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.

δ13C of bulk organic matter and cellulose reveal post-photosynthetic fractionation during ontogeny in C4 grass leaves.

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 increasing leaf age class, the 12C/13C fractionation of leaf organic matter relative to the δ13C of atmosphere CO2 (ΔDM) increased while that of cellulose (Δcel) was almost constant. The divergence between ΔDM and Δcel increased with leaf age class, with a maximum value of 1.6‰, indicating the accumulation of 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 ΔDM among leaves, pseudostems, daughter tillers, and roots indicate that post-photosynthetic fractionation happens at the whole-plant level. Compared with ΔDM of old leaves, ΔDM of young leaves and Δcel are more reliable proxies for predicting physiological parameters due to the lower sensitivity to post-photosynthetic fractionation and the similar sensitivity in responses to environmental changes.

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.

Natural alleles of the abscisic acid catabolism gene ZmAbh4 modulate water use efficiency and carbon isotope discrimination in maize.

Altering plant water use efficiency (WUE) is a promising approach for achieving sustainable crop production in changing climate scenarios. Here, we show that WUE can be tuned by alleles of a single gene discovered in elite maize (Zea mays) breeding material. Genetic dissection of a genomic region affecting WUE led to the identification of the gene ZmAbh4 as causative for the effect. CRISPR/Cas9-mediated ZmAbh4 inactivation increased WUE without growth reductions in well-watered conditions. ZmAbh4 encodes an enzyme that hydroxylates the phytohormone abscisic acid (ABA) and initiates its catabolism. Stomatal conductance is regulated by ABA and emerged as a major link between variation in WUE and discrimination against the heavy carbon isotope (Δ13C) during photosynthesis in the C4 crop maize. Changes in Δ13C persisted in kernel material, which offers an easy-to-screen proxy for WUE. Our results establish a direct physiological and genetic link between WUE and Δ13C through a single gene with potential applications in maize breeding.

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.

Accounting for mesophyll conductance substantially improves 13 C-based estimates of intrinsic water-use efficiency.

Carbon isotope discrimination (Δ) has been used widely to infer intrinsic water-use efficiency (iWUE) of C3 plants, a key parameter linking carbon and water fluxes. Despite the essential role of mesophyll conductance (gm ) in photosynthesis and Δ, its effect on Δ-based predictions of iWUE has generally been neglected. Here, we derive a mathematical expression of iWUE as a function of Δ that includes gm (iWUEmes ) and exploits the gm -stomatal conductance (gsc ) relationship across drought-stress levels and plant functional groups (deciduous or semideciduous woody, evergreen woody and herbaceous species) in a global database. iWUEmes was further validated with an independent dataset of online-Δ and CO2 and H2 O gas exchange measurements with seven species. Drought stress reduced gsc and gm by nearly one-half across all plant functional groups, but had no significant effect on the gsc  : gm ratio, with a well supported value of 0.79 ± 0.07 (95% CI, n = 198). gm was negatively correlated to iWUE. Incorporating the gsc  : gm ratio greatly improved estimates of iWUE, compared with calculations that assumed infinite gm . The inclusion of the gsc  : gm ratio, fixed at 0.79 when gm was unknown, proved desirable to eliminate significant errors in estimating iWUE from Δ across various C3 vegetation types.

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.

Gross and net nitrogen export from leaves of a vegetative C4 grass.

