Understanding the Role of Chloroplast Signalling in Plant Development and Drought Tolerance

3’-phosphoadenosine 5’-phosphate (PAP) was recently proposed as a new chloroplast retrograde signal that accumulates during drought stress. PAP is a by-product of secondary sulfur assimilation, generated by SULFOTRANSFERASES (SOTs) upon transferring of the sulfate group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to sulfate acceptor molecules. PAP is efficiently degraded by the SAL1 phosphatase into inorganic phosphate and adenosine monophosphate (AMP). Consequently, PAP levels are normally kept at very low during plant development. Constitutive over-accumulation of this retrograde signal resulted in plants with pleiotropic altered phenotypes as demonstrated by the sal1 mutant in Arabidopsis, namely improved drought tolerance, altered rosette morphology and delayed development. These multifaceted altered phenotypes of sal1 correlate well with the many transcriptional changes, which are mainly due to the inhibition of 5’- 3’ EXORIBONUCLEASES (XRNs) by PAP. However, the details of downstream changes that links SAL1-PAP-XRN to the many altered phenotypes of sal1 remains poorly understood. This thesis investigates the possibility of conferring drought tolerance to plants by manipulating the SAL1 gene expression with minimal negative effects on growth, while unraveling the signalling pathway(s) contributing to sal1 altered development and drought tolerance. I hypothesised that PAP accumulation at early developmental stages is detrimental for plant growth and development while its accumulation at later stages of development or prior to drought stress is beneficiary for conferring plant drought tolerance. To test this hypothesis, two different strategies for easy manipulation of SAL1 expression were attempted: inducible silencing of SAL1 in wild-type Arabidopsis and inducible complementation of SAL1 in the sal1 mutant background. Surprisingly, efficient silencing of SAL1 could not be achieved even with a strong constitutive promoter driving the expression of SAL1-hair-pin RNA interference (hpRNAi) or SAL1-artificial microRNA (amiRNA) and inducible-silencing was similarly inefficient. On the other hand, inducible complementation of SAL1 in the sal1 mutant background allowed for better manipulation of SAL1 expression and PAP levels. This provides the platform for exploring the engineering of drought tolerant plants without compromising plant development by manipulating PAP levels via regulating SAL1 expression. Since hormones are key regulators of plant development, the possible interaction between PAP and hormone signalling was investigated to study the basis of sal1 developmental phenotypes. Significantly, a comprehensive hormonal profiling showed reduced gibberellins (GAs) content in sal1 and improved growth rate was achieved when sal1 was treated with GAs and brassinosteroids (BRs), which are interdependent hormones that promote growth. Additionally, sal1 germination was hyper-responsive to treatments with abscisic acid (ABA) and/or GAs biosynthetic inhibitor - paclobutrazol (PAC). Furthermore, this observation can be reproduced by feeding wild-type seeds with PAP in addition to ABA and PAC, suggesting that PAP could act as secondary messenger for both GAs and ABA signalling. Meanwhile, upon taking into consideration both the findings of this thesis and literature available, it is proposed that SAL1-PAP-XRN stabilises DELLA accumulation by reducing transcription of key GA biosynthetic genes, allowing the DELLA protein to interact and affect the key transcription factors of light and other hormonal signalling pathways such as BRs, jasmonic acid (JA) and ethylene. It is likely that the overall interactions and feedback between 1) altered sulfur metabolism, 2) altered hormonal homeostasis and 3) inhibitory effects of PAP on XRNs and other nucleotide-binding proteins eventually culminate in the pleitropic developmental phenotype and drought tolerance of sal1. The ultimate application from this project will be to engineer drought tolerance in Brassicaceae by manipulating the SAL1-PAP pathway. This thesis has generated the tools that will aid in the above engineering and uncovered the potential connections between the sal1 altered phenotypes and SAL1-PAP-XRNs, linking chloroplast signalling to plant hormonal homeostasis in regulating plant development and drought tolerance

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Master'sMASTER OF SOCIAL SCIENCE

Secondary sulfur metabolism in cellular signalling and oxidative stress responses

The sulfur metabolism pathway in plants produces a variety of compounds that are central to the acclimation response to oxidative stresses such as drought and high light. Primary sulfur assimilation provides the amino acid cysteine, which is utilized in protein synthesis and as a precursor for the cellular redox buffer glutathione. In contrast, the secondary sulfur metabolism pathway produces sulfated compounds such as glucosinolates and sulfated peptides, as well as a corresponding by-product 3'-phosphoadenosine 5'-phosphate (PAP). Emerging evidence over the past decade has shown that secondary sulfur metabolism also has a crucial engagement during oxidative stress. This occurs across various cellular, tissue and organismal levels including chloroplast-to-nucleus retrograde signalling events mediated by PAP, modulation of hormonal signalling by sulfated compounds and PAP, control of physiological responses such as stomatal closure, and potential regulation of plant growth. In this review, we examine the contribution of the different components of plant secondary metabolism to oxidative stress homeostasis, and how this pathway is metabolically regulated. We further outline the key outstanding questions in the field that are necessary to understand how and why this 'specialized' metabolic pathway plays significant roles in plant oxidative stress tolerance

Reconsidering the nature and mode of action of metabolite retrograde signals from the chloroplast

Plant organelles produce retrograde signals to alter nuclear gene expression in order to coordinate their biogenesis, maintain homeostasis, or optimize their performance under adverse conditions. Many signals of different chemical nature have been described in the past decades, including chlorophyll intermediates, reactive oxygen species (ROS), and adenosine derivatives. While the effects of retrograde signaling on gene expression are well understood, the initiation and transport of the signals and their mode of action have either not been resolved, or are a matter of speculation. Moreover, retrograde signaling should be considered as part of a broader cellular network, instead of as separate pathways, required to adjust to changing physiologically relevant conditions. Here we summarize current plastid retrograde signaling models in plants, with a focus on new signaling pathways, SAL1-PAP, methylerythritol cyclodiphosphate (MEcPP), and β-cyclocitral (β-CC), and outline missing links or future areas of research that we believe need to be addressed to have a better understanding of plant intracellular signaling networks

