Sensory plastid-associated PsbP DOMAIN-CONTAINING PROTEIN 3 triggers plant growth- and defense-related epigenetic responses.

Sensory plastids are important in plant responses to environmental changes. Previous studies show that MutS HOMOLOG 1 (MSH1) perturbation in sensory plastids induces heritable epigenetic phenotype adjustment. Previously, the PsbP homolog DOMAIN-CONTAINING PROTEIN 3 (PPD3), a protein of unknown function, was postulated to be an interactor with MSH1. This study investigates the relationship of PPD3 with MSH1 and with plant environmental sensing. The ppd3 mutant displays a whole-plant phenotype variably altered in growth rate, flowering time, reactive oxygen species (ROS) modulation and response to salt, with effects on meristem growth. Present in both chloroplasts and sensory plastids, PPD3 colocalized with MSH1 in root tips but not in leaf tissues. The suppression or overexpression of PPD3 affected the plant growth rate and stress tolerance, and led to a heritable, heterogenous 'memory' state with both dwarfed and vigorous growth phenotypes. Gene expression and DNA methylome data sets from PPD3-OX and derived memory states showed enrichment in growth versus defense networks and meristem effects. Our results support a model of sensory plastid influence on nuclear epigenetic behavior and ppd3 as a second trigger, functioning within meristem plastids to recalibrate growth plasticity.

Specialized Plastids Trigger Tissue-Specific Signaling for Systemic Stress Response in Plants.

Plastids comprise a complex set of organelles in plants that can undergo distinctive patterns of differentiation and redifferentiation during their lifespan. Plastids localized to the epidermis and vascular parenchyma are distinctive in size, structural features, and functions. These plastids are termed "sensory" plastids, and here we show their proteome to be distinct from chloroplasts, with specialized stress-associated features. The distinctive sensory plastid proteome in Arabidopsis (Arabidopsis thaliana) derives from spatiotemporal regulation of nuclear genes encoding plastid-targeted proteins. Perturbation caused by depletion of the sensory plastid-specific protein MutS HOMOLOG1 conditioned local, programmed changes in gene networks controlling chromatin, stress-related phytohormone, and circadian clock behavior and producing a global, systemic stress response in the plant. We posit that the sensory plastid participates in sensing environmental stress, integrating this sensory function with epigenetic and gene expression circuitry to condition heritable stress memory.

Epigenomic plasticity of Arabidopsis msh1 mutants under prolonged cold stress.

Dynamic transcriptional and epigenetic changes enable rapid adaptive benefit to environmental fluctuations. However, the underlying mechanisms and the extent to which this occurs are not well known. MutS Homolog 1 (MSH1) mutants cause heritable developmental phenotypes accompanied by modulation of defense, phytohormone, stress-response, and circadian rhythm genes, as well as heritable changes in DNA methylation patterns. Consistent with gene expression changes, msh1 mutants display enhanced tolerance for abiotic stress including drought and salt stress, while showing increased susceptibility to freezing temperatures. Despite changes in defense and biotic stress-response genes, msh1 mutants showed increasing susceptibility to the bacterial pathogen Pseudomonas syringae. Our results suggest that chronic cold and low light stress (10°C, 150 µmol m-2 s-1) influences non-CG methylation to a greater degree in msh1 mutants compared to wild-type Col-0. Furthermore, CHG changes are more closely pericentromeric, whereas CHH changes are generally more dispersed. This increased variation in non-CG methylation pattern does not significantly affect the msh1-derived enhanced growth behavior after mutants are crossed with isogenic wild type, reiterating the importance of CG methylation changes in msh1-derived enhanced vigor. These results indicate that msh1methylome is hyper-responsive to environmental stress in a manner distinct from the wild-type response, but CG methylation changes are potentially responsible for growth vigor changes in the crossed progeny.

Laser Scanning Confocal Microscopy for Arabidopsis Epidermal, Mesophyll and Vascular Parenchyma Cells.

Investigation of protein targeting to plastids in plants by confocal laser scanning microscopy (CLSM) can be complicated by numerous sources of artifact, ranging from misinterpretations from in vivo protein over-expression, false fluorescence in cells under stress, and organellar mis-identification. Our studies have focused on the plant-specific gene MSH1, which encodes a dual targeting protein that is regulated in its expression and resides within the nucleoid of a specialized plastid type ( Virdi et al., 2016 ). Therefore, our methods have been optimized to study protein dual targeting to mitochondria and plastids, spatial and temporal regulation of protein expression, and sub-organellar localization, producing a protocol and set of experimental standards that others may find useful for such studies.