Nitrogen (N) mobilization from mature leaves plays a key role in supplying amino acids to vegetative and reproductive sinks. However, it is unknown if the mobilized N is predominantly sourced by net N-export (a senescence-related process) or other source of N-export from leaves. We used a new approach to partition gross and net N-export from leaf blades at different developmental stages in Cleistogenes squarrosa (a perennial C4 grass). Net N-export was determined as net loss of leaf N with age, while gross N-export was quantified from isotopic mass balances obtained following 24 h-long 15N-labeling with nitrate on 10-12 developmentally distinct (mature and senescing) leaves of individual major tillers. Net N-export was apparent only in older leaves (leaf no. > 7, with leaves numbered basipetally from the tip of the tiller and leaf no. 2 the youngest fully-expanded leaf), while gross N-export was largely independent of leaf age category and was ∼8.4 times greater than the net N-export of a tiller. At whole-tiller level, N import compensated 88 ±â€¯14 (SE) % of gross N-export of all mature blades leading to a net N-export of 0.51 ±â€¯0.07 (SE) µg h-1 tiller-1. N-import was equivalent to 0.09 ±â€¯0.01 (SE) d-1 of total leaf N, similar to reported rates of leaf protein turnover. Gross N-export from all mature blades of a tiller was ∼1.9-times the total demand of the immature tissues of the same (vegetative) tiller. Significant N-export is evident in all mature blades, and is not limited to senescence conditions, implying a much shorter mean residence time of leaf N than that calculated from net N-export. Gross N-export contributes not only to the N demand of the immature tissues of the same tiller but also to N supply of other sinks, such as newly formed tillers. N dynamics at tiller level is integrated with that of the remainder of the shoot, thus highlights the importance of integration of leaf-, tiller-, and plant-scale N dynamics.

Determination of leaf respiration in the light: comparison between an isotopic disequilibrium method and the Laisk method.

Quantification of leaf respiration is important for understanding plant physiology and ecosystem biogeochemical processes. Leaf respiration continues in the light (RL ) but supposedly at a lower rate than in the dark (RDk ). However, there is no method for direct measurement of RL and the available methods require nonphysiological measurement conditions. A method based on isotopic disequilibrium quantified RL (RL13C ) and mesophyll conductance of young and old fully expanded leaves of six species. RL13C was compared to RL determined by the Laisk method (RL Laisk ) on the very same leaves with a minimum time lag. RL 13C and RL Laisk were generally lower than RDk , and were not significantly affected by leaf ageing. RL Laisk and RL 13C were positively correlated (r2  = 0.35), and both were positively correlated with RDk (r2  ≥ 0.6). RL Laisk was systematically lower than RL 13C by 0.4 µmol m-2  s-1 . Using A/Cc instead of A/Ci curves, a higher photocompensation point Γ* (by 5 µmol mol-1 ) was found but no influence on RL Laisk estimates was observed. The results imply that the Laisk method underestimates actual RL significantly, probably related to the measurement condition of low CO2 and irradiance. The isotopic disequilibrium method is useful for assessing responses of RL to irradiance and CO2 , improving our mechanistic understanding of RL .

Ambient Conditions and Feeding Strategy Influence δ18O of Milk Water in Cows (Bos taurus).

There are increasing concerns by consumers regarding agricultural product traceability and authenticity. Oxygen isotope composition (δ18O) has been used in this context based on the relationship between δ18O of animal products and annual precipitation. However, in dairy products this relationship is affected by the seasonality of δ18O in milk water which in turn depends on the feeding system used. We measured 608 milk samples from 28 farms with various feeding strategies in southern Germany throughout the year, investigating the influences of ambient conditions, drinking water source, and feeding strategies on seasonal variation of δ18O in milk water (δmilk). The mechanistic Munich-Kohn model reflecting these influences predicted the seasonal and farm-specific variation of δmilk well. The relationship between δ18O of precipitation and δmilk varied in different feeding strategies. The interplay of ambient conditions and feeding strategy on δmilk should thus be carefully considered when identifying the origin of milk.

An oxygen isotope chronometer for cellulose deposition: the successive leaves formed by tillers of a C4 perennial grass.

