FTSH PROTEASE 3 facilitates Complex I degradation through a direct interaction with the Complex I subunit PSST.

Complex I (CI) (NADH dehydrogenase), the largest complex involved in mitochondrial oxidative phosphorylation, is composed of nuclear- and mitochondrial-encoded subunits. CI assembly occurs via the sequential addition of subdomains and modules. As CI is prone to oxidative damage, its subunits continually undergo proteolysis and turnover. We describe the mechanism by which CI abundance is regulated in a CI-deficient Arabidopsis thaliana mutant. Using a forward genetic approach, we determined that the CI Q-module domain subunit PSST interacts with FTSH PROTEASE 3 (FTSH3) to mediate the disassembly of the matrix arm domain for proteolysis and turnover as a means of protein quality control. We demonstrated the direct interaction of FTSH3 with PSST and identified the amino acid residues required for this interaction. The ATPase function of FTSH3, rather than its proteolytic activity, is required for this interaction, as its mutation was compensated for by a proteolytically inactive form of FTSH3. This study reveals the mechanistic process by which FTSH3 recognizes CI for degradation at amino acid resolution.

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.

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/.

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.

The function of the Medicago truncatula ZIP transporter MtZIP14 is linked to arbuscular mycorrhizal fungal colonization.

Soil micronutrient availability, including zinc (Zn), is a limiting factor for crop yield. Arbuscular mycorrhizal (AM) fungi can improve host plant growth and nutrition through the mycorrhizal pathway of nutrient uptake. Although the physiology of Zn uptake through the mycorrhizal pathway is well established, the identity of the related molecular components are unknown. Here, RNA-seq analysis was used to identify genes differentially-regulated by AM colonization and soil Zn concentration in roots of Medicago truncatula. The putative Zn transporter gene MtZIP14 was markedly up-regulated in M. truncatula roots when colonized by Rhizophagus irregularis. MtZIP14 restored yeast growth under low Zn availability. Loss-of-function mutant plants (mtzip14) had reduced shoot biomass compared to the wild-type when colonized by AM fungi and grown under low and sufficient soil Zn concentration; at high soil Zn concentration, there were no genotypic differences in shoot biomass. The vesicular and arbuscular colonization of roots was lower in the mtzip14 plants regardless of soil Zn concentration. We propose that MtZIP14 is linked to AM colonization in M. truncatula plants, with the possibility that MtZIP14 function with AM colonization is linked to plant Zn nutrition.

RNA-seq analysis of laser microdissected Arabidopsis thaliana leaf epidermis, mesophyll and vasculature defines tissue-specific transcriptional responses to multiple stress treatments.

Acclimation of plants to adverse conditions requires the coordination of gene expression and signalling pathways between tissues and cell types. As the energy and carbon capturing organs, leaves are significantly affected by abiotic and biotic stresses. However, tissue- or cell type-specific analyses of stress responses have focussed on the Arabidopsis root. Here, we comparatively explore the transcriptomes of three leaf tissues (epidermis, mesophyll, vasculature) after induction of diverse stress pathways by chemical stimuli (antimycin A, 3-amino-1,2,4-triazole, methyl viologen, salicylic acid) and ultraviolet light in Arabidopsis using laser capture microdissection followed by RNA sequencing. Stimulation of stress pathways caused an overall reduction in the number of genes expressed in a tissue-specific manner, though a small subset gained or changed their tissue specificity. We find no evidence of a common stress response, with only a few genes consistently responsive to two or more treatments in the analysed tissues. However, differentially expressed genes overlap between tissues for individual treatments. A focussed analysis provided evidence for an interaction of auxin and ethylene that mediates retrograde signalling during mitochondrial dysfunction specifically in the epidermis, and a gene regulatory network defined the hierarchy of interactions. Taken together, we have generated an extensive reference dataset that will be valuable for future experiments analysing transcriptional responses on a tissue or single-cell level. Our results will enable the tailoring of the tissue-specific engineering of stress-tolerant plants.

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.

The mitochondrial AAA protease FTSH3 regulates Complex I abundance by promoting its disassembly.

