Novel markers for high-throughput protoplast-based analyses of phytohormone signaling

Abstract

Phytohormones mediate most diverse processes in plants, ranging from organ development to immune responses. Receptor protein complexes perceive changes in intracellular phytohormone levels and trigger a signaling cascade to effectuate downstream responses. The in planta analysis of elements involved in phytohormone signaling can be achieved through transient expression in mesophyll protoplasts, which are a fast and versatile alternative to generating plant lines that stably express a transgene. While promoter-reporter constructs have been used successfully to identify internal or external factors that change phytohormone signaling, the range of available marker constructs does not meet the potential of the protoplast technique for large scale approaches. The aim of our study was to provide novel markers for phytohormone signaling in the Arabidopsis mesophyll protoplast system. We validated 18 promoter::luciferase constructs towards their phytohormone responsiveness and specificity and suggest an experimental setup for high-throughput analyses. We recommend novel markers for the analysis of auxin, abscisic acid, cytokinin, salicylic acid and jasmonic acid responses that will facilitate future screens for biological elements and environmental stimuli affecting phytohormone signaling.

Mediator Subunits MED16, MED14, and MED2 Are Required for Activation of ABRE-Dependent Transcription in Arabidopsis

“…To ascertain whether MED16, MED2, and MED14 are required for ABA-responsive transcriptional activation of drought gene promoters, we adopted a protoplast transformation system ( Lehmann et al, 2020 ). This allowed transient expression of promoter reporter constructs in wild type and mutant backgrounds to be measured and facilitated a direct assessment of promoter activation, eliminating any possible confounding effects of differential transcript stability.…”

“…This allowed transient expression of promoter reporter constructs in wild type and mutant backgrounds to be measured and facilitated a direct assessment of promoter activation, eliminating any possible confounding effects of differential transcript stability. Protoplasts of all four genotypes were transformed with reporter constructs in which the native promoter of LTI78 and RAB18 fused to a coding sequence for luciferase ( Lehmann et al, 2020 ). Consistent with whole seedling data, ABA treatment increased the activation of the LTI78 and RAB18 promoters in Col-0 to significant levels ( p < 0.001 for both gene constructs, ANOVA) but the levels of expression in the three Mediator subunit mutants were significantly lower than those observed in wild type ( Figure 2 ).…”

“…Native promoter sequences of RAB18 and LTI78 coupled to a luciferase coding sequence were constructed previously using the plasmid pFRK1:LUC ( Lehmann et al, 2020 ). The transfection control plasmid was pAtUBQ10:GUS .…”

“…The isolation of mesophyll protoplasts was performed as described previously ( Wu et al, 2009 ). The isolated protoplasts were transfected, and the expression of the reporter constructs was analyzed according to Lehmann et al (2020) .…”

Biosensors: A Sneak Peek into Plant Cell’s Immunity

“…So far, this has been used in combination with either luciferase or GUS due to its low strength [ 10 ]. Lately, some new promoters responding to ABA, auxin, cytokinin, SA and JA were identified in A. thaliana as promising candidates for transcriptional reporters [ 62 ].…”