Variable expression of cyanide detoxification and tolerance genes in cyanogenic and acyanogenic white clover (Trifolium repens).

PREMISE: ß-Cyanoalanine synthase (ß-CAS) and alternative oxidase (AOX) play important roles in the ability of plants to detoxify and tolerate hydrogen cyanide (HCN). These functions are critical for all plants because HCN is produced at low levels during basic metabolic processes, and especially for cyanogenic species, which release high levels of HCN following tissue damage. However, expression of ß-CAS and Aox genes has not been examined in cyanogenic species, nor compared between cyanogenic and acyanogenic genotypes within a species. METHODS: We used a natural polymorphism for cyanogenesis in white clover to examine ß-CAS and Aox gene expression in relation to cyanogenesis-associated HCN exposure. We identified all ß-CAS and Aox gene copies present in the genome, including members of the Aox1, Aox2a, and Aox2d subfamilies previously reported in legumes. Expression levels were compared between cyanogenic and acyanogenic genotypes and between damaged and undamaged leaf tissue. RESULTS: ß-CAS and Aox2a expression was differentially elevated in cyanogenic genotypes, and tissue damage was not required to induce this increased expression. Aox2d, in contrast, appeared to be upregulated as a generalized wounding response. CONCLUSIONS: These findings suggest a heightened constitutive role for HCN detoxification (via elevated ß-CAS expression) and HCN-toxicity mitigation (via elevated Aox2a expression) in plants that are capable of cyanogenesis. As such, freezing-induced cyanide autotoxicity is unlikely to be the primary selective factor in the evolution of climate-associated cyanogenesis clines.

Dual-species origin of an adaptive chemical defense polymorphism.

Allopolyploid speciation and chemical defense diversification are two of the most characteristic features of plant evolution; although the former has likely shaped the latter, this has rarely been documented. Here we document allopolyploidy-mediated chemical defense evolution in the origin of cyanogenesis (HCN release upon tissue damage) in white clover (Trifolium repens). We combined linkage mapping of the loci that control cyanogenesis (Ac, controlling production of cyanogenic glucosides; and Li, controlling production of their hydrolyzing enzyme linamarase) with genome sequence comparisons between white clover, a recently evolved allotetraploid, and its diploid progenitors (Trifolium pallescens, Trifolium occidentale). The Ac locus (a three-gene cluster comprising the cyanogenic glucoside pathway) is derived from T. occidentale; it maps to linkage group 2O (occidentale subgenome) and is orthologous to a highly similar cluster in the T. occidentale reference genome. By contrast, Li maps to linkage group 4P (pallescens subgenome), indicating an origin in the other progenitor species. These results indicate that cyanogenesis evolved in white clover as a product of the interspecific hybridization that created the species. This allopolyploidization-derived chemical defense, together with subsequent selection on intraspecific cyanogenesis variation, appears to have contributed to white clover's ecological success as a globally distributed weed species.

Reaching the Hard-to-Reach: Outcomes of the Severe Hypertension Outreach Intervention.

INTRODUCTION: Severe hypertension (≥180 mmHg systolic or ≥110 mmHg diastolic) is associated with a twofold increase in the relative risk of death. At the authors' Federally Qualified Health Center in the Southeast, 39% of adults (n=8,695) had hypertension, and 3% (n=235) were severe. The purpose of this project was to lower blood pressure and improve the proportion of patients achieving the Agency for Healthcare Research and Quality goal for blood pressure. METHODS: This quality improvement project was performed in 2017 in three 3-month Plan, Do, Study, Act cycles using a multidisciplinary outreach model in a community-based primary care setting. A clinical team including physicians, nurses, patient navigators, behavioral health counselors, and pharmacists contacted adult patients with severe hypertension (≥180/110 mmHg), scheduled visits, and established blood pressure and medication management goals. The data review and analysis concluded in 2019. RESULTS: Among patients with blood pressure ≥180/110 mmHg (n=235), the average age was 57 years (SD=12 years), 37% (n=87) were male, 82% (n=193) were Black, and 46% (n=108) were uninsured. The majority of those contacted attended a follow-up appointment within the 9-month project (77%, n=181) and achieved an improved systolic blood pressure (87%, n=167) and diastolic blood pressure (76%, n=146). Target blood pressure of <140/90 mmHg was achieved in 29% of patients (n=53). Medication possession ratio improved from 23% to 40% among patients reached by pharmacists (n=30). Fewer deaths occurred in those reached by the intervention than in those not reached (n=1 vs n=3). CONCLUSIONS: Multidisciplinary outreach and use of evidence-based guidelines (Eighth Joint National Committee) were associated with lower blood pressure in patients with severe hypertension.

Micro- and macroevolutionary adaptation through repeated loss of a complete metabolic pathway.