ATP is generated in mitochondria by oxidative phosphorylation. Complex I (NADH:ubiquinone oxidoreductase or NADH dehydrogenase) is the first multisubunit protein complex of this pathway, oxidizing NADH and transferring electrons to the ubiquinone pool. Typically, Complex I mutants display a slow growth rate compared to wild-type plants. Here, using a forward genetic screen approach for restored growth of a Complex I mutant, we have identified the mitochondrial ATP-dependent metalloprotease, Filamentous Temperature Sensitive H 3 (FTSH3), as a factor that is required for the disassembly of Complex I. An ethyl methanesulfonate-induced mutation in FTSH3, named as rmb1 (restoration of mitochondrial biogenesis 1), restored Complex I abundance and plant growth. Complementation could be achieved with FTSH3 lacking proteolytic activity, suggesting the unfoldase function of FTSH3 has a role in Complex I disassembly. The introduction of the rmb1 to an additional, independent, and extensively characterized Complex I mutant, ndufs4, resulted in similar increases to Complex I abundance and a partial restoration of growth. These results show that disassembly or degradation of Complex I plays a role in determining its steady-state abundance and thus turnover may vary under different conditions.

Arabidopsis DGD1 SUPPRESSOR1 Is a Subunit of the Mitochondrial Contact Site and Cristae Organizing System and Affects Mitochondrial Biogenesis.

Mitochondrial and plastid biogenesis requires the biosynthesis and assembly of proteins, nucleic acids, and lipids. In Arabidopsis (Arabidopsis thaliana), the mitochondrial outer membrane protein DGD1 SUPPRESSOR1 (DGS1) is part of a large multi-subunit protein complex that contains the mitochondrial contact site and cristae organizing system 60-kD subunit, the translocase of outer mitochondrial membrane 40-kD subunit (TOM40), the TOM20s, and the Rieske FeS protein. A point mutation in DGS1, dgs1-1, altered the stability and protease accessibility of this complex. This altered mitochondrial biogenesis, mitochondrial size, lipid content and composition, protein import, and respiratory capacity. Whole plant physiology was affected in the dgs1-1 mutant as evidenced by tolerance to imposed drought stress and altered transcriptional responses of markers of mitochondrial retrograde signaling. Putative orthologs of Arabidopsis DGS1 are conserved in eukaryotes, including the Nuclear Control of ATP Synthase2 (NCA2) protein in yeast (Saccharomyces cerevisiae), but lost in Metazoa. The genes encoding DGS1 and NCA2 are part of a similar coexpression network including genes encoding proteins involved in mitochondrial fission, morphology, and lipid homeostasis. Thus, DGS1 links mitochondrial protein and lipid import with cellular lipid homeostasis and whole plant stress responses.

Enhanced reactive oxygen detoxification occurs in salt-stressed soybean roots expressing GmSALT3.

Soybean (Glycine max) is an important crop globally for food and edible oil production. Soybean plants are sensitive to salinity (NaCl), with significant yield decreases reported under saline conditions. GmSALT3 is the dominant gene underlying a major QTL for salt tolerance in soybean. GmSALT3 encodes a transmembrane protein belonging to the plant cation/proton exchanger (CHX) family, and is predominately expressed in root phloem and xylem associated cells under both saline and non-saline conditions. It is currently unknown through which molecular mechanism(s) the ER-localised GmSALT3 contributes to salinity tolerance, as its localisation excludes direct involvement in ion exclusion. In order to gain insights into potential molecular mechanism(s), we used RNA-seq analysis of roots from two soybean NILs (near isogenic lines); NIL-S (salt-sensitive, Gmsalt3), and NIL-T (salt-tolerant, GmSALT3), grown under control and saline conditions (200 mM NaCl) at three time points (0 h, 6 h, and 3 days). Gene ontology (GO) analysis showed that NIL-T has greater responses aligned to oxidation reduction. ROS were less abundant and scavenging enzyme activity was greater in NIL-T, consistent with the RNA-seq data. Further analysis indicated that genes related to calcium signalling, vesicle trafficking and Casparian strip (CS) development were upregulated in NIL-T following salt treatment. We propose that GmSALT3 improves the ability of NIL-T to cope with saline stress through preventing ROS overaccumulation in roots, and potentially modulating Ca2+ signalling, vesicle trafficking and formation of diffusion barriers.