Multiannual time series of (palaeo)hydrological information can be reconstructed from the oxygen isotope composition of cellulose (δ18 OCel ) in biological archives, for example, tree rings, but our ability to temporally resolve information at subannual scale is limited. We capitalized on the short and predictable leaf appearance interval (2.4 d) of a perennial C4 grass (Cleistogenes squarrosa), to assess its potential for providing highly time-resolved δ18 OCel records of vapour pressure deficit (VPD). Plants grown at low (0.63 kPa) or high (1.58 kPa) VPD were swapped between VPD environments and exposed to the new environment for 7 d with simultaneous 13 CO2 labelling. Then, leaves were sampled by age/position along individual tillers. Five leaves at different developmental stages were growing simultaneously. The period of most-active leaf elongation, from 10 to 90% of final length, lasted 6.6 d, and ~80% of all carbon and oxygen incorporation in whole-leaf cellulose occurred within 7 d. Cellulose deposition stopped at (or shortly after) full leaf expansion. The direction of change, low-to-high or high-to-low VPD, had no differential effect on new oxygen and carbon incorporation in cellulose. Successive leaves produced by tillers of C. squarrosa provide a δ18 OCel record useful for reconstructions of short-term hydrological dynamics.

The δ18 O and δ2 H of water in the leaf growth-and-differentiation zone of grasses is close to source water in both humid and dry atmospheres.

The oxygen and hydrogen isotope composition of water in the leaf growth-and-differentiation zone, LGDZ, (δ18 OLGDZ , δ2 HLGDZ ) of grasses influences the isotopic composition of leaf cellulose (oxygen) and wax (hydrogen) - important for understanding (paleo)environmental and physiological information contained in these biological archives - but is presently unknown. This work determined δ18 OLGDZ and δ2 HLGDZ , 18 O- and 2 H-enrichment of LGDZ (∆18 OLGDZ and ∆2 HLGDZ ), and the 18 O- and 2 H-enrichment of leaf blade water (∆18 OLW, ∆2 HLW ) in two C3 and three C4 grasses grown at high and low vapor pressure deficit (VPD). The proportion of unenriched water (px ) in the LGDZ ranged from 0.9 to 1.0 for 18 O and 1.0 to 1.2 for 2 H. VPD had no effect on the proportion of 18 O- and 2 H-enriched water in the LGDZ, and species effects were small or nonsignificant. Deuterium discrimination caused depletion of 2 H in LGDZ water, increasing (apparent) px -values > 1.0 in some cases. The isotopic composition of water in the LGDZ was close to that of source water, independent of VPD and much less enriched than previously supposed, but similar to reported xylem water in trees. The well-constrained px will be useful in future investigations of oxygen and hydrogen isotopic fractionation during cellulose and wax synthesis, respectively.

Bundle-sheath leakiness and intrinsic water use efficiency of a perennial C4 grass are increased at high vapour pressure deficit during growth.

Bundle-sheath leakiness (ϕ) is a key parameter of the CO2-concentrating mechanism of C4 photosynthesis and is related to leaf-level intrinsic water use efficiency (WUEi). This work studied short-term dynamic responses of ϕ to alterations of atmospheric CO2 concentration in Cleistogenes squarrosa, a perennial grass, grown at high (1.6 kPa) or low (0.6 kPa) vapour pressure deficit (VPD) combined with high or low N supply in controlled environment experiments. ϕ was determined by concurrent measurements of photosynthetic gas exchange and on-line carbon isotope discrimination, using a new protocol. Growth at high VPD led to an increase of ϕ by 0.13 and a concurrent increase of WUEi by 14%, with similar effects at both N levels. ϕ responded dynamically to intercellular CO2 concentration (C i), increasing with C i Across treatments, ϕ was negatively correlated to the ratio of CO2 saturated assimilation rate to carboxylation efficiency (a proxy of the relative activities of Rubisco and phosphoenolpyruvate carboxylase) indicating that the long-term environmental effect on ϕ was related to the balance between C3 and C4 cycles. Our study revealed considerable dynamic and long-term variation in ϕ of C. squarrosa, suggesting that ϕ should be determined when carbon isotope discrimination is used to assess WUEi Also, the data indicate a trade-off between WUEi and energetic efficiency in C. squarrosa.

Atmospheric CO2 mole fraction affects stand-scale carbon use efficiency of sunflower by stimulating respiration in light.

