Spatially resolved transcriptomic analysis of the germinating barley grain.

Seeds are a vital source of calories for humans and a unique stage in the life cycle of flowering plants. During seed germination, the embryo undergoes major developmental transitions to become a seedling. Studying gene expression in individual seed cell types has been challenging due to the lack of spatial information or low throughput of existing methods. To overcome these limitations, a spatial transcriptomics workflow was developed for germinating barley grain. This approach enabled high-throughput analysis of spatial gene expression, revealing specific spatial expression patterns of various functional gene categories at a sub-tissue level. This study revealed over 14 000 genes differentially regulated during the first 24 h after imbibition. Individual genes, such as the aquaporin gene family, starch degradation, cell wall modification, transport processes, ribosomal proteins and transcription factors, were found to have specific spatial expression patterns over time. Using spatial autocorrelation algorithms, we identified auxin transport genes that had increasingly focused expression within subdomains of the embryo over time, suggesting their role in establishing the embryo axis. Overall, our study provides an unprecedented spatially resolved cellular map for barley germination and identifies specific functional genomics targets to better understand cellular restricted processes during germination. The data can be viewed at https://spatial.latrobe.edu.au/.

Cross-species transcriptomic analyses reveals common and opposite responses in Arabidopsis, rice and barley following oxidative stress and hormone treatment.

BACKGROUND: For translational genomics, a roadmap is needed to know the molecular similarities or differences between species, such as model species and crop species. This knowledge is invaluable for the selection of target genes and pathways to alter downstream in response to the same stimuli. Here, the transcriptomic responses to six treatments including hormones (abscisic acid - ABA and salicylic acid - SA); treatments that cause oxidative stress (3-amino-1,2,4-triazole - 3AT, methyl viologen - MV); inhibit respiration (antimycin A - AA) or induce genetic damage (ultraviolet radiation -UV) were analysed and compared between Arabidopsis (Arabidopsis thaliana), barley (Hordeum vulgare) and rice (Oryza sativa). RESULTS: Common and opposite responses were identified between species, with the number of differentially expressed genes (DEGs) varying greatly between treatments and species. At least 70% of DEGs overlapped with at least one other treatment within a species, indicating overlapping response networks. Remarkably, 15 to 34% of orthologous DEGs showed opposite responses between species, indicating diversity in responses, despite orthology. Orthologous DEGs with common responses to multiple treatments across the three species were correlated with experimental data showing the functional importance of these genes in biotic/abiotic stress responses. The mitochondrial dysfunction response was revealed to be highly conserved in all three species in terms of responsive genes and regulation via the mitochondrial dysfunction element. CONCLUSIONS: The orthologous DEGs that showed a common response between species indicate conserved transcriptomic responses of these pathways between species. However, many genes, including prominent salt-stress responsive genes, were oppositely responsive in multiple-stresses, highlighting fundamental differences in the responses and regulation of these genes between species. This work provides a resource for translation of knowledge or functions between species.

Mitochondrial signalling is critical for acclimation and adaptation to flooding in Arabidopsis thaliana.

Mitochondria have critical functions in the acclimation to abiotic and biotic stresses. Adverse environmental conditions lead to increased demands in energy supply and metabolic intermediates, which are provided by mitochondrial ATP production and the tricarboxylic acid (TCA) cycle. Mitochondria also play a role as stress sensors to adjust nuclear gene expression via retrograde signalling with the transcription factor (TF) ANAC017 and the kinase CDKE1 key components to integrate various signals into this pathway. To determine the importance of mitochondria as sensors of stress and their contribution in the tolerance to adverse growth conditions, a comparative phenotypical, physiological and transcriptomic characterisation of Arabidopsis mitochondrial signalling mutants (cdke1/rao1 and anac017/rao2) and a set of contrasting accessions was performed after applying the complex compound stress of submergence. Our results showed that impaired mitochondrial retrograde signalling leads to increased sensitivity to the stress treatments. The multi-factorial approach identified a network of 702 co-expressed genes, including several WRKY TFs, overlapping in the transcriptional responses in the mitochondrial signalling mutants and stress-sensitive accessions. Functional characterisation of two WRKY TFs (WRKY40 and WRKY45), using both knockout and overexpressing lines, confirmed their role in conferring tolerance to submergence. Together, the results revealed that acclimation to submergence is dependent on mitochondrial retrograde signalling, and underlying transcriptional re-programming is used as an adaptation mechanism.

