to improved predictions of canopy-level carbon and water fluxes. Overall, modelled Δ13 C trends vary strongly between regions during the recent (1979-2016) historical period but stay nearly constant when averaged over the globe. Photorespiratory and mesophyll effects modulate the simulated global Δ13 C trend by 0.0015 ± 0.005‰ and -0.0006 ± 0.001‰ ppm-1 , respectively. These predictions contrast with previous findings based on atmospheric carbon isotope measurements. Predicted Δ13 C and GPP tend to be negatively correlated in wet-humid and cold regions, and in tropical African forests, but positively related elsewhere. The negative correlation between Δ13 C and GPP is partly due to the strong dominant influences of temperature on GPP and vapor pressure deficit on Δ13 C in those forests. Our results demonstrate that the combined analysis of Δ13 C and GPP can help understand the drivers of photosynthesis changes in different climatic regions.

The genetic basis of water-use efficiency and yield in lettuce.

BACKGROUND: Water supply limits agricultural productivity of many crops including lettuce. Identifying cultivars within crop species that can maintain productivity with reduced water supply is a significant challenge, but central to developing resilient crops for future water-limited climates. We investigated traits known to be related to water-use efficiency (WUE) and yield in lettuce, a globally important leafy salad crop, in a recombinant inbred line (RIL) lettuce mapping population, produced from a cross between the cultivated Lactuca sativa L. cv. Salinas and its wild progenitor L. serriola L. RESULTS: Wild and cultivated lettuce differed in their WUE and we observed transgressive segregation in yield and water-use traits in the RILs. Quantitative trait loci (QTL) analysis identified genomic regions controlling these traits under well-watered and droughted conditions. QTL were detected for carbon isotope discrimination, transpiration, stomatal conductance, leaf temperature and yield, controlling 4-23 % of the phenotypic variation. A QTL hotspot was identified on chromosome 8 that controlled carbon isotope discrimination, stomatal conductance and yield under drought. Several promising candidate genes in this region were associated with WUE, including aquaporins, late embryogenesis abundant proteins, an abscisic acid-responsive element binding protein and glutathione S-transferases involved in redox homeostasis following drought stress were also identified. CONCLUSIONS: For the first time, we have characterised the genetic basis of WUE of lettuce, a commercially important and water demanding crop. We have identified promising candidate genomic regions determining WUE and yield under well-watered and water-limiting conditions, providing important pre-breeding data for future lettuce selection and breeding where water productivity will be a key target.

Transgenic chickpea (Cicer arietinum L.) harbouring AtDREB1a are physiologically better adapted to water deficit.

BACKGROUND: Chickpea (Cicer arietinum L.) is the second most widely grown pulse and drought (limiting water) is one of the major constraints leading to about 40-50% yield losses annually. Dehydration responsive element binding proteins (DREBs) are important plant transcription factors that regulate the expression of many stress-inducible genes and play a critical role in improving the abiotic stress tolerance. Transgenic chickpea lines harbouring transcription factor, Dehydration Responsive Element-Binding protein 1A from Arabidopsis thaliana (AtDREB1a gene) driven by stress inducible promoter rd29a were developed, with the intent of enhancing drought tolerance in chickpea. Performance of the progenies of one transgenic event and control were assessed based on key physiological traits imparting drought tolerance such as plant water relation characteristics, chlorophyll retention, photosynthesis, membrane stability and water use efficiency under water stressed conditions. RESULTS: Four transgenic chickpea lines harbouring stress inducible AtDREB1a were generated with transformation efficiency of 0.1%. The integration, transmission and regulated expression were confirmed by Polymerase Chain Reaction (PCR), Southern Blot hybridization and Reverse Transcriptase polymerase chain reaction (RT-PCR), respectively. Transgenic chickpea lines exhibited higher relative water content, longer chlorophyll retention capacity and higher osmotic adjustment under severe drought stress (stress level 4), as compared to control. The enhanced drought tolerance in transgenic chickpea lines were also manifested by undeterred photosynthesis involving enhanced quantum yield of PSII, electron transport rate at saturated irradiance levels and maintaining higher relative water content in leaves under relatively severe soil water deficit. Further, lower values of carbon isotope discrimination in some transgenic chickpea lines indicated higher water use efficiency. Transgenic chickpea lines exhibiting better OA resulted in higher seed yield, with progressive increase in water stress, as compared to control. CONCLUSIONS: Based on precise phenotyping, involving non-invasive chlorophyll fluorescence imaging, carbon isotope discrimination, osmotic adjustment, higher chlorophyll retention and membrane stability index, it can be concluded that AtDREB1a transgenic chickpea lines were better adapted to water deficit by modifying important physiological traits. The selected transgenic chickpea event would be a valuable resource that can be used in pre-breeding or directly in varietal development programs for enhanced drought tolerance under parched conditions.

