Callitriche as a potential model system for evolutionary studies on the dorsiventral distribution of stomata

ABSTRACT

Controlling the distribution of stomata is crucial for the adaptation of plants to new, or changing environments. While many plant species produce stomata predominantly on the abaxial leaf surface (hypostomy), some produce stomata on both surfaces (amphistomy), and the remaining few produce them only on the adaxial surface (hyperstomy). Various selective pressures have driven the evolution of these three modes of stomatal distribution. Despite recent advances in our understanding of stomatal development and dorsiventral leaf polarity, the genetic basis for the evolution of different stomatal distributions is still unclear. Here, we propose the genus Callitriche as a new model system to investigate patterns in the evolution of stomatal distribution. Callitriche comprises species with diverse lifestyles, including terrestrial, amphibious, and obligately aquatic plants. We found that species in this genus cover all three modes of dorsiventral stomatal distribution, making it a desirable model for comparative and evolutionary analyses on distribution modes. We further characterized the genetic basis of the different distribution modes, focusing on the stomatal key transcription factor SPEECHLESS. Future research using the promising model system Callitriche would open a new direction for evolutionary developmental biology studies on stomata.

Plant stomata are the gas-exchange machinery of plants, contributing to efficient water usage and carbon fixation when arranged and distributed optimally.^1,2 Stomata are also a primary route of pathogen entry into inner plant tissues,³which further highlights the importance of stomata distribution control in plants. Here we focus on the distribution of stomata on the adaxial and abaxial sides of leaves. While some plant species can plastically change their adaxial-abaxial distribution of stomata in response to changing environments,^4,5 the distribution pattern also varies greatly among species.⁶ The stomata of many plant species are almost exclusively found on the abaxial (lower) leaf surface (hypostomy), but there are also plant species with stomata on both sides of the leaf (amphistomy),^7,8 including the model plant Arabidopsis.^9,10 Very few plant species have stomata only on their upper (adaxial) surface, and those that do tend to be plants with floating leaves.¹¹ Plants evolved to exhibit one of the three stomatal distribution modes in response to various selective pressures.¹² However, the direct genetic mechanisms underlying the evolutionary transition from one stomatal distribution mode into another are still unknown. Although the genetic mechanisms for both stomatal development¹³ and specification of the dorsiventrality of leaves¹⁴ in model species are currently well understood, the mechanisms determining the adaxial-abaxial distribution of stomata are less well-characterized. This is in part due to the lack of a model system, in which we can genetically trace the evolutionary processes of the three stomata distribution modes.

Here, we propose a new model system for investigating the evolution of dorsiventral stomatal distribution: the genus Callitriche (Plantaginaceae; Lamiales). The cosmopolitan genus Callitriche includes 50–75 herbaceous species that have diverse lifestyles, ranging from terrestrial to amphibious to completely aquatic.¹⁵ Callitriche is of particular interest for evolutionary developmental (evo-devo) studies of stomata, because of the ability of amphibious Callitriche species to block stomatal differentiation under submerged conditions.^16,17 Our recent study revealed that stomatal development occurs via different processes in amphibious and terrestrial Callitriche species.¹⁸ While the stomatal precursor cells of terrestrial species divide multiple times before differentiating into stomata, those of amphibious species skip the division stage and directly differentiate into stomata. In addition, we found significant variation in adaxial-abaxial stomatal distributions throughout the Callitriche genus, which has species that exhibit amphistomatous, hypostomatous, and hyperstomatous distribution modes (Figure 1a). Along with the established experimental tools, including laboratory cultures and transcriptomic data,^16,18,20 the presence of all three stomata distribution modes makes this genus a promising model for the evolution of stomatal distribution.

Figure 1.

The distribution of stomata in Callitriche species. (a) Summary of the stomatal distribution and lifestyle of each species. The phylogenetic tree was constructed based on previous studies.^15,19 See our recent study¹⁸ for quantitative data of the stomatal distribution. (b–i) Traced images of the adaxial and abaxial epidermis in each species; images were generated using microscopic images taken as previously described.¹⁸ Plants were grown under aerial conditions, except C. hermaphroditica, which was grown in a water-filled aquarium. Stomata are colored red and hair cells are colored black. Bars = 100 μm. (b–c) Adaxial (b) and abaxial (c) epidermis of C. japonica. (d–e) Adaxial (d) and abaxial (e) epidermis of C. palustris. (f–g) Adaxial (f) and abaxial (g) epidermis of C. terrestris. (h–i) Adaxial (h) and abaxial (i) epidermis of C. hermaphroditica.

