Abstract. The oxygen isotope composition (δ18O) of leaf water (δ18Oleaf) is an important determinant of environmental and physiological information found in biological archives, but the system-scale understanding of the propagation of the δ18O of rain through soil and xylem water to δ18Oleaf has not been verified for grassland. Here we report a unique and comprehensive dataset of fortnightly δ18O observations in soil, stem and leaf waters made over seven growing seasons in a temperate, drought-prone, mixed-species grassland. Using the ecohydrology part of a physically based, 18O-enabled soil–plant–atmosphere transfer model (MuSICA), we evaluated our ability to predict the dynamics of δ18O in soil water, the depth of water uptake, and the effects of soil and atmospheric moisture on 18O enrichment of leaf water (Δ18Oleaf) in this ecosystem. The model accurately predicted the δ18O dynamics of the different ecosystem water pools, suggesting that the model generated realistic predictions of the vertical distribution of soil water and root water uptake dynamics. Observations and model predictions indicated that water uptake occurred predominantly from shallow (<20 cm) soil depths throughout dry and wet periods in all years, presumably due (at least in part) to the effects of high grazing pressure on root system turnover and placement. Δ18Oleaf responded to both soil and atmospheric moisture contents and was best described in terms of constant proportions of unenriched and evaporatively enriched water (two-pool model). The good agreement between model predictions and observations is remarkable as model parameters describing the relevant physical features or functional relationships of soil and vegetation were held constant with one single value for the entire mixed-species ecosystem.

 We explore our mechanistic understanding of the environmental and physiological processes that determine the oxygen isotope composition of leaf cellulose (δ 18 O cellulose) in a drought-prone, temperate grassland ecosystem.  A new allocation-and-growth model was designed and added to an 18 O-enabled soilvegetation-atmosphere transfer model (MuSICA) to predict seasonal (April-October) and multiannual (2007-2012) variation of δ 18 O cellulose and 18 O-enrichment of leaf cellulose ( 18 O cellulose) based on the Barbour-Farquhar model.  Modelled δ 18 O cellulose agreed best with observations when integrated over ~400 growingdegree-days, similar to the average leaf-lifespan observed at the site. Over the integration time, air temperature ranged from 7 to 22 °C and midday relative humidity from 47 to 73%. Model agreement with observations of δ 18 O cellulose (R²=0.57) and  18 O cellulose (R²=0.74), and their negative relationship with canopy conductance, was improved significantly when both (1) the biochemical 18 O-fractionation between water and substrate for cellulose synthesis ( bio , range 26-30‰) was temperature-sensitive, as previously reported for aquatic plants and heterotrophically-grown wheat seedlings, and (2) the proportion of oxygen in cellulose reflecting leaf water 18 O-enrichment (1-p ex p x , range 0.23-0.63) was dependent on air relative humidity, as observed in independent controlled experiments with grasses.  Understanding physiological information in δ 18 O cellulose requires quantitative knowledge of climatic effects on p ex p x and  bio .

Summary We explored the effects of atmospheric CO2 concentration (Ca) and vapor pressure deficit (VPD) on putative mechanisms controlling leaf elongation in perennial ryegrass. Plants were grown in stands at a Ca of 200, 400 or 800 μmol mol−1 combined with high (1.17 kPa) or low (0.59 kPa) VPD during the 16 h‐day in well‐watered conditions with reduced nitrogen supply. We measured day : night‐variation of leaf elongation rate (LERday : LERnight), final leaf length and width, epidermal cell number and length, stomatal conductance, transpiration, leaf water potential and water‐soluble carbohydrates and osmotic potential in the leaf growth‐and‐differentiation zone (LGDZ). Daily mean LER or morphometric parameters did not differ between treatments, but LERnight strongly exceeded LERday, particularly at low Ca and high VPD. Across treatments LERday was negatively related to transpiration (R2 = 0.75) and leaf water potential (R2 = 0.81), while LERnight was independent of leaf water potential or turgor. Enhancement of LERnight over LERday was proportional to the turgor‐change between day and night (R2 = 0.93). LGDZ sugar concentration was high throughout diel cycles, providing no evidence of source limitation in any treatment. Our data indicate a mechanism of diel cycling between daytime hydraulic and night‐time stored‐growth controls of LER, buffering Ca and daytime VPD effects on leaf elongation.