MutS HOMOLOG1 silencing mediates ORF220 substoichiometric shifting and causes male sterility in Brassica juncea.

Cytoplasmic male sterility (CMS) has consistently been associated with the expression of mitochondrial open reading frames (ORFs) that arise from genomic rearrangements. Spontaneous fertility reversion in CMS has been observed in several cases, but a clear understanding of fertility reversion controlled by nuclear genetic influences has been lacking. Here, we identified spontaneous fertile revertant lines for Brassica juncea CMS cytoplasm in which the mitochondrial genome has undergone substoichiometric shifting (SSS) to suppress ORF220 copy number. We placed ORF220, with or without a mitochondrial targeting presequence, under the control of the CaMV35S and AP3 promoters in Arabidopsis to confirm that ORF220 causes male sterility when mitochondrially localized. We found that copy number of the ORF220 gene was altered under conditions that suppress MSH1, a nuclear gene that controls illegitimate recombination in plant mitochondria. MSH1-RNAi lines with increased ORF220 copy number were male sterile compared with wild type. We found that a wide range of genes involved in anther development were up- and down-regulated in revertant and MSH1-RNAi lines, respectively. The system that we have developed offers valuable future insight into the interplay of MSH1 and SSS in CMS induction and fertility reversion as a mediator of nuclear-mitochondrial crosstalk.

MSH1 Is a Plant Organellar DNA Binding and Thylakoid Protein under Precise Spatial Regulation to Alter Development.

As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant-specific protein involved in organellar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the msh1 phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppression lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, but fractionation and protein-protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochondria results in 7%-10% of plants altered in leaf morphology, heat tolerance, and mitochondrial genome stability. MSH1 does not localize within the nucleus directly, but plastid depletion produces non-genetic changes in flowering time, maturation, and growth rate that are heritable independent of MSH1. MSH1 depletion alters non-photoactive redox behavior in plastids and a sub-set of mitochondrially altered lines. Ectopic expression produces deleterious effects, underlining its strict expression control. Unraveling the complexity of the MSH1 effect offers insight into triggers of plant-specific, transgenerational adaptation behaviors.

Arabidopsis MSH1 mutation alters the epigenome and produces heritable changes in plant growth.

Plant phenotypes respond to environmental change, an adaptive capacity that is at least partly transgenerational. However, epigenetic components of this interplay are difficult to measure. Depletion of the nuclear-encoded protein MSH1 causes dramatic and heritable changes in plant development, and here we show that crossing these altered plants with isogenic wild type produces epi-lines with heritable, enhanced growth vigour. Pericentromeric DNA hypermethylation occurs in a subset of msh1 mutants, indicative of heightened transposon repression, while enhanced growth epi-lines show large chromosomal segments of differential CG methylation, reflecting genome-wide reprogramming. When seedlings are treated with 5-azacytidine, root growth of epi-lines is restored to wild-type levels, implicating hypermethylation in enhanced growth. Grafts of wild-type floral stems to mutant rosettes produce progeny with enhanced growth and altered CG methylation strikingly similar to epi-lines, indicating a mobile signal when MSH1 is downregulated, and confirming the programmed nature of methylome and phenotype changes.

MSH1-induced non-genetic variation provides a source of phenotypic diversity in Sorghum bicolor.

MutS Homolog 1 (MSH1) encodes a plant-specific protein that functions in mitochondria and chloroplasts. We showed previously that disruption or suppression of the MSH1 gene results in a process of developmental reprogramming that is heritable and non-genetic in subsequent generations. In Arabidopsis, this developmental reprogramming process is accompanied by striking changes in gene expression of organellar and stress response genes. This developmentally reprogrammed state, when used in crossing, results in a range of variation for plant growth potential. Here we investigate the implications of MSH1 modulation in a crop species. We found that MSH1-mediated phenotypic variation in Sorghum bicolor is heritable and potentially valuable for crop breeding. We observed phenotypic variation for grain yield, plant height, flowering time, panicle architecture, and above-ground biomass. Focusing on grain yield and plant height, we found some lines that appeared to respond to selection. Based on amenability of this system to implementation in a range of crops, and the scope of phenotypic variation that is derived, our results suggest that MSH1 suppression provides a novel approach for breeding in crops.

The Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in Arabidopsis.