Temporal tissue-specific regulation of transcriptomes during barley (Hordeum vulgare) seed germination.

The distinct functions of individual cell types require cells to express specific sets of genes. The germinating seed is an excellent model to study genome regulation between cell types since the majority of the transcriptome is differentially expressed in a short period, beginning from a uniform, metabolically inactive state. In this study, we applied laser-capture microdissection RNA-sequencing to small numbers of cells from the plumule, radicle tip and scutellum of germinating barley seeds every 8 h, over a 48 h time course. Tissue-specific gene expression was notably common; 25% (910) of differentially expressed transcripts in plumule, 34% (1876) in radicle tip and 41% (2562) in scutellum were exclusive to that organ. We also determined that tissue-specific storage of transcripts occurs during seed development and maturation. Co-expression of genes had strong spatiotemporal structure, with most co-expression occurring within one organ and at a subset of specific time points during germination. Overlapping and distinct enrichment of functional categories were observed in the tissue-specific profiles. We identified candidate transcription factors amongst these that may be regulators of spatiotemporal gene expression programs. Our findings contribute to the broader goal of generating an integrative model that describes the structure and function of individual cells within seeds during germination.

Mitochondrial function modulates touch signalling in Arabidopsis thaliana.

Plants respond to short- and long-term mechanical stimuli, via altered transcript abundance and growth respectively. Jasmonate, gibberellic acid and calcium have been implicated in mediating responses to mechanical stimuli. Previously it has been shown that the transcript abundance for the outer mitochondrial membrane protein of 66 kDa (OM66), is induced several fold after 30 min in response to touch. Therefore, the effect of mitochondrial function on the response to mechanical stimulation by touch at 30 min was investigated. Twenty-five mutants targeting mitochondrial function or regulators revealed that all affected the touch transcriptome. Double and triple mutants revealed synergistic or antagonistic effects following the observed responses in the single mutants. Changes in the touch-responsive transcriptome were localised, recurring with repeated rounds of stimulus. The gene expression kinetics after repeated touch were complex, displaying five distinct patterns. These transcriptomic responses were altered by some regulators of mitochondrial retrograde signalling, such as cyclic dependent protein kinase E1, a kinase protein in the mediator complex, and KIN10 (SnRK1 - sucrose non-fermenting related protein kinase 1), revealing an overlap between the touch response and mitochondrial stress signalling and alternative mitochondrial metabolic pathways. Regulatory network analyses revealed touch-induced stress responses and suppressed growth and biosynthetic processes. Interaction with the phytohormone signalling pathways indicated that ethylene and gibberellic acid had the greatest effect. Hormone measurements revealed that mutations of genes that encoded mitochondrial proteins altered hormone concentrations. Mitochondrial function modulates touch-induced changes in gene expression directly through altered regulatory networks, and indirectly via altering hormonal levels.

ANAC017 Coordinates Organellar Functions and Stress Responses by Reprogramming Retrograde Signaling.

Mitochondria adjust their activities in response to external and internal stimuli to optimize growth via the mitochondrial retrograde response signaling pathway. The Arabidopsis (Arabidopsis thaliana) NAC domain transcription factor ANAC017 has previously been identified as a regulator of the mitochondrial retrograde response. We show here that overexpression of ANAC017 in Arabidopsis leads to growth retardation, altered leaf development with decreased cell size and viability, and early leaf senescence. RNA sequencing analyses revealed that increased ANAC017 expression leads to higher expression of genes related to mitochondrial stress, cell death/autophagy, and leaf senescence under nonlimiting growth conditions as well as extensive repression of chloroplast function. Gene regulatory network analysis indicated that a complex hierarchy of transcription factors exists downstream of ANAC017. These involve a set of up-regulated ANAC and WRKY transcription factors associated with organellar signaling and senescence. The network also includes a number of ethylene- and gibberellic acid-related transcription factors with established functions in stress responses and growth regulation, which down-regulate their target genes. A number of BASIC LEUCINE-ZIPPER MOTIF transcription factors involved in the endoplasmic reticulum unfolded protein response or balancing of energy homeostasis via the SNF1-RELATED PROTEIN KINASE1 were also down-regulated by ANAC017 overexpression. Our results show that the endoplasmic reticulum membrane tethering of the constitutively expressed ANAC017, and its controlled release, are crucial to fine-tune a fast reactive but potentially harmful signaling cascade. Thus, ANAC017 is a master regulator of cellular responses with mitochondria acting as central sensors.

