A high diversity of non-target site resistance mechanisms to acetolactate-synthase (ALS) inhibiting herbicides has evolved within and among field populations of common ragweed (Ambrosia artemisiifolia L.).

BACKGROUND: Non-target site resistance (NTSR) to herbicides is a polygenic trait that threatens the chemical control of agricultural weeds. NTSR involves differential regulation of plant secondary metabolism pathways, but its precise genetic determinisms remain fairly unclear. Full-transcriptome sequencing had previously been implemented to identify NTSR genes. However, this approach had generally been applied to a single weed population, limiting our insight into the diversity of NTSR mechanisms. Here, we sought to explore the diversity of NTSR mechanisms in common ragweed (Ambrosia artemisiifolia L.) by investigating six field populations from different French regions where NTSR to acetolactate-synthase-inhibiting herbicides had evolved. RESULTS: A de novo transcriptome assembly (51,242 contigs, 80.2% completeness) was generated as a reference to seek genes differentially expressed between sensitive and resistant plants from the six populations. Overall, 4,609 constitutively differentially expressed genes were identified, of which none were common to all populations, and only 197 were shared by several populations. Similarly, population-specific transcriptomic response was observed when investigating early herbicide response. Gene ontology enrichment analysis highlighted the involvement of stress response and regulatory pathways, before and after treatment. The expression of 121 candidate constitutive NTSR genes including CYP71, CYP72, CYP94, oxidoreductase, ABC transporters, gluco and glycosyltransferases was measured in 220 phenotyped plants. Differential expression was validated in at least one ragweed population for 28 candidate genes. We investigated whether expression patterns at some combinations of candidate genes could predict phenotype. Within populations, prediction accuracy decreased when applied to an additional, independent plant sampling. Overall, a wide variety of genes linked to NTSR was identified within and among ragweed populations, of which only a subset was captured in our experiments. CONCLUSION: Our results highlight the complexity and the diversity of NTSR mechanisms that can evolve in a weed species in response to herbicide selective pressure. They strongly point to a non-redundant, population-specific evolution of NTSR to ALS inhibitors in ragweed. It also alerts on the potential of common ragweed for rapid adaptation to drastic environmental or human-driven selective pressures.

Primary Aldosteronism: Spatial Multiomics Mapping of Genotype-Dependent Heterogeneity and Tumor Expansion of Aldosterone-Producing Adenomas.

BACKGROUND: Primary aldosteronism is frequently caused by an adrenocortical aldosterone-producing adenoma (APA) carrying a somatic mutation that drives aldosterone overproduction. APAs with a mutation in KCNJ5 (APA-KCNJ5MUT) are characterized by heterogeneous CYP11B2 (aldosterone synthase) expression, a particular cellular composition and larger tumor diameter than those with wild-type KCNJ5 (APA-KCNJ5WT). We exploited these differences to decipher the roles of transcriptome and metabolome reprogramming in tumor pathogenesis. METHODS: Consecutive adrenal cryosections (7 APAs and 7 paired adjacent adrenal cortex) were analyzed by spatial transcriptomics (10x Genomics platform) and metabolomics (in situ matrix-assisted laser desorption/ionization mass spectrometry imaging) co-integrated with CYP11B2 immunohistochemistry. RESULTS: We identified intratumoral transcriptional heterogeneity that delineated functionally distinct biological pathways. Common transcriptomic signatures were established across all APA specimens which encompassed 2 distinct transcriptional profiles in CYP11B2-immunopositive regions (CYP11B2-type 1 or 2). The CYP11B2-type 1 signature was characterized by zona glomerulosa gene markers and was detected in both APA-KCNJ5MUT and APA-KCNJ5WT. The CYP11B2-type 2 signature displayed markers of the zona fasciculata or reticularis and predominated in APA-KCNJ5MUT. Metabolites that promote oxidative stress and cell death accumulated in APA-KCNJ5WT. In contrast, antioxidant metabolites were abundant in APA-KCNJ5MUT. Finally, APA-like cell subpopulations-negative for CYP11B2 gene expression-were identified in adrenocortical tissue adjacent to APAs suggesting the existence of tumor precursor states. CONCLUSIONS: Our findings provide insight into intra- and intertumoral transcriptional heterogeneity and support a role for prooxidant versus antioxidant systems in APA pathogenesis highlighting genotype-dependent capacities for tumor expansion.

