Single-Leaf Plantlet Bioassays for the Study of Root Morphogenesis and Rhizobium-Legume Nodulation

Rolfe, B. G.; McIver, J.

doi:10.1071/pp9960271

Cited by 19 publications

(4 citation statements)

References 35 publications

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“…Axenically grown rooted leaves were generated from mature plants (Rolfe and McIver, 1996) using Jensen's medium (Jensen, 1942). The roots were kept dark by covering the lower half of the plates with brown paper or specifically designed black plastic boxes.…”

Section: Generation Of Rooted Leavesmentioning

confidence: 99%

Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides

Mathesius¹,

Schlaman²,

Spaink³

et al. 1998

The Plant Journal

425

362

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SummaryThe expression of the auxin responsive reporter construct, GH3:gusA, was examined in transgenic white clover plants to assess changes in the auxin balance during the earliest stages of root nodule formation. Reporter gene expression was monitored at marked locations after the application of bacteria or signal molecules using two precise inoculation techniques: spot-inoculation and a novel method for ballistic microtargeting. Changes in GH3:gusA expression were monitored after the inoculation of Rhizobium leguminosarum biovar trifolii, non-host rhizobia, lipo-chitin oligosaccharides (LCOs), chitin oligosaccharides, a synthetic auxin transport inhibitor (naphthylphthalamic acid; NPA), auxin, the ENOD40-1 peptide or different flavonoids. The results show that clover-nodulating rhizobia induce a rapid, transient and local downregulation of GH3:gusA expression during nodule initiation followed by an upregulation of reporter gene expression at the site of nodule initiation. Microtargeting of auxin caused a local and acropetal upregulation of GH3:gusA expression, whereas NPA caused local and acropetal downregulation of expression. Both spot-inoculation and microtargeting of R. l. bv. trifolii LCOs or flavonoid aglycones induced similar changes to GH3:gusA expression as NPA. O-acetylated chitin oligosaccharides caused similar changes to GH3:gusA expression as R. l. bv. trifolii spot-inoculation, but only after delivery by microtargeting. Non-Oacetylated chitin oligosaccharides, flavonoid glucosides or the ENOD40-1 peptide failed to induce any detectable Received 10 June 1997; revised 17 November 1997; accepted 8 January 1998. *For correspondence (fax ϩ61 6 249 0754; e-mail Michael@rsbs.anu.edu.au).© 1998 Blackwell Science Ltd 23 changes in GH3:gusA expression. GH3:gusA expression patterns during the later stages of nodule and lateral root development were similar. These results support the hypothesis that LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation, and that endogenous flavonoids could mediate this response.

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Section: Generation Of Rooted Leavesmentioning

confidence: 99%

Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides

Mathesius¹,

Schlaman²,

Spaink³

et al. 1998

The Plant Journal

425

362

View full text Add to dashboard Cite

show abstract

“…White clover plants (Trifolium repens cv. Haifa) were grown as described elsewhere (Rolfe and McIver 1996;Mathesius et al 1998a;, under sterile nitrogen-free medium conditions in all experiments unless otherwise stated. Nitrate was added as KNO 3 at 15 mM to the medium where stated.…”

Section: Plant Growth Conditions and Inoculationsmentioning

confidence: 99%

Temporal and Spatial Order of Events During the Induction of Cortical Cell Divisions in White Clover byRhizobium leguminosarumbv.trifoliiInoculation or Localized Cytokinin Addition

Mathesius¹,

Charon²,

Rolfe³

et al. 2000

MPMI

134

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We examined the timing and location of several early root responses to Rhizobium leguminosarum bv. trifolii infection, compared with a localized addition of cytokinin in white clover, to study the role of cytokinin in early signaling during nodule initiation. Induction of ENOD40 expression by either rhizobia or cytokinin was similar in timing and location and occurred in nodule progenitor cells in the inner cortex. Inoculation of rhizobia in the mature root failed to induce ENOD40 expression and cortical cell divisions (ccd). Nitrate addition at levels repressing nodule formation inhibited ENOD40 induction by rhizobia but not by cytokinin. ENOD40 expression was not induced by auxin, an auxin transport inhibitor, or an ethylene precursor. In contrast to rhizobia, cytokinin addition was not sufficient to induce a modulation of the auxin flow, the induction of specific chalcone synthase genes, and the accumulation of fluorescent compounds associated with nodule initiation. However, cytokinin addition was sufficient for the localized induction of auxin-induced GH3 gene expression and the initiation of ccd. Our results suggest that rhizobia induce cytokinin-mediated events in parallel to changes in auxin-related responses during nodule initiation and support a role of ENOD40 in regulating ccd. We propose a model for the interactions of cytokinin with auxin, ENOD40, flavonoids, and nitrate during nodulation.

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“…Rooted leaves of Trifolium repens cv. Haifa were generated following a technique by Rolfe and McIver (1996) under growth conditions described elsewhere (Mathesius et al 1998). Roots of wild-type rooted leaves were used for fluorescence measurements, and rooted leaves of transgenic plants for β-glucuronidase (GUS) assays.…”

Section: Rooted Leaf Assaymentioning

confidence: 99%

Flavonoids Synthesized in Cortical Cells During Nodule Initiation Are Early Developmental Markers in White Clover

Mathesius¹,

Bayliss²,

Weinman³

et al. 1998

MPMI

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We examined the site-specific induction of the flavonoid pathway before and during nodule initiation in white clover with transgenic plants, fluorescence microscopy, and microspectrofluorometry to test if flavonoids play a role in nodule organogenesis. A chalcone synthase regulated β-glucuronidase (GUS) transgene (CHS3:gusA) was up-regulated from 3 h post inoculation (p.i.) until cell division (around 40 h p.i.) in inner cortex cells underlying the inoculation site. Intracellular fluorescence occurred in vacuoles of those inner cortex cells from 13 h p.i. until the fluorescent cells divided. Fluorescence emission spectra of contents of individual fluorescing cortex cells were measured in situ and compared with emission spectra of compounds purified from root extracts. The fluorescing compound located in cells of the inner cortex after Rhizobium leguminosarum bv. trifolii infection was identified as a water-soluble derivative of 7,4′-dihydroxyflavone. Nodule primordium cells contained a different fluorescent compound, identified as the isoflavonoid formononetin. CHS3:gusA expression and flavonoid accumulation were only induced in inner cortex cells by a nodulating Rhizo-bium strain and by clover-specific lipo-chitinoligosac-charides, but not by non-nodulating rhizobia. Fluorescence was also induced by compatible rhizobia in other legumes such as alfalfa, pea, and siratro in the cells that participate in nodule initiation. Our results show that fluorescent flavonoids are useful markers in nodule or-ganogenesis in clover and may have direct roles in nodule formation.

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Single-Leaf Plantlet Bioassays for the Study of Root Morphogenesis and Rhizobium-Legume Nodulation

Cited by 19 publications

References 35 publications

Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides

Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides

Temporal and Spatial Order of Events During the Induction of Cortical Cell Divisions in White Clover byRhizobium leguminosarumbv.trifoliiInoculation or Localized Cytokinin Addition

Flavonoids Synthesized in Cortical Cells During Nodule Initiation Are Early Developmental Markers in White Clover

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