Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3′-phosphoadenosine 5′-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5′ to 3′ exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.

Plants require daily coordinated regulation of energy metabolism for optimal growth and survival and therefore need to integrate cellular responses with both mitochondrial and plastid retrograde signaling. Using a forward genetic screen to characterize regulators of alternative oxidase1a (rao) mutants, we identified RAO2/Arabidopsis NAC domain-containing protein17 (ANAC017) as a direct positive regulator of AOX1a. RAO2/ANAC017 is targeted to connections and junctions in the endoplasmic reticulum (ER) and F-actin via a C-terminal transmembrane (TM) domain. A consensus rhomboid protease cleavage site is present in ANAC017 just prior to the predicted TM domain. Furthermore, addition of the rhomboid protease inhibitor N-p-Tosyl-l-Phe chloromethyl abolishes the induction of AOX1a upon antimycin A treatment. Simultaneous fluorescent tagging of ANAC017 with N-terminal red fluorescent protein (RFP) and C-terminal green fluorescent protein (GFP) revealed that the N-terminal RFP domain migrated into the nucleus, while the C-terminal GFP tag remained in the ER. Genome-wide analysis of the transcriptional network regulated by RAO2/ANAC017 under stress treatment revealed that RAO2/ANAC017 function was necessary for >85% of the changes observed as a primary response to cytosolic hydrogen peroxide (H₂O₂), but only ∼33% of transcriptional changes observed in response to antimycin A treatment. Plants with mutated rao2/anac017 were more stress sensitive, whereas a gain-of-function mutation resulted in plants that had lower cellular levels of H₂O₂ under untreated conditions.

Summary: Mitochondria host vital cellular functions, including oxidative phosphorylation and co‐factor biosynthesis, which are reflected in their proteome. At the cellular level plant mitochondria are organized into hundreds of discrete functional entities, which undergo dynamic fission and fusion. It is the individual organelle that operates in the living cell, yet biochemical and physiological assessments have exclusively focused on the characteristics of large populations of mitochondria. Here, we explore the protein composition of an individual average plant mitochondrion to deduce principles of functional and structural organisation. We perform proteomics on purified mitochondria from cultured heterotrophic Arabidopsis cells with intensity‐based absolute quantification and scale the dataset to the single organelle based on criteria that are justified by experimental evidence and theoretical considerations. We estimate that a total of 1.4 million protein molecules make up a single Arabidopsis mitochondrion on average. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40 000 for Voltage‐Dependent Anion Channel 1 to sub‐stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat proteins that modify mitochondrial transcripts. For our analysis, we consider the physical and chemical constraints of the single organelle and discuss prominent features of mitochondrial architecture, protein biogenesis, oxidative phosphorylation, metabolism, antioxidant defence, genome maintenance, gene expression, and dynamics. While assessing the limitations of our considerations, we exemplify how our understanding of biochemical function and structural organization of plant mitochondria can be connected in order to obtain global and specific insights into how organelles work.

Germination represents a rapid transition from dormancy to a high level of metabolic activity. In-depth transcriptomic profiling at 10 time points in Arabidopsis (Arabidopsis thaliana), including fresh seed, ripened seed, during stratification, germination, and postgermination per se, revealed specific temporal expression patterns that to our knowledge have not previously been identified. Over 10,000 transcripts were differentially expressed during cold stratification, with subequal numbers up-regulated as down-regulated, revealing an active period in preparing seeds for germination, where transcription and RNA degradation both play important roles in regulating the molecular sequence of events. A previously unidentified transient expression pattern was observed for a group of genes, whereby a significant rise in expression was observed at the end of stratification and significantly lower expression was observed 6 h later. These genes were further defined as germination specific, as they were most highly expressed at this time in germination, in comparison with all developmental tissues in the AtGenExpress data set. Functional analysis of these genes using genetic inactivation revealed that they displayed a significant enrichment for embryo-defective or -arrested phenotype. This group was enriched in genes encoding mitochondrial and nuclear RNA-processing proteins, including more than 45% of all pentatricopeptide domain-containing proteins expressed during germination. The presence of mitochondrial DNA replication factors and RNA-processing functions in this germination-specific subset represents the earliest events in organelle biogenesis, preceding any changes associated with energy metabolism. Green fluorescent protein analysis also confirmed organellar localization for 65 proteins, largely showing germination-specific expression. These results suggest that mitochondrial biogenesis involves a two-step process to produce energetically active organelles: an initial phase at the end of stratification involving mitochondrial DNA synthesis and RNA processing, and a later phase for building the better-known energetic functions. This also suggests that signals with a mitochondrial origin and retrograde signals may be crucial for successful germination.

Diurnal regulation of transcripts encoding proteins located in mitochondria, plastids, and peroxisomes is important for adaptation of organelle biogenesis and metabolism to meet cellular requirements. We show this regulation is related to diurnal changes in promoter activities and the presence of specific cis-acting regulatory elements in the proximal promoter region [TGGGC(C/T)], previously defined as site II elements, and leads to diurnal changes in organelle protein abundances. These site II elements can act both as activators or repressors of transcription, depending on the night/day period and on the number and arrangement of site II elements in the promoter tested. These elements bind to the TCP family of transcriptions factors in Arabidopsis thaliana, which nearly all display distinct diurnal patterns of cycling transcript abundance. TCP2, TCP3, TCP11, and TCP15 were found to interact with different components of the core circadian clock in both yeast two-hybrid and direct protein–protein interaction assays, and tcp11 and tcp15 mutant plants showed altered transcript profiles for a number of core clock components, including LATE ELONGATED HYPOCOTYL1 and PSEUDO RESPONSE REGULATOR5. Thus, site II elements in the promoter regions of genes encoding mitochondrial, plastid, and peroxisomal proteins provide a direct mechanism for the coordination of expression for genes involved in a variety of organellar functions, including energy metabolism, with the time-of-day specific needs of the organism.

To obtain a global overview of how mitochondria respond to stress, we aimed to define the plant mitochondrial stress response (MSR). By combining a set of 1196 Arabidopsis thaliana genes that putatively encode mitochondrial proteins with 16 microarray experiments on stress-related conditions, 45 nuclear encoded genes were defined as widely stress-responsive. Using green fluorescent protein (GFP) fusion assays, the mitochondrial targeting of a large number of these proteins was tested, confirming in total 26 proteins as mitochondrially targeted. Several of these proteins were observed to be dual targeted to mitochondria and plastids, including the small heat shock proteins sHSP23.5 and sHSP23.6. In addition to the well defined stress components of mitochondria, such as alternative oxidases, nicotinamide adenine dinucleotide (NAD(P)H) dehydrogenases, and heat shock proteins, a variety of other proteins, many with unknown function, were identified. The mitochondrial carrier protein family was over-represented in the stress-responsive genes, suggesting that stress induces altered needs for metabolite transport across the mitochondrial inner membrane. Although the genes encoding many of these proteins contain common cis-acting regulatory elements, it was apparent that a number of distinct regulatory processes or signals likely triggered the MSR. Therefore, these genes provide new model systems to study mitochondrial retrograde regulation, in addition to the widely used alternative oxidase model. Additionally, as changes in proteins responsive to stress did not correlate well with changes at a transcript level, it suggests that post-transcriptional mechanisms also play an important role in defining the MSR.