Atmospheric carbon dioxide, one of the most relevant greenhouse gases has been increasing continuously since pre-industrial times from 280 ppm in 1750, and is predicted to exceed 700 ppm by the end of 21st century (IPCC, 2021). This accumulation of CO₂ - among other air pollutants in the atmosphere - leads to a changed re-radiative effect and thus to an increase in global mean surface temperature - widely known as global warming. Besides that, high-pressure “blocking” weather systems (Davini and D’Andrea, 2020), an altered wind frequency and a shifting precipitation pattern are also consequences of a worldwide changing climate with an increasing intensity of extreme weather events (Manning and Tiedemann, 1995).

Plant and ecosystem performance is influenced by increasing CO₂ levels leading to a modified plant physiology and thus to altered plant growth as well as developmental changes. For most of C₃ plant species, elevated CO₂ improves the photosynthetic apparatus resulting in an increased plant biomass production (Reddy et al., 2010) – in both – vegetative and reproductive performance. Besides agricultural crops, various CO₂ enrichment experiments have been conducted worldwide for various plant types with CO₂ effects on plant growth and ecosystems via a multitude of mechanisms (Ainsworth and Long, 2005). The up-regulation of photosynthesis under elevated CO₂ as one main outcome is reported for most plant types. Likewise, water use efficiency, which is referred to net assimilation related to either transpiration or stomatal conductance, is shown to be improved under eCO₂ conditions. Carbon metabolism in C₃ plants is promoted under eCO₂ due to higher carboxylation rates by RuBisCO and together with higher net assimilation rates are accountable for an enhanced biomass production.

Field studies on grapevines under elevated CO₂ conditions that have been conducted are rare, showing higher yield and vegetative growth due to enhanced net assimilation rates (Bindi et al., 2001; Moutinho-Pereira et al., 2009; Edwards et al., 2017). Furthermore, in a previous study emerged from the VineyardFACE, Riesling and Cabernet Sauvignon resulted in higher lateral leaf area and leaf biomass, as well as increased bunch and berry weight under elevated CO₂ concentrations (Wohlfahrt et al., 2018). Crop yield of Riesling showed a 10.4% (2015) and 17.8% (2016) increase under eCO₂ and Cabernet Sauvignon gained 17.3% (2015) and 10.1% (2016) higher yield under eCO₂. Effects on grapevine leaf transpiration and stomatal conductance are distinct, but most of the times the water demand decreased under eCO₂ conditions when vines were mature at an age of 9 up to 20 years (Bindi et al., 2005; Tognetti et al., 2005; Moutinho-Pereira et al., 2009; Edwards et al., 2016; Edwards et al., 2017). Younger vines, at an age of 4 to 6 years showed a higher water consumption under eCO₂ and therefore an increased leaf transpiration and stomatal conductance (Wohlfahrt et al., 2018). Nevertheless, independent of vine age, all previous studies observed an eCO₂ effect on vine water use efficiency, which was shown to improve and has been supported by higher photosynthetic capacity under eCO₂. As leaf photosynthesis occurs in chloroplasts of the mesophyll (palisade and spongy parenchyma) it is likely that an increased photosynthesis rate leads to an adaptation in morphological characteristics of leaves. Furthermore, spongy parenchyma has larger intercellular space for gas transportation, while palisade parenchyma is higher in chloroplast number and thus more beneficial to increase leaf photosynthesis.

Morphological alteration of leaves under eCO₂ has been reported for several tree and agricultural C₃ species, e.g. increase in leaf thickness and layers, extension of leaf cells and chloroplast development (Thomas and Harvey, 1983; Robertson and Leech, 1995; Saxe et al., 1998). The increase in leaf thickness of the grapevine cultivar Touriga Franca was derived from an extended spongy parenchyma and only partially due to an increase in palisade parenchyma under eCO₂ conditions (Moutinho-Pereira et al., 2009).