Conserved and Opposite Transcriptome Patterns during Germination in Hordeum vulgare and Arabidopsis thaliana.

Seed germination is a critical process for completion of the plant life cycle and for global food production. Comparing the germination transcriptomes of barley (Hordeum vulgare) to Arabidopsis thaliana revealed the overall pattern was conserved in terms of functional gene ontology; however, many oppositely responsive orthologous genes were identified. Conserved processes included a set of approximately 6000 genes that peaked early in germination and were enriched in processes associated with RNA metabolism, e.g., pentatricopeptide repeat (PPR)-containing proteins. Comparison of orthologous genes revealed more than 3000 orthogroups containing almost 4000 genes that displayed similar expression patterns including functions associated with mitochondrial tricarboxylic acid (TCA) cycle, carbohydrate and RNA/DNA metabolism, autophagy, protein modifications, and organellar function. Biochemical and proteomic analyses indicated mitochondrial biogenesis occurred early in germination, but detailed analyses revealed the timing involved in mitochondrial biogenesis may vary between species. More than 1800 orthogroups representing 2000 genes displayed opposite patterns in transcript abundance, representing functions of energy (carbohydrate) metabolism, photosynthesis, protein synthesis and degradation, and gene regulation. Differences in expression of basic-leucine zippers (bZIPs) and Apetala 2 (AP2)/ethylene-responsive element binding proteins (EREBPs) point to differences in regulatory processes at a high level, which provide opportunities to modify processes in order to enhance grain quality, germination, and storage as needed for different uses.

Dynamic and rapid changes in the transcriptome and epigenome during germination and in developing rice (Oryza sativa) coleoptiles under anoxia and re-oxygenation.

Detailed molecular profiling of Oryza sativa (rice) was carried out to uncover the features that are essential for germination and early seedling growth under anoxic conditions. Temporal analysis of the transcriptome and methylome from germination to young seedlings under aerobic and anaerobic conditions revealed 82% similarity in the transcriptome and no differences in the epigenome up to 24 h. Following germination, significant changes in the transcriptome and DNA methylation were observed between 4-day aerobically and anaerobically grown coleoptiles. A link between the epigenomic state and cell division versus cell elongation is suggested, as no differences in DNA methylation were observed between 24-h aerobically and anaerobically germinating embryos, when there is little cell division. After that, epigenetic changes appear to correlate with differences between cell elongation (anaerobic conditions) versus cell division (aerobic conditions) in the coleoptiles. Re-oxygenation of 3-day anaerobically grown seedlings resulted in rapid transcriptomic changes in DNA methylation in these coleoptiles. Unlike the transcriptome, changes in DNA methylation upon re-oxygenation did not reflect those seen in aerobic coleoptiles, but instead, reverted to a pattern similar to dry seeds. Reversion to the 'dry seed' state of DNA methylation upon re-oxygenation may act to 'reset the clock' for the rapid molecular changes and cell division that result upon re-oxygenation.

Dissecting the Metabolic Role of Mitochondria during Developmental Leaf Senescence.

