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 (H 2 O 2 ), 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 H 2 O 2 under untreated conditions. INTRODUCTIONMitochondria and plastids (chloroplasts) are composed of ;1500 and ;3000 proteins, respectively, with >95% of these proteins encoded by nuclear-located genes (Woodson and Chory, 2008). It has been shown that two-way communication pathways exist between the nucleus and mitochondria and chloroplasts, called anterograde and retrograde signaling pathways (Rhoads and Subbaiah, 2007;Woodson and Chory, 2008). Anterograde regulation refers to a top-down regulatory pathway, where signals have a direct impact on gene expression in the nucleus. Conversely, nuclear gene expression is also influenced by signals that originate from within the organelles, mitochondria, or chloroplasts and is referred to as retrograde regulation.Several components involved in plastid retrograde signaling have been identified, with at least five different pathways characterized: reactive oxygen species (ROS), redox signals, plastidial gene expression, pigment biosynthesis, and specific signaling metabolites (Pfannschmidt, 2010). The most intensively studied retrograde signaling pathway is in the genomes uncoupled (gun) mutants that uncouple the expression of nuclear-encoded chloroplastic proteins from the functional state of chloroplasts (Susek et al., 1993). A recently identified plastid-bound transcription factor, PTM (plant homeodomaintype transcription factor with transmembrane [TM] domains), was also identified as a regulator for plastid retrograde signaling and acts do...

Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.

The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of β-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.

Mechanical stimuli, such as wind, rain, and touch affect plant development, growth, pest resistance, and ultimately reproductive success. Using water spray to simulate rain, we demonstrate that jasmonic acid (JA) signaling plays a key role in early gene-expression changes, well before it leads to developmental changes in flowering and plant architecture. The JA-activated transcription factors MYC2/MYC3/MYC4 modulate transiently induced expression of 266 genes, most of which peak within 30 min, and control 52% of genes induced >100-fold. Chromatin immunoprecipitation-sequencing analysis indicates that MYC2 dynamically binds >1,300 promoters and trans-activation assays show that MYC2 activates these promoters. By mining our multiomic datasets, we identified a core MYC2/MYC3/MYC4-dependent “regulon” of 82 genes containing many previously unknown MYC2 targets, including transcription factors bHLH19 and ERF109. bHLH19 can in turn directly activate the ORA47 promoter, indicating that MYC2/MYC3/MYC4 initiate a hierarchical network of downstream transcription factors. Finally, we also reveal that rapid water spray-induced accumulation of JA and JA-isoleucine is directly controlled by MYC2/MYC3/MYC4 through a positive amplification loop that regulates JA-biosynthesis genes.

Background:Mitochondria send signals to the nucleus to modulate gene expression when mitochondrial function is perturbed. Results: Cyclin-dependent kinase E1 (CDKE1) was identified as an essential component in regulation of responses to perturbation of mitochondrial electron transport. Conclusion: Mitochondrial regulation is integrated with growth, energy, and other cellular stress signaling pathways. Significance: The identification of a molecular link between mitochondrial retrograde regulation and growth and stress signaling pathways.

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.

SummaryA variety of approaches were used to predict dual-targeted proteins in Arabidopsis thaliana. These predictions were experimentally tested using GFP fusions. Twelve new dual-targeted proteins were identified: five that were dual-targeted to mitochondria and plastids, six that were dual-targeted to mitochondria and peroxisomes, and one that was dual-targeted to mitochondria and the nucleus. Two methods to predict dual-targeted proteins had a high success rate: (1) combining the AraPerox database with a variety of subcellular prediction programs to identify mitochondrial-and peroxisomal-targeted proteins, and (2) using a variety of prediction programs on a biochemical pathway or process known to contain at least one dualtargeted protein. Several technical parameters need to be taken into account before assigning subcellular localization using GFP fusion proteins. The position of GFP with respect to the tagged polypeptide, the tissue or cells used to detect subcellular localization, and the portion of a candidate protein fused to GFP are all relevant to the expression and targeting of a fusion protein. Testing all gene models for a chromosomal locus is required if more than one model exists.

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