The aim of this study was to investigate the effects of eCO₂ on leaf physiology and morphological characteristics of the two Vitis vinifera L. cultivars Riesling and Cabernet Sauvignon grown in the VineyardFACE system and under temperate oceanic climate conditions.

The net assimilation rates were significantly stimulated under eCO₂ for both cultivars and seasons, which are presented in Figure 2 and have previously been described for stomatal conductance, water use efficiency, pre-dawn leaf water potential as well as for pruning weight or leaf area (Wohlfahrt et al., 2018). Additionally, results of the statistical output are shown in Table 1 . Cabernet Sauvignon net assimilation rate increased from 18% up to 41% in 2015 under eCO₂ conditions, and showed +31% on a seasonal average. In 2016, the increase was 25% up to 63% with an average of +42% under eCO₂. Net assimilation of Riesling was 19% to 62% higher under eCO₂ in 2015 showing a seasonal average of a 41% increase. The gain in 2016 ranged between 31% to 46% with a seasonal average of +40%. Overall, Riesling was stimulated higher in net assimilation under eCO₂ in 2015, whereas in 2016 cultivars did not differ in their rate of increase (approx. 40%). It was obvious that in both cultivars the year as well as the measuring date have to be considered as independent factors. For Cabernet Sauvignon an interaction between the treatment and year and date occurred ( Table 1 ).

Optical leaf clip meter indices did not differ between treatments or years for both cultivars ( Table 2 ). Only for Riesling a trend to higher Chl index under CO₂ enrichment over the two years (P=0.0629) was observed. Whereas NBI index was higher, Flav index was lower in leaves of Cabernet Sauvignon compared to Riesling in both years. As shown in Table 2 , leaf pigments (Chl a, Chl b, total Chl and Car) were affected by the year and not by eCO₂.

Total leaf thickness and width of spongy parenchyma of Cabernet Sauvignon ( Figure 3A ) and Riesling ( Figure 3B ) remained less affected under eCO₂ conditions ( Table 3 ). However, significant differences were found in histological analyses of the leaf cross-sections between the two CO₂ treatments in upper and lower epidermis and the palisade parenchyma of Cabernet Sauvignon ( Figure 3A ). Whereas under eCO₂ the palisade parenchyma increased, the epidermal tissue decreased in thickness. Also, palisade parenchyma in Riesling showed a trend in increase under eCO₂ ( Table 3 ), no significance difference was detected. However, the ratio between palisade and spongy parenchyma hardly differed between the CO₂ treatments in Riesling whilst in Cabernet Sauvignon the treatment effect was significantly pronounced (P=0.017) with an increasing ratio under eCO₂. Leaf layer thickness of both cultivars was affected by the year, like the epidermis and palisade parenchyma, the latter appeared to have higher values in 2015 ( Table 3 ). Additionally, total leaf thickness and ratio between palisade and spongy parenchyma showed an effect by the year in Cabernet Sauvignon. Block effects occurred for both cultivars in total thickness of the leaf and the spongy parenchyma thickness.

Responses of two different grapevine cultivars grown in the VineyardFACE-system indicate that an increase in atmospheric CO₂ predicted for the mid-century affects leaf gas exchange, and especially enhances net assimilation. This is in accordance with results obtained from previous studies on field-grown grapevines (Bindi et al., 2001; Moutinho-Pereira et al., 2009; Edwards et al., 2017) and a multitude of other C₃ crop species under elevated CO₂ concentrations. In a previous VineyardFACE trial both, Riesling and Cabernet Sauvignon had frequently higher photosynthetic rates in their early years of adaptation and increased in leaf as well as fruit biomass production. Hence, an impact on single berry weight, cluster weight and bunch architecture has been shown (Wohlfahrt et al., 2018 and Wohlfahrt et al., 2020). Even though net assimilation was highly stimulated for both cultivars under a relative low CO₂ increase (+39% net assimilation vs. +20% CO₂ increase), no impact was found in chlorophyll content nor lead to changes in other leaf pigments or leaf nitrogen status.

