Abstract
Homeostasis of metabolism and regulation of stress‐signaling pathways are important for plant growth. The metabolite 3′‐phosphoadenosine‐5′‐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.
Highlights
Chloroplasts are one of the key organelles in plant cells, acting as the site of oxygenic photosynthesis while housing the biosynthesis of various important metabolites including amino acids, nucleotides, fatty acids, phytohormones, and sulfur assimilation (Bobik & BurchSmith, 2015)
We investigated if the GUS staining intensities for each line is correlated with their corresponding SAL1-silencing efficiencies by quantifying SAL1 transcript levels and PAP levels (Figure 1b,c; Figure S2b,c)
To avoid confounding effects from poorer silencing efficiency in subsequent generations, which was observed in the dex-inducible SAL1-Hairpin RNA interference (hpRNAi) system, we studied the first generation (T1) transformants carrying the 35S:SAL1hpRNAi construct
Summary
Chloroplasts are one of the key organelles in plant cells, acting as the site of oxygenic photosynthesis while housing the biosynthesis of various important metabolites including amino acids, nucleotides, fatty acids, phytohormones, and sulfur assimilation (Bobik & BurchSmith, 2015). Multiple sRNAs can be produced from the hpRNAi approach as opposed to the amiRNA approach that generates only a single sRNA (Ossowski, Schwab, & Weigel, 2008) Both strategies have been extensively used in plants; they can be driven by inducible promoters, such as a dexamethasone (dex)-inducible promoter (Wielopolska, Townley, Moore, Waterhouse, & Helliwell, 2005), which allows targeted manipulation of the gene of interest. We investigate the potential of genetically manipulating SAL1 expression in wild-type Arabidopsis, including the utilization of hpRNAi and amiRNA strategies under inducible and constitutive promoters, for adjusting the levels of the chloroplast retrograde signal PAP. Complementary strategies utilizing chemical-inducible and developmental stage-specific complementation of sal were tested The efficiencies of these various strategies in enabling the alteration of PAP levels in Arabidopsis and the corresponding developmental effects are presented. |3 assessed on selected plants and insights into SAL1/PAP interaction with rosette growth and drought tolerance are discussed
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