Comparative genomics of flowering behavior in Cannabis sativa.

Cannabis sativa L. is a phenotypically diverse and multi-use plant used in the production of fiber, seed, oils, and a class of specialized metabolites known as phytocannabinoids. The last decade has seen a rapid increase in the licit cultivation and processing of C. sativa for medical end-use. Medical morphotypes produce highly branched compact inflorescences which support a high density of glandular trichomes, specialized epidermal hair-like structures that are the site of phytocannabinoid biosynthesis and accumulation. While there is a focus on the regulation of phytocannabinoid pathways, the genetic determinants that govern flowering time and inflorescence structure in C. sativa are less well-defined but equally important. Understanding the molecular mechanisms that underly flowering behavior is key to maximizing phytocannabinoid production. The genetic basis of flowering regulation in C. sativa has been examined using genome-wide association studies, quantitative trait loci mapping and selection analysis, although the lack of a consistent reference genome has confounded attempts to directly compare candidate loci. Here we review the existing knowledge of flowering time control in C. sativa, and, using a common reference genome, we generate an integrated map. The co-location of known and putative flowering time loci within this resource will be essential to improve the understanding of C. sativa phenology.

Repurposed inhibitor of bacterial dihydrodipicolinate reductase exhibits effective herbicidal activity.

Herbicide resistance represents one of the biggest threats to our natural environment and agricultural sector. Thus, new herbicides are urgently needed to tackle the rise in herbicide-resistant weeds. Here, we employed a novel strategy to repurpose a 'failed' antibiotic into a new and target-specific herbicidal compound. Specifically, we identified an inhibitor of bacterial dihydrodipicolinate reductase (DHDPR), an enzyme involved in lysine biosynthesis in plants and bacteria, that exhibited no antibacterial activity but severely attenuated germination of the plant Arabidopsis thaliana. We confirmed that the inhibitor targets plant DHDPR orthologues in vitro, and exhibits no toxic effects against human cell lines. A series of analogues were then synthesised with improved efficacy in germination assays and against soil-grown A. thaliana. We also showed that our lead compound is the first lysine biosynthesis inhibitor with activity against both monocotyledonous and dicotyledonous weed species, by demonstrating its effectiveness at reducing the germination and growth of Lolium rigidum (rigid ryegrass) and Raphanus raphanistrum (wild radish). These results provide proof-of-concept that DHDPR inhibition may represent a much-needed new herbicide mode of action. Furthermore, this study exemplifies the untapped potential of repurposing 'failed' antibiotic scaffolds to fast-track the development of herbicide candidates targeting the respective plant enzymes.

A dual-target herbicidal inhibitor of lysine biosynthesis.

Herbicides with novel modes of action are urgently needed to safeguard global agricultural industries against the damaging effects of herbicide-resistant weeds. We recently developed the first herbicidal inhibitors of lysine biosynthesis, which provided proof-of-concept for a promising novel herbicide target. In this study, we expanded upon our understanding of the mode of action of herbicidal lysine biosynthesis inhibitors. We previously postulated that these inhibitors may act as proherbicides. Here, we show this is not the case. We report an additional mode of action of these inhibitors, through their inhibition of a second lysine biosynthesis enzyme, and investigate the molecular determinants of inhibition. Furthermore, we extend our herbicidal activity analyses to include a weed species of global significance.

Draft Genome Sequence of Enterobacter asburiae NCR1, a Plant Growth-Promoting Rhizobacterium Isolated from a Cadmium-Contaminated Environment.

Enterobacter asburiae NCR1 is a plant growth-promoting rhizobacterium isolated from the rhizosphere of Carpobrotus rossii. We report the draft genome sequence of E. asburiae strain NCR1, which revealed many genes facilitating beneficial interactions with plant hosts.

Draft Genome Sequence of Enterobacter mori Strain NSE2, Isolated from the Rhizosphere of a Sedum sp.

Enterobacter mori is an important plant pathogen. Here, we report the draft genome sequence of the plant-associated strain Enterobacter mori NSE2, which was found to harbor genes for promotive and pathogenic interactions with plants.

Towards novel herbicide modes of action by inhibiting lysine biosynthesis in plants.

