Phloem loading is the first step in photoassimilate translocation from source leaves to heterotrophic sink organs (e.g., roots, flowers, and fruits), comprising the transport of photoassimilates from photosynthetic mesophyll cells to the long-distance transport tissue, the phloem (Rennie and Turgeon, 2009; Ainsworth and Lemonnier, 2018). Depending on the species, plants use primarily two distinct routes to load sugars (mainly sucrose) into the phloem, involving either an apoplastic or a symplastic pathway. In apoplastic loading, sucrose moves from mesophyll cells to the cell wall space (apoplast) and is subsequently loaded energetically into the phloem by the action of specific transport proteins, including members of the SWEET and sucrose transporter (SUCs or SUTs) protein families. In symplastic loading, sucrose moves cell-to-cell towards phloem through numerous narrow cytoplasmic channels, called plasmodesmata (Rennie and Turgeon, 2009; Braun et al., 2014; Ainsworth and Lemonnier, 2018).

Current evidence suggests that several features associated with phloem loading and carbon export are subject to environmental control to balance source supply with sink demand (Amiard et al., 2005; Adams et al., 2007; Bishop et al., 2018; Xu et al., 2018). These responses to environmental cues involve anatomical and molecular changes and depend on the mode of phloem loading. For example, growth under high light conditions resulted in a higher number of cell wall invaginations in minor vein companion cells to facilitate more sugar transporters (apoplastic loaders), or in a higher vein density to increase total plasmodesmatal frequency (symplastic loaders), ensuring in both cases greater delivery of photoassimilates to heterotrophic organs as a result of higher photosynthetic rates (Adams et al., 2007). Similarly, environmental stresses that limit carbon assimilation rate reduced SUTs expression in various apoplastic species (Xu et al., 2018).

A current gap in our knowledge is how plants with different phloem loading mechanisms respond to elevated CO₂ with only few studies having dealt with this question so far (Körner et al., 1995; Bishop et al., 2018). Körner et al. (1995) investigated the accumulation of non-structural carbohydrates in leaves of apoplastic and symplastic species to test the hypothesis that symplastic species are less efficient than apoplastic species in exporting carbohydrates into the phloem for long distance transport to heterotrophic organs. The authors additionally hypothesized that if symplastic species were limited in their ability to export carbohydrates into the phloem, then they would have exhibited an excess of non-structural carbohydrates in their leaves when subjected to elevated CO₂ conditions (Körner et al., 1995). Indeed, the authors found a higher accumulation of non-structural carbohydrates in the leaves of most symplastic species, but they did not investigate further what would be the impact of this differential accumulation of non-structural carbohydrates between symplastic and apoplastic species on their photosynthesis or other important agronomic parameters such as yield. Likewise, Bishop et al. (2018) examined the impact of elevated CO₂ levels on photosynthesis and carbohydrate accumulation in leaves of three apoplastic (pea, beet, and sugar beet) and three symplastic species (strawberry, melon and peony). The authors concluded that species differing in phloem loading mechanism had similar photosynthetic responses to elevated CO₂, while, contrarily to Körner et al. (1995), they did not observe a higher sucrose build up in the leaves of symplastic species compared to apoplastic species. Again, however, there was no information about the distribution of photoassimilates to different sink organs or changes in yield in response to elevated CO₂.

Strawberry (Fragaria × ananassa) and tomato (Solanum lycopersicum) are two important fruit crops that use different mechanisms to load sugars into the phloem – strawberry is a symplastic loader (Rennie and Turgeon, 2009; Bishop et al., 2018) and tomato is an apoplastic loader (Osorio et al., 2014) – yet both species benefit from CO₂ enrichment by increasing their yields (Mamatha et al., 2014; Tagawa et al., 2022). In this study, we investigated the long-term effects of CO₂ enrichment on strawberry and tomato plants to determine the morphological and physiological adaptations that enable these two contrasting crops to adapt and benefit from elevated CO₂ levels. Determining how these two species respond to elevated CO₂ levels could eventually inform practices involving CO₂-enrichment in the glasshouse and reveal potential barriers that may limit their productivity.

One of the key findings of this study is that elevated CO₂ treatment promoted growth and reproductive development in both tomato and strawberry. In tomato, the higher yield ( Figure 1E ) can be attributed to the higher number of inflorescences ( Figure 1B ), but not to the number of fruits per inflorescence ( Figure 1C ). In strawberry, the higher yield at elevated CO₂ conditions ( Figure 2D ) can be attributed to increased crown growth ( Figure 2B ), which enabled the formation of more flowers ( Figure 2C ), and presumably the number of fruits per plant. The findings agree with those of other studies that found an increase in flower and fruit number in both tomato and strawberry under elevated CO₂ (Deng and Woodward, 1998; Mamatha et al., 2014; Rangaswamy et al., 2021; Tagawa et al., 2022). Overall, these data suggest that elevated CO₂ levels in the atmosphere will have a positive impact on the yield of field-grown tomato and strawberry crops in the future and support current glasshouse management practices entailing CO₂ enrichment.

