SPEECHLESS duplication in grasses expands potential for environmental regulation of stomatal development

Abstract

Plants acquire atmospheric carbon dioxide for photosynthesis while minimizing water loss and do so by regulating stomatal function and development. The ancestral basic helix-loop-helix transcription factor (TF) gene that drove stomata production in early land plants diversified in sequence and function to become paralogs SPEECHLESS (SPCH), MUTE, and FAMA. Extant angiosperms use these three TFs and their heterodimer partners to regulate stomatal cell identities. Grasses exhibit a particularly interesting set of duplications and losses of SPCH. Using phylogenetic methods, we tracked the duplication of SPCH to the Poaceae-specific rho whole genome duplication and demonstrated that both paralogs remain under selection. By following responses to environmental change in B. distachyon plants bearing mutations in either BdSPCH1 or BdSPCH2, we reveal paralog-specific divergence in response to light or temperature shifts, and further show this behavior is conserved O. sativa SPCH paralogs. Plausible molecular mechanisms underpinning paralog divergence, and cellular mechanisms driving the stomatal phenotypes are supported by analyses of RNA and protein expression in B. distachyon and sequence variation among grasses. These studies suggest ways in which a duplication of a key stomatal regulator enables adaptation and could inform genetic strategies to mitigate anticipated stressors in agronomically important plants.

Keywords: Poaceae; SPEECHLESS; Stomata; WGD; evolution; gene duplication; subfunctionalization.

Figure 1:. SPCH is retained following the rho whole genome duplication in grasses.

A) Riparian plot showing blocks of synteny containing SPCH1 (teal) and SPCH2 (red) across multiple grass species. Only chromosomes with synteny to B. distachyon’s Chr1 and Chr3 are shown and are scaled by the number of genes. B) Scaled density plot for Ks between paralogs colored by species. Dashed lines represent the Ks value between SPCH1 and SPCH2 for the species colored. C) Scaled density plot of Ka. D) Amino acid alignment of SPCH1 and SPCH2 proteins across multiple grass species. Consensus sequences for each paralog are colored by Ka/Ks ratio for that amino acid. Paralogs from each species are shaded by similarity across paralogs for that amino acid. See also Fig. S1 and Table S2

Figure 2:. Plants bearing mutations that leave only one SPCH paralog functional cannot adjust stomatal density to environmental changes.

A) DIC images of B. distachyon abaxial leaf epidermis. Stomata are false-colored green. Images are arranged in rows by environmental conditions and columns by genotype. B) Stomatal density on the abaxial leaf epidermis for plants grown under 22°C or 28°C, 50μE conditions. BdSPCH1 knockout (KO) plants are not sensitive to the temperature change. C) Stomata density on the abaxial leaf epidermis for plants grown under 50μE or 300μE, 22°C conditions. BdSPCH2 KO plants are not sensitive to the light intensity change. D) Average space (interval) between two stomata in a row. Stomatal spacing decreases with light intensity except in BdSPCH2 KO plants. E) Row density of stomata cell files. Row density increases with increased temperature except in BdSPCH1 KO plants. In B-E each point represents a measured region (n > 15 regions per genotype and condition). Significance measured by two-way ANOVA with Tukey post-hoc reporting the effect of condition while controlling for leaf identity of each region (* < 0.05, **** < 1e-04). See also Fig. S2

Figure 3:. Differential environmental regulation appears to act at the level of SPCH protein, not SPCH expression.

A) RT-qPCR quantification of BdSPCH1 and BdSPCH2 RNA expression after moving B. distachyon WT plants to either 300μE or 28°C for time specified on X-axis. Quantification was normalized to a control gene and then to the expression of SPCH1/2 isolated from unshifted plants. B) Micrograph of BdSPCH2:BdSPCH2-YFP expression (yellow) in WT B.distachyon epidermis highlighting the first ACD within the stomatal cell file, cells outlined in magenta. C-D) Length measurements of the zone of BdSPCH1:BdSPCH1-YFP or BdSPCH2:BdSPCH2-YFP fluorescence within a cell file. Dots represent cell files across multiple leaves (n > 8 per reporter per condition). Significance measured by two-way ANOVA with Tukey post-hoc reporting the effect of condition while controlling for leaf identity of each cell file (** < 0.01, *** < 1e-3, **** < 1e-4). See also Figs. S3 and S40

Figure 4:. Loss of function OsSPCH mutants exhibit the same environmental insensitivities seen with mutations in B. distachyon SPCH paralogs

A) DIC images of O. sativa abaxial leaf epidermis. Stomata are false colored green. Images are arranged in columns by environmental conditions and rows by genotype. B-E) Stomatal density on the abaxial leaf epidermis for plants grown under different light or temperature regimes. OsSPCH1 knockout (KO) and Kitaake plants are not sensitive to the temperature change and OsSPCH2 KO plants are not sensitive to the light intensity change. Dots represent regions across multiple leaves (Kitaake n > 7, others n > 16). Significance measured by two-way ANOVA with Tukey post-hoc reporting the effect of condition while controlling for leaf identity of each region (* < 0.05,** < 0.01, *** < 1e-3, **** < 1e-4). See also Fig. S5