Spruce beetle outbreak was not driven by drought stress: Evidence from a tree-ring iso-demographic approach indicates temperatures were more important.

Climate change has amplified eruptive bark beetle outbreaks over recent decades, including spruce beetle (Dendroctonus rufipennis). However, for projecting future bark beetle dynamics there is a critical lack of evidence to differentiate how outbreaks have been promoted by direct effects of warmer temperatures on beetle life cycles versus indirect effects of drought on host susceptibility. To diagnose whether drought-induced host-weakening was important to beetle attack success we used an iso-demographic approach in Engelmann spruce (Picea engelmannii) forests that experienced widespread mortality caused by spruce beetle outbreaks in the 1990s, during a prolonged drought across the central and southern Rocky Mountain region. We determined tree death date demography during this outbreak to differentiate early- and late-dying trees in stands distributed across a landscape within this larger regional mortality event. To directly test for a role of drought stress during outbreak initiation we determined whether early-dying trees had greater sensitivity of tree-ring carbon isotope discrimination (∆13 C) to drought compared to late-dying trees. Rather, evidence indicated the abundance and size of host trees may have modified ∆13 C responses to drought. ∆13 C sensitivity to drought did not differ among early- versus late-dying trees, which runs contrary to previously proposed links between spruce beetle outbreaks and drought. Overall, our results provide strong support for the view that irruptive spruce beetle outbreaks across North America have primarily been driven by warming-amplified beetle life cycles whereas drought-weakened host defenses appear to have been a distant secondary driver of these major disturbance events.

Forest restoration treatments in a ponderosa pine forest enhance physiological activity and growth under climatic stress.

As the climate warms, drought will increasingly occur under elevated temperatures, placing forest ecosystems at growing risk of extensive dieback and mortality. In some cases, increases in tree density following early 20th-century fire suppression may exacerbate this risk. Treatments designed to restore historical stand structure and enhance resistance to high-severity fire might also alleviate drought stress by reducing competition, but the duration of these effects and the underlying mechanisms remain poorly understood. To elucidate these mechanisms, we evaluate tree growth, mortality, and tree-ring stable-carbon isotope responses to stand-density reduction treatments with and without prescribed fire in a ponderosa pine forest of western Montana. Moderate and heavier cutting experiments (basal area reductions of 35% and 56%, respectively) were initiated in 1992, followed by prescribed burning in a subset of the thinned units. All treatments led to a growth release that persisted to the time of resampling. The treatments had little effect on climate-growth relationships, but they markedly altered seasonal carbon isotope signals and their relationship to climate. In burned and unburned treatments, carbon isotope discrimination (Δ13 C) increased in the earlywood (EW) and decreased in the latewood (LW) relative to the control. The sensitivity of LW Δ13 C to late-summer climate also increased in all treatments, but not in the control. Such increased sensitivity indicates that the reduction in competition enabled trees to continue to fix carbon for new stem growth, even when the climate became sufficiently stressful to stop new assimilation in slower-growing trees in untreated units. These findings would have been masked had we not separated EW and LW. The importance of faster growth and enhanced carbon assimilation under late-summer climatic stress became evident in the second decade post-treatment, when mountain pine beetle activity increased locally, and tree mortality rates in the controls of both experiments increased to more than twice those in their respective treatments. These findings highlight that, when thinning is used to restore historical forest structure or increase resistance to high-severity fire, there will likely be additional benefits of enhanced growth and physiological activity under climatic stress, and the effects may persist for more than two decades.

The temperature response of mesophyll conductance, and its component conductances, varies between species and genotypes.

The temperature response of mesophyll conductance to CO2 diffusion (gm) has been shown to vary considerably between species but remains poorly understood. Here, we tested the hypothesis that increases in chloroplast surface area with increasing temperature, due to the formation of chloroplast protrusions, caused observed positive responses of gm to temperature. We found no evidence of chloroplast protrusions. Using simultaneous measurements of carbon and oxygen isotope discrimination during photosynthesis to separate total gm (gm13) into cell wall and plasma membrane conductance (gm18) and chloroplast membrane conductance (gcm) components, we explored the temperature response in genotypes of soybean and barley, and sunflower plants grown at differing CO2 concentrations. Differences in the temperature sensitivity of gm18 were found between genotypes and between plants grown at differing CO2 concentration but did not relate to measured anatomical features such as chloroplast surface area or cell wall thickness. The closest fit of modelled gm13 to estimated values was found when cell wall thickness was allowed to decline at higher temperatures and transpiration rates, but it remains to be tested if this decline is realistic. The temperature response of gcm (calculated from the difference between 1/gm13 and 1/gm18) varied between barley genotypes, and was best fitted by an optimal response in sunflower. Taken together, these results indicate that gm is a highly complex trait with unpredictable sensitivity to temperature that varies between species, between genotypes within a single species, with growth environment, between replicate leaves, and even with age for an individual leaf.