The functions of mitochondria during leaf senescence, a type of programmed cell death aimed at the massive retrieval of nutrients from the senescing organ to the rest of the plant, remain elusive. Here, combining experimental and analytical approaches, we showed that mitochondrial integrity in Arabidopsis (Arabidopsis thaliana) is conserved until the latest stages of leaf senescence, while their number drops by 30%. Adenylate phosphorylation state assays and mitochondrial respiratory measurements indicated that the leaf energy status also is maintained during this time period. Furthermore, after establishing a curated list of genes coding for products targeted to mitochondria, we analyzed in isolation their transcript profiles, focusing on several key mitochondrial functions, such as the tricarboxylic acid cycle, mitochondrial electron transfer chain, iron-sulfur cluster biosynthesis, transporters, as well as catabolic pathways. In tandem with a metabolomic approach, our data indicated that mitochondrial metabolism was reorganized to support the selective catabolism of both amino acids and fatty acids. Such adjustments would ensure the replenishment of α-ketoglutarate and glutamate, which provide the carbon backbones for nitrogen remobilization. Glutamate, being the substrate of the strongly up-regulated cytosolic glutamine synthase, is likely to become a metabolically limiting factor in the latest stages of developmental leaf senescence. Finally, an evolutionary age analysis revealed that, while branched-chain amino acid and proline catabolism are very old mitochondrial functions particularly enriched at the latest stages of leaf senescence, auxin metabolism appears to be rather newly acquired. In summation, our work shows that, during developmental leaf senescence, mitochondria orchestrate catabolic processes by becoming increasingly central energy and metabolic hubs.

Plant-Specific Preprotein and Amino Acid Transporter Proteins Are Required for tRNA Import into Mitochondria.

A variety of eukaryotes, in particular plants, do not contain the required number of tRNAs to support the translation of mitochondria-encoded genes and thus need to import tRNAs from the cytosol. This study identified two Arabidopsis (Arabidopsis thaliana) proteins, Tric1 and Tric2 (for tRNA import component), which on simultaneous inactivation by T-DNA insertion lines displayed a severely delayed and chlorotic growth phenotype and significantly reduced tRNA import capacity into isolated mitochondria. The predicted tRNA-binding domain of Tric1 and Tric2, a sterile-α-motif at the C-terminal end of the protein, was required to restore tRNA uptake ability in mitochondria of complemented plants. The purified predicted tRNA-binding domain binds the T-arm of the tRNA for alanine with conserved lysine residues required for binding. T-DNA inactivation of both Tric proteins further resulted in an increase in the in vitro rate of in organello protein synthesis, which was mediated by a reorganization of the nuclear transcriptome, in particular of genes encoding a variety of proteins required for mitochondrial gene expression at both the transcriptional and translational levels. The characterization of Tric1/2 provides mechanistic insight into the process of tRNA import into mitochondria and supports the theory that the tRNA import pathway resulted from the repurposing of a preexisting protein import apparatus.

Molecular interaction between PHO2 and GIGANTEA reveals a new crosstalk between flowering time and phosphate homeostasis in Oryza sativa.

Plants are often confronted to nutrient limiting conditions, such as inorganic phosphate (Pi) deficiency, resulting in a reduction in growth and yield. PHO2, encoding a ubiquitin-conjugating E2 enzyme, is a central component of the Pi-starvation response signalling pathway. A yeast-two-hybrid screen using Oryza sativa (rice) PHO2 as bait, revealed an interaction between OsPHO2 and OsGIGANTEA, a key regulator of flowering time, which was confirmed using bimolecular fluorescence complementation (BiFC). Characterization of rice Osgi and Ospho2 mutants revealed that they displayed several similar phenotypic features supporting a physiological role for this interaction. Reduced growth, leaf tip necrosis, delayed flowering and over-accumulation of Pi in leaves compared to wild type were shared features of Osgi and Ospho2 plants. Pi analysis of individual leaves demonstrated that Osgi, similar to Ospho2 mutants, were impaired in Pi remobilization from old to young leaves, albeit to a lesser extent. Transcriptome analyses revealed more than 55% of the genes differentially expressed in Osgi plants overlapped with the set of differentially expressed genes in Ospho2 plants. The interaction between OsPHO2 and OsGI links high-level regulators of Pi homeostasis and development in rice.

Mechanisms of growth and patterns of gene expression in oxygen-deprived rice coleoptiles.

