Sink strength, nutrient allocation, cannabinoid yield and associated transcript profiles vary in two drug-type Cannabis chemovars.

Cannabis sativa L. is one of the oldest domesticated crops. Hemp-type cultivars, which predominantly produce non-intoxicating cannabidiol (CBD), have been selected for their fast growth, seed, and fibre production, while drug-type chemovars were bred for high accumulation of tetrahydrocannabinol (THC). We investigated how the generation of CBD-dominant chemovars by introgression of hemp- into drug-type Cannabis impacted plant performance. The THC-dominant chemovar showed superior sink strength, higher flower biomass and demand-driven control of nutrient uptake. By contrast, the CBD-dominant chemovar hyperaccumulated phosphate in sink organs leading to reduced carbon and nitrogen assimilation in leaves, which limited flower biomass and cannabinoid yield. RNA-seq analyses determined organ- and chemovar-specific differences in expression of genes associated with nitrate and phosphate homeostasis as well as growth-regulating transcription factors that were correlated with measured traits. Among these were genes positively selected for during Cannabis domestication encoding an inhibitor of the phosphate starvation response SPX DOMAIN GENE3, nitrate reductase and two nitrate transporters. Altered nutrient sensing, acquisition or distribution are likely a consequence of adaption to growth on marginal, low-nutrient input lands in hemp. Our data provide evidence that such ancestral traits may become detrimental for female flower development and consequently overall CBD yield in protected cropping environments.

Establishment of single-cell transcriptional states during seed germination.

Germination involves highly dynamic transcriptional programs as the cells of seeds reactivate and express the functions necessary for establishment in the environment. Individual cell types have distinct roles within the embryo, so must therefore have cell type-specific gene expression and gene regulatory networks. We can better understand how the functions of different cell types are established and contribute to the embryo by determining how cell type-specific transcription begins and changes through germination. Here we describe a temporal analysis of the germinating Arabidopsis thaliana embryo at single-cell resolution. We define the highly dynamic cell type-specific patterns of gene expression and how these relate to changing cellular function as germination progresses. Underlying these are unique gene regulatory networks and transcription factor activity. We unexpectedly discover that most embryo cells transition through the same initial transcriptional state early in germination, even though cell identity has already been established during embryogenesis. Cells later transition to cell type-specific gene expression patterns. Furthermore, our analyses support previous findings that the earliest events leading to the induction of seed germination take place in the vasculature. Overall, our study constitutes a general framework with which to characterize Arabidopsis cell transcriptional states through seed germination, allowing investigation of different genotypes and other plant species whose seed strategies may differ.

Overexpression of the transcription factor ANAC017 results in a genomes uncoupled phenotype under lincomycin.

Over-expression (OE) lines for the ER-tethered NAC transcription factor ANAC017 displayed de-repression of gun marker genes when grown on lincomycin (lin). RNA-seq revealed that ANAC017OE2 plants constitutively expressed greater than 40% of the genes induced in wild-type with lin treatment, including plastid encoded genes ycf1.2 and the gene cluster ndhH-ndhA-ndhI-ndhG-ndhE-psaC-ndhD, documented as direct RNA targets of GUN1. Genes encoding components involved in organelle translation were enriched in constitutively expressed genes in ANAC017OE2. ANAC017OE resulted in constitutive location in the nucleus and significant constitutive binding of ANAC017 was detected by ChIP-Seq to target genes. ANAC017OE2 lines maintained the ability to green on lin, were more ABA sensitive, did not show photo-oxidative damage after exposure of de-etiolated seedlings to continuous light and the transcriptome response to lin were as much as 80% unique compared to gun1-1. Both double mutants, gun1-1:ANAC017OE and bzip60:ANAC017OE (but not single bzip60), have a gun molecular gene expression pattern and result in variegated and green plants, suggesting that ANAC017OE may act through an independent pathway compared to gun1. Over-expression of ANAC013 or rcd1 did not produce a GUN phenotype or green plants on lin. Thus, constitutive ANAC017OE2 establishes an alternative transcriptional program that likely acts through a number of pathways, that is, maintains plastid gene expression, and induction of a variety of transcription factors involved in reactive oxygen species metabolism, priming plants for lin tolerance to give a gun phenotype.

Same, Yet Different: Towards Understanding Nutrient Use In Hemp- And Drug-Type Cannabis.

Cannabis sativa L., one of humanity's oldest cultivated crops, has a complex domestication history due to its diverse uses for fibre, seed, oil and drugs, and its wide geographic distribution. This review explores how human selection has shaped the biology of hemp and drug-type Cannabis, focusing on acquisition and utilisation of nitrogen and phosphorus, and how resulting changes in source-sink relations shape their contrasting phenology. Hemp has been optimized for rapid, slender growth and nutrient efficiency, whereas drug-type cultivars have been selected for compact growth with large phytocannabinoid producing female inflorescences. Understanding these nutrient use and ontogenetic differences will enhance our general understanding of resource allocation in plants. Knowledge gained in comparison with other model species, such as tomato, rice or Arabidopsis thaliana can help inform crop improvement and sustainability in the Cannabis industry.