Background The anthropogenic increase of atmospheric CO2 concentration (ca) is impacting carbon (C), water, and nitrogen (N) cycles in grassland and other terrestrial biomes. Plant canopy stomatal conductance is a key player in these coupled cycles: it is a physiological control of vegetation water use efficiency (the ratio of C gain by photosynthesis to water loss by transpiration), and it responds to photosynthetic activity, which is influenced by vegetation N status. It is unknown if the ca-increase and climate change over the last century have already affected canopy stomatal conductance and its links with C and N processes in grassland. Results Here, we assessed two independent proxies of (growing season-integrating canopy-scale) stomatal conductance changes over the last century: trends of δ18O in cellulose (δ18Ocellulose) in archived herbage from a wide range of grassland communities on the Park Grass Experiment at Rothamsted (U.K.) and changes of the ratio of yields to the CO2 concentration gradient between the atmosphere and the leaf internal gas space (ca – ci). The two proxies correlated closely (R2 = 0.70), in agreement with the hypothesis. In addition, the sensitivity of δ18Ocellulose changes to estimated stomatal conductance changes agreed broadly with published sensitivities across a range of contemporary field and controlled environment studies, further supporting the utility of δ18Ocellulose changes for historical reconstruction of stomatal conductance changes at Park Grass. Trends of δ18Ocellulose differed strongly between plots and indicated much greater reductions of stomatal conductance in grass-rich than dicot-rich communities. Reductions of stomatal conductance were connected with reductions of yield trends, nitrogen acquisition, and nitrogen nutrition index. Although all plots were nitrogen-limited or phosphorus- and nitrogen-co-limited to different degrees, long-term reductions of stomatal conductance were largely independent of fertilizer regimes and soil pH, except for nitrogen fertilizer supply which promoted the abundance of grasses. Conclusions Our data indicate that some types of temperate grassland may have attained saturation of C sink activity more than one century ago. Increasing N fertilizer supply may not be an effective climate change mitigation strategy in many grasslands, as it promotes the expansion of grasses at the disadvantage of the more CO2 responsive forbs and N-fixing legumes.

The 18O enrichment (Δ18O) of leaf water affects the Δ18O of photosynthetic products such as sucrose, generating an isotopic archive of plant function and past climate. However, uncertainty remains as to whether leaf water compartmentation between photosynthetic and nonphotosynthetic tissue affects the relationship between Δ18O of bulk leaf water (Δ18OLW) and leaf sucrose (Δ18OSucrose). We grew Lolium perenne (a C3 grass) in mesocosm‐scale, replicated experiments with daytime relative humidity (50% or 75%) and CO2 level (200, 400 or 800 μmol mol−1) as factors, and determined Δ18OLW, Δ18OSucrose and morphophysiological leaf parameters, including transpiration (Eleaf), stomatal conductance (gs) and mesophyll conductance to CO2 (gm). The Δ18O of photosynthetic medium water (Δ18OSSW) was estimated from Δ18OSucrose and the equilibrium fractionation between water and carbonyl groups (εbio). Δ18OSSW was well predicted by theoretical estimates of leaf water at the evaporative site (Δ18Oe) with adjustments that correlated with gas exchange parameters (gs or total conductance to CO2). Isotopic mass balance and published work indicated that nonphotosynthetic tissue water was a large fraction (~0.53) of bulk leaf water. Δ18OLW was a poor proxy for Δ18OSucrose, mainly due to opposite Δ18O responses of nonphotosynthetic tissue water (Δ18Onon‐SSW) relative to Δ18OSSW, driven by atmospheric conditions.