Coleoptiles of rice (Oryza sativa) seedlings grown under water commonly elongate by up to 1 mm h(-1) to reach the atmosphere. We initially analysed this highly specialized phenomenon by measuring epidermal cell lengths along the coleoptile axis to determine elongation rates. This revealed a cohort of cells in the basal zone that elongated rapidly following emergence from the embryo, reaching 200 µm within 12 h. After filming coleoptiles in vivo for a day, kinematic analysis was applied. Eight time-sliced 'segments' were defined by their emergence from the embryo at four-hourly intervals, revealing a mathematically simple growth model. Each segment entering the coleoptile from the embryo elongated at a constant velocity, resulting in accelerating growth for the entire organ. Consistent with the epidermal cell lengths, relative rates of elongation (mm mm(-1) h(-1)) were tenfold greater in the small, newly emerged basal segments than the older distal tip segments. This steep axial gradient defined two contrasting growth zones (bases versus tips) in which we measured ATP production and protein, RNA and DNA content, and analysed the global transcriptome under steady-state normoxia, hypoxia (3% O2) and anoxia. Determination of the transcriptome revealed tip-specific induction of genes encoding TCP [Teosinte Branched1 (Tb1) of maize, Cycloidea (Cyc), and Proliferating Cell Factor (Pcf)] transcription factors, RNA helicases, ribosomal proteins and proteins involved in protein folding, whilst expression of F-box domain-containing proteins in the ubiquitin E3-SCF complex (Skp, Cullin, F-box containing complex) was induced specifically in bases under low oxygen conditions. We ascribed the sustained elongation under hypoxia to hypoxia-specific responses such as controlled suppression of photosystem components and induction of RNA binding/splicing functions, indicating preferential allocation of energy to cell extension.

Decreasing electron flux through the cytochrome and/or alternative respiratory pathways triggers common and distinct cellular responses dependent on growth conditions.

Diverse signaling pathways are activated by perturbation of mitochondrial function under different growth conditions.Mitochondria have emerged as an important organelle for sensing and coping with stress in addition to being the sites of important metabolic pathways. Here, responses to moderate light and drought stress were examined in different Arabidopsis (Arabidopsis thaliana) mutant plants lacking a functional alternative oxidase (alternative oxidase1a [aox1a]), those with reduced cytochrome electron transport chain capacity (T3/T7 bacteriophage-type RNA polymerase, mitochondrial, and plastidial [rpoTmp]), and double mutants impaired in both pathways (aox1a:rpoTmp). Under conditions considered optimal for growth, transcriptomes of aox1a and rpoTmp were distinct. Under adverse growth conditions, however, transcriptome changes in aox1a and rpoTmp displayed a highly significant overlap and were indicative of a common mitochondrial stress response and down-regulation of photosynthesis. This suggests that the role of mitochondria to support photosynthesis is provided through either the alternative pathway or the cytochrome pathway, and when either pathway is inhibited, such as under environmental stress, a common, dramatic, and succinct mitochondrial signal is activated to alter energy metabolism in both organelles. aox1a:rpoTmp double mutants grown under optimal conditions showed dramatic reductions in biomass production compared with aox1a and rpoTmp and a transcriptome that was distinct from aox1a or rpoTmp. Transcript data indicating activation of mitochondrial biogenesis in aox1a:rpoTmp were supported by a proteomic analysis of over 200 proteins. Under optimal conditions, aox1a:rpoTmp plants seemed to switch on many of the typical mitochondrial stress regulators. Under adverse conditions, aox1a:rpoTmp turned off these responses and displayed a biotic stress response. Taken together, these results highlight the diverse signaling pathways activated by the perturbation of mitochondrial function under different growth conditions.

Characterization of a novel ß-barrel protein (AtOM47) from the mitochondrial outer membrane of Arabidopsis thaliana.