Weeds are becoming increasingly resistant to our current herbicides, posing a significant threat to agricultural production. Therefore, new herbicides with novel modes of action are urgently needed. In this study, we exploited a novel herbicide target, dihydrodipicolinate synthase (DHDPS), which catalyses the first and rate-limiting step in lysine biosynthesis. The first class of plant DHDPS inhibitors with micromolar potency against Arabidopsis thaliana DHDPS was identified using a high-throughput chemical screen. We determined that this class of inhibitors binds to a novel and unexplored pocket within DHDPS, which is highly conserved across plant species. The inhibitors also attenuated the germination and growth of A. thaliana seedlings and confirmed their pre-emergence herbicidal activity in soil-grown plants. These results provide proof-of-concept that lysine biosynthesis represents a promising target for the development of herbicides with a novel mode of action to tackle the global rise of herbicide-resistant weeds.

Differential lysine-mediated allosteric regulation of plant dihydrodipicolinate synthase isoforms.

Lysine biosynthesis in plants occurs via the diaminopimelate pathway. The first committed and rate-limiting step of this pathway is catalysed by dihydrodipicolinate synthase (DHDPS), which is allosterically regulated by the end product, l-lysine (lysine). Given that lysine is a common nutritionally limiting amino acid in cereal crops, there has been much interest in probing the regulation of DHDPS. Interestingly, knockouts in Arabidopsis thaliana of each isoform (AtDHDPS1 and AtDHDPS2) result in different phenotypes, despite the enzymes sharing > 85% protein sequence identity. Accordingly, in this study, we compared the catalytic activity, lysine-mediated inhibition and structures of both A. thaliana DHDPS isoforms. We found that although the recombinantly produced enzymes have similar kinetic properties, AtDHDPS1 is 10-fold more sensitive to lysine. We subsequently used X-ray crystallography to probe for structural differences between the apo- and lysine-bound isoforms that could account for the differential allosteric inhibition. Despite no significant changes in the overall structures of the active or allosteric sites, we noted differences in the rotamer conformation of a key allosteric site residue (Trp116) and proposed that this could result in differences in lysine dissociation. Microscale thermophoresis studies supported our hypothesis, with AtDHDPS1 having a ~ 6-fold tighter lysine dissociation constant compared to AtDHDPS2, which agrees with the lower half minimal inhibitory concentration for lysine observed. Thus, we highlight that subtle differences in protein structures, which could not have been predicted from the primary sequences, can have profound effects on the allostery of a key enzyme involved in lysine biosynthesis in plants. DATABASES: Structures described are available in the Protein Data Bank under the accession numbers 6VVH and 6VVI.

Review: amino acid biosynthesis as a target for herbicide development.

There are three amino acid biosynthesis pathways that are targeted by current herbicides, namely those leading to the production of aromatic amino acids, branched chain amino acids and glutamine. However, their efficacy is diminishing as a result of the increasing number of resistant weeds. Indeed, resistance to most classes of herbicides is on the rise, posing a significant threat to the utility of current herbicides to sustain effective weed management. This review provides an overview of potential herbicide targets within amino acid biosynthesis that remain unexploited commercially, and recent inhibitor discovery efforts. Despite contemporary approaches to herbicide discovery, such as chemical repurposing and the use of omics technologies, there have been no new products introduced to the market that inhibit amino acid biosynthesis over the past three decades. This highlights the chasm that exists between identifying a potent inhibitor and introducing a commercial herbicide. The unpredictability of a mode of action at the systemic level, as well as poor physicochemical properties, often contribute to a lack of progression beyond the target inhibition stage. Nevertheless, it will be important to overcome these obstacles for the development of new herbicides to protect our agricultural industry and ensure food security for an increasing world population. © 2020 Society of Chemical Industry.

Soil greenhouse gas emissions from Australian sports fields.

