Ongoing climate change and the depletion of water resources are posing serious threats to crop productivity, making yield stability under drought conditions a growing challenge. Innovative strategies in plant breeding are essential to develop crop varieties with improved resilience and stable yield under water deficit. The TOLERANT project (PRIN 2022 PNRR, P20228HKHM) aims to enhance water use efficiency and yield stability in tomato under water-limiting conditions by targeting abscisic acid (ABA)-mediated stress responses. ABA plays a central role in drought adaptation by modulating stomatal conductance and maintaining water homeostasis. Among ABA-related transcriptional regulators, SlMYB60 (Solyc10g081490.1.1) stands out as the tomato ortholog of AtMYB60, a well-characterized guard cell-specific factor in Arabidopsis involved in integrating multiple stress signals. To investigate the role of SlMYB60, CRISPR-Cas9 editing was applied to two commercial tomato cultivars (Ailsa Craig and Red Setter), generating knockout alleles through multiple sgRNAs. T1 slmyb60-edited plants were assessed for stomatal aperture and drought response. Unexpectedly, mutant lines showed a slight but significative increase in stomatal pore opening in both leaves and cotyledons compared to wild type. Under dry-down conditions, leaf area, fresh weight, and relative water content were significantly affected by both genotype and treatment. RNA-seq analyses confirmed the strong downregulation of SlMYB60 in the edited lines and identified 186 differentially expressed genes, many of which involved in cell wall or cuticle biosynthesis. Chlorophyll leaching assays indicated increased permeability in mutant lines, while electron microscopy revealed defects in epidermal cell surface structure and patterning. Notably, slmyb60 mutants exhibited reduced guard cell density and thinner stomatal ledges compared to wild type, suggesting defects in stomatal architecture. These anatomical alterations likely contribute to increased water loss and reduced drought tolerance, due to impaired stomatal closure and epidermal sealing. To verify the spatial domains of the SlMYB60 expression, we generated a construct comprising a 1.5 kb putative promoter of SlMYB60 fused to GFPGUS. Transient expression in Nicotiana benthamiana revealed widespread GUS activity, indicating that SlMYB60 is not exclusively expressed in guard cells. Confocal microscopy confirmed expression in epidermal cells, implying a broader regulatory role besides guard cells. Our findings reveal that SlMYB60, unlike its Arabidopsis homolog, is not confined to guard cells and plays broader roles in epidermal development, indicating poor conservation of the MYB60 regulatory network between Arabidopsis and tomato.
BEYOND GUARD CELLS: SLMYB60 EDITING REVEALS A BROADER ROLE IN EPIDERMAL REGULATION AND CUTICLE PERMEABILITY IN TOMATO
GERVASONI D.;BALDONI E.;PIRONA R.;GALBIATI M.
2025
Abstract
Ongoing climate change and the depletion of water resources are posing serious threats to crop productivity, making yield stability under drought conditions a growing challenge. Innovative strategies in plant breeding are essential to develop crop varieties with improved resilience and stable yield under water deficit. The TOLERANT project (PRIN 2022 PNRR, P20228HKHM) aims to enhance water use efficiency and yield stability in tomato under water-limiting conditions by targeting abscisic acid (ABA)-mediated stress responses. ABA plays a central role in drought adaptation by modulating stomatal conductance and maintaining water homeostasis. Among ABA-related transcriptional regulators, SlMYB60 (Solyc10g081490.1.1) stands out as the tomato ortholog of AtMYB60, a well-characterized guard cell-specific factor in Arabidopsis involved in integrating multiple stress signals. To investigate the role of SlMYB60, CRISPR-Cas9 editing was applied to two commercial tomato cultivars (Ailsa Craig and Red Setter), generating knockout alleles through multiple sgRNAs. T1 slmyb60-edited plants were assessed for stomatal aperture and drought response. Unexpectedly, mutant lines showed a slight but significative increase in stomatal pore opening in both leaves and cotyledons compared to wild type. Under dry-down conditions, leaf area, fresh weight, and relative water content were significantly affected by both genotype and treatment. RNA-seq analyses confirmed the strong downregulation of SlMYB60 in the edited lines and identified 186 differentially expressed genes, many of which involved in cell wall or cuticle biosynthesis. Chlorophyll leaching assays indicated increased permeability in mutant lines, while electron microscopy revealed defects in epidermal cell surface structure and patterning. Notably, slmyb60 mutants exhibited reduced guard cell density and thinner stomatal ledges compared to wild type, suggesting defects in stomatal architecture. These anatomical alterations likely contribute to increased water loss and reduced drought tolerance, due to impaired stomatal closure and epidermal sealing. To verify the spatial domains of the SlMYB60 expression, we generated a construct comprising a 1.5 kb putative promoter of SlMYB60 fused to GFPGUS. Transient expression in Nicotiana benthamiana revealed widespread GUS activity, indicating that SlMYB60 is not exclusively expressed in guard cells. Confocal microscopy confirmed expression in epidermal cells, implying a broader regulatory role besides guard cells. Our findings reveal that SlMYB60, unlike its Arabidopsis homolog, is not confined to guard cells and plays broader roles in epidermal development, indicating poor conservation of the MYB60 regulatory network between Arabidopsis and tomato.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
