RESUMEN
Epidermal Patterning Factor Like 9 (EPFL9), also known as STOMAGEN, is a cysteine-rich peptide that induces stomata formation in vascular plants, acting antagonistically to other epidermal patterning factors (EPF1, EPF2). In grapevine there are two EPFL9 genes, EPFL9-1 and EPFL9-2 sharing 82% identity at protein level in the mature functional C-terminal domain. In this study, CRISPR/Cas9 system was applied to functionally characterize VvEPFL9-1 in 'Sugraone', a highly transformable genotype. A set of plants, regenerated after gene transfer in embryogenic calli via Agrobacterium tumefaciens, were selected for evaluation. For many lines, the editing profile in the target site displayed a range of mutations mainly causing frameshift in the coding sequence or affecting the second cysteine residue. The analysis of stomata density revealed that in edited plants the number of stomata was significantly reduced compared to control, demonstrating for the first time the role of EPFL9 in a perennial fruit crop. Three edited lines were then assessed for growth, photosynthesis, stomatal conductance, and water use efficiency in experiments carried out at different environmental conditions. Intrinsic water-use efficiency was improved in edited lines compared to control, indicating possible advantages in reducing stomatal density under future environmental drier scenarios. Our results show the potential of manipulating stomatal density for optimizing grapevine adaptation under changing climate conditions.
RESUMEN
BACKGROUND: The vertebrate clade diverged into Chondrichthyes (sharks, rays, and chimeras) and Osteichthyes fishes (bony fishes) approximately 420 mya, with each group accumulating vast anatomical and physiological differences, including skin properties. The skin of Chondrichthyes fishes is covered in dermal denticles, whereas Osteichthyes fishes are covered in scales and are mucous rich. The divergence time among these two fish groups is hypothesized to result in predictable variation among symbionts. Here, using shotgun metagenomics, we test if patterns of diversity in the skin surface microbiome across the two fish clades match predictions made by phylosymbiosis theory. We hypothesize (1) the skin microbiome will be host and clade-specific, (2) evolutionary difference in elasmobranch and teleost will correspond with a concomitant increase in host-microbiome dissimilarity, and (3) the skin structure of the two groups will affect the taxonomic and functional composition of the microbiomes. RESULTS: We show that the taxonomic and functional composition of the microbiomes is host-specific. Teleost fish had lower average microbiome within clade similarity compared to among clade comparison, but their composition is not different among clade in a null based model. Elasmobranch's average similarity within clade was not different than across clade and not different in a null based model of comparison. In the comparison of host distance with microbiome distance, we found that the taxonomic composition of the microbiome was related to host distance for the elasmobranchs, but not the teleost fishes. In comparison, the gene function composition was not related to the host-organism distance for elasmobranchs but was negatively correlated with host distance for teleost fishes. CONCLUSION: Our results show the patterns of phylosymbiosis are not consistent across both fish clades, with the elasmobranchs showing phylosymbiosis, while the teleost fish are not. The discrepancy may be linked to alternative processes underpinning microbiome assemblage, including possible historical host-microbiome evolution of the elasmobranchs and convergent evolution in the teleost which filter specific microbial groups. Our comparison of the microbiomes among fishes represents an investigation into the microbial relationships of the oldest divergence of extant vertebrate hosts and reveals that microbial relationships are not consistent across evolutionary timescales. Video abstract.