Managed turf is a potential net source of greenhouse gas (GHG) emissions. While most studies to date have focused on non-sports turf, sports turf may pose an even greater risk of high GHG emissions due to the generally more intensive fertiliser, irrigation and mowing regimes. This study used manual and automated chambers to measure nitrous oxide (N2O) and methane (CH4) emissions from three sports fields and an area of non-sports turf in southern Australia. Over 213 days (autumn to late spring), the average daily N2O emission was 37.6 g N ha-1day-1 at a sports field monitored at least weekly and cumulative N2O emission was 2.5 times higher than the adjacent non-sports turf. Less frequent seasonal sampling at two other sports fields showed average N2O daily emission ranging from 26 to 90 g N ha-1 day-1. Management practices associated with periods of relatively high N2O emissions were surface renovation and herbicide application. CH4 emissions at all of the sports fields were generally negligible with the exception of brief periods when soil was waterlogged following heavy rainfall where emissions of up to 1.3 kg C ha-1 day-1 were recorded. Controlled release and nitrification inhibitor containing fertilisers didn't reduce N2O, CH4 or CO2 emissions relative to urea in a short term experiment. The N2O emissions from the sports fields, and even the lower emissions from the non-sports turf, were relatively high compared to other land uses in Australia highlighting the importance of accounting for these emissions at a national level and investigating mitigation practices.

Artificial selection causes significant linkage disequilibrium among multiple unlinked genes in Australian wheat.

Australia has one of the oldest modern wheat breeding programs worldwide although the crop was first introduced to the country in 1788. Breeders selected wheat with high adaptation to different Australian climates, while ensuring satisfactory yield and quality. This artificial selection left distinct genomic signatures that can be used to retrospectively understand breeding targets, and to detect economically important alleles. To study the effect of artificial selection on modern cultivars and cultivars released in different Australian states, we genotyped 482 Australian cultivars representing the history of wheat breeding in Australia since 1840. Computer simulation showed that 86 genomic regions were significantly affected by artificial selection. Characterization of 18 major genes known to affect wheat adaptation, yield, and quality revealed that many were affected by artificial selection and contained within regions under selection. Similarly, many reported QTL and genes for yield, quality, and adaptation were also contained in regions affected by artificial selection. These included TaCwi-A1, TaGw2-6A, Sus-2B, TaSus1-7A, TaSAP1-7A, Glu-A1, Glu-B1, Glu-B3, PinA, PinB, Ppo-D1, Psy-A1, Psy-A2, Rht-A1, Rht-B1, Ppd-D1, Vrn-A1, Vrn-B1, and Cre8. Interestingly, 17 regions affected by artificial selection were in moderate-to-high linkage disequilibrium with each other with an average r 2 value of 0.35 indicating strong simultaneous selection on specific alleles. These regions included Glu-B1, TaGw2-6A, Cre8, Ppd-D1, Rht-B1, Vrn-B1, TaSus1-7A, TaSAP1-7A, and Psy-A1 plus multiple QTL affecting wheat yield and yield components. These results highlighted the effects of the long-term artificial selection on Australian wheat germplasm and identified putative regions underlying important traits in wheat.

NHX-type Na+(K+)/H+ antiporters are required for TGN/EE trafficking and endosomal ion homeostasis in Arabidopsis thaliana.

The regulation of ion and pH homeostasis of endomembrane organelles is critical for functional protein trafficking, sorting and modification in eukaryotic cells. pH homeostasis is maintained through the activity of vacuolar H+-ATPases (V-ATPases) pumping protons (H+) into the endomembrane lumen, and counter-action by cation/proton exchangers, such as the NHX family of Na+(K+)/H+ exchangers. In plants, V-ATPase activity at the trans-Golgi network/early endosome (TGN/EE) is important for secretory and endocytic trafficking; however, the role of the endosomal antiporters NHX5 and NHX6 in endomembrane trafficking is unclear. Here we show through genetic, pharmacological and live-cell imaging approaches that double knockout of NHX5 and NHX6 results in the impairment of endosome motility and protein recycling at the TGN/EE, but not in the secretion of integral membrane proteins. Furthermore, we report that nhx5 nhx6 mutants are partially insensitive to osmotic swelling of TGN/EE induced by the monovalent cation ionophore monensin, and to late endosomal swelling by the phosphatidylinositol 3/4-kinase inhibitor wortmannin, demonstrating that NHX5 and NHX6 function to regulate the luminal cation composition of endosomes.