It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. Here, we demonstrate that Arabidopsis thaliana uses for that purpose two separate biosynthetic branches stemming from phenylalanine and tyrosine. Gene network modeling and characterization of T-DNA mutants indicated that acyl-activating enzyme encoded by At4g19010 contributes to the biosynthesis of ubiquinone specifically from phenylalanine. CoA ligase assays verified that At4g19010 prefers para-coumarate, ferulate, and caffeate as substrates. Feeding experiments demonstrated that the at4g19010 knockout cannot use para-coumarate for ubiquinone biosynthesis and that the supply of 4-hydroxybenzoate, the side-chain shortened version of para-coumarate, can bypass this blockage. Furthermore, a trans-cinnamate 4-hydroxylase mutant, which is impaired in the conversion of trans-cinnamate into para-coumarate, displayed similar defects in ubiquinone biosynthesis to that of the at4g19010 knockout. Green fluorescent protein fusion experiments demonstrated that At4g19010 occurs in peroxisomes, resulting in an elaborate biosynthetic architecture where phenylpropanoid intermediates have to be transported from the cytosol to peroxisomes and then to mitochondria where ubiquinone is assembled. Collectively, these results demonstrate that At4g19010 activates the propyl side chain of para-coumarate for its subsequent ß-oxidative shortening. Evidence is shown that the peroxisomal ABCD transporter (PXA1) plays a critical role in this branch.

Functional modeling identifies paralogous solanesyl-diphosphate synthases that assemble the side chain of plastoquinone-9 in plastids.

It is a little known fact that plastoquinone-9, a vital redox cofactor of photosynthesis, doubles as a precursor for the biosynthesis of a vitamin E analog called plastochromanol-8, the physiological significance of which has remained elusive. Gene network reconstruction, GFP fusion experiments, and targeted metabolite profiling of insertion mutants indicated that Arabidopsis possesses two paralogous solanesyl-diphosphate synthases, AtSPS1 (At1g78510) and AtSPS2 (At1g17050), that assemble the side chain of plastoquinone-9 in plastids. Similar paralogous pairs were detected throughout terrestrial plant lineages but were not distinguished in the literature and genomic databases from mitochondrial homologs involved in the biosynthesis of ubiquinone. The leaves of the atsps2 knock-out were devoid of plastochromanol-8 and displayed severe losses of both non-photoactive and photoactive plastoquinone-9, resulting in near complete photoinhibition at high light intensity. Such a photoinhibition was paralleled by significant damage to photosystem II but not to photosystem I. In contrast, in the atsps1 knock-out, a small loss of plastoquinone-9, restricted to the non-photoactive pool, was sufficient to eliminate half of the plastochromanol-8 content of the leaves. Taken together, these results demonstrate that plastochromanol-8 originates from a subfraction of the non-photoactive pool of plastoquinone-9. In contrast to other plastochromanol-8 biosynthetic mutants, neither the single atsps knock-outs nor the atsps1 atsps2 double knock-out displayed any defects in tocopherols accumulation or germination.

Gene network reconstruction identifies the authentic trans-prenyl diphosphate synthase that makes the solanesyl moiety of ubiquinone-9 in Arabidopsis.

Ubiquinone (coenzyme Q) is the generic name of a class of lipid-soluble electron carriers formed of a redox active benzoquinone ring attached to a prenyl side chain. The length of the latter varies among species, and depends upon the product specificity of a trans-long-chain prenyl diphosphate synthase that elongates an allylic diphosphate precursor. In Arabidopsis, this enzyme is assumed to correspond to an endoplasmic reticulum-located solanesyl diphosphate synthase, although direct genetic evidence was lacking. In this study, the reconstruction of the functional network of Arabidopsis genes linked to ubiquinone biosynthesis singled out an unsuspected solanesyl diphosphate synthase candidate--product of gene At2g34630--that, extraordinarily, had been shown previously to be targeted to plastids and to contribute to the biosynthesis of gibberellins. Green fluorescent protein (GFP) fusion experiments in tobacco and Arabidopsis, and complementation of a yeast coq1 knockout lacking mitochondrial hexaprenyl diphosphate synthase demonstrated that At2g34630 is also targeted to mitochondria. At2g34630 is the main--if not sole--contributor to solanesyl diphosphate synthase activity required for the biosynthesis of ubiquinone, as demonstrated by the dramatic (75-80%) reduction of the ubiquinone pool size in corresponding RNAi lines. Overexpression of At2g34630 gave up to a 40% increase in ubiquinone content compared to wild-type plants. None of the silenced or overexpressing lines, in contrast, displayed altered levels of plastoquinone. Phylogenetic analyses revealed that At2g34630 is the only Arabidopsis trans-long-chain prenyl diphosphate synthase that clusters with the Coq1 orthologs involved in the biosynthesis of ubiquinone in other eukaryotes.