In this study, we closely investigated the diversity in the dorsiventral stomatal distributions of Callitriche species. We cultured five Callitriche species found in Japan in the laboratory (Figure 1a), and examined the epidermis of the mature leaves as previously described¹⁸ (Figure 1b–i). Terrestrial C. japonica had typical hypostomatous leaves, with many stomata on the abaxial surface and few, if any, stomata on the adaxial surface (Figure 1b,c). By contrast, the two amphibious species, C. palustris and C. stagnalis, had more stomata on the adaxial side of the aerial leaf. This tendency of having greater numbers of adaxial stomata was most evident in C. palustris, in which stomata were rarely found on the abaxial leaf surface; therefore, this species can be regarded as having hyperstomatous leaves (Figure 1d,e). Previous studies mentioned hyperstomy in the floating morph of amphibious Callitriche.²¹ Based on our result, hyperstomy seems to be a fixed trait for the leaves of C. palustris, independent of whether they are floating or not, because the plant shoot was kept in air in our growth conditions. The terrestrial species C. terrestris had typical amphistomatous leaves with dorsiventrally symmetrical distributions of stomata (Figure 1f,g), which is consistent with previous descriptions of terrestrial Callitriche.²¹ Notably, C. hermaphroditica, the only obligate aquatic species that was included in our analysis, represented the other extreme of symmetry; the leaves of this species completely lacked stomata (Figure 1h,i). Leaves lacking stomata are common among obligate aquatic plant species. C. hermaphroditica might have lost its stomata developmental genes, similar to Zostera marina (Zosteraceae; Alismatales).²² Another possibility is that C. hermaphroditica retains the latent genetic potential to form stomata, but with a blocked upstream signaling pathway under normal growth conditions, as reported in Potamogeton perfoliatus (Potamogetonaceae; Alismatales).²³ Future genomic and physiological analyses of C. hermaphroditica are needed to determine which of these hypotheses is correct.

We also characterized the expression patterns of a key stomatal development gene, which is involved in determining the mode of stomatal distribution. Orthologs of the Arabidopsis transcription factor SPEECHLESS (SPCH) regulate the natural variation of the adaxial-abaxial stomatal distribution in tomato²⁴ and poplar²⁵ species. In our recent work, we applied whole-mount insitu hybridization (WISH) and found that CpSPCH was expressed exclusively in the adaxial epidermis in hyperstomatous C. palustris¹⁸ (Figure 2a). This led us to speculate that the spatial expression patterns of SPCH orthologs might underlie the different stomatal distributions found in Callitriche species (Figure 1). To explore this hypothesis, we analyzed the expression pattern of the SPCH ortholog in the amphistomatous species C. terrestris. We applied WISH to C. terrestris leaf primordia using a full-length CtSPCH probe and found that CtSPCH was expressed in both adaxial and abaxial epidermal cells (Figure 2b,c). The CtSPCH expression pattern contrasted sharply with that of C. palustris CpSPCH (Figure 2a), and corresponded to the amphistomatous nature of C. terrestris. Our results suggest that the stomatal distribution modes in Callitriche species are characterized by the differential localization of SPCH mRNA. Comparing the regulatory sequences of SPCH in the genomes of various Callitriche species may provide insight into the dorsiventral polarity-dependent regulation of SPCH and the molecular mechanisms behind the evolution of different stomatal distribution modes.

Figure 2.

The expression of SPCH orthologs in amphistomatous C. terrestris and hyperstomatous C. palustris. WISH-stained leaf primordia (a–c) samples were cross-sectioned (a’–c’; white arrowheads in a–c indicate the positions where the sections were made). Bars = 100 μm. To detect the expression of SPCH orthologs in each species, C. palustris samples were treated with the antisense (as) probe for CpSPCH (a). C. terrestris samples were treated with the antisense probe for CtSPCH (b) or the sense (se) probe for CtSPCH (c), as previously described.¹⁸ Black arrowheads in (a’) indicate abaxial expression of CtSPCH.

To summarize, we characterized the stomata of Callitriche species that exhibit different stomatal distribution modes, namely amphistomy (C. terrestris), hyperstomy (C. palustris, C. stagnalis), and hypostomy (C. japonica) (Figure 1). The occurrence of all three distribution modes in the same genus will facilitate evolutionary analyses of stomatal distribution. For example, future investigations could focus on how the different lifestyles of Callitriche species have affected the evolution of stomatal distribution. Various factors including light, growth rate, leaf thickness, and herbaceous or woody nature influenced the evolution of stomatal distributions.^6–8,12 However, previous studies examined few or no hyperstomatous species, many of which have amphibious or floating lifestyles. Therefore, the mechanisms by which the transition to aquatic lifestyles affected the evolution of stomatal distribution are currently unclear. The genus Callitriche could be an excellent model system to bridge this knowledge gap. Moreover, genetic pathways involved in stomata development could be investigated further. Here, we found that the spatial expression pattern of SPCH corresponds with the different stomatal distribution modes (Figure 2). Recently, the regulation and expression profiles of SPCH were investigated not only in Arabidopsis²⁶ but also in tomato,²⁷ another potential model for the evolution of stomatal distribution.²⁴ A comparison of Callitriche SPCH sequences and functions with those of such other emerging model systems could provide insight into evo-devo studies of stomatal distribution. Collectively, this study illuminates the potential of the genus Callitriche as a new model system for studying the evolution of plant stomata.

Funding Statement

This work was supported by Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) Fellows (20J20446, to Y.D.), JSPS Grant-in-Aid for Research Activity start-up (16H06733, to H.K.), and the Ministry of Education, Culture, Sports, Science and Technology (MEXT)  Grant-in-Aid for Scientific Research on Innovative Areas (19H05672, to H.T.).

Disclosure of potential conflicts of interest

No potential conflict of interest was reported by the author(s).