Development of strategies for genetic manipulation and fine‐tuning of a chloroplast retrograde signal 3′‐phosphoadenosine 5′‐phosphate

Abstract

Homeostasis of metabolism and regulation of stress-signaling pathways are important for plant growth. The metabolite 3 0 -phosphoadenosine-5 0 -phosphate (PAP)plays dual roles as a chloroplast retrograde signal during drought and high light stress, as well as a toxic by-product of secondary sulfur metabolism, and thus, its levels are regulated by the chloroplastic phosphatase, SAL1. Constitutive PAP accumulation in sal1 mutants improves drought tolerance but can impair growth and alter rosette morphology. Therefore, it is of interest to derive strategies to enable controlled and targeted PAP manipulation that could enhance drought tolerance while minimizing the negative effects on plant growth. We systematically tested the potential and efficiency of multiple established transgenic manipulation tools in altering PAP levels in Arabidopsis. Dexamethasone (dex)-inducible silencing of SAL1 via hpRNAi [pOpOff:SAL1hpRNAi] yielded reduction in SAL1 transcript and protein levels, yet failed to significantly induce PAP accumulation. Surprisingly, this was not due to insufficient silencing of the inducible system, as constitutive silencing using a strong promoter to drive hpRNAi and amiRNA targeting the SAL1 transcript also failed to increase PAP content or induce a sal1-like plant morphology despite significantly reducing the SAL1 transcript levels. In contrast, using dex-inducible expression of SAL1 cDNA to complement an Arabidopsis sal1 mutant successfully modulated PAP levels and restored rosette growth in a dosage-dependent manner.Results from this inducible complementation system indicate that plants with intermediate PAP levels could have improved rosette growth without compromising its drought tolerance. Additionally, preliminary evidence suggests that SAL1 cDNA driven by promoters of genes expressed specifically during early developmental stages such as ABA-Insensitive 3 (ABI3) could be another potential strategy for studying and optimizing PAP levels and drought tolerance while alleviating the negative impact of PAP on plant growth in sal1. Thus, we have identified ways that can allow future dissection into multiple aspects of stress and developmental regulation mediated by this chloroplast signal.--

Secondary sulfur metabolism in cellular signalling and oxidative stress responses

“…Inducible or strong constitutive promoter-driven artificial micro RNA and RNA interference silencing lines of SAL1 show up to 90% reduction in SAL1 transcript abundance, but do not have a corresponding increase in PAP levels (Phua et al, 2018a). Furthermore, while PAP accumulation extends the circadian period, the expression of SAL1 is diurnal rather than circadian (Litthauer et al, 2018).…”

“…PAP-accumulating mice are non-viable (Frederick et al, 2008;Hudson et al, 2013). In contrast, in plants PAP is not simply a toxic metabolite: although sal1 mutants in Arabidopsis show a dwarfed phenotype, PAP accumulation is not lethal and the mutants are also drought-tolerant (Estavillo et al, 2011;Phua et al, 2018a;Phua et al, 2018b;Wilson et al, 2009). Indeed, PAP acts as a chloroplast-to-nucleus retrograde signal during oxidative stresses such as drought and high light.…”

The Arabidopsis SAL1-PAP Pathway: A Case Study for Integrating Chloroplast Retrograde, Light and Hormonal Signaling in Modulating Plant Growth and Development?

“…Soil grown sal1-6 (fry1-6 allele) mutant were sprayed with different hormones (50 μM of GA 3 , 50 μM of IAA, or 1 μM of EBR individually, or all of the three hormones together) or blank control [containing 0.5% (v/v) ethanol, which was used to dissolve the commercially available hormones, and 0.1% (v/v) Tween-20] from 2 weeks old onwards (three times per week). The rosette growth since treatment were quantified via LemnaTec Scanalyzer for 3.5 weeks (until bolting stage) based on rosette area (full method see, Phua et al, 2018 ). Average rosette area ± SD of at least 10 plants per treatment are shown.…”