Hans Schnyder

Fructan stores constitute the most important reserve carbohydrate pools of cool season (C3) grasses, but it is largely unknown how the variation of atmospheric CO2 concentration ([CO2]) between past and projected future levels affects their function in source (photosynthesis) – sink (growth and respiration) relationships. This study addressed this question with perennial ryegrass (Lolium perenne L.) grown at [CO2] of 200, 400 or 800 μmol mol-1 with growth-limiting nitrogen fertilization. Sixty-six days-old vegetative stands were labelled with 13CO2/12CO2 mixtures for 7 days and the 13C-tracer dynamics in whole-shoot sucrose, fructans, glucose and fructose pools evaluated with a four-pool compartmental model of central carbohydrate metabolism. Increasing [CO2] from 200 to 800 μmol mol-1 increased shoot mass 1.6-fold and fructan concentration from 35% to 50% of dry weight, while decreasing the nitrogen nutrition status of stands. Conversely, [CO2] had no effect on the half-lives of water-soluble carbohydrate pools (fructans, ∼7.7 days; sucrose, glucose and fructose, 2.3-4.5 hours). Carbon cycling through the fructan pool was enhanced by [CO2], increasing the mean residence time of carbon in the carbohydrate system from 9 to 14 days between 200 and 800 μmol mol-1 [CO2]. Sucrose resynthesis from breakdown products of fructans (fructose) was extensive and corresponded to 65% and 113% of concurrent sucrose neo-synthesis (or sucrose consumption in growth and respiration) at 200 and 800 μmol mol-1 [CO2], respectively. These results imply a strong constitutive buffering of sucrose availability in the source-sink system of perennial ryegrass (and likely most other C3 grasses) by fructan metabolism.

The C18OO fractionation during photosynthesis (Δ18OA), a powerful isotopic proxy, is strongly influenced by the 18O exchange between CO2 and leaf water. Δ18OA is believed to relate to physiological parameters such as the evaporative 18O enrichment of leaf water (Δe), CO2 influx and efflux; however, the key mechanism remains unclear. Here, we investigated the response of Δ18OA to short-term changes in CO2 levels in three C3 species (Helianthus annuus, Vigna unguiculata and Triticum aestivum) and one C4 species (Cleistogenes squarrosa) grown under different levels of vapor pressure deficit (VPD) and nitrogen supply. We found a significant CO2 effect on Δ18OA for C. squarrosa, but not for the C3 species. Δ18OA was not significantly correlated with Δe in the C3 species, and Δ18OA of the C4 species was not sensitive to changes in Δe driven by VPD. The gross CO2 efflux and Δ18OA were significantly correlated for both C3 and C4 species (r2 = 0.92), demonstrating its role in regulating Δ18OA. We also found that the C3 species had significantly higher Δ18OA than the C4 species, due to the lower ratio of intercellular to atmospheric CO2 (Ci/Ca) in the latter. Δ18OA was significantly (r2 = 0.94) correlated with Ci/Ca, supported by a compiled literature dataset that included 31 species. Our study reveals the distinct difference in Δ18OA between C3 and C4 species and the remarkable relationship between Δ18OA and Ci/Ca, providing new insights into how Δ18OA can be used to infer carbon cycle processes from leaf to ecosystem scales.

Drought events can have long‐lasting effects on plant performance and progeny traits. We investigated how an early, severe drought at the seedling stage affected plant fitness and seed traits in Lolium multiflorum , and whether these responses were modulated by symbiosis with the vertically transmitted fungal endophyte Epichloë occultans . Drought caused significant mortality, and the symbiosis with the endophyte improved survival independently of plant biomass. Surviving plants fully recovered aboveground biomass and seed production only in the presence of the endophyte. Isotopic analyses indicated that only non‐symbiotic plants showed reduced stomatal conductance during seed set, which likely explains their lower seed production. Seeds from drought‐exposed symbiotic plants had higher concentrations of compatible solutes (mannitol and sorbitol) and starch. However, symbiotic seeds from drought‐exposed plants showed reduced germination under intermediate water potential. This response was associated with a drought‐induced increase in the base water potential (Ψ _b ). Alternatively, constant hydrotime was positively associated with starch content. Our results suggest that endophyte symbiosis enables recovery from early drought via osmotic adjustment and photosynthetic maintenance, with intergenerational responses mediated by changes in seed biochemical composition and germination. These findings highlight the role of vertically transmitted endophytes in plant memory of stress and drought resilience across generations.