The genetic origin of evolidine, the first cyclopeptide discovered in plants, and related orbitides.

Cyclic peptides are reported to have antibacterial, antifungal, and other bioactivities. Orbitides are a class of cyclic peptides that are small, head-to-tail cyclized, composed of proteinogenic amino acids and lack disulfide bonds; they are also known in several genera of the plant family Rutaceae. Melicope xanthoxyloides is the Australian rain forest tree of the Rutaceae family in which evolidine, the first plant cyclic peptide, was discovered. Evolidine (cyclo-SFLPVNL) has subsequently been all but forgotten in the academic literature, so to redress this we used tandem MS and de novo transcriptomics to rediscover evolidine and decipher its biosynthetic origin from a short precursor just 48 residues in length. We also identified another six M. xanthoxyloides orbitides using the same techniques. These peptides have atypically diverse C termini consisting of residues not recognized by either of the known proteases plants use to macrocyclize peptides, suggesting new cyclizing enzymes await discovery. We examined the structure of two of the novel orbitides by NMR, finding one had a definable structure, whereas the other did not. Mining RNA-seq and whole genome sequencing data from other species of the Rutaceae family revealed that a large and diverse family of peptides is encoded by similar sequences across the family and demonstrates how powerful de novo transcriptomics can be at accelerating the discovery of new peptide families.

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.

Knockdown of Succinate Dehydrogenase Assembly Factor 2 Induces Reactive Oxygen Species-Mediated Auxin Hypersensitivity Causing pH-Dependent Root Elongation.

Metabolism, auxin signaling and reactive oxygen species (ROS) all contribute to plant growth, and each is linked to plant mitochondria and the process of respiration. Knockdown of mitochondrial succinate dehydrogenase assembly factor 2 (SDHAF2) in Arabidopsis thaliana lowered succinate dehydrogenase activity and led to pH-inducible root inhibition when the growth medium pH was poised at different points between 7.0 and 5.0, but this phenomenon was not observed in wildtype (WT). Roots of sdhaf2 mutants showed high accumulation of succinate, depletion of citrate and malate and up-regulation of ROS-related and stress-inducible genes at pH 5.5. A change of oxidative status in sdhaf2 roots at low pH was also evidenced by low ROS staining in root tips and altered root sensitivity to H2O2. sdhaf2 had low auxin activity in root tips via DR5-GUS staining but displayed increased indole-3-acetic acid (IAA, auxin) abundance and IAA hypersensitivity, which is most likely caused by the change in ROS levels. On this basis, we conclude that knockdown of SDHAF2 induces pH-related root elongation and auxin hyperaccumulation and hypersensitivity, mediated by altered ROS homeostasis. This observation extends the existing evidence of associations between mitochondrial function and auxin by establishing a cascade of cellular events that link them through ROS formation, metabolism and root growth at different pH values.

Alternative Splicing Plays a Critical Role in Maintaining Mineral Nutrient Homeostasis in Rice (Oryza sativa).

Alternative splicing (AS) of pre-mRNAs promotes transcriptome and proteome diversity and plays important roles in a wide range of biological processes. However, the role of AS in maintaining mineral nutrient homeostasis in plants is largely unknown. To clarify this role, we obtained whole transcriptome RNA sequencing data from rice (Oryza sativa) roots grown in the presence or absence of several mineral nutrients (Fe, Zn, Cu, Mn, and P). Our systematic analysis revealed 13,291 alternatively spliced genes, representing ∼53.3% of the multiexon genes in the rice genome. As the overlap between differentially expressed genes and differentially alternatively spliced genes is small, a molecular understanding of the plant's response to mineral deficiency is limited by analyzing differentially expressed genes alone. We found that the targets of AS are highly nutrient-specific. To verify the role of AS in mineral nutrition, we characterized mutants in genes encoding Ser/Arg (SR) proteins that function in AS. We identified several SR proteins as critical regulators of Zn, Mn, and P nutrition and showed that three SR protein-encoding genes regulate P uptake and remobilization between leaves and shoots of rice, demonstrating that AS has a key role in regulating mineral nutrient homeostasis in rice.

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.

SPX4 Acts on PHR1-Dependent and -Independent Regulation of Shoot Phosphorus Status in Arabidopsis.