Involvement of glutamine synthetase 2 (GS2) amplification and overexpression in Amaranthus palmeri resistance to glufosinate.

MAIN CONCLUSION: Amplification and overexpression of the target site glutamine synthetase, specifically the plastid-located isoform, confers resistance to glufosinate in Amaranthus palmeri. This mechanism is novel among glufosinate-resistant weeds. Amaranthus palmeri has recently evolved resistance to glufosinate herbicide. Several A. palmeri populations from Missouri and Mississippi, U.S.A. had survivors when sprayed with glufosinate-ammonium (GFA, 657 g ha-1). One population, MO#2 (fourfold resistant) and its progeny (sixfold resistant), were used to study the resistance mechanism, focusing on the herbicide target glutamine synthetase (GS). We identified four GS genes in A. palmeri; three were transcribed: one coding for the plastidic protein (GS2) and two coding for cytoplasmic isoforms (GS1.1 and GS1.2). These isoforms did not contain mutations associated with resistance. The 17 glufosinate survivors studied showed up to 21-fold increase in GS2 copies. GS2 was expressed up to 190-fold among glufosinate survivors. GS1.1 was overexpressed > twofold in only 3 of 17, and GS1.2 in 2 of 17 survivors. GS inhibition by GFA causes ammonia accumulation in susceptible plants. Ammonia level was analyzed in 12 F1 plants. GS2 expression was negatively correlated with ammonia level (r = - 0.712); therefore, plants with higher GS2 expression are less sensitive to GFA. The operating efficiency of photosystem II (ϕPSII) of Nicotiana benthamiana overexpressing GS2 was four times less inhibited by GFA compared to control plants. Therefore, increased copy and overexpression of GS2 confer resistance to GFA in A. palmeri (or other plants). We present novel understanding of the role of GS2 in resistance evolution to glufosinate.

Can double PPO mutations exist in the same allele and are such mutants functional?

BACKGROUND: Resistance to protoporphyrinogen oxidase (PPO)-inhibiting herbicides is endowed primarily by target-site mutations at the PPX2 gene that compromise binding of the herbicide to the catalytic domain. In Amaranthus spp. PPX2, the most prevalent target mutations are deletion of the G210 codon, and the R128G and G339A substitutions. These mutations strongly affect the dynamic of the PPO2 binding pocket, resulting in reduced affinity with the ligand. Here we investigated the likelihood of co-occurrence of the most widespread target site mutations in the same PPX2 allele. RESULTS: Plants carrying R128G+/+ ΔG210+/-, where + indicates presence of the mutation, were crossed with each other. The PPX2 of the offspring was subjected to pyrosequencing and E. coli-based Sanger sequencing to determine mutation frequencies and allele co-occurrence. The data show that R128G ΔG210 can occur in one allele only; the second allele carries only one mutation. Double mutation in both alleles is less likely because of significant loss of enzyme activity. The segregation of offspring populations derived from a cross between heterozygous plants carrying ΔG210 G399A also showed no co-occurrence in the same allele. The offspring exhibited the expected mutation distribution patterns with few exceptions. CONCLUSIONS: Homozygous double-mutants are not physiologically viable. Double-mutant plants can only exist in a heterozygous state. Alternatively, if two mutations are detected in one plant, each mutation would occur in a separate allele. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Lab meets field: Accelerated selection and field monitoring concur that non-target-site-based resistance evolves first in the dicotyledonous, allergenic weed Ambrosia artemisiifolia.