Sodium chloride decreases cadmium accumulation and changes the response of metabolites to cadmium stress in the halophyte Carpobrotus rossii.

Background and Aims: Salinity affects the bioavailability of cadmium (Cd) in soils and Cd accumulation in plants, but the associated mechanisms remain unclear. This study aimed to assess the metabolic response to NaCl and Cd and the relationship between metabolites and Cd accumulation in the halophyte Carpobrotus rossii, which has potential for Cd phytoextraction. Methods: Plants were grown in nutrient solution with 0-400 mm NaCl in the presence of 5 or 15 µm Cd, with varied or constant solution Cd2+ activity. Plant growth and Cd uptake were measured, and the accumulation of peptides, and organic and amino acids in plant tissues were assessed. Key Results: The addition of NaCl to Cd-containing solutions improved plant growth along with 70-87 % less shoot Cd accumulation, resulting from decreases in Cd root uptake and root-to-shoot translocation irrespective of Cd2+ activity in solutions. Moreover, Cd exposure increased the concentration of phytochelatins, which correlated positively with Cd concentrations in plants regardless of NaCl addition. In comparison, Cd inhibited the synthesis of organic acids in shoots and roots in the absence of NaCl, but increased it in shoots in the presence of NaCl. While Cd increased the concentrations of amino acids in plant shoots, the effect of NaCl on the synthesis of amino acids was inconsistent. Conclusions: Our data provide the first evidence that NaCl decreased Cd shoot accumulation in C. rossii by decreasing Cd root uptake and root-to-shoot translocation even under constant Cd2+ activity. The present study also supports the important role of peptides and organic acids, particular of phytochelatins, in Cd tolerance and accumulation although the changes of those metabolites was not the main reason for the decreased Cd accumulation.

Two Endosomal NHX-Type Na+/H+ Antiporters are Involved in Auxin-Mediated Development in Arabidopsis thaliana.

In Arabidopsis thaliana, the endosomal-localized Na+/H+ antiporters NHX5 and NHX6 regulate ion and pH homeostasis and are important for plant growth and development. However, the mechanism by which these endosomal NHXs function in plant development is not well understood. Auxin modulates plant growth and development through the formation of concentration gradients in plant tissue to control cell division and expansion. Here, we identified a role for NHX5 and NHX6 in the establishment and maintenance of auxin gradients in embryo and root tissues. We observed developmental impairment and abnormal cell division in embryo and root tissues in the double knockout nhx5 nhx6, consistent with these tissues showing high expression of NHX5 and NHX6. Through confocal microscopy imaging with the DR5::GFP auxin reporter, we identify defects in the perception, accumulation and redistribution of auxin in nhx5 nhx6 cells. Furthermore, we find that the steady-state levels of the PIN-FORMED (PIN) auxin efflux carriers PIN1 and PIN2 are reduced in nhx5 nhx6 root cells. Our results demonstrate that NHX5 and NHX6 function in auxin-mediated plant development by maintaining PIN abundance at the plasma membrane, and provide new insight into the regulation of plant development by endosomal NHX antiporters.

Trafficking to the seed protein storage vacuole.

The processing and subcellular trafficking of seed storage proteins is a critical area of physiological, agricultural and biotechnological research. Trafficking to the lytic vacuole has been extensively discussed in recent years, without substantial distinction from trafficking to the protein storage vacuole (PSV). However, despite some overlap between these pathways, there are several examples of unique processing and machinery in the PSV pathway. Moreover, substantial new data has recently come to light regarding the important players in this pathway, in particular, the intracellular NHX proteins and their role in regulating lumenal pH. In some cases, these new data are limited to genetic evidence, with little mechanistic understanding. As such, the implications of these data in the current paradigm of PSV trafficking is perhaps yet unclear. Although it has generally been assumed that the major classes of storage proteins are trafficked via the same pathway, there is mounting evidence that the 12S globulins and 2S albumins may be trafficked independently. Advances in identification of vacuolar targeting signals, as well as an improved mechanistic understanding of various vacuolar sorting receptors, may reveal the differences in these trafficking pathways.