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/.

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 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.

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.

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.

Emerging trends in genomic and epigenomic regulation of plant specialised metabolism.

Regulation of specialised metabolism genes is multilayered and complex, influenced by an array of genomic, epigenetic and epigenomic mechanisms. Here, we review the most recent knowledge in this field, drawing from discoveries in several plant species. Our aim is to improve understanding of how plant genome structure and function influence specialised metabolism. We also highlight key areas for future exploration. Gene regulatory mechanisms influencing specialised metabolism include gene duplication and neo-functionalization, conservation of operon-like clusters of specialised metabolism genes, local chromatin modifications, and the organisation of higher order chromatin structures within the nucleus. Genomic and epigenomic research to-date in the discipline have focused on a relatively small number of plant species, primarily at whole organ or tissue level. This is largely due to the technical demands of the experimental methods needed. However, a high degree of cell-type specificity of function exists in specialised metabolism, driven by similarly specific gene regulation. In this review we focus on the genomic characteristics of genes that are found in different types of clusters within the genome. We propose that acquisition of cell-resolution epigenomic datasets in emerging models, such as the glandular trichomes of Cannabis sativa, will yield important advances. Data such as chromatin accessibility and histone modification profiles can pinpoint which regulatory sequences are active in individual cell types and at specific times in development. These could provide fundamental biological insight as well as novel targets for genetic engineering and crop improvement.

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.

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.

scCloudMine: A cloud-based app for visualization, comparison, and exploration of single-cell transcriptomic data.

scCloudMine is a cloud-based application for visualization, comparison, and exploration of single-cell transcriptome data. It does not require an on-site, high-power computing server, installation, or associated expertise and expense. Users upload their own or publicly available scRNA-seq datasets after pre-processing for visualization using a web browser. The data can be viewed in two color modes-Cluster, representing cell identity, and Values, showing levels of expression-and data can be queried using keywords or gene identification number(s). Using the app to compare studies, we determined that some genes frequently used as cell-type markers are in fact study specific. The apparent cell-specific expression of PHO1;H3 differed between GFP-tagging and scRNA-seq studies. Some phosphate transporter genes were induced by protoplasting, but they retained cell specificity, suggesting that cell-specific responses to stress (i.e., protoplasting) can occur. Examination of the cell specificity of hormone response genes revealed that 132 hormone-responsive genes display restricted expression and that the jasmonate response gene TIFY8 is expressed in endodermal cells, in contrast to previous reports. It also appears that JAZ repressors have cell-type-specific functions. These features identified using scCloudMine highlight the need for resources to enable biological researchers to compare their datasets of interest under a variety of parameters. scCloudMine enables researchers to form new hypotheses and perform comparative studies and allows for the easy re-use of data from this emerging technology by a wide variety of users who may not have access or funding for high-performance on-site computing and support.

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.

Applications of hyperspectral imaging in plant phenotyping.

Our ability to interrogate and manipulate the genome far exceeds our capacity to measure the effects of genetic changes on plant traits. Much effort has been made recently by the plant science research community to address this imbalance. The responses of plants to environmental conditions can now be defined using a variety of imaging approaches. Hyperspectral imaging (HSI) has emerged as a promising approach to measure traits using a wide range of wavebands simultaneously in 3D to capture information in lab, glasshouse, or field settings. HSI has been applied to define abiotic, biotic, and quality traits for optimisation of crop management.

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.

Extracellular Vesicles from Fusarium graminearum Contain Protein Effectors Expressed during Infection of Corn.

Fusarium graminearum (Fgr) is a devastating filamentous fungal pathogen that causes diseases in cereals, while producing mycotoxins that are toxic for humans and animals, and render grains unusable. Low efficiency in managing Fgr poses a constant need for identifying novel control mechanisms. Evidence that fungal extracellular vesicles (EVs) from pathogenic yeast have a role in human disease led us to question whether this is also true for fungal plant pathogens. We separated EVs from Fgr and performed a proteomic analysis to determine if EVs carry proteins with potential roles in pathogenesis. We revealed that protein effectors, which are crucial for fungal virulence, were detected in EV preparations and some of them did not contain predicted secretion signals. Furthermore, a transcriptomic analysis of corn (Zea mays) plants infected by Fgr revealed that the genes of some of the effectors were highly expressed in vivo, suggesting that the Fgr EVs are a mechanism for the unconventional secretion of effectors and virulence factors. Our results expand the knowledge on fungal EVs in plant pathogenesis and cross-kingdom communication, and may contribute to the discovery of new antifungals.

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.