Assessing weed capacity to evolve herbicide resistance before resistance occurs in the field is of major interest for chemical weed control. We used herbicide selection followed by controlled crosses to provoke accelerated evolution of resistance to imazamox (imidazolinones) and tribenuron (sulfonyurea), two acetolactate-synthase (ALS) inhibitors targeting Ambrosia artemisiifolia. In natural populations with no herbicide application records, some plants were initially resistant to metsulfuron (sulfonylurea), a cereal herbicide. Non-target-site-based resistance (NTSR) to metsulfuron was substantially increased from these plants within two generations. NTSR to imazamox and/or tribenuron emerged in metsulfuron-selected G1 progenies and was strongly reinforced in G2 progenies selected by imazamox or tribenuron. NTSR to the herbicides assayed was endowed by partly overlapping and partly specific pathways. Herbicide sensitivity bioassays conducted over 62 ALS-inhibitor-sprayed fields identified emerging resistance to imazamox and/or tribenuron in 14 A. artemisiifolia populations. Only NTSR was detected in 13 of these populations. In the last population, NTSR was present together with a mutant, herbicide-resistant ALS allele bearing an Ala-205-Thr substitution. NTSR was thus by far the predominant type of resistance to ALS inhibitors in France. This confirmed accelerated selection results and demonstrated the relevance of this approach to anticipate resistance evolution in a dicotyledonous weed.

Single-Center Prospective Cohort Study on the Histopathology, Genotype, and Postsurgical Outcomes of Patients With Primary Aldosteronism.

[Figure: see text].

Angiotensin II Type 1 Receptor Autoantibodies in Primary Aldosteronism.

Primary aldosteronism (PA) is the most common form of endocrine hypertension. Agonistic autoantibodies against the angiotensin II type 1 receptor (AT1R-Abs) have been described in transplantation medicine and women with pre-eclampsia and more recently in patients with PA. Any functional role of AT1R-Abs in either of the two main subtypes of PA (aldosterone-producing adenoma or bilateral adrenal hyperplasia) requires clarification. In this review, we discuss the studies performed to date on AT1R-Abs in PA.

Mass Spectrometry Imaging Establishes 2 Distinct Metabolic Phenotypes of Aldosterone-Producing Cell Clusters in Primary Aldosteronism.

Aldosterone-producing adenomas (APAs) are one of the main causes of primary aldosteronism and the most prevalent surgically correctable form of hypertension. Aldosterone-producing cell clusters (APCCs) comprise tight nests of zona glomerulosa cells, strongly positive for CYP11B2 (aldosterone synthase) in immunohistochemistry. APCCs have been suggested as possible precursors of APAs because they frequently carry driver mutations for constitutive aldosterone production, and a few adrenal lesions with histopathologic features of both APCCs and APAs have been identified. Our objective was to investigate the metabolic phenotypes of APCCs (n=27) compared with APAs (n=6) using in situ matrix-assisted laser desorption/ionization mass spectrometry imaging of formalin-fixed paraffin-embedded adrenals from patients with unilateral primary aldosteronism. Specific distribution patterns of metabolites were associated with APCCs and classified 2 separate APCC subgroups (subgroups 1 and 2) indistinguishable by CYP11B2 immunohistochemistry. Metabolic profiles of APCCs in subgroup 1 were tightly clustered and distinct from subgroup 2 and APAs. Multiple APCCs from the same adrenal displayed metabolic profiles of the same subgroup. Metabolites of APCC subgroup 2 were highly similar to the APA group and indicated enhanced metabolic pathways favoring cell proliferation compared with APCC subgroup 1. In conclusion, we demonstrate specific subgroups of APCCs with strikingly divergent distribution patterns of metabolites. One subgroup displays a metabolic phenotype convergent with APAs and may represent the progression of APCCs to APAs.

Timeline of Advances in Genetics of Primary Aldosteronism.