In plant cells, mitochondria are major providers of energy and building blocks for growth and development as well as abiotic and biotic stress responses. They are encircled by two lipid membranes containing proteins that control mitochondrial function through the import of macromolecules and metabolites. Characterization of a novel ß-barrel protein, OUTER MEMBRANE PROTEIN 47 (OM47), unique to the green lineage and related to the voltage-dependent anion channel (VDAC) protein family, showed that OM47 can complement a VDAC mutant in yeast. Mutation of OM47 in Arabidopsis thaliana by T-DNA insertion had no effect on the import of proteins, such as the ß-barrel proteins translocase of the outer membrane 40 (TOM40) or sorting and assembly machinery 50 (SAM50), into mitochondria. Molecular and physiological analyses revealed a delay in chlorophyll breakdown, higher levels of starch, and a delay in the induction of senescence marker genes in the mutant lines. While there was a reduction of >90% in OM47 protein in mitochondria isolated from 3-week-old om47 mutants, in mitochondria isolated from 8-week-old plants OM47 levels were similar to that of the wild type. This recovery was achieved by an up-regulation of OM47 transcript abundance in the mutants. Combined, these results highlight a role in leaf senescence for this plant-specific ß-barrel protein, probably mediating the recovery and recycling of chloroplast breakdown products by transporting metabolic intermediates into and out of mitochondria.

The plant mitochondrial protein import apparatus - the differences make it interesting.

BACKGROUND: Mitochondria play essential roles in the life and death of almost all eukaryotic cells, ranging from single-celled to multi-cellular organisms that display tissue and developmental differentiation. As mitochondria only arose once in evolution, much can be learned from studying single celled model systems such as yeast and applying this knowledge to other organisms. However, two billion years of evolution have also resulted in substantial divergence in mitochondrial function between eukaryotic organisms. SCOPE OF REVIEW: Here we review our current understanding of the mechanisms of mitochondrial protein import between plants and yeast (Saccharomyces cerevisiae) and identify a high level of conservation for the essential subunits of plant mitochondrial import apparatus. Furthermore, we investigate examples whereby divergence and acquisition of functions have arisen and highlight the emerging examples of interactions between the import apparatus and components of the respiratory chain. MAJOR CONCLUSIONS: After more than three decades of research into the components and mechanisms of mitochondrial protein import of plants and yeast, the differences between these systems are examined. Specifically, expansions of the small gene families that encode the mitochondrial protein import apparatus in plants are detailed, and their essential role in seed viability is revealed. GENERAL SIGNIFICANCE: These findings point to the essential role of the inner mitochondrial protein translocases in Arabidopsis, establishing their necessity for seed viability and the crucial role of mitochondrial biogenesis during germination. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

MPIC: a mitochondrial protein import components database for plant and non-plant species.

In the 2 billion years since the endosymbiotic event that gave rise to mitochondria, variations in mitochondrial protein import have evolved across different species. With the genomes of an increasing number of plant species sequenced, it is possible to gain novel insights into mitochondrial protein import pathways. We have generated the Mitochondrial Protein Import Components (MPIC) Database (DB; http://www.plantenergy.uwa.edu.au/applications/mpic) providing searchable information on the protein import apparatus of plant and non-plant mitochondria. An in silico analysis was carried out, comparing the mitochondrial protein import apparatus from 24 species representing various lineages from Saccharomyces cerevisiae (yeast) and algae to Homo sapiens (human) and higher plants, including Arabidopsis thaliana (Arabidopsis), Oryza sativa (rice) and other more recently sequenced plant species. Each of these species was extensively searched and manually assembled for analysis in the MPIC DB. The database presents an interactive diagram in a user-friendly manner, allowing users to select their import component of interest. The MPIC DB presents an extensive resource facilitating detailed investigation of the mitochondrial protein import machinery and allowing patterns of conservation and divergence to be recognized that would otherwise have been missed. To demonstrate the usefulness of the MPIC DB, we present a comparative analysis of the mitochondrial protein import machinery in plants and non-plant species, revealing plant-specific features that have evolved.

The mitochondrial protein import component, TRANSLOCASE OF THE INNER MEMBRANE17-1, plays a role in defining the timing of germination in Arabidopsis.