To determine the underlying mechanisms responsible for yield improvements under elevated CO₂ conditions and to identify potential barriers limiting yield, we examined tomato and strawberry photosynthetic responses to ambient and elevated CO₂ conditions. Interestingly, gas exchange analysis showed that carbon assimilation rate was higher in strawberry plants under elevated CO₂, but there was no difference between tomato plants grown under ambient and elevated CO₂ ( Figure 3 ), despite the higher levels of CO₂ in the mesophyll cells of elevated-CO₂-treated tomato plants ( Figure 3 ). The latter could be explained by the decrease in Rubisco’s carboxylation efficiency of tomato plants subjected to elevated CO₂ compared with plants grown under ambient CO₂, as evidenced by the lower CO₂ assimilation rate of the former to increasing CO₂ levels ( Figure 4 ). In contrast, strawberry plants subjected to elevated CO₂ conditions not only had higher CO₂ concentration in the mesophyll space of their leaves, but also higher Rubisco carboxylation efficiency compared with plants grown under ambient CO₂ conditions. The latter observation indicates that the symplastic loader, strawberry, can better adjust its photosynthesis to elevated CO₂ levels than the apoplastic loader, tomato. Ainsworth and Lemonnier (2018) reported the results of a meta-analysis in which they compared the photosynthetic acclimation of apoplastic and symplastic species that had been studied to date in Free Air CO₂ Enrichment (FACE) experiments. They found that the light-saturated photosynthetic rate, the maximum rate of Rubisco carboxylation (V _{c, max}), and the maximum rate of electron transport (J _max) of symplastic species were significantly higher than that of apoplastic species, and they attributed those differences to the better adaptation of passive loaders to high mesophyll sugar concentrations, which in turn renders them less susceptible to carbohydrate-mediated downregulation of photosynthesis. The findings of this study agree with those of Ainsworth and Lemonnier (2018).

In agreement with this study, Bishop et al. (2018) observed an increase in carbon assimilation rate in strawberry plants grown at elevated CO₂ conditions; however, they also found an increase in carbon assimilation rate in all three apoplastic species as a result of elevated CO₂ levels in the atmosphere. This contrasts with the findings of our work, in which we observed a similar carbon assimilation rate between tomato plants grown under either ambient or elevated CO₂ conditions. One reason for that discrepancy might be associated with differences among apoplastic species in altering their photosynthetic responses to elevated CO₂ levels. Another reason could be related to the fact that tomato plants in this study were only lightly pruned, resulting in higher shoot-to-root ratio and thus lower sink strength. This alteration in source-sink relationship in tomatoes may have disrupted the balance between production of sugars in source leaves and their utilization in sink organs. Indeed, as Arp (1991) indicated, whereas photosynthesis of C3 plants is stimulated when the CO₂ levels in the atmosphere increase, their photosynthetic capacity is often reduced after long-term exposure to elevated CO₂ levels, especially under conditions of low sink strength. The latter explanation is further supported by the data from ¹³C-labelling experiment in which we found that the distribution of newly assimilated carbon to sink organs was similar between tomato plants grown under ambient and elevated CO₂ conditions ( Figure 5B ), suggesting that tomato plants are sink limited under elevated CO₂. Hence, future breeding efforts, focusing on improving tomato productivity at elevated CO₂ conditions, could revolve around enhancing sink strength, particularly that of flowers and fruits, and therefore the utilization of photoassimilates by harvestable organs.

Photosynthesis is generally considered a major determinant of yield, providing heterotrophic organs with carbon skeletons and energy for growth and development (Jones et al., 2013; Ort et al., 2015; Petridis et al., 2018). Here, we found that carbon assimilation rate was unaffected in tomato, but increased in strawberry, in response to long-term CO₂ enrichment ( Figure 3 ). Hence, based on their photosynthetic response, one would expect that tomato plants grown under elevated CO₂ would have had similar yields to those grown under ambient CO₂, whereas CO₂ enriched strawberry plants would have had higher yields compared to those grown under ambient CO₂. However, we found that not only strawberry plants, but also tomato plants had higher yields as a result of CO₂ enrichment ( Figures 1 , 2 ). In addition, we also observed that carbon distribution across organs was similar between treatments in both species ( Figure 5B ). If both species increase their yield in response to elevated CO₂, irrespective of carbon assimilation rate and carbon distribution, then a question that arises is what triggers them. Although further research is needed to answer this question, we speculate that the yield benefits may arise from early developmental events, probably prior to photosynthetic acclimation to elevated CO₂, promoting floral initiation and differentiation and thus the formation of more flowers. If this hypothesis is true, then it would be interesting to investigate the connection between photosynthesis and early stages of development, especially in relation to the maturation rate of shoot apical meristem, as this process can be a major driver of yield by altering inflorescence architecture and the number of flowers on inflorescences (Lippman et al., 2008; Park et al., 2012, 2014).

Our work confirms previous studies indicating that CO₂ enrichment enhances strawberry and tomato yields (Mamatha et al., 2014; Tagawa et al., 2022). For both species this improvement in yield was the result of greater vegetative growth and flower formation. We also found that at elevated CO₂ conditions carbon assimilation rate in tomato was limited by Rubisco’s carboxylation efficiency, but this limitation was not observed in strawberry. Furthermore, in both species most newly assimilated carbon had been allocated to fruits during fruit ripening; however, neither species allocated more carbon to sink organs of CO₂-enriched plants, suggesting that the observed yield improvements in response to CO₂ may have resulted from differences between treatments at earlier developmental stages. The similar response between strawberry and tomato plants, that is both species had similar levels of newly assimilated carbon in their sink organs between the two CO₂ treatments, may additionally suggest that symplastic and apoplastic species are limited by their ability to export carbon at elevated CO₂ conditions, at least at later developmental stages. Finally, based on this as well as previous studies (Bishop et al., 2018), we conclude that the phloem loading mechanism does not seem to affect crop adaptation to elevated CO₂ per se, but any potential differences among species should be attributed to the individual ability of a species to adapt to elevated CO₂.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AP: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft. MD: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. SK: Data curation, Formal analysis, Investigation, Writing – review & editing.