The evolution of stomatal traits along the trajectory toward C4 photosynthesis

Abstract

C4 photosynthesis optimizes plant carbon and water relations, allowing high photosynthetic rates with low stomatal conductance. Stomata have long been considered a part of the C4 syndrome. However, it remains unclear how stomatal traits evolved along the path from C3 to C4. Here, we examined stomata in the Flaveria genus, a model used for C4 evolutionary study. Comparative, transgenic, and semi-in vitro experiments were performed to study the molecular basis that underlies the changes of stomatal traits in C4 evolution. The evolution from C3 to C4 species is accompanied by a gradual rather than an abrupt change in stomatal traits. The initial change appears near the Type I intermediate stage. Co-evolution of the photosynthetic pathway and stomatal traits is supported. On the road to C4, stomata tend to be fewer in number but larger in size and stomatal density dominates changes in anatomical maximum stomatal conductance (gsmax). Reduction of FSTOMAGEN expression underlies decreased gsmax in Flaveria and likely occurs in other C4 lineages. Decreased gsmax contributes to the increase in intrinsic water-use efficiency in C4 evolution. This work highlights the stomatal traits in the current C4 evolutionary model. Our study provides insights into the pattern, mechanism, and role of stomatal evolution along the road toward C4.

Figure 1

g_smax of Flaveria species. A, Phylogeny of the Flaveria genus according to McKown et al. (2005) and Lyu et al. (2015). Different colors represent different photosynthetic types, orange: C₃, purple: Type I, gray: Type II, green: C₄-like, blue: C₄. Scale bar = 10 μm. B, The trend of g_smax on the abaxial surface in Flaveria species with different photosynthetic types from C₃ to C₄ grown in phytotron (C) (n = 4). The abbreviation of the species name is the first three letters of the species name. C, g_smax of the abaxial surface between species with different photosynthetic types in the Flaveria genus grown in phytotron. C3 (n = 8), Type I (n = 20), Type II (n = 12), C4-like (n = 12), and C4 (n = 16). D, Correlation of g_smax and g_s for Flaveria species with different photosynthetic types. The data of g_s were from Vogan and Sage (2011) (n ≥ 12). The species used are pri, pub, opp, flo, son, chl, ang, ram, flo, vag, bro, pal, tri, bid, and koc. The colors for photosynthetic types refer to (B). E, The difference in g_s measured under 500 μmol m⁻²s⁻¹ PPFD between C₃ (rob) and C₄ (bid) (n = 6). F, The difference in iWUE (A/g_s) measured under 500 μmol m⁻²s⁻¹ PPFD between C₃ (rob) and C₄ (bid) (n = 6). Specific experimental details are in the “Materials and method.” Error bar for g_s (D) indicates se. Error bar in other graphs indicates sd. The asterisks represent statistically significant differences between different photosynthetic types (one-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001); different letters represent statistically significant differences between two groups (Tukey’s multiple test, α = 0.05). Pearson correlation coefficient and the significance were noted in each panel.

Figure 2

SD and its patterning. A, The trend of SD of abaxial surface from C₃ to C₄ in Flaveria species grown in phytotron. B, SD and guard cell length (l) of abaxial surface between species with different photosynthetic types in Flaveria genus grown in phytotron. C, Correlation of g_smax and SD for different Flaveria species. Different color indicates different photosynthetic type (see Figure 1 for details). D, Correlation of g_smax and l for different Flaveria species. E, Correlation of l and SD for different Flaveria species. F, The photographs of stomatal patterning on abaxial surface in rob (left), ram (middle) and bid (right). Scale bar = 20 μm. G, Comparison of SI between rob, ram, and bid. SD, (n = 4). l, (n = 20). SI, (n = 3). Error bar indicates sd. The asterisks represent statistically significant differences between different photosynthetic types (one-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001); different letters represent statistically significant differences between two groups (Tukey’s multiple test, α = 0.05). Pearson correlation coefficient and the significance are noted in each panel.