That the NBI index in leaves differs within different grapevine cultivars and that Chl index is used as indicator for leaf nitrogen content was reported by Cerovic et al. (2015), and could further provide information about the nutrition status of berries. Interestingly, the differences found between the leaves of the two cultivars for Chl index and NBI were also detected earlier during berry ripening in 2015 and 2016 by higher amino acid concentration in berries of Cabernet Sauvignon in comparison to Riesling (Wohlfahrt et al., 2020). These cultivar dependent differences, probably influenced by the choice of rootstock and the scion-rootstock combination as well, were found for various plant growth parameters, e.g. lateral leaf area or perennial wood growth (Wohlfahrt et al., 2018). Differences in leaf nutrition status by using optical leaf clip meter indices or leaf pigment content have not been found between the two CO₂ treatments and for neither of the two cultivars, which corroborates the results of Moutinho-Pereira et al. (2009) when using a SPAD meter. Leaf nitrogen status relates to the photosynthetic capacity and that is why leaves form the highest growth demand for nitrogen (Evans, 1989), while under elevated CO₂ leaf nitrogen content generally decreases by a N-dilution effect caused by the increase in carbohydrate accumulation through enhanced net assimilation (Feng et al., 2015). Thus, it remains unclear if the two cultivars within the VineyardFACE will decrease in leaf nitrogen under eCO₂ in future as variations in nitrogen content are also depending on the initial nitrogen limitation status of the single plant (Stitt and Krapp, 1999; Ainsworth and Long, 2005).

Leaf pigments (Chl a, Chl b, total Chl and Car) were not altered under eCO₂ which is in accordance with results of total chlorophyll and carotenoid content in beech leaves, where eCO₂ revealed no effects (Polle et al., 1997). Only a varying nutrient supply caused significant differences in leaf pigments of beech. The seasonal differences in leaf pigments shown for both cultivars were expected due to their dependence on environmental factors such as water availability (Fanizza et al., 1991), which differed in rainfall 2015 (230 mm) and 2016 (369 mm) during growing season. Leaf chlorophyll pigments (Chl a, Chl b, Chl total) were reduced about 50% and carotenoids by 30% in 2015, when precipitation was shortened in comparison to 2016.

