Altering Stomatal Density for Manipulating Transpiration and Photosynthetic Traits in Rice through CRISPR/Cas9 Mutagenesis

Abstract

Stomata regulates conductance, transpiration and photosynthetic traits in plants. Increased stomatal density may contribute to enhanced water loss and thereby help improve the transpirational cooling process and mitigate the high temperature-induced yield losses. However, genetic manipulation of stomatal traits through conventional breeding still remains a challenge due to problems involved in phenotyping and the lack of suitable genetic materials. Recent advances in functional genomics in rice identified major effect genes determining stomatal traits, including its number and size. Widespread applications of CRISPR/Cas9 in creating targeted mutations paved the way for fine tuning the stomatal traits for enhancing climate resilience in crops. In the current study, attempts were made to create novel alleles of OsEPF1 (Epidermal Patterning Factor), a negative regulator of stomatal frequency/density in a popular rice variety, ASD 16, using the CRISPR/Cas9 approach. Evaluation of 17 T₀ progenies identified varying mutations (seven multiallelic, seven biallelic and three monoallelic mutations). T₀ mutant lines showed a 3.7-44.3% increase in the stomatal density, and all the mutations were successfully inherited into the T₁ generation. Evaluation of T₁ progenies through sequencing identified three homozygous mutants for one bp insertion. Overall, T₁ plants showed 54-95% increased stomatal density. The homozygous T₁ lines (# E1-1-4, # E1-1-9 and # E1-1-11) showed significant increase in the stomatal conductance (60-65%), photosynthetic rate (14-31%) and the transpiration rate (58-62%) compared to the nontransgenic ASD 16. Results demonstrated that the genetic alterations in OsEPF1 altered the stomatal density, stomatal conductance and photosynthetic efficiency in rice. Further experiments are needed to associate this technology with canopy cooling and high temperature tolerance.

Keywords: OsEPF1; photosynthetic efficiency; rice; stomatal density.

Figure 1

Development of the rice lines harboring mutations in OsEPF1: (a) Structure of OsEPF1 (LOC_Os04g54490 on Chromosome 4) showing three exons and two introns; (b) The highlighted sequence shown in the red box represents the guide RNA, whereas the inner box represents the PAM; (c) T-DNA cassette of pRGEB31 harboring OsEPF1 guide RNA. The expression of guide RNA scaffold is driven by OsU3 small nuclear RNA promoter; Cas9 gene is driven by CaMV35S promoter; hygromycin gene is driven by enhanced CaMV35S promoter; LB-left border RB-right border of T-DNA (d) PCR screening of transgenic plants (1–6 in the left panel and 1–11 in the right panel) using vector-specific primers (Table 1) 1 kb, DNA ladder, NC, negative control and \NT, Non transgenic ASD 16.

Figure 2

Sequence analysis of targeted region among the gene-edited lines (T₀) using ICE CRISPR tool. Putative mutations were detected upon comparison against the reference sequence i.e., non-transgenic ASD 16 on the top. Sequence analysis of events E2-1 and E2-3 showed monoallelic mutation; E1-1 and E2-2 showed biallelic mutation, and E1-3, E1-8 and E2-18 showed multiallelic mutation. The vertical, black-dotted line represents the cut site of cas9 whereas the horizontal black hyphen denotes deletions and N indicates the insertion.

Figure 3

Adaxial and abaxial row density, stomatal density and stomatal size among the putative transgenic lines (T₀) compared to the non-transgenic ASD 16 (NT). Gene edited lines showed altered stomatal parameters on both adaxial and abaxial side of leaves. Each data point represents an average of ten replications and error bar represents ± standard error of the mean data. Statistical significance was tested using Duncan’s multiple range test (DMRT). Different alphabets indicate the significance at p < 0.05.

Figure 4

Sequence analysis of the target region among the T₁ progenies of # E1-1 using ICE CRISPR tool. Mutants #E1-1-4, E1-1-9, E1-1-11 showed homozygous monoallelic mutation and the progenies E1-1-1, E1-1-3, E1-1-6 showed biallelic mutation on sequence analysis. The vertical, black-dotted line represents the cut site of cas9 whereas the horizontal black hyphen denotes the deletions and N indicates the insertion.

Figure 5

OsEPF1 edited mutants showed altered stomatal density and stomatal size. (A). Stomatal distribution in the T₁ progenies of OsEPF1 edited lines against its wild type ASD 16. Homozygous mutants showed increased stomatal density and altered stomatal file density compared to non-transgenic ASD 16. The clustered stomata showed reduction in stomatal size. The top row represents the stomatal density in abaxial side, whereas the bottom row represents the stomatal density in adaxial leaf surface. Scale bar = 100 µm. (B). Graphical representation of stomatal density and stomatal size in the T₁ transgenic lines compared against the non-transgenic ASD 16 (NT). Each data point represents an average of 10 replications and error bar represents ± standard error of the mean data. Statistical significance was tested using Duncan’s multiple range test (DMRT). Different alphabets indicate the significance at p < 0.05.

Figure 5

OsEPF1 edited mutants showed altered stomatal density and stomatal size. (A). Stomatal distribution in the T₁ progenies of OsEPF1 edited lines against its wild type ASD 16. Homozygous mutants showed increased stomatal density and altered stomatal file density compared to non-transgenic ASD 16. The clustered stomata showed reduction in stomatal size. The top row represents the stomatal density in abaxial side, whereas the bottom row represents the stomatal density in adaxial leaf surface. Scale bar = 100 µm. (B). Graphical representation of stomatal density and stomatal size in the T₁ transgenic lines compared against the non-transgenic ASD 16 (NT). Each data point represents an average of 10 replications and error bar represents ± standard error of the mean data. Statistical significance was tested using Duncan’s multiple range test (DMRT). Different alphabets indicate the significance at p < 0.05.

Figure 6

Evaluation of the homozygous mutant lines for stomatal conductance, photosynthetic rate and transpiration rate. NT, nontransgenic ASD 16; #E1-1-4, E1-1-9 and #E1-1-11 represented the OsEPF1 gene edited lines. (a). transpiration rate; (b). stomatal conductance, (c). carbon dioxide assimilation rate (d). water use efficiency (A/gs). All values of stomatal density and stomatal size are mean ± standard error; each value represents the mean of ten replications and different letters indicates significance at p < 0.05 compared.

Figure 7

Correlation analysis between the stomatal traits and physiological parameters. Stomatal density showed positive correlation with stomatal conductance, photosynthetic rate and transpiration rate and negative correlation to stomatal size.