Flavone Limitations to Root Nodulation and Symbiotic Nitrogen Fixation in Alfalfa

Kapulnik, Yoram; Joseph, Cecillia M.; Phillips, Donald A.

doi:10.1104/pp.84.4.1193

Cited by 106 publications

(46 citation statements)

References 19 publications

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“…Given the fact that increasing the number of rhizobial cells promotes root nodule formation (reviewed in ref. 1), the growth-enhancing effects reported here may have significant agricultural implications and could partially explain several previous reports (14,26).…”

Section: Discussionsupporting

confidence: 79%

Flavonoids Released Naturally from Alfalfa Seeds Enhance Growth Rate of Rhizobium meliloti

Hartwig¹,

Joseph²,

Phillips³

1991

Plant Physiol.

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192

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Alfalfa (Medicago sativa L.) releases different flavonoids from seeds and roots. Imbibing seeds discharge 3',4',5,7-substituted flavonoids; roots exude 5-deoxy molecules. Many, but not all, of these flavonoids induce nodulation (nod) genes in Rhizobium meliloti. The dominant flavonoid released from alfalfa seeds is identified here as quercetin-3-O-galactoside, a molecule that does not induce nod genes. Low concentrations (1-10 micromolar) of this compound, as well as luteolin-7-O-glucoside, another major flavonoid released from germinating seeds, and the aglycones, quercetin and luteolin, increase growth rate of R. meliloti in a defined minimal medium. Tests show that the 5,7-dihydroxyl substitution pattern on those molecules was primarily responsible for the growth effect, thus explaining how 5-deoxy flavonoids in root exudates fail to enhance growth of R. meliloti. Luteolin increases growth by a mechanism separate from its capacity to induce rhizobial nod genes, because it still enhanced growth rate of R. meliloti lacking functional copies of the three known nodD genes. Quercetin and luteolin also increased growth rate of Pseudomonas putida. They had no effect on growth rate of Bacillus subtilis or Agrobacterium tumefaciens, but they slowed growth of two fungal pathogens of alfalfa. These results suggest that alfalfa can create ecochemical zones for controlling soil microbes by releasing structurally different flavonoids from seeds and roots.

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Section: Discussionsupporting

confidence: 79%

Flavonoids Released Naturally from Alfalfa Seeds Enhance Growth Rate of Rhizobium meliloti

Hartwig¹,

Joseph²,

Phillips³

1991

Plant Physiol.

Self Cite

192

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“…It was reported that the flavone luteolin from seed extracts of Medicago can specifically induce nod gene expression in S. meliloti (Peters et al, 1986). Since then, more detailed analysis has revealed that luteolin, apigenin, 7,4#-dihydroxflavone, and 4,4#-dihydroxy 2#-methoxy-chalcone are the most potent inducers of S. meliloti nod genes (Kapulnik et al, 1987;Orgambide et al, 1994). Of these compounds, only the 5-deoxy flavones have been identified in Medicago roots so far Coronado et al, 1995).…”

mentioning

confidence: 99%

Flavone Synthases from Medicago truncatula Are Flavanone-2-Hydroxylases and Are Important for Nodulation

et al. 2007

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Flavones are important copigments found in the flowers of many higher plants and play a variety of roles in plant adaptation to stress. In Medicago species, flavones also act as signal molecules during symbiotic interaction with the diazotropic bacterium Sinorhizobium meliloti. They are the most potent nod gene inducers found in root exudates. However, flavone synthase II (FNS II), the key enzyme responsible for flavone biosynthesis, has not been characterized in Medicago species. We cloned two FNS II genes from Medicago truncatula using known FNS II sequences from other species and named them MtFNSII-1 and MtFNSII-2. Functional assays in yeast (Saccharomyces cerevisiae) suggested that the catalytic mechanisms of both cytochrome P450 monooxygenases were similar to the other known legume FNS II from licorice (Glycyrrhiza echinata). MtFNSII converted flavanones to 2-hydroxyflavanones instead of flavones whereas FNS II from the nonlegume Gerbera hybrida, converted flavanones to flavones directly. The two MtFNSII genes had distinct tissue-specific expression patterns. MtFNSII-1 was highly expressed in roots and seeds whereas MtFNSII-2 was highly expressed in flowers and siliques. In addition, MtFNSII-2 was inducible by S. meliloti and methyl jasmonate treatment, whereas MtFNSII-1 was not. Histochemical staining of transgenic hairy roots carrying the promoter-reporter constructs indicated that the MtFNSII-2 induction was tissue specific, mostly localized to vascular tissues and root hairs. RNA interference-mediated suppression of MtFNSII genes resulted in flavone depleted roots and led to significantly reduced nodulation when inoculated with S. meliloti. Our results provide genetic evidence supporting that flavones are important for nodulation in M. truncatula.