Plant carbon-use-efficiency (CUE), a key parameter in carbon cycle and plant growth models, quantifies the fraction of fixed carbon that is converted into net primary production rather than respired. CUE has not been directly measured, partly because of the difficulty of measuring respiration in light. Here, we explore if CUE is affected by atmospheric CO2 . Sunflower stands were grown at low (200 µmol mol-1 ) or high CO2 (1000 µmol mol-1 ) in controlled environment mesocosms. CUE of stands was measured by dynamic stand-scale 13 C labelling and partitioning of photosynthesis and respiration. At the same plant age, growth at high CO2 (compared with low CO2 ) led to 91% higher rates of apparent photosynthesis, 97% higher respiration in the dark, yet 143% higher respiration in light. Thus, CUE was significantly lower at high (0.65) than at low CO2 (0.71). Compartmental analysis of isotopic tracer kinetics demonstrated a greater commitment of carbon reserves in stand-scale respiratory metabolism at high CO2 . Two main processes contributed to the reduction of CUE at high CO2 : a reduced inhibition of leaf respiration by light and a diminished leaf mass ratio. This work highlights the relevance of measuring respiration in light and assessment of the CUE response to environment conditions.

Effects of nitrogen and vapour pressure deficit on phytomer growth and development in a C4 grass.

Phytomers are basic morphological units of plants. Knowledge of phytomer development is essential for understanding morphological plasticity, functional-structural modelling of plant growth and the usage of leaf characteristics to indicate growth conditions at the time of production (e.g. stable isotope signals). Yet, systematic analysis on the process of phytomer development is unavailable for wild or perennial C4 grasses. Also, effects of environmental factors, such as nitrogen nutrition or vapour pressure deficit (VPD), on coordination events of developmental processes of C4 grasses have not been studied. This study investigates phytomer growth and development in Cleistogenes squarrosa, a predominant C4 grass in the Eurasian steppe, grown at low (0.63 kPa) or high (1.58 kPa) VPD with low or high nitrogen supply in controlled environments. Elongation of phytomers on marked tillers was measured daily for 13 days. Then lengths of immature and mature phytomer components (blade, sheath and internode) of all phytomers were measured following dissection. Nitrogen nutrition and VPD had no effects on coordination of growth within and between phytomers: phytomer tips emerged when phytomers reached 26 % of their final length, coincident with the acceleration phase of its elongation; blade elongation stopped when phytomers reached ∼75 % of their final length and elongation of the preceding phytomer was confined to the internode. The relationship between fraction of final phytomer length and days after tip emergence for all treatments was well described by a sigmoidal function: y = 1/{1 + exp[(1.82 - x)/1.81]}. C. squarrosa exhibited little morphological plasticity at phytomer-level in response to nitrogen supply and VPD, but a clear increase in tillering under high N supply. Also, the invariant coordination of elongation within and between phytomers was a stable developmental feature, thus the quantitative coordination rules are applicable for predicting morphological development of C. squarrosa under contrasting levels of nitrogen nutrition or VPD.

Nitrogen fertilization and δ18 O of CO2 have no effect on 18 O-enrichment of leaf water and cellulose in Cleistogenes squarrosa (C4 ) - is VPD the sole control?

The oxygen isotope composition of cellulose (δ18 OCel ) archives hydrological and physiological information. Here, we assess previously unexplored direct and interactive effects of the δ18 O of CO2 (δ18 OCO2 ), nitrogen (N) fertilizer supply and vapour pressure deficit (VPD) on δ18 OCel , 18 O-enrichment of leaf water (Δ18 OLW ) and cellulose (Δ18 OCel ) relative to source water, and pex px , the proportion of oxygen in cellulose that exchanged with unenriched water at the site of cellulose synthesis, in a C4 grass (Cleistogenes squarrosa). δ18 OCO2 and N supply, and their interactions with VPD, had no effect on δ18 OCel , Δ18 OLW , Δ18 OCel and pex px . Δ18 OCel and Δ18 OLW increased with VPD, while pex px decreased. That VPD-effect on pex px was supported by sensitivity tests to variation of Δ18 OLW and the equilibrium fractionation factor between carbonyl oxygen and water. N supply altered growth and morphological features, but not 18 O relations; conversely, VPD had no effect on growth or morphology, but controlled 18 O relations. The work implies that reconstructions of VPD from Δ18 OCel would overestimate amplitudes of VPD variation, at least in this species, if the VPD-effect on pex px is ignored. Progress in understanding the relationship between Δ18 OLW and Δ18 OCel will require separate investigations of pex and px and of their responses to environmental conditions.