Background: Quantitative understanding of plant carbon (C) metabolism by ¹³CO₂/¹²CO₂-labelling studies requires absence (or knowledge) of C-isotopic contamination artefacts during tracer application and sample processing. Surprisingly, this concern has not been addressed systematically and comprehensively yet is especially crucial in experiments at different atmospheric CO₂ concentrations ([CO₂]), when experimental protocols require frequent access to the labelling chambers. Here, we used a plant growth chamber-based ¹³CO₂/¹²CO₂ gas exchange-facility to address this topic. The facility comprised four independent units, with two chambers routinely operated in parallel under identical conditions except for the isotopic composition of CO₂ supplied to them (δ¹³C_CO2 −43.5‰ versus −5.6‰). In this setup, dδ¹³C_X (the measurements-based δ¹³C-difference between matching samples X collected from the parallel chambers) is expected to equal dδ¹³C_Ref (the predictable, non-contaminated δ¹³C-difference ), if sample-C is completely derived from the contrasting CO₂ sources. Accordingly, contamination (f _contam) was determined as f _contam = 1– dδ¹³C_X/dδ¹³C_Ref in this experimental setup. Determinations were made for biomass fractions, water-soluble carbohydrate (WSC) components and dark respiration of Lolium perenne (perennial ryegrass) stands following growth for ∼9 weeks at 200, 400 or 800 µmol mol^− 1 CO₂, with a terminal two weeks-long period of extensive experimental disturbance of the chambers. Results: Contamination was small and similar (average 3.3% ±0.9% SD, n = 18) for shoot and root biomass and WSC fractions (fructan, sucrose, glucose, fructose) at every [CO₂] level. [CO₂] had no significant effect on contamination of these samples. There was no evidence for any contamination of WSC components during extraction, separation and analysis. At 200 and 400 µmol mol^− 1 CO₂, contamination of respiratory CO₂ was close to that of biomass- and WSC-C, suggesting it originated primarily from in vivo-contaminated respiratory substrate. Surprisingly, we found no evidence of contamination of respiratory CO₂ at 800 µmol mol^− 1 CO₂. Overall, contamination likely resulted overwhelmingly from photosynthetic fixation of extraneous contaminating CO₂ which entered chambers primarily during daytime experimental activities. Conclusions: The labelling facility enables months-long, quantitative ¹³CO₂/¹²CO₂-labelling of large numbers of plants with accuracy and precision across contrasts of [CO₂], empowering eco-physiological study of climate change scenarios. Effective protocols for contamination avoidance are discussed.

Summary: Carbon isotope discrimination (∆) in leaf biomass (∆_BL) and tree rings (∆_TR) provides important proxies for plant responses to climate change, specifically in terms of intrinsic water‐use efficiency (iWUE). However, the nonphotosynthetic ¹²C/¹³C fractionation in plant tissues has rarely been quantified and its influence on iWUE estimation remains uncertain. We derived a comprehensive, ∆ based iWUE model (iWUE_com) which includes nonphotosynthetic fractionations (d) and characterized tissue‐specific d‐values based on global compilations of data of ∆_BL, ∆_TR and real‐time ∆ in leaf photosynthesis (∆_online). iWUE_com was further validated with independent datasets. ∆_BL was larger than ∆_online by 2.53‰, while ∆_BL and ∆_TR showed a mean offset of 2.76‰, indicating that ∆_TR is quantitatively very similar to ∆_online. Applying the tissue‐specific d‐values (d_BL = 2.5‰, d_TR = 0‰), iWUE estimated from ∆_BL aligned well with those estimated from ∆_TR or gas exchange. ∆_BL and ∆_TR showed a consistent iWUE trend with an average CO₂ sensitivity of 0.15 ppm ppm⁻¹ during 1975–2015. Accounting for nonphotosynthetic fractionations improves the estimation of iWUE based on isotope records in leaf biomass and tree rings, which is ultimate for inferring changes in carbon and water cycles under historical and future climate.