Histological analyses of the grapevine leaf cross-sections revealed no increase in total leaf thickness under elevated CO₂. Other C₃ species, particularly soybean, loblolly pine and sweet gum showed an increase in leaf thickness under different CO₂ enrichment scenarios (Thomas and Harvey, 1983), and in different poplar clones in the early phase of growth (Radoglou and Jarvis, 1990). Furthermore, leaves of crop species were reported to exhibit greater increases in leaf thickness compared to wild species (Pritchard et al., 1999), but in this review only experiments conducted in chambers (growth chamber and open top chamber), glass houses and phytotrons have been considered. However, effects of elevated CO₂ on leaf anatomy were summarized to depend on leaf development stage, soil fertility, and again, season of the year (Pritchard et al., 1999). The latter is in accordance with the total thickness of epidermis and palisade parenchyma of Riesling and Cabernet Sauvignon, which were enlarged in 2015 compared to 2016 and thus affected by the season. The differences in leaf thickness could be attributed to extreme temperatures in the growing season 2015 (29 heat days (≥30°C) compared to 17 heat days in 2016) since under high temperature conditions an increase in thickness of grapevine leaves was reported (Salem-Fnayou et al., 2011). Still, both types of ground tissue, palisade and spongy parenchyma contain chloroplasts. Even though the palisade parenchyma contains a high number of chloroplasts compared to the spongy parenchyma, the latter is very prominent in terms of the intercellular air space in the lower mesophyll. Chlorenchyma and aerenchyma are both of utmost importance for the photosynthetic rate which in parts may help to explain that under eCO₂ the photosynthetic activity will be further stimulated, since a higher internal leaf surface enhances the ability to absorb CO₂ to a larger extent. In a previous study on grapevines (cv. Touriga Franca) under open top chamber conditions authors assumed that an increased leaf and therefore parenchyma thickness under eCO₂ was due to an enlargement of cells rather than increased cell division (Moutinho-Pereira et al., 2009), which was previously suggested by Pritchard et al. (1999). This could be explained by the same amount of parenchyma layers in both CO₂ treatments ( Figure 3 , data not shown). Nevertheless, thickness of palisade parenchyma increased, at least for Cabernet Sauvignon under eCO₂. These morphological alterations of leaf layers and extension of cells under eCO₂ were found in other agricultural C₃ species (Thomas and Harvey, 1983; Robertson and Leech, 1995). Surprisingly, instead of an expansion in leaf thickness Cabernet Sauvignon epidermal thickness decreased under higher CO₂ concentration. That an increase in leaf tissues within the mesophyll happens at the expense of epidermis (Garnier et al., 1999), and could therefore lead to increasing foliage photosynthetic potentials was proposed by Niinemets (1999) and approved in this study. Contrary to the leaf morphology of the red cultivar Touriga Franca, which resulted in thicker spongy parenchyma and thus lower or unchanged palisade to spongy parenchyma ratio (Moutinho-Pereira et al., 2009), the palisade to spongy parenchyma ratio increased under eCO₂ within Cabernet Sauvignon under open field conditions. This leads to the assumption that chamber experiments are not fundamentally comparable with studies conducted under field conditions on the one hand, and cultivar specific leaf characteristics and responses on the other hand (Boso et al., 2010). Also, different ‘climatic’ effects are possibly responsible for the differences in parenchyma responses. In addition, Riesling (cool to intermediate) and Cabernet Sauvignon (warm) belong to different climate maturity groupings based on average growing season temperatures (Jones et al., 2005). Under these requirements, different plant reaction of the two cultivars are expected with the accessory climatic changes apparent from season to season which were recently shown (Wohlfahrt et al., 2018). In a study based on climate and developmental plasticity with regards to the seasonal variability in grapevine leaf morphology, results demonstrated that besides environmental, genetic and developmental effects influence the leaf shape in a way largely independent of each other (Chitwood et al., 2016).

Eventually, free air CO₂ enrichment studies are essential to understand plant responses to a changing climate, especially for permanent plant crops and obtained results are likely to improve the current understanding of physiological and structural responses of plants to future environmental conditions, e.g. elevated CO₂ levels.

Results observed on leaf physiology and morphological characteristics of cvs. Riesling and Cabernet Sauvignon can provide first insights to seasonal adaptation strategies of grapevines under a changing climate and in particular to future elevated CO₂ concentrations. However, regardless of the CO₂ treatment the effect of the season and in particularly high temperature and low precipitation can modify the plant response to eCO₂. Thus, the plant water as well as nutrition status may have a large impact on leaf morphology too. For these reasons, field studies on the effect of elevated CO₂, especially by using non-herbaceous perennial plants, are complex and difficult to execute and thus need a long-term investigation over at least two decades. Therefore, studies like the present one are welcome to improve our knowledge about the response of plants to future environmental conditions under realistic conditions.

Furthermore, as the plant nutrient status is suggested to be linked to the antioxidative enzyme response under elevated CO₂ concentrations (Schwanz et al., 1996) the nutritional status of the leaves and the whole plant needs to be intensified in further VineyardFACE studies. In addition, investigations should be carried out in the direction of carbon sink and in regards to the C/N ratio in the soil if it is assumed that a higher surface litter input due to more leaf biomass under eCO₂ could also stimulate the rate of mineralization.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

Conceptualization: YW. Methodology: YW, KK, DP, and ST. Formal analysis: YW. Investigation: YW and ST. Resources: MS. Data curation: YW and ST. Writing—original draft preparation: YW, KK, DP, ST, and MS. Writing—review and editing: YW and MS. Visualization: YW, KK, and DP. Supervision: MS. Project administration: YW, ST, and MS. Correspondence with the journal’s editor: YW. All authors contributed to the article and approved the submitted version.