Figure 3

Transcript abundance of stomatal development-related genes between C₃ and C₄Flaveria species. A, A model of the signaling pathway for the stomatal development in Arabidopsis (Zoulias et al., 2018). In the signaling pathway, the arrow represents activation, other represents inhibition. A protodermal cell (PDC, white) becomes a meristemoid mother cell (MMC, orange), which divides asymmetrically into two daughter cells, with one being a meristemoid (M, red) and the other being a stomatal-lineage ground cell (SLGC). The M cell develops into a guard mother cell (GMC, yellow); the SLGC develops into a pavement cell (PC). The GMC conducts a symmetrical division to produce two equal-sized guard cells (GC, green). EPF1, EPF2, and STOMAGEN competitively bind to receptors ER and TMM, which can deliver a signal to the YDA MAPK cascade. SPCH, MUTE, and FAMA ultimately are inactivated through phosphorylation by the YDA MAPK cascade. A subtilisin-like protease SDD1 negatively regulates stomatal development. SCRM interacts with SPCH, MUTE, and FAMA to regulate stomatal development. Flp negatively regulates GMC cell division and delay the transition from GMC to GC. B, Comparison of the expression of stomatal developmental related genes between juvenile and mature leaves. Data are from 1KP (One Thousand Plant Transcriptomes, 2019). Each gene was standardized by z score. Cro, pri, tri, and bid, respectively, indicates Flaveria cronquistii (C₃), Flaveria pringlei (C₃), Flaveria trinervia (C₄), and Flaveria bidentis (C₄). C, The foldchanges of stomatal development-related genes between the C₃ and C₄Flaveria species. Foldchange means the ratio between the gene expression in a C₃Flaveria species and gene expression in a C₄Flaveria species under the same leaf development stage and growth condition as the C₃Flaveria species. The expression data were from Billakurthi et al. (2018), Mallmann et al. (2014), Gowik et al. (2011), and 1KP (One Thousand Plant Transcriptomes, 2019). The asterisks represent statistically significant differences between C₃ and C₄Flaveria species (*P < 0.05, **P < 0.01, Mann–Whitney U test, two-tailed distribution, unpaired). The red lines indicate median. D, Relative expression level determined by RT-qPCR and expression level determined by RNA-seq of FSTO (FSTOMAGEN) in rob and bid. (n = 4, biologically independent replicates.) RPKM indicates reads per kilobase per million mapped reads. Error bar indicates sd. The asterisks represent statistically significant differences (P < 0.05, t test, two-tailed distribution, unpaired).

Figure 4

The Function of FSTOMAGEN in controlling the stomatal development. A, Protein conservation analysis of the homologs of FSTOMAGEN in different species. The amino acid regions marked by blue line and red line represent the signal peptide and the functional domain of FSTOMAGEN, respectively. Red letters represent conserved amino acids in most species, white letters with red backgrounds represent amino acid conserved across all species. B, The SD for Arabidopsis leaves with FSTOMAGEN applied at different concentrations (n = 8). C, The SD for bid leaves with FSTOMAGEN applied at different concentrations (n = 8). D, The SI for bid leaves with FSTOMAGEN applied at different concentrations (Mock and 10 μmol: n = 6, 1 μmol: n = 4). Error bar indicates sd. The asterisks represent statistically significant differences (P < 0.05, t test, two-tailed distribution, unpaired). E, The structure of recombinant genes used to transform Arabidopsis. AFSTO represents the recombination of the signal peptide of STOMAGEN from Arabidopsis and the functional domain of FSTOMAGEN from F. robusta. FSTO represents the intact STOMAGEN from F. robusta. F and I, The SD (F) and RNA-level (I) of the different transgenic Arabidopsis (FSTO) lines (n = 3). G and J, The SD (G) and RNA-level (J) of the different transgenic Arabidopsis (AFSTO) lines (n = 3). H, Comparison of SD between the transgenic FSTO lines and AFSTO lines. K, Photographs of stomata in a leaf from Arabidopsis with FSTO and AFSTO over-expressed. Scar bar represents 20 μm. Error bar indicates sd. The n.s. represent statistically no significant differences (P > 0.05, t test, two-tailed distribution, unpaired).