Reconsidering the nature and mode of action of metabolite retrograde signals from the chloroplast

Plant organelles produce retrograde signals to alter nuclear gene expression in order to coordinate their biogenesis, maintain homeostasis, or optimize their performance under adverse conditions. Many signals of different chemical nature have been described in the past decades, including chlorophyll intermediates, reactive oxygen species (ROS), and adenosine derivatives. While the effects of retrograde signaling on gene expression are well understood, the initiation and transport of the signals and their mode of action have either not been resolved, or are a matter of speculation. Moreover, retrograde signaling should be considered as part of a broader cellular network, instead of as separate pathways, required to adjust to changing physiologically relevant conditions. Here we summarize current plastid retrograde signaling models in plants, with a focus on new signaling pathways, SALl-PAP, methylerythritol cyclodiphosphate (MEcPP), and beta-cyclocitral (beta-CC), and outline missing links or future areas of research that we believe need to be addressed to have a better understanding of plant intracellular signaling networks

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

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

The arabidopsis SAL1-PAP pathway: A case study for integrating chloroplast retrograde, light and hormonal signaling in modulating plant growth and development?

Plant growth and development are dependent on chloroplast development and function. Constitutive high level accumulation of a chloroplast stress signal, 3'-phosphoadenosine-5'-phosphate (PAP), confers drought tolerance to plants, but slow downs and alters plant growth and development. PAP, a by-product of sulfur metabolism, is maintained at very low levels by the SAL1 phosphatase during vegetative growth of Arabidopsis and accumulates in rosettes during drought and excess light. Eight independent forward genetic screens in Arabidopsis identified SAL1 as the regulator of multiple phenotypes related to stress responses, hormonal signaling and/or perception. In this perspective article, we collate all the sal1 phenotypes published in the past two decades, and distill the different pathways affected. Our meta-analysis of publicly available sal1 microarray data coupled to preliminary hormonal treatment and profiling results on sal1 indicate that homeostasis and responses to multiple hormones in sal1 are altered during rosette growth, suggesting a potential connection between SAL1-PAP stress retrograde pathway and hormonal signaling. We propose the SAL1-PAP pathway as a case study for integrating chloroplast retrograde signaling, light signaling and hormonal signaling in plant growth and morphogenesis.We received financial support from the ARC Centre of Excellence in Plant Energy Biology (CE140100008) and scholarships to SYP (ANU), and KXC (ANU). KXC was also funded by a Postdoctoral Fellowship from Research Foundation-Flanders (FWO)

MARÍA DE MESA Y LÓPEZ [Material gráfico]

ÁLBUM FAMILIAR CASA DE COLÓNCopia digital. Madrid : Ministerio de Educación, Cultura y Deporte. Subdirección General de Coordinación Bibliotecaria, 201

Evolution of chloroplast retrograde signaling facilitates green plant adaptation to land

Chloroplast retrograde signaling networks are vital for chloroplast biogenesis, operation, and signaling, including excess light and drought stress signaling. To date, retrograde signaling has been considered in the context of land plant adaptation, but not regarding the origin and evolution of signaling cascades linking chloroplast function to stomatal regulation. We show that key elements of the chloroplast retrograde signaling process, the nucleotide phosphatase (SAL1) and 3'-phosphoadenosine-5'-phosphate (PAP) metabolism, evolved in streptophyte algae-the algal ancestors of land plants. We discover an early evolution of SAL1-PAP chloroplast retrograde signaling in stomatal regulation based on conserved gene and protein structure, function, and enzyme activity and transit peptides of SAL1s in species including flowering plants, the fern Ceratopteris richardii, and the moss Physcomitrella patens. Moreover, we demonstrate that PAP regulates stomatal closure via secondary messengers and ion transport in guard cells of these diverse lineages. The origin of stomata facilitated gas exchange in the earliest land plants. Our findings suggest that the conquest of land by plants was enabled by rapid response to drought stress through the deployment of an ancestral SAL1-PAP signaling pathway, intersecting with the core abscisic acid signaling in stomatal guard cells

Evolution of chloroplast retrograde signaling facilitates green plant adaptation to land

Chloroplast retrograde signaling networks are vital for chloroplast biogenesis, operation, and signaling, including excess light and drought stress signaling. To date, retrograde signaling has been considered in the context of land plant adaptation, but not regarding the origin and evolution of signaling cascades linking chloroplast function to stomatal regulation. We show that key elements of the chloroplast retrograde signaling process, the nucleotide phosphatase (SAL1) and 3'-phosphoadenosine-5'-phosphate (PAP) metabolism, evolved in streptophyte algae-the algal ancestors of land plants. We discover an early evolution of SAL1-PAP chloroplast retrograde signaling in stomatal regulation based on conserved gene and protein structure, function, and enzyme activity and transit peptides of SAL1s in species including flowering plants, the fern Ceratopteris richardii, and the moss Physcomitrella patens Moreover, we demonstrate that PAP regulates stomatal closure via secondary messengers and ion transport in guard cells of these diverse lineages. The origin of stomata facilitated gas exchange in the earliest land plants. Our findings suggest that the conquest of land by plants was enabled by rapid response to drought stress through the deployment of an ancestral SAL1-PAP signaling pathway, intersecting with the core abscisic acid signaling in stomatal guard cells