In Arabidopsis (Arabidopsis thaliana), small gene families encode multiple isoforms for many of the components of the mitochondrial protein import apparatus. There are three isoforms of the TRANSLOCASE OF THE INNER MEMBRANE17 (Tim17). Transcriptome analysis indicates that AtTim17-1 is only detectable in dry seed. In this study, two independent transfer DNA insertional mutant lines of tim17-1 exhibited a germination-specific phenotype, showing a significant increase in the rate of germination. Microarray analyses revealed that Attim17-1 displayed alterations in the temporal sequence of transcriptomic events during germination, peaking earlier compared with the wild type. Promoter analysis of AtTim17-1 further identified an abscisic acid (ABA)-responsive element, which binds ABA-responsive transcription factors, acting to repress the expression of AtTim17-1. Attim17-1 dry seeds contained significantly increased levels of ABA and gibberellin, 2- and 5-fold, respectively. These results support the model that mitochondrial biogenesis is regulated in a tight temporal sequence of events during germination and that altering mitochondrial biogenesis feeds back to alter the germination rate, as evidenced by the altered levels of the master regulatory hormones that define germination.

A Functional Antagonistic Relationship between Auxin and Mitochondrial Retrograde Signaling Regulates Alternative Oxidase1a Expression in Arabidopsis.

The perception and integration of stress stimuli with that of mitochondrion function are important during periods of perturbed cellular homeostasis. In a continuous effort to delineate these mitochondrial/stress-interacting networks, forward genetic screens using the mitochondrial stress response marker alternative oxidase 1a (AOX1a) provide a useful molecular tool to identify and characterize regulators of mitochondrial stress signaling (referred to as regulators of alternative oxidase 1a [RAOs] components). In this study, we reveal that mutations in genes coding for proteins associated with auxin transport and distribution resulted in a greater induction of AOX1a in terms of magnitude and longevity. Three independent mutants for polarized auxin transport, rao3/big, rao4/pin-formed1, and rao5/multidrug-resistance1/abcb19, as well as the Myb transcription factor rao6/asymmetric leaves1 (that displays altered auxin patterns) were identified and resulted in an acute sensitivity toward mitochondrial dysfunction. Induction of the AOX1a reporter system could be inhibited by the application of auxin analogs or reciprocally potentiated by blocking auxin transport. Promoter activation studies with AOX1a::GUS and DR5::GUS lines further confirmed a clear antagonistic relationship between the spatial distribution of mitochondrial stress and auxin response kinetics, respectively. Genome-wide transcriptome analyses revealed that mitochondrial stress stimuli, such as antimycin A, caused a transient suppression of auxin signaling and conversely, that auxin treatment repressed a part of the response to antimycin A treatment, including AOX1a induction. We conclude that mitochondrial stress signaling and auxin signaling are reciprocally regulated, balancing growth and stress response(s).

Dual location of the mitochondrial preprotein transporters B14.7 and Tim23-2 in complex I and the TIM17:23 complex in Arabidopsis links mitochondrial activity and biogenesis.

Interactions between the respiratory chain and protein import complexes have been previously reported in Saccharomyces cerevisiae, but the biological significance of such interactions remains unknown. Characterization of two mitochondrial preprotein and amino acid transport proteins from Arabidopsis thaliana, NADH dehydrogenase B14.7 like (B14.7 [encoded by At2g42210]) and Translocase of the inner membrane subunit 23-2 (Tim23-2 [encoded by At1g72750]), revealed both proteins are present in respiratory chain complex I and the Translocase of the Inner Membrane 17:23. Whereas depletion of B14.7 by T-DNA insertion is lethal, Tim23-2 can be depleted without lethality. Subtle overexpression of Tim23-2 results in a severe delayed growth phenotype and revealed an unexpected, inverse correlation between the abundance of Tim23-2 and the abundance of respiratory complex I. This newly discovered relationship between protein import and respiratory function was confirmed through the investigation of independent complex I knockout mutants, which were found to have correspondingly increased levels of Tim23-2. This increase in Tim23-2 was also associated with delayed growth phenotypes, increased abundance of other import components, and an increased capacity for mitochondrial protein import. Analysis of the Tim23-2-overexpressing plants through global quantitation of transcript abundance and in-organelle protein synthesis assays revealed widespread alterations in transcript abundance of genes encoding mitochondrial proteins and altered rates of mitochondrial protein translation, indicating a pivotal relationship between the machinery of mitochondrial biogenesis and mitochondrial function.