Figure 5

Expression level of FSTOMAGEN at different stages of C₄ evolution. A, Protein conservation analysis of the homologs of FSTOMAGEN between different species in the Flaveria genus. The amino acid regions marked by blue line and red line represent the signal peptide and the functional domain of STOMAGEN, respectively. Red letters represent conserved amino acids in most species; white letters with red background represent amino acids conserved across all species. B, Expression levels of FSTOMAGEN determined by RNA-seq at the different developmental stages of leaves in rob and bid. RNA-seq data was from Billakurthi et al. (2018). P denotes leaf primordia. Approximate leaf length for the different leaf stages was 0.3 cm (stage 1), 0.5 cm (stage 2), 1.5 cm (stage 3), 2 cm (stage 4), 3 cm (stage 5), 4 cm (stage 6), 6 cm (stage7), 8 cm (stage 8), and 10 cm (stage 9). RPKM indicates reads per kilobase per million mapped reads (n = 4). C, Comparison of relative expression levels of FSTOMAGEN between rob, ram, and bid (n = 3) by RT-qPCR. The asterisks represent statistically significant differences (P < 0.05, t test, two-tailed distribution, unpaired). D, Comparison of the expression levels of FSTOMAGEN between different species with different photosynthetic types in Flaveria genus. For RT-qPCR, C₃ species include pri (n = 5) and rob (n = 13), intermediate species includes ang (n = 3), ram (n = 5), and vag (n = 3), C₄ species include tri (n = 4), aus (n = 3), bid (n = 4), and koc (n = 3). For RNA-seq, C₃ species include pri (n = 4) and rob (n = 4), intermediate species includes ano (n = 4), pub (n = 4), chl (n = 4), bro (n = 4), C₄ species include tri (n = 4) and bid (n = 4). RNA-seq data was from Mallmann et al. (2014). Dashed lines indicate median. Dotted lines indicate lower quartile and upper quartile. E, Phylogenetic tree for FSTOMAGEN. Orange represents the Flaveria species. The gene tree of STOMAGEN based on ML method. The relative expression indicates the measurements with RT-qPCR and the others were measured with RNA-seq. The geometric means of EF1a and ACT7 were used as the reference in the calculation of expression levels for other genes. Error bars indicate sd. The asterisks represent statistically significant differences, one-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; different letters represent statistically significant differences, Tukey’s multiple test (α = 0.05).

Figure 6

Comparison of stomatal pattern and expression levels of stomatal development-related genes in the C₄ lineages other than Flaveria. A–D, Stomatal characteristics for rice and maize. SD (n = 8); B, stomatal length (l) (n = 25); C, g_s (n = 8). D, iWUE (n = 8). Error bar indicates sd. The asterisks represent statistically significant differences (****P < 0.0001, t test, two-tailed distribution, unpaired). E, Photographs of stomatal pattern in rice and maize. Arrows point to stomata. Scale bars represent 50 μm. F, Comparison of the expression levels of STOMAGEN homologs between rice and maize. There are one and two putative paralogs of STOMAGEN in rice and maize, respectively. OsSTO represents the homologs of STOMAGEN in rice. ZmSTO represents the homologs of STOMAGEN in maize. The expression was determined by RNA-seq (RPKM) in rice and maize. The RNA-seq data were from Wang et al. (2014). G, Comparison of the expression of STOMAGEN homologs between C₃ species, intermediate and C₄ species from different C₄ lineages (shown in Supplemental Figure S15a). Dashed lines indicate median. Dotted lines indicate lower quartile and upper quartile. The asterisks represent statistically significant differences (P < 0.05, t test, two-tailed distribution, unpaired).

Figure 7

Schematic of the tactic change in the C₄ syndrome in the current C₄ evolution model. The positions of the C₄ syndromes in the current C₄ evolution model are shown. The arrows represent the gradual enhancement or weakening of features over the entire evolutionary gradient. Color deepening means enhancement, lighter color means weakening. The parts related to the stomatal traits are indicated by a pictorial stoma. The rest of the content originates from previous research on photosynthesis in Flaveria genus (McKown and Dengler, 2007; Kocacinar et al., 2008; Vogan and Sage, 2011; Schulze et al., 2013; Mallmann et al., 2014; Sage et al., 2014; Stata et al., 2016, 2019;Taniguchi et al., 2021).