Cytokinin Accumulation and an Altered Ethylene Response Mediate the Pleiotropic Phenotype of the Pea Nodulation Mutant R50 (\u3cem\u3esym16\u3c/em\u3e)

R50 (sym16), a pleiotropic mutant of Pisum sativum L., is short, has thickened internodes and roots, and has a reduced number of lateral roots and nodules. Its low nodule phenotype can be restored with the application of ethylene inhibitors; furthermore, it can be mimicked by applying cytokinins (CKs) to the roots of the parent line #8216;Sparkle’. Here, we report on the etiolation phenotypes of R50 and ‘Sparkle’, and on the interactive roles of ethylene and CKs in these lines. R50 displayed an altered etiolation phenotype, as it was shorter and thicker, and had more developed leaves than dark-grown ‘Sparkle’. Shoot morphological differences induced by exogenous ethylene or CKs were found to be less severe for R50. Ethylene inhibitor application induced root and shoot elongation and encouraged apical hook opening in both etiolated lines. Liquid chromatography–tandem mass spectrometry analysis indicated that CK concentrations in R50 were higher than in ‘Sparkle’, particularly in mature shoots where the levels were maintained at elevated concentrations. These differences indicate a reduction in the CK catabolism of R50. The accumulation of CKs can be directly related to several traits of R50, with the reduced number of nodules and altered shoot ethylene response being likely indirect effects

Impacts of Smooth Pigweed (Amaranthus hybridus) on Cover Crops in Southern Ontario

Amaranthus hybridus is a noxious weed in Ontario, with demonstrated allelopathic properties that can lead to decreased agricultural production. We tested the germination and growth of five cover crop species exposed to A. hybridus extracts, and to dried or fresh materials in soil. A germination index was calculated, and the dry weight of plant organs were measured to quantify responses to treatments. All species had reduced germination (≤29%) in 100% extract. Trifolium pratense had significant root weight reductions in extract (52%) and dried (72%) treatments, whereas shoot weight only decreased (48%) in dried treatment. Medicago sativa shoot weight decreased (52%) in 20g fresh treatment, while root weight decreased (62%) in dried treatment. Shoot weight of Raphanus sativus increased (32%) at mid-extract concentrations, while root weight increased (33%) only with dried treatment; however, both its shoot and root weight decreased (\u3e40%) in fresh treatment. Only the shoot weight of Lolium multiflorum increased (41% in 75% extract and 55% in dried treatment). Both Cichorium intybus shoot and root weights decreased (~50%) in fresh treatment. Crop responses to A. hybridus are complex, and material and species-dependant. Further testing in the field may provide a more comprehensive understanding of how to improve the management of A. hybridus

E151 (sym15), A Pleiotropic Mutant of Pea (Pisum sativum L.), Displays Low Nodule Number, Enhanced Mycorrhizae, Delayed Lateral Root Emergence, and High Root Cytokinin Levels

In legumes, the formation of rhizobial and mycorrhizal root symbioses is a highly regulated process which requires close communication between plant and microorganism. Plant mutants that have difficulties establishing symbioses are valuable tools for unravelling the mechanisms by which these symbioses are formed and regulated. Here E151, a mutant of Pisum sativum cv. Sparkle, was examined to characterize its root growth and symbiotic defects. The symbioses in terms of colonization intensity, functionality of micro-symbionts, and organ dominance were compared between the mutant and wild type. The endogenous cytokinin (CK) and abscisic acid (ABA) levels and the effect of the exogenous application of these two hormones were determined. E151 was found to be a low and delayed nodulator, exhibiting defects in both the epidermal and cortical programmes though a few mature and functional nodules develop. Mycorrhizal colonization of E151 was intensified, although the fungal functionality was impaired. Furthermore, E151 displayed an altered lateral root (LR) phenotype compared with that of the wild type whereby LR emergence is initially delayed but eventually overcome. No differences in ABA levels were found between the mutant and the wild type, but non-inoculated E151 exhibited significantly high CK levels. It is hypothesized that CK plays an essential role in differentially mediating the entry of the two micro-symbionts into the cortex; whereas it would inhibit the entry of the rhizobia in that tissue, it would promote that of the fungus. E151 is a developmental mutant which may prove to be a useful tool in further understanding the role of hormones in the regulation of beneficial root symbioses

Reproducible hairy root transformation and spot-inoculation methods to study root symbioses of pea

Pea has lagged behind other model legumes in the molecular study of nodulation and mycorrhizae-formation because of the difficulty to transform its roots and its poor growth on agar plates. Here we describe for pea 1) a transformation technique which permits the complementation of two known non-nodulating pea mutants, 2) a rhizobial inoculation method which allows the study of early cellular events giving rise to nodule primordia, and 3) a targeted fungal inoculation method which allows us to study short segments of mycorrhizal roots assured to be infected. These tools are certain to advance our knowledge of pea root symbioses