There is growing evidence for the convergent evolution of physically linked gene clusters encoding chemical defense pathways. Metabolic clusters are proposed to evolve because they ensure co-inheritance of all required genes where the defense is favored, and prevent inheritance of toxic partial pathways where it is not. This hypothesis rests on the assumption that clusters evolve in species where selection favors intraspecific polymorphism for the defense; however, they have not been examined in polymorphic species. We examined metabolic cluster evolution in relation to an adaptive polymorphism for cyanogenic glucoside (CNglc) production in clover. Using 163 accessions, we performed CNglc assays, BAC sequencing, Southern hybridizations and molecular evolutionary analyses. We find that the CNglc pathway forms a 138-kb cluster in white clover, and that the adaptive polymorphism occurs through presence/absence of the complete cluster. Component genes are orthologous to those in the distantly related legume Lotus japonicus. These findings provide empirical support for the co-inheritance hypothesis, and they indicate that adaptive CNglc variation in white clover evolves through recurrent deletions of the entire pathway. They further indicate that the shared ancestor of many important legume crops was likely cyanogenic and that this defense was lost repeatedly over the last 50 Myr.

Long-term balancing selection at the Phosphorus Starvation Tolerance 1 (PSTOL1) locus in wild, domesticated and weedy rice (Oryza).

BACKGROUND: The ability to grow in phosphorus-depleted soils is an important trait for rice cultivation in many world regions, especially in the tropics. The Phosphorus Starvation Tolerance 1 (PSTOL1) gene has been identified as underlying the ability of some cultivated rice varieties to grow under low-phosphorus conditions; however, the gene is absent from other varieties. We assessed PSTOL1 presence/absence in a geographically diverse sample of wild, domesticated and weedy rice and sequenced the gene in samples where it is present. RESULTS: We find that the presence/absence polymorphism spans cultivated, weedy and wild Asian rice groups. For the subset of samples that carry PSTOL1, haplotype sequences suggest long-term selective maintenance of functional alleles, but with repeated evolution of loss-of-function alleles through premature stops and frameshift mutations. The loss-of-function alleles have evolved convergently in multiple rice species and cultivated rice varieties. Greenhouse assessments of plant growth under low- and high-phosphorus conditions did not reveal significant associations with PSTOL1 genotype variation; however, the striking signature of balancing selection at this locus suggests that further phenotypic characterizations of PSTOL1 allelic variants is warranted and may be useful for crop improvement. CONCLUSIONS: These findings suggest balancing selection for both functional and non-functional PSTOL1 alleles that predates and transcends Asian rice domestication, a pattern that may reflect fitness tradeoffs associated with geographical variation in soil phosphorus content.

Adaptive gains through repeated gene loss: parallel evolution of cyanogenesis polymorphisms in the genus Trifolium (Fabaceae).

Variation in cyanogenesis (hydrogen cyanide release following tissue damage) was first noted in populations of white clover more than a century ago, and subsequent decades of research have established this system as a classic example of an adaptive chemical defence polymorphism. Here, we document polymorphisms for cyanogenic components in several relatives of white clover, and we determine the molecular basis of this trans-specific adaptive variation. One hundred and thirty-nine plants, representing 13 of the 14 species within Trifolium section Trifoliastrum, plus additional species across the genus, were assayed for cyanogenic components (cyanogenic glucosides and their hydrolysing enzyme, linamarase) and for the presence of underlying cyanogenesis genes (CYP79D15 and Li, respectively). One or both cyanogenic components were detected in seven species, all within section Trifoliastrum; polymorphisms for the presence/absence (PA) of components were detected in six species. In a pattern that parallels our previous findings for white clover, all observed biochemical polymorphisms correspond to gene PA polymorphisms at CYP79D15 and Li. Relationships of DNA sequence haplotypes at the cyanogenesis loci and flanking genomic regions suggest independent evolution of gene deletions within species. This study thus provides evidence for the parallel evolution of adaptive biochemical polymorphisms through recurrent gene deletions in multiple species.

Recurrent gene deletions and the evolution of adaptive cyanogenesis polymorphisms in white clover (Trifolium repens L.).