The overwhelming majority of cases of primary aldosteronism (PA) occur sporadically due to a unilateral aldosterone-producing adenoma (APA) or bilateral idiopathic adrenal hyperplasia. Familial forms of PA are rare with four subtypes defined to date (familial hyperaldosteronism types I-IV). The molecular basis of familial hyperaldosteronism type I (FH type I or glucocorticoid-remediable aldosteronism) was established in 1992; two decades later the genetic variant causing FH type III was identified and germline mutations causing FH type IV and FH type II were determined soon after. Effective diagnostic protocols and methods to detect the overactive gland in unilateral PA by adrenal venous sampling followed by laparoscopic adrenalectomy have made available APAs for scientific studies. In rapid succession, following the widespread use of next-generation sequencing, recurrent somatic driver mutations in APAs were identified in genes encoding ion channels and transporters. The development of highly specific monoclonal antibodies against key enzymes in adrenal steroidogenesis has unveiled the heterogeneous features of the diseased adrenal in PA and helped reveal the high proportion of APAs with driver mutations. We discuss what is known about the genetics of PA that has led to a clearer understanding of the disease pathophysiology.

Primary Aldosteronism: KCNJ5 Mutations and Adrenocortical Cell Growth.

Aldosterone-producing adenomas with somatic mutations in the KCNJ5 G-protein-coupled inwardly rectifying potassium channel are a cause of primary aldosteronism. These mutations drive aldosterone excess, but their role in cell growth is undefined. Our objective was to determine the role of KCNJ5 mutations in adrenal cell proliferation and apoptosis. The Ki67 proliferative index was positively correlated with adenoma diameter in aldosterone-producing adenomas with a KCNJ5 mutation (r=0.435, P=0.007), a negative correlation was noted in adenomas with no mutation detected (r=-0.548, P=0.023). Human adrenocortical cell lines were established with stable expression of cumate-inducible wild-type or mutated KCNJ5. Increased cell proliferation was induced by low-level induction of KCNJ5-T158A expression compared with control cells (P=0.009), but increased induction ablated this difference. KCNJ5-G151R displayed no apparent proliferative effect, but KCNJ5-G151E and L168R mutations each resulted in decreased cell proliferation (difference P<0.0001 from control cells, both comparisons). Under conditions tested, T158A had no effect on apoptosis, but apoptosis increased with expression of G151R (P<0.0001), G151E (P=0.008), and L168R (P<0.0001). We generated a specific KCNJ5 monoclonal antibody which was used in immunohistochemistry to demonstrate strong KCNJ5 expression in adenomas without a KCNJ5 mutation and in the zona glomerulosa adjacent to adenomas irrespective of genotype as well as in aldosterone-producing cell clusters. Double immunofluorescence staining for KCNJ5 and CYP11B2 (aldosterone synthase) showed markedly decreased KCNJ5 immunostaining in CYP11B2-positive cells compared with CYP11B2-negative cells in aldosterone-producing adenomas with a KCNJ5 mutation. Together, these findings support the concept that cell growth effects of KCNJ5 mutations are determined by the expression level of the mutated channel.

Immunohistopathology and Steroid Profiles Associated With Biochemical Outcomes After Adrenalectomy for Unilateral Primary Aldosteronism.

Unilateral primary aldosteronism (PA) is the most common surgically curable form of hypertension that must be accurately differentiated from bilateral PA for therapeutic management (surgical versus medical). Adrenalectomy results in biochemical cure (complete biochemical success) in almost all patients diagnosed with unilateral PA; the remaining patients with partial or absent biochemical success comprise those with persisting aldosteronism who were misdiagnosed as unilateral PA preoperatively. To identify determinants of postsurgical biochemical outcomes, we compared the adrenal histopathology and the peripheral venous steroid profiles of patients with partial and absent or complete biochemical success after adrenalectomy for unilateral PA. A large multicenter cohort of adrenals from patients with absent and partial biochemical success (n=43) displayed a higher prevalence of hyperplasia (49% versus 21%; P=0.004) and a lower prevalence of solitary functional adenoma (44% versus 79%; P<0.001) compared with adrenals from age- and sex-matched patients with PA with complete biochemical success (n=52). We measured the peripheral plasma steroid concentrations in a subgroup of these patients (n=43) and in a group of patients with bilateral PA (n=27). Steroid profiling was associated with histopathologic phenotypes (solitary functional adenoma, hyperplasia, and aldosterone-producing cell clusters) and classified patients according to biochemical outcome or diagnosis of bilateral PA. If validated, peripheral venous steroid profiling may be a useful tool to guide the decision to perform surgery based on expectations of biochemical outcome after the procedure.

