Coordinated regulation of the mitochondrial retrograde response by circadian clock regulators and ANAC017.

Mitochondrial retrograde signaling (MRS) supports photosynthetic function under a variety of conditions. Induction of mitochondrial dysfunction with myxothiazol (a specific inhibitor of the mitochondrial bc1 complex) or antimycin A (an inhibitor of the mitochondrial bc1 complex and cyclic electron transport in the chloroplast under light conditions) in the light and dark revealed diurnal control of MRS. This was evidenced by (1) significantly enhanced binding of ANAC017 to promoters in the light compared with the dark in Arabidopsis plants treated with myxothiazol (but not antimycin A), (2) overlap in the experimentally determined binding sites for ANAC017 and circadian clock regulators in the promoters of ANAC013 and AOX1a, (3) a diurnal expression pattern for ANAC017 and transcription factors it regulates, (4) altered expression of ANAC017-regulated genes in circadian clock mutants with and without myxothiazol treatment, and (5) a decrease in the magnitude of LHY and CCA1 expression in an ANAC017-overexpressing line and protein-protein interaction between ANAC017 and PIF4. This study also shows a large difference in transcriptome responses to antimycin A and myxothiazol in the dark: these responses are ANAC017 independent, observed in shoots and roots, similar to biotic challenge and salicylic acid responses, and involve ERF and ZAT transcription factors. This suggests that antimycin A treatment stimulates a second MRS pathway that is mediated or converges with salicylic acid signaling and provides a merging point with chloroplast retrograde signaling.

Co-regulation of mitochondrial and chloroplast function: Molecular components and mechanisms.

The metabolic interdependence, interactions, and coordination of functions between chloroplasts and mitochondria are established and intensively studied. However, less is known about the regulatory components that control these interactions and their responses to external stimuli. Here, we outline how chloroplastic and mitochondrial activities are coordinated via common components involved in signal transduction pathways, gene regulatory events, and post-transcriptional processes. The endoplasmic reticulum emerges as a point of convergence for both transcriptional and post-transcriptional pathways that coordinate chloroplast and mitochondrial functions. Although the identification of molecular components and mechanisms of chloroplast and mitochondrial signaling increasingly suggests common players, this raises the question of how these allow for distinct organelle-specific downstream pathways. Outstanding questions with respect to the regulation of post-transcriptional pathways and the cell and/or tissue specificity of organelle signaling are crucial for understanding how these pathways are integrated at a whole-plant level to optimize plant growth and its response to changing environmental conditions.

The retrograde signaling regulator ANAC017 recruits the MKK9-MPK3/6, ethylene, and auxin signaling pathways to balance mitochondrial dysfunction with growth.

In plant cells, mitochondria are ideally positioned to sense and balance changes in energy metabolism in response to changing environmental conditions. Retrograde signaling from mitochondria to the nucleus is crucial for adjusting the required transcriptional responses. We show that ANAC017, the master regulator of mitochondrial stress, directly recruits a signaling cascade involving the plant hormones ethylene and auxin as well as the MAP KINASE KINASE (MKK) 9-MAP KINASE (MPK) 3/6 pathway in Arabidopsis thaliana. Chromatin immunoprecipitation followed by sequencing and overexpression demonstrated that ANAC017 directly regulates several genes of the ethylene and auxin pathways, including MKK9, 1-AMINO-CYCLOPROPANE-1-CARBOXYLATE SYNTHASE 2, and YUCCA 5, in addition to genes encoding transcription factors regulating plant growth and stress responses such as BASIC REGION/LEUCINE ZIPPER MOTIF (bZIP) 60, bZIP53, ANAC081/ATAF2, and RADICAL-INDUCED CELL DEATH1. A time-resolved RNA-seq experiment established that ethylene signaling precedes the stimulation of auxin signaling in the mitochondrial stress response, with a large part of the transcriptional regulation dependent on ETHYLENE-INSENSITIVE 3. These results were confirmed by mutant analyses. Our findings identify the molecular components controlled by ANAC017, which integrates the primary stress responses to mitochondrial dysfunction with whole plant growth via the activation of regulatory and partly antagonistic feedback loops.

GWAS on multiple traits identifies mitochondrial ACONITASE3 as important for acclimation to submergence stress.

Flooding causes severe crop losses in many parts of the world. Genetic variation in flooding tolerance exists in many species; however, there are few examples for the identification of tolerance genes and their underlying function. We conducted a genome-wide association study (GWAS) in 387 Arabidopsis (Arabidopsis thaliana) accessions. Plants were subjected to prolonged submergence followed by desubmergence, and seven traits (score, water content, Fv/Fm, and concentrations of nitrate, chlorophyll, protein, and starch) were quantified to characterize their acclimation responses. These traits showed substantial variation across the range of accessions. A total of 35 highly significant single-nucleotide polymorphisms (SNPs) were identified across the 20 GWA datasets, pointing to 22 candidate genes, with functions in TCA cycle, DNA modification, and cell division. Detailed functional characterization of one candidate gene, ACONITASE3 (ACO3), was performed. Chromatin immunoprecipitation followed by sequencing showed that a single nucleotide polymorphism in the ACO3 promoter co-located with the binding site of the master regulator of retrograde signaling ANAC017, while subcellular localization of an ACO3-YFP fusion protein confirmed a mitochondrial localization during submergence. Analysis of mutant and overexpression lines determined changes in trait parameters that correlated with altered submergence tolerance and were consistent with the GWAS results. Subsequent RNA-seq experiments suggested that impairing ACO3 function increases the sensitivity to submergence by altering ethylene signaling, whereas ACO3 overexpression leads to tolerance by metabolic priming. These results indicate that ACO3 impacts submergence tolerance through integration of carbon and nitrogen metabolism via the mitochondrial TCA cycle and impacts stress signaling during acclimation to stress.

Identification of regulatory networks and hub genes controlling soybean seed set and size using RNA sequencing analysis.

To understand the gene expression networks controlling soybean seed set and size, transcriptome analyses were performed in three early seed developmental stages, using two genotypes with contrasting seed size. The two-dimensional data set provides a comprehensive and systems-level view on dynamic gene expression networks underpinning soybean seed set and subsequent development. Using pairwise comparisons and weighted gene coexpression network analyses, we identified modules of coexpressed genes and hub genes for each module. Of particular importance are the discoveries of specific modules for the large seed size variety and for seed developmental stages. A large number of candidate regulators for seed size, including those involved in hormonal signaling pathways and transcription factors, were transiently and specifically induced in the early developmental stages. The soybean homologs of a brassinosteroid signaling receptor kinase, a brassinosteroid-signaling kinase, were identified as hub genes operating in the seed coat network in the early seed maturation stage. Overexpression of a candidate seed size regulatory gene, GmCYP78A5, in transgenic soybean resulted in increased seed size and seed weight. Together, these analyses identified a large number of potential key regulators controlling soybean seed set, seed size, and, consequently, yield potential, thereby providing new insights into the molecular networks underlying soybean seed development.