Molecular evolution of an oligomeric biocatalyst functioning in lysine biosynthesis.

Dihydrodipicolinate synthase (DHDPS) is critical to the production of lysine through the diaminopimelate (DAP) pathway. Elucidation of the function, regulation and structure of this key class I aldolase has been the focus of considerable study in recent years, given that the dapA gene encoding DHDPS has been found to be essential to bacteria and plants. Allosteric inhibition by lysine is observed for DHDPS from plants and some bacterial species, the latter requiring a histidine or glutamate at position 56 (Escherichia coli numbering) over a basic amino acid. Structurally, two DHDPS monomers form the active site, which binds pyruvate and (S)-aspartate ß-semialdehyde, with most dimers further dimerising to form a tetrameric arrangement around a solvent-filled centre cavity. The architecture and behaviour of these dimer-of-dimers is explored in detail, including biophysical studies utilising analytical ultracentrifugation, small-angle X-ray scattering and macromolecular crystallography that show bacterial DHDPS tetramers adopt a head-to-head quaternary structure, compared to the back-to-back arrangement observed for plant DHDPS enzymes. Finally, the potential role of pyruvate in providing substrate-mediated stabilisation of DHDPS is considered.

Genetic Diversity, Population Structure and Ancestral Origin of Australian Wheat.

Since the introduction of wheat into Australia by the First Fleet settlers, germplasm from different geographical origins has been used to adapt wheat to the Australian climate through selection and breeding. In this paper, we used 482 cultivars, representing the breeding history of bread wheat in Australia since 1840, to characterize their diversity and population structure and to define the geographical ancestral background of Australian wheat germplasm. This was achieved by comparing them to a global wheat collection using in-silico chromosome painting based on SNP genotyping. The global collection involved 2,335 wheat accessions which was divided into 23 different geographical subpopulations. However, the whole set was reduced to 1,544 accessions to increase the differentiation and decrease the admixture among different global subpopulations to increase the power of the painting analysis. Our analysis revealed that the structure of Australian wheat germplasm and its geographic ancestors have changed significantly through time, especially after the Green Revolution. Before 1920, breeders used cultivars from around the world, but mainly Europe and Africa, to select potential cultivars that could tolerate Australian growing conditions. Between 1921 and 1970, a dependence on African wheat germplasm became more prevalent. Since 1970, a heavy reliance on International Maize and Wheat Improvement Center (CIMMYT) germplasm has persisted. Combining the results from linkage disequilibrium, population structure and in-silico painting revealed that the dependence on CIMMYT materials has varied among different Australian States, has shrunken the germplasm effective population size and produced larger linkage disequilibrium blocks. This study documents the evolutionary history of wheat breeding in Australia and provides an understanding for how the wheat genome has been adapted to local growing conditions. This information provides a guide for industry to assist with maintaining genetic diversity for long-term selection gains and to plan future breeding programs.

Using Transcriptomics to Identify Differential Gene Expression in Response to Salinity among Australian Phragmites australis Clones.

Common Reed (Phragmites australis) is a frequent component of inland and coastal wetlands in temperate zones worldwide. Ongoing environmental changes have resulted in the decline of this species in many areas and invasive expansion in others. In the Gippsland Lakes coastal waterway system in south-eastern Australia, increasing salinity is thought to have contributed to the loss of fringing P. australis reed beds leading to increased shoreline erosion. A major goal of restoration in this waterway is to address the effect of salinity by planting a genetically diverse range of salt-tolerant P. australis plants. This has prompted an interest in examining the variation in salinity tolerance among clones and the underlying basis of this variation. Transcriptomics is an approach for identifying variation in genes and their expression levels associated with the exposure of plants to environmental stressors. In this paper we present initial results of the first comparative culm transcriptome analysis of P. australis clones. After sampling plants from sites of varied surface water salinity across the Gippsland Lakes, replicates from three clones from highly saline sites (>18 g L(-1) TDS) and three from low salinity sites (<6 g L(-1)) were grown in containers irrigated with either fresh (<0.1 g L(-1)) or saline water (16 g L(-1)). An RNA-Seq protocol was used to generate sequence data from culm tissues from the 12 samples allowing an analysis of differential gene expression. Among the key findings, we identified several genes uniquely up- or down-regulated in clones from highly saline sites when irrigated with saline water relative to clones from low salinity sites. These included the higher relative expression levels of genes associated with photosynthesis and lignan biosynthesis indicative of a greater ability of these clones to maintain growth under saline conditions. Combined with growth data from a parallel study, our data suggests local adaptation of certain clones to salinity and provides a basis for more detailed studies.