Double-strand break repair processes drive evolution of the mitochondrial genome in Arabidopsis.

BACKGROUND: The mitochondrial genome of higher plants is unusually dynamic, with recombination and nonhomologous end-joining (NHEJ) activities producing variability in size and organization. Plant mitochondrial DNA also generally displays much lower nucleotide substitution rates than mammalian or yeast systems. Arabidopsis displays these features and expedites characterization of the mitochondrial recombination surveillance gene MSH1 (MutS 1 homolog), lending itself to detailed study of de novo mitochondrial genome activity. In the present study, we investigated the underlying basis for unusual plant features as they contribute to rapid mitochondrial genome evolution. RESULTS: We obtained evidence of double-strand break (DSB) repair, including NHEJ, sequence deletions and mitochondrial asymmetric recombination activity in Arabidopsis wild-type and msh1 mutants on the basis of data generated by Illumina deep sequencing and confirmed by DNA gel blot analysis. On a larger scale, with mitochondrial comparisons across 72 Arabidopsis ecotypes, similar evidence of DSB repair activity differentiated ecotypes. Forty-seven repeat pairs were active in DNA exchange in the msh1 mutant. Recombination sites showed asymmetrical DNA exchange within lengths of 50- to 556-bp sharing sequence identity as low as 85%. De novo asymmetrical recombination involved heteroduplex formation, gene conversion and mismatch repair activities. Substoichiometric shifting by asymmetrical exchange created the appearance of rapid sequence gain and loss in association with particular repeat classes. CONCLUSIONS: Extensive mitochondrial genomic variation within a single plant species derives largely from DSB activity and its repair. Observed gene conversion and mismatch repair activity contribute to the low nucleotide substitution rates seen in these genomes. On a phenotypic level, these patterns of rearrangement likely contribute to the reproductive versatility of higher plants.

The majority of the type III effector inventory of Pseudomonas syringae pv. tomato DC3000 can suppress plant immunity.

The Pseudomonas syringae type III protein secretion system (T3SS) and the type III effectors it injects into plant cells are required for plant pathogenicity and the ability to elicit a hypersensitive response (HR). The HR is a programmed cell death that is associated with effector-triggered immunity (ETI). A primary function of P. syringae type III effectors appears to be the suppression of ETI and pathogen-associated molecular pattern-triggered immunity (PTI), which is induced by conserved molecules on microorganisms. We reported that seven type III effectors from P. syringae pv. tomato DC3000 were capable of suppressing an HR induced by P. fluorescens(pHIR11) and have now tested 35 DC3000 type III effectors in this assay, finding that the majority of them can suppress the HR induced by HopA1. One newly identified type III effector with particularly strong HR suppression activity was HopS2. We used the pHIR11 derivative pLN1965, which lacks hopA1, in related assays and found that a subset of the type III effectors that suppressed HopA1-induced ETI also suppressed an ETI response induced by AvrRpm1 in Arabidopsis thaliana. A. thaliana plants expressing either HopAO1 or HopF2, two type III effectors that suppressed the HopA1-induced HR, were reduced in the flagellin-induced PTI response as well as PTI induced by other PAMPs and allowed enhanced in planta growth of P. syringae. Collectively, our results suggest that the majority of DC3000 type III effectors can suppress plant immunity. Additionally, the construct pLN1965 will likely be a useful tool in determining whether other type III effectors or effectors from other types of pathogens can suppress either ETI, PTI, or both.

Participation of leaky ribosome scanning in protein dual targeting by alternative translation initiation in higher plants.

Postendosymbiotic evolution has given rise to proteins that are multiply targeted within the cell. Various mechanisms have been identified to permit the expression of proteins encoding distinct N termini from a single gene. One mechanism involves alternative translation initiation (aTI). We previously showed evidence of aTI activity within the Arabidopsis thaliana organellar DNA polymerase gene POLgamma2. Translation initiates at four distinct sites within this gene, two non-AUG, to produce distinct plastid and mitochondrially targeted forms of the protein. To understand the regulation of aTI in higher plants, we used Polgamma2 as a model to investigate both cis- and trans-acting features of the process. Here, we show that aTI in Polgamma2 and other plant genes involves ribosome scanning dependent on sequence context at the multiple initiation sites to condition specific binding of at least one trans-acting factor essential for site recognition. Multiple active translation initiation sites appear to operate in several plant genes, often to expand protein targeting. In plants, where the mitochondrion and plastid must share a considerable portion of their proteomes and coordinate their functions, leaky ribosome scanning behavior provides adaptive advantage in the evolution of protein dual targeting and translational regulation.