Understanding the molecular evolution of genes that underlie intraspecific polymorphisms can provide insights into the process of adaptive evolution. For adaptive polymorphisms characterized by gene presence/absence (P/A) variation, underlying loci commonly show signatures of long-term balancing selection, with gene-presence and gene-absence alleles maintained as two divergent lineages. We examined the molecular evolution of two unlinked P/A polymorphisms that underlie a well-documented adaptive polymorphism for cyanogenesis (hydrogen cyanide release with tissue damage) in white clover. Both cyanogenic and acyanogenic plants occur in this species, and the ecological forces that maintain this chemical defence polymorphism have been studied for several decades. Using a sample of 65 plants, we investigated the molecular evolution of sequences flanking the two underlying cyanogenesis genes: Ac/ac (controlling the presence/absence of cyanogenic glucosides) and Li/li (controlling the presence/absence of their hydrolysing enzyme, linamarase). A combination of genome walking, PCR assays, DNA sequence analysis and Southern blotting was used to test whether these adaptive P/A polymorphisms show evidence of long-term balancing selection, or whether gene-absence alleles have evolved repeatedly through independent deletion events. For both loci, we detect no signatures of balancing selection in the closest flanking genomic sequences. Instead, we find evidence for variation in the size of the deletions characterizing gene-absence alleles. These observations strongly suggest that both of these polymorphisms have been evolving through recurrent gene deletions over time. We discuss the genetic mechanisms that could account for this surprising pattern and the implications of these findings for mechanisms of rapid adaptive evolution in white clover.

Evidence on the molecular basis of the Ac/ac adaptive cyanogenesis polymorphism in white clover (Trifolium repens L).

White clover is polymorphic for cyanogenesis, with both cyanogenic and acyanogenic plants occurring in nature. This chemical defense polymorphism is one of the longest-studied and best-documented examples of an adaptive polymorphism in plants. It is controlled by two independently segregating genes: Ac/ac controls the presence/absence of cyanogenic glucosides; and Li/li controls the presence/absence of their hydrolyzing enzyme, linamarase. Whereas Li is well characterized at the molecular level, Ac has remained unidentified. Here we report evidence that Ac corresponds to a gene encoding a cytochrome P450 of the CYP79D protein subfamily (CYP79D15), and we describe the apparent molecular basis of the Ac/ac polymorphism. CYP79D orthologs catalyze the first step in cyanogenic glucoside biosynthesis in other cyanogenic plant species. In white clover, Southern hybridizations indicate that CYP79D15 occurs as a single-copy gene in cyanogenic plants but is absent from the genomes of ac plants. Gene-expression analyses by RT-PCR corroborate this finding. This apparent molecular basis of the Ac/ac polymorphism parallels our previous findings for the Li/li polymorphism, which also arises through the presence/absence of a single-copy gene. The nature of these polymorphisms may reflect white clover's evolutionary origin as an allotetraploid derived from cyanogenic and acyanogenic diploid progenitors.

Community-partnered approaches to enhance chronic kidney disease awareness, prevention, and early intervention.

There is a need to increase community involvement in addressing the growing burden of chronic kidney disease (CKD). Community-partnered participatory research (CPPR) is a collaborative approach that equitably involves academic, community, and professional partners in research, and the development of shared goals and of interventional programs to attain these goals. We present a case study of the processes, strategies, and activities concerning the interface of World Kidney Day goals and community-academic partnerships using a CPPR model focused on CKD. We show that CPPR methods can be used to (1) bring together community and academic leaders around goal sharing and research agenda development, (2) convene a community/professional conference aimed at knowledge transfer and data collection among partners, and (3) develop workgroups from a diverse group of participants to collaborate in community partnered strategies to reduce the burden of CKD. Participants included health care professionals, patients, faith-based professionals, government employees and officials, academics, caregivers, and community members. Follow-up workgroups developed action plans to address shared concerns. Using CPPR practices and principles, we were able to incorporate World Kidney Day objectives with community-derived goals to develop a community-partnered infrastructure, shared objectives, and workgroups to reduce the burden of chronic kidney disease.

Between-species analysis of short-repeat modules in cell wall and sex-related hydroxyproline-rich glycoproteins of Chlamydomonas.

Protein diversification is commonly driven by single amino acid changes at random positions followed by selection, but, in some cases, the structure of the gene itself favors the occurrence of particular kinds of mutations. Genes encoding hydroxyproline-rich glycoproteins (HRGPs) in green organisms, key protein constituents of the cell wall, carry short-repeat modules that are posited to specify proline hydroxylation and/or glycosylation events. We show here, in a comparison of two closely related Chlamydomonas species-Chlamydomonas reinhardtii (CC-621) and Chlamydomonas incerta (CC-1870/3871)-that these modules are prone to misalignment and hence to both insertion/deletion and endoduplication events, and that the dynamics of the rearrangements are constrained by purifying selection on the repeat patterns themselves, considered either as helical or as longitudinal face modules. We suggest that such dynamics may contribute to evolutionary diversification in cell wall architecture and physiology. Two of the HRGP genes analyzed (SAG1 and SAD1) encode the mating-type plus and minus sexual agglutinins, displayed only by gametes, and we document that these have undergone far more extensive divergence than two HRGP genes (GP1 and VSP3) that encode cell wall components-an example of the rapid evolution that characterizes sex-related proteins in numerous lineages. Strikingly, the central regions of the agglutinins of both mating types have diverged completely, by selective endoduplication of repeated motifs, since the two species last shared a common ancestor, suggesting that these events may have participated in the speciation process.