Arabidopsis Intracellular NHX-Type Sodium-Proton Antiporters are Required for Seed Storage Protein Processing.

The Arabidopsis intracellular sodium-proton exchanger (NHX) proteins AtNHX5 and AtNHX6 have a well-documented role in plant development, and have been used to improve salt tolerance in a variety of species. Despite evidence that intracellular NHX proteins are important in vacuolar trafficking, the mechanism of this role is poorly understood. Here we show that NHX5 and NHX6 are necessary for processing of the predominant seed storage proteins, and also influence the processing and activity of a vacuolar processing enzyme. Furthermore, we show by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) technology that the C-terminal tail of NHX6 interacts with a component of Retromer, another component of the cell sorting machinery, and that this tail is critical for NHX6 activity. These findings demonstrate that NHX5 and NHX6 are important in processing and activity of vacuolar cargo, and suggest a mechanism by which NHX intracellular (IC)-II antiporters may be involved in subcellular trafficking.

Acceleration of flowering in Arabidopsis thaliana by Cape Verde Islands alleles of FLOWERING H is dependent on the floral promoter FD.

Flowering time in the model plant Arabidopsis thaliana is regulated by both external environmental signals and internal developmental pathways. Natural variation at the FLOWERING H (FLH) locus has previously been described, with alleles present in the Cape Verde Islands accession causing early flowering, particularly after vernalization. The mechanism of FLH-induced early flowering is not understood. Here, the integration of FLH activity into the known flowering time pathways is described using molecular and genetic approaches. The identification of molecular markers that co-segregated with the FLH locus allowed the generation of multiple combinations of FLH alleles with mutations in flowering time genes in different flowering pathways. Combining an early flowering FLH allele with mutations in vernalization pathway genes that regulate FLC expression revealed that FLH appears to act in parallel to FLC. Surprisingly, the early flowering allele of FLH requires the floral integrator FD, but not FT, to accelerate flowering. This suggests a model in which some alleles of FLH are able to affect the FD-dependent activity of the floral activator complex.

Identification and characterization of orthologs of AtNHX5 and AtNHX6 in Brassica napus.

Improving crop species by breeding for salt tolerance or introducing salt tolerant traits is one method of increasing crop yields in saline affected areas. Extensive studies of the model plant species Arabidopsis thaliana has led to the availability of substantial information regarding the function and importance of many genes involved in salt tolerance. However, the identification and characterization of A. thaliana orthologs in species such as Brassica napus (oilseed rape) can prove difficult due to the significant genomic changes that have occurred since their divergence approximately 20 million years ago (MYA). The recently released Brassica rapa genome provides an excellent resource for comparative studies of A. thaliana and the cultivated Brassica species, and facilitates the identification of Brassica species orthologs which may be of agronomic importance. Sodium hydrogen antiporter (NHX) proteins transport a sodium or potassium ion in exchange for a hydrogen ion in the other direction across a membrane. In A. thaliana there are eight members of the NHX family, designated AtNHX1-8, that can be sub-divided into three clades, based on their subcellular localization: plasma membrane (PM), intracellular class I (IC-I) and intracellular class II (IC-II). In plants, many NHX proteins are primary determinants of salt tolerance and act by transporting Na(+) out of the cytosol where it would otherwise accumulate to toxic levels. Significant work has been done to determine the role of both PM and IC-I clade members in salt tolerance in a variety of plant species, but relatively little analysis has been described for the IC-II clade. Here we describe the identification of B. napus orthologs of AtNHX5 and AtNHX6, using the B. rapa genome sequence, macro- and micro-synteny analysis, comparative expression and promoter motif analysis, and highlight the value of these multiple approaches for identifying true orthologs in closely related species with multiple paralogs.