Comparative Genomics and Transcriptome Profiling in Primary Aldosteronism.

Primary aldosteronism is the most common form of endocrine hypertension with a prevalence of 6% in the general population with hypertension. The genetic basis of the four familial forms of primary aldosteronism (familial hyperaldosteronism FH types I-IV) and the majority of sporadic unilateral aldosterone-producing adenomas has now been resolved. Familial forms of hyperaldosteronism are, however, rare. The sporadic forms of the disease prevail and these are usually caused by either a unilateral aldosterone-producing adenoma or bilateral adrenal hyperplasia. Aldosterone-producing adenomas frequently carry a causative somatic mutation in either of a number of genes with the KCNJ5 gene, encoding an inwardly rectifying potassium channel, a recurrent target harboring mutations at a prevalence of more than 40% worldwide. Other than genetic variations, gene expression profiling of aldosterone-producing adenomas has shed light on the genes and intracellular signalling pathways that may play a role in the pathogenesis and pathophysiology of these tumors.

New gSSR and EST-SSR markers reveal high genetic diversity in the invasive plant Ambrosia artemisiifolia L. and can be transferred to other invasive Ambrosia species.

Ambrosia artemisiifolia L., (common ragweed), is an annual invasive and highly troublesome plant species originating from North America that has become widespread across Europe. New sets of genomic and expressed sequence tag (EST) based simple sequence repeats (SSRs) markers were developed in this species using three approaches. After validation, 13 genomic SSRs and 13 EST-SSRs were retained and used to characterize the genetic diversity and population genetic structure of Ambrosia artemisiifolia populations from the native (North America) and invasive (Europe) ranges of the species. Analysing the mating system based on maternal families did not reveal any departure from complete allogamy and excess homozygosity was mostly due the presence of null alleles. High genetic diversity and patterns of genetic structure in Europe suggest two main introduction events followed by secondary colonization events. Cross-species transferability of the newly developed markers to other invasive species of the Ambrosia genus was assessed. Sixty-five percent and 75% of markers, respectively, were transferable from A. artemisiifolia to Ambrosia psilostachya and Ambrosia tenuifolia. 40% were transferable to Ambrosia trifida, this latter species being seemingly more phylogenetically distantly related to A. artemisiifolia than the former two.

Ecology and genetics affect relative invasion success of two Echium species in southern Australia.

Echium plantagineum and E. vulgare are congeneric exotics first introduced to Australia in the early 1800 s. There, E. plantagineum is now highly invasive, whereas E. vulgare has a limited distribution. Studies were conducted to evaluate distribution, ecology, genetics and secondary chemistry to shed light on factors associated with their respective invasive success. When sampled across geographically diverse locales, E. plantagineum was widespread and exhibited a small genome size (1 C = 0.34 pg), an annual life cycle, and greater genetic diversity as assessed by DNA sequence analysis. It was found frequently in areas with temperature extremes and low rainfall. In contrast, E. vulgare exhibited a larger genome size (1 C = 0.43 pg), a perennial lifecycle, less chloroplast genetic diversity, and occurred in areas with lower temperatures and higher rainfall. Twelve chloroplast haplotypes of E. plantagineum were evident and incidence aligned well with reported historical introduction events. In contrast, E. vulgare exhibited two haplotypes and was found only sporadically at higher elevations. Echium plantagineum possessed significantly higher levels of numerous pyrrolizidine alkaloids involved in plant defence. We conclude that elevated genetic diversity, tolerance to environmental stress and capacity for producing defensive secondary metabolites have contributed to the successful invasion of E. plantagineum in Australia.