Routing of Fatty Acids from Fresh Grass to Milk Restricts the Validation of Feeding Information Obtained by Measuring (13)C in Milk.

Dairy production systems vary widely in their feeding and livestock-keeping regimens. Both are well-known to affect milk quality and consumer perceptions. Stable isotope analysis has been suggested as an easy-to-apply tool to validate a claimed feeding regimen. Although it is unambiguous that feeding influences the carbon isotope composition (δ(13)C) in milk, it is not clear whether a reported feeding regimen can be verified by measuring δ(13)C in milk without sampling and analyzing the feed. We obtained 671 milk samples from 40 farms distributed over Central Europe to measure δ(13)C and fatty acid composition. Feeding protocols by the farmers in combination with a model based on δ(13)C feed values from the literature were used to predict δ(13)C in feed and subsequently in milk. The model considered dietary contributions of C3 and C4 plants, contribution of concentrates, altitude, seasonal variation in (12/13)CO2, Suess's effect, and diet-milk discrimination. Predicted and measured δ(13)C in milk correlated closely (r(2) = 0.93). Analyzing milk for δ(13)C allowed validation of a reported C4 component with an error of <8% in 95% of all cases. This included the error of the method (measurement and prediction) and the error of the feeding information. However, the error was not random but varied seasonally and correlated with the seasonal variation in long-chain fatty acids. This indicated a bypass of long-chain fatty acids from fresh grass to milk.

(13) CO2 /(12) CO2 exchange fluxes in a clamp-on leaf cuvette: disentangling artefacts and flux components.

Leaks and isotopic disequilibria represent potential errors and artefacts during combined measurements of gas exchange and carbon isotope discrimination (Δ). This paper presents new protocols to quantify, minimize, and correct such phenomena. We performed experiments with gradients of CO2 concentration (up to ±250 µmol mol(-1) ) and δ(13) CCO2 (34‰), between a clamp-on leaf cuvette (LI-6400) and surrounding air, to assess (1) leak coefficients for CO2 , (12) CO2 , and (13) CO2 with the empty cuvette and with intact leaves of Holcus lanatus (C3 ) or Sorghum bicolor (C4 ) in the cuvette; and (2) isotopic disequilibria between net photosynthesis and dark respiration in light. Leak coefficients were virtually identical for (12) CO2 and (13) CO2 , but ∼8 times higher with leaves in the cuvette. Leaks generated errors on Δ up to 6‰ for H. lanatus and 2‰ for S. bicolor in full light; isotopic disequilibria produced similar variation of Δ. Leak errors in Δ in darkness were much larger due to small biological : leak flux ratios. Leak artefacts were fully corrected with leak coefficients determined on the same leaves as Δ measurements. Analysis of isotopic disequilibria enabled partitioning of net photosynthesis and dark respiration, and indicated inhibitions of dark respiration in full light (H. lanatus: 14%, S. bicolor: 58%).

Isotope evidence for preferential dispersal of fast-spreading invasive gobies along man-made river bank structures.

Invasive round goby Neogobius melanostomus and bighead goby Ponticola kessleri have successfully colonized freshwater and coastal habitats worldwide. The objective was to use stable isotope analyses to study the foraging and movement of both species at small spatial scales in the Upper Danube River, considering 861 samples from two different years, seasons and sides of the river in an area where limited mixing at a confluence occurs. A difference in δ(13)C of 1 ‰ between gobies from both river sides was observed in both species and reflected the isotope spacing in their dominant benthic prey Dikerogammarus villosus. These results suggest an absence of goby movement across the Danube River which was unexpected, given the fast spread of gobies at invasion fronts. It can be concluded that their dispersal is highly preferential with longitudinal movement likely being facilitated by artificial rip-rap structures along river banks, which provide shelter and food.