A Stimulatory Role for Cytokinin in the Arbuscular Mycorrhizal Symbiosis of Pea

The arbuscular mycorrhizal (AM) symbiosis between terrestrial plants and AM fungi is regulated by plant hormones. For most of these, a role has been clearly assigned in this mutualistic interaction; however, there are still contradictory reports for cytokinin (CK). Here, pea plants, the wild type (WT) cv. Sparkle and its mutant E151 (Pssym15), were inoculated with the AM fungus Rhizophagus irregularis. E151 has previously been characterized as possessing high CK levels in non-mycorrhizal (myc-) roots and exhibiting high number of fungal structures in mycorrhizal (myc+) roots. Myc- and myc+ plants were treated 7, 9, and 11 days after inoculation (DAI) with synthetic compounds known to alter CK status. WT plants were treated with a synthetic CK [6-benzylaminopurine (BAP)] or the CK degradation inhibitor INCYDE, whereas E151 plants were treated with the CK receptor antagonist PI-55. At 13 DAI, plant CK content was analyzed by mass spectrometry. The effects of the synthetic compounds on AM colonization were assessed at 28 (WT) or 35 (E151) DAI via a modified magnified intersections method. The only noticeable difference seen between myc- and myc+ plants in terms of CK content was in the levels of nucleotides (NTs). Whereas WT plants responded to fungi by lowering their NT levels, E151 plants did not. Since NTs are thought to be converted into active CK forms, this result suggests that active CKs were synthesized more effectively in WT than in E151. In general, myc+ and myc- WT plants responded similarly to INCYDE by lowering significantly their NT levels and increasing slightly their active CK levels; these responses were less obvious in BAP-treated WT plants. In contrast, the response of E151 plants to PI-55 depended on the plant mycorrhizal status. Whereas treated myc- plants exhibited high NT and low active CK levels, treated myc+ plants displayed low levels of both NTs and active CKs. Moreover, treated WT plants were more colonized than treated E151 plants. We concluded that CKs have a stimulatory role in AM colonization because increased active CK levels were paralleled with increased AM colonization while decreased CK levels corresponded to reduced AM colonization

A Stimulatory Role for Cytokinin in the Arbuscular Mycorrhizal Symbiosis of Pea

The arbuscular mycorrhizal (AM) symbiosis between terrestrial plants and AM fungi is regulated by plant hormones. For most of these, a role has been clearly assigned in this mutualistic interaction; however, there are still contradictory reports for cytokinin (CK). Here, pea plants, the wild type (WT) cv. Sparkle and its mutant E151 (Pssym15), were inoculated with the AM fungus Rhizophagus irregularis. E151 has previously been characterized as possessing high CK levels in non-mycorrhizal (myc-) roots and exhibiting high number of fungal structures in mycorrhizal (myc+) roots. Myc- and myc+ plants were treated 7, 9, and 11 days after inoculation (DAI) with synthetic compounds known to alter CK status. WT plants were treated with a synthetic CK [6-benzylaminopurine (BAP)] or the CK degradation inhibitor INCYDE, whereas E151 plants were treated with the CK receptor antagonist PI-55. At 13 DAI, plant CK content was analyzed by mass spectrometry. The effects of the synthetic compounds on AM colonization were assessed at 28 (WT) or 35 (E151) DAI via a modified magnified intersections method. The only noticeable difference seen between myc- and myc+ plants in terms of CK content was in the levels of nucleotides (NTs). Whereas WT plants responded to fungi by lowering their NT levels, E151 plants did not. Since NTs are thought to be converted into active CK forms, this result suggests that active CKs were synthesized more effectively in WT than in E151. In general, myc+ and myc- WT plants responded similarly to INCYDE by lowering significantly their NT levels and increasing slightly their active CK levels; these responses were less obvious in BAP-treated WT plants. In contrast, the response of E151 plants to PI-55 depended on the plant mycorrhizal status. Whereas treated myc- plants exhibited high NT and low active CK levels, treated myc+ plants displayed low levels of both NTs and active CKs. Moreover, treated WT plants were more colonized than treated E151 plants. We concluded that CKs have a stimulatory role in AM colonization because increased active CK levels were paralleled with increased AM colonization while decreased CK levels corresponded to reduced AM colonization

Mini-review: Fostering comprehension and integration in mycorrhiza biology: conceptual scaffolding as an aid in teaching and exploration

The last decade has witnessed extraordinary progress in understanding molecular dialogues between partners of plant root mutualisms and, as such, a considerable amount of new information now needs to be integrated into an already significant body of literature. The topic of symbiosis has become difficult to explore in a teaching venue, as there is seemingly so much to discuss, and yet students are truly interested in the discipline because of its potential applications in conservation, sustainable agriculture and forestry. In this review targeted to instructors, senior students and scientists, we offer a means of teaching the symbioses between mycorrhizal fungi and vascular plants, whereby we propose a conceptual staircase with three levels of incremental learning difficulty. At the first level, we describe the fundamentals of mycorrhizas with special emphasis on the plant-fungus interface. At the second level, we focus on the pre-communication between the two partners. At the third level, we discuss the physiology of the interface in terms of agriculture and forestry. At the end of each level, we provide a short summary where the most important concepts have been outlined for an instructor. As well, throughout the text, we raise questions of interest to the field at large.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author