Half of the 18O enrichment of leaf sucrose is conserved in leaf cellulose of a C3 grass across atmospheric humidity and CO2 levels.

The 18O enrichment (Δ18O) of cellulose (Δ18OCel) is recognized as a unique archive of past climate and plant function. However, there is still uncertainty regarding the proportion of oxygen in cellulose (pex) that exchanges post-photosynthetically with medium water of cellulose synthesis. Particularly, recent research with C3 grasses demonstrated that the Δ18O of leaf sucrose (Δ18OSuc, the parent substrate for cellulose synthesis) can be much higher than predicted from daytime Δ18O of leaf water (Δ18OLW), which could alter conclusions on photosynthetic versus post-photosynthetic effects on Δ18OCel via pex. Here, we assessed pex in leaves of perennial ryegrass (Lolium perenne) grown at different atmospheric relative humidity (RH) and CO2 levels, by determinations of Δ18OCel in leaves, Δ18OLGDZW (the Δ18O of water in the leaf growth-and-differentiation zone) and both Δ18OSuc and Δ18OLW (adjusted for εbio, the biosynthetic fractionation between water and carbohydrates) as alternative proxies for the substrate for cellulose synthesis. Δ18OLGDZW was always close to irrigation water, and pex was similar (0.53 ± 0.02 SE) across environments when determinations were based on Δ18OSuc. Conversely, pex was erroneously and variably underestimated (range 0.02-0.44) when based on Δ18OLW. The photosynthetic signal fraction in Δ18OCel is much more constant than hitherto assumed, encouraging leaf physiological reconstructions.

18 O enrichment of sucrose and photosynthetic and nonphotosynthetic leaf water in a C3 grass-atmospheric drivers and physiological relations.

The 18 O enrichment (Δ18 O) of leaf water affects the Δ18 O 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 Δ18 O of bulk leaf water (Δ18 OLW ) and leaf sucrose (Δ18 OSucrose ). 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 Δ18 OLW , Δ18 OSucrose and morphophysiological leaf parameters, including transpiration (Eleaf ), stomatal conductance (gs ) and mesophyll conductance to CO2 (gm ). The Δ18 O of photosynthetic medium water (Δ18 OSSW ) was estimated from Δ18 OSucrose and the equilibrium fractionation between water and carbonyl groups (εbio ). Δ18 OSSW was well predicted by theoretical estimates of leaf water at the evaporative site (Δ18 Oe ) 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. Δ18 OLW was a poor proxy for Δ18 OSucrose , mainly due to opposite Δ18 O responses of nonphotosynthetic tissue water (Δ18 Onon-SSW ) relative to Δ18 OSSW , driven by atmospheric conditions.

Do 2 H and 18 O in leaf water reflect environmental drivers differently?

We compiled hydrogen and oxygen stable isotope compositions (δ2 H and δ18 O) of leaf water from multiple biomes to examine variations with environmental drivers. Leaf water δ2 H was more closely correlated with δ2 H of xylem water or atmospheric vapour, whereas leaf water δ18 O was more closely correlated with air relative humidity. This resulted from the larger proportional range for δ2 H of meteoric waters relative to the extent of leaf water evaporative enrichment compared with δ18 O. We next expressed leaf water as isotopic enrichment above xylem water (Δ2 H and Δ18 O) to remove the impact of xylem water isotopic variation. For Δ2 H, leaf water still correlated with atmospheric vapour, whereas Δ18 O showed no such correlation. This was explained by covariance between air relative humidity and the Δ18 O 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 2 H and 18 O 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.

Stomatal conductance limited the CO2 response of grassland in the last century.

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.

Temperature-sensitive biochemical 18 O-fractionation and humidity-dependent attenuation factor are needed to predict δ18 O of cellulose from leaf water in a grassland ecosystem.

We explore here our mechanistic understanding of the environmental and physiological processes that determine the oxygen isotope composition of leaf cellulose (δ18 Ocellulose ) in a drought-prone, temperate grassland ecosystem. A new allocation-and-growth model was designed and added to an 18 O-enabled soil-vegetation-atmosphere transfer model (MuSICA) to predict seasonal (April-October) and multi-annual (2007-2012) variation of δ18 Ocellulose and 18 O-enrichment of leaf cellulose (Δ18 Ocellulose ) based on the Barbour-Farquhar model. Modelled δ18 Ocellulose agreed best with observations when integrated over c. 400 growing-degree-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 δ18 Ocellulose (R2  = 0.57) and Δ18 Ocellulose (R2  = 0.74), and their negative relationship with canopy conductance, was improved significantly when both the biochemical 18 O-fractionation between water and substrate for cellulose synthesis (εbio , range 26-30‰) was temperature-sensitive, as previously reported for aquatic plants and heterotrophically grown wheat seedlings, and the proportion of oxygen in cellulose reflecting leaf water 18 O-enrichment (1 - pex px , range 0.23-0.63) was dependent on air relative humidity, as observed in independent controlled experiments with grasses. Understanding physiological information in δ18 Ocellulose requires quantitative knowledge of climatic effects on pex px and εbio .

Atmospheric CO2 and VPD alter the diel oscillation of leaf elongation in perennial ryegrass: compensation of hydraulic limitation by stored-growth.

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.