The ¹⁸O enrichment (Δ¹⁸O) of cellulose (Δ¹⁸O_Cel) is recognized as a unique archive of past climate and plant function. However, there is still uncertainty regarding the proportion of oxygen in cellulose (p_ex) that exchanges post‐photosynthetically with medium water of cellulose synthesis. Particularly, recent research with C₃ grasses demonstrated that the Δ¹⁸O of leaf sucrose (Δ¹⁸O_Suc, the parent substrate for cellulose synthesis) can be much higher than predicted from daytime Δ¹⁸O of leaf water (Δ¹⁸O_LW), which could alter conclusions on photosynthetic versus post‐photosynthetic effects on Δ¹⁸O_Cel via p_ex. Here, we assessed p_ex in leaves of perennial ryegrass (Lolium perenne) grown at different atmospheric relative humidity (RH) and CO₂ levels, by determinations of Δ¹⁸O_Cel in leaves, Δ¹⁸O_LGDZW (the Δ¹⁸O of water in the leaf growth‐and‐differentiation zone) and both Δ¹⁸O_Suc and Δ¹⁸O_LW (adjusted for ε_bio, the biosynthetic fractionation between water and carbohydrates) as alternative proxies for the substrate for cellulose synthesis. Δ¹⁸O_LGDZW was always close to irrigation water, and p_ex was similar (0.53 ± 0.02 SE) across environments when determinations were based on Δ¹⁸O_Suc. Conversely, p_ex was erroneously and variably underestimated (range 0.02–0.44) when based on Δ¹⁸O_LW. The photosynthetic signal fraction in Δ¹⁸O_Cel is much more constant than hitherto assumed, encouraging leaf physiological reconstructions.

The ¹³C isotope composition (δ¹³C) of leaf dry matter is a useful tool for physiological and ecological studies. However, how post-photosynthetic fractionation associated with respiration and carbon export influences δ¹³C remains uncertain. We investigated the effects of post-photosynthetic fractionation on δ¹³C of mature leaves of Cleistogenes squarrosa, a perennial C₄ grass, in controlled experiments with different levels of vapour pressure deficit and nitrogen supply. With the increase of leaf age classes, the ¹²C/¹³C fractionation of leaf organic matter relative to the δ¹³C of atmosphere CO₂ (Δ_DM) increased while that of cellulose (Δ_cel) was almost constant. The divergence between Δ_DM and Δ_cel increased with leaf age classes with a maximum value of 1.6‰, indicating the accumulation post-photosynthetic fractionation. Applying a new mass balance model that accounts for respiration and export of photosynthates, we found an apparent ¹²C/¹³C fractionation associated with carbon export of –0.5 to –1.0‰. Different Δ_DM among leaves, pseudostems, daughter tillers and roots indicate that post-photosynthetic fractionation happens at the whole-plant level. Compared with Δ_DM of old leaves, Δ_DM of young leaves and Δ_cel are more reliable proxies for predicting physiological parameters due to the smaller sensitivity to post-photosynthetic fractionation and the similar sensitivity in responses to environmental changes.

Summary: We compiled hydrogen and oxygen stable isotope compositions (δ²H and δ¹⁸O) of leaf water from multiple biomes to examine variations with environmental drivers. Leaf water δ²H was more closely correlated with δ²H of xylem water or atmospheric vapour, whereas leaf water δ¹⁸O was more closely correlated with air relative humidity. This resulted from the larger proportional range for δ²H of meteoric waters relative to the extent of leaf water evaporative enrichment compared with δ¹⁸O. We next expressed leaf water as isotopic enrichment above xylem water (Δ²H and Δ¹⁸O) to remove the impact of xylem water isotopic variation. For Δ²H, leaf water still correlated with atmospheric vapour, whereas Δ¹⁸O showed no such correlation. This was explained by covariance between air relative humidity and the Δ¹⁸O of atmospheric vapour. This is consistent with a previously observed diurnal correlation between air relative humidity and the deuterium excess of atmospheric vapour across a range of ecosystems. We conclude that ²H and ¹⁸O in leaf water do indeed reflect the balance of environmental drivers differently; our results have implications for understanding isotopic effects associated with water cycling in terrestrial ecosystems and for inferring environmental change from isotopic biomarkers that act as proxies for leaf water.