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“…Kapulnik et al (14) observed significant increases in nodulation, N2 fixation, and seedling growth of 'Hairy Peruvian' alfalfa when the rhizosphere was supplemented with luteolin. This is consistent with evidence that some N2-dependent alfalfa seedlings are initially N-limited due to insufficient root nodule formation (10) and suggests that more nodules might be formed if more rhizobial cells are induced to initiate the infection process.…”

mentioning

confidence: 99%

A Chalcone and Two Related Flavonoids Released from Alfalfa Roots Induce nod Genes of Rhizobium meliloti

Maxwell¹,

Hartwig²,

Joseph³

et al. 1989

Plant Physiol.

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219

167

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Flavonoid signals from alfalfa (Medicago sativa L.) induce transcription of nodulation (nod) genes in Rhizobium melioti. Previous investigations identified the flavone luteolin as an active inducer in alfalfa seed extracts, but the nature of nod inducers released from roots has not been reported. Root exudate from 3-day-old alfalfa seedlings was purified and then assayed for biological activity with a nodABC-IacZ fusion in R. meliloti. Indentities of major nod inducers were established by spectroscopic analyses (ultraviolet/visible, proton nuclear magnetic resonance, and mass spectroscopy) and comparison with authentic standards. Major nod inducers, which were identified as 4',7-dihydroxyflavone, 4'-7-dihydroxyflavanone, and 4,4'-dihydroxy-2'-methoxychalcone, were released from seedling roots at 54, 22, and 20 picomole.plantr'. day-1, respectively. Luteolin was not found in these root exudates. The 4,4'-dihydroxy-2'-methoxychalcone induced nod genes at a concentration one order of magnitude lower than luteolin and is the first naturally released chalcone reported to have this function. Moderate and weak nod-inducing activity was associated, respectively, with 4',7-dihydroxyflavone and 4',7-dihydroxyflavanone.Alfalfa (Medicago sativa L.), an important leguminous forage crop throughout the world, forms N2-fixing root nodules in association with the soil bacterium Rhizobium meliloti. The earliest events of alfalfa nodule formation require expression ofthe nodulation (nod) DABC genes on the megaplasmid (pSym) of R. meliloti (8,16). Transcription of nodABC is induced through the cooperative action of the constitutive nodD product and components of root and seed exudates (22). Luteolin, 3',4',5,7-tetrahydroxyflavone, was isolated from alfalfa seed extracts and shown to participate in nod induction (23) evidence that some N2-dependent alfalfa seedlings are initially N-limited due to insufficient root nodule formation (10) and suggests that more nodules might be formed if more rhizobial cells are induced to initiate the infection process.The presence of active nod inducers in plants does not guarantee release into the rhizosphere. Yelton et al. (30) observed that extracts from some plants induced nod transcription in R. meliloti even when exudates ofthe same plants did not. In contrast, both extracts and exudates of alfalfa induced nod genes, but it is unclear if luteolin was solely responsible for nod induction.In order to better understand the process of nod-inducer release, it is necessary to identify active compounds actually exuded into the rhizosphere. The purpose ofthis study was to identify, quantify, and characterize the major nod inducers exuded by roots of young, unnodulated alfalfa seedlings. MATERIALS AND METHODS Plant CultureOne g of alfalfa (Medicago sativa L.) seed (cv 'Moapa 69') containing about 400 seeds (94% viable) was scarified, surfacesterilized 3 min with 70% ethanol, rinsed with sterile water, and imbibed in sterile, aerated water. Imbibing solutions were changed after 4 and 8 h to rem...

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Flavone Limitations to Root Nodulation and Symbiotic Nitrogen Fixation in Alfalfa

Cited by 106 publications

References 19 publications

Flavonoids Released Naturally from Alfalfa Seeds Enhance Growth Rate of Rhizobium meliloti

Flavonoids Released Naturally from Alfalfa Seeds Enhance Growth Rate of Rhizobium meliloti

Flavone Synthases from Medicago truncatula Are Flavanone-2-Hydroxylases and Are Important for Nodulation

A Chalcone and Two Related Flavonoids Released from Alfalfa Roots Induce nod Genes of Rhizobium meliloti

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