Phosphorus (P) is an essential macronutrient for all living organisms and limits plant growth. Four proteins comprising a single SYG1/Pho81/XPR1 (SPX) domain, SPX1 to SPX4, are putative phosphate-dependent inhibitors of Arabidopsis (Arabidopsis thaliana) PHOSPHATE STARVATION RESPONSE1 (PHR1), the master transcriptional activator of phosphate starvation responses. This work demonstrated that SPX4 functions as a negative regulator not only of PHR1-dependent but also of PHR1-independent responses in P-replete plants. Transcriptomes of P-limited spx4 revealed that, unlike SPX1 and SPX2, SPX4 modulates the shoot phosphate starvation response but not short-term recovery after phosphate resupply. In roots, transcriptional regulation of P status is SPX4 independent. Genes misregulated in spx4 shoots intersect with both PHR1-dependent and PHOSPHATE2-dependent signaling networks associated with plant development, senescence, and ion/metabolite transport. Gene regulatory network analyses suggested that SPX4 interacts with transcription factors other than PHR1, such as SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN55, known regulators of shoot development. Transient expression studies in protoplasts indicated that PHR1 retention in the cytosol by SPX4 occurs in a dose- and P-status-dependent manner. Using a luciferase reporter in vivo, SPX4 expression kinetics and stability revealed that SPX4 is a short-lived protein with P-status-dependent turnover. SPX4 protein levels were quickly restored by phosphate resupply to P-limited plants. Unlike its monocot ortholog, AtSPX4 was not stabilized by the phosphate analog phosphite, implying that intracellular P status is sensed by its SPX domain via phosphate-rich metabolite signals.

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.

Molecular and physiological responses during thermal acclimation of leaf photosynthesis and respiration in rice.

To further our understanding of how sustained changes in temperature affect the carbon economy of rice (Oryza sativa), hydroponically grown plants of the IR64 cultivar were developed at 30°C/25°C (day/night) before being shifted to 25/20°C or 40/35°C. Leaf messenger RNA and protein abundance, sugar and starch concentrations, and gas-exchange and elongation rates were measured on preexisting leaves (PE) already developed at 30/25°C or leaves newly developed (ND) subsequent to temperature transfer. Following a shift in growth temperature, there was a transient adjustment in metabolic gene transcript abundance of PE leaves before homoeostasis was reached within 24 hr, aligning with Rdark (leaf dark respiratory CO2 release) and An (net CO2 assimilation) changes. With longer exposure, the central respiratory protein cytochrome c oxidase (COX) declined in abundance at 40/35°C. In contrast to Rdark , An was maintained across the three growth temperatures in ND leaves. Soluble sugars did not differ significantly with growth temperature, and growth was fastest with extended exposure at 40/35°C. The results highlight that acclimation of photosynthesis and respiration is asynchronous in rice, with heat-acclimated plants exhibiting a striking ability to maintain net carbon gain and growth when exposed to heat-wave temperatures, even while reducing investment in energy-conserving respiratory pathways.

Transcriptional and biochemical analyses of gibberellin expression and content in germinated barley grain.

Mobilization of reserves in germinated cereal grains is critical for early seedling vigour, global crop productivity, and hence food security. Gibberellins (GAs) are central to this process. We have developed a spatio-temporal model that describes the multifaceted mechanisms of GA regulation in germinated barley grain. The model was generated using RNA sequencing transcript data from tissues dissected from intact, germinated grain, which closely match measurements of GA hormones and their metabolites in those tissues. The data show that successful grain germination is underpinned by high concentrations of GA precursors in ungerminated grain, the use of independent metabolic pathways for the synthesis of several bioactive GAs during germination, and a capacity to abort bioactive GA biosynthesis. The most abundant bioactive form is GA1, which is synthesized in the scutellum as a glycosyl conjugate that diffuses to the aleurone, where it stimulates de novo synthesis of a GA3 conjugate and GA4. Synthesis of bioactive GAs in the aleurone provides a mechanism that ensures the hormonal signal is relayed from the scutellum to the distal tip of the grain. The transcript data set of 33 421 genes used to define GA metabolism is available as a resource to analyse other physiological processes in germinated grain.