Defective long-distance auxin transport regulation in the Medicago truncatula super numeric nodules mutant

Long-distance auxin transport was examined in Medicago truncatula and in its supernodulating mutant sunn (super numeric nodules) to investigate the regulation of auxin transport during autoregulation of nodulation (AON). A method was developed to monitor the transport of auxin from the shoot to the root in whole seedlings. Subsequently, the transport was monitored after inoculation of roots with the nodulating symbiont Sinorhizobium meliloti. The sunn mutant showed an increased amount of auxin transported from the shoot to the root compared to the wild type. The auxin transport capacity of excised root segments was similar in wild type and sunn, suggesting that the difference in long-distance auxin transfer between them is due to loading in the shoot. After inoculation, wild-type seedlings showed decreased auxin loading from the shoot to the root; however, the sunn mutant failed to reduce the amount of auxin loaded. The time of reduced auxin loading correlated with the onset of AON. Quantification of endogenous auxin levels at the site of nodule initiation showed that sunn contained three times more auxin than wild type. Inoculation of sunn failed to reduce the level of auxin within 24 h, as was observed in the wild type. We propose a model for the role of auxin during AON of indeterminate legumes: 1) high levels of endogenous auxin are correlated with increased numbers of nodules, 2) inoculation of roots reduces auxin loading from the shoot to the root, and 3) subsequent reduction of auxin levels in the root inhibits further nodule initiation.     

The Ethylene-insensitive sickle mutant of Medicago truncatula shows altered auxin transport regulation during nodulation

We studied the ethylene-insensitive, hypernodulating mutant, sickle (skl), to investigate the interaction of ethylene with auxin transport during root nodulation in Medicago truncatula. Grafting experiments demonstrated that hypernodulation in skl is root controlled. Long distance transport of auxin from shoot to root was reduced by rhizobia after 24 h in wild type but not in skl. Similarly, the ethylene precursor 1-amino cyclopropane-1-carboxylic acid inhibited auxin transport in wild type but not in skl. Auxin transport at the nodule initiation zone was significantly reduced by rhizobia after 4 h in both wild type and skl. After 24 h, auxin transport significantly increased at the nodule initiation zone in skl compared to wild type, accompanied by an increase in the expression of the MtPIN1 and MtPIN2 (pin formed) auxin efflux transporters. Response assays to different auxins did not show any phenotype that would suggest a defect of auxin uptake in skl. The auxin transport inhibitor N-1-naphthylphtalamic acid inhibited nodulation in wild type but not skl, even though N-1-naphthylphtalamic acid still inhibited auxin transport in skl. Our results suggest that ethylene signaling modulates auxin transport regulation at certain stages of nodule development, partially through PIN gene expression, and that an increase in auxin transport relative to the wild type is correlated with higher nodule numbers. We also discuss the regulation of auxin transport in skl in comparison to previously published data on the autoregulation mutant, super numerary nodules (van Noorden et al., 2006).     

Gibberellins are involved in nodulation of Sesbania rostrata

Upon submergence, Azorhizobium caulinodans infects the semiaquatic legume Sesbania rostrata via the intercellular crack entry process, resulting in lateral root-based nodules. A gene encoding a gibberellin (GA) 20-oxidase, SrGA20ox1, involved in GA biosynthesis, was transiently up-regulated during lateral root base nodulation. Two SrGA20ox1 expression patterns were identified, one related to intercellular infection and a second observed in nodule meristem descendants. The infection-related expression pattern depended on bacterially produced nodulation (Nod) factors. Pharmacological studies demonstrated that GAs were involved in infection pocket and infection thread formation, two Nod factor-dependent events that initiate lateral root base nodulation, and that they were also needed for nodule primordium development. Moreover, GAs inhibited the root hair curling process. These results show that GAs are Nod factor downstream signals for nodulation in hydroponic growth.     

Hormone modulation of legume‐rhizobial symbiosis

Leguminous plants can establish symbiotic associations with diazotropic rhizobia to form nitrogenfixating nodules, which are classified as determinate or indeterminate based on the persistence of nodule meristem. The formation of nitrogen-fixing nodules requires coordinating rhizobial infection and root nodule organogenesis. The formation of an infection thread and the extent of nodule formation are largely under plant control, but vary with environmental conditions and the physiological state of the host plants. Many achievements in these two areas have been made in recent decades.Phytohormone signaling pathways have gradually emerged as important regulators of root nodule symbiosis. Cytokinin, strigolactones(SLs) and local accumulation of auxin can promote nodule development. Ethylene,jasmonic acid(JA), abscisic acid(ABA) and gibberellic acid(GA) all negatively regulate infection thread formation and nodule development. However, salicylic acid(SA) and brassinosteroids(BRs) have different effects on the formation of these two nodule types. Some peptide hormones are also involved in nodulation. This review summarizes recent findings on the roles of these plant hormones in legume-rhizobial symbiosis, and we propose that DELLA proteins may function as a node to integrate plant hormones to regulate nodulation.     

Strigolactones promote nodulation in pea

Strigolactones are recently defined plant hormones with roles in mycorrhizal symbiosis and shoot and root architecture. Their potential role in controlling nodulation, the related symbiosis between legumes and Rhizobium bacteria, was explored using the strigolactone-deficient rms1 mutant in pea (Pisum sativum L.). This work indicates that endogenous strigolactones are positive regulators of nodulation in pea, required for optimal nodule number but not for nodule formation per se. rms1 mutant root exudates and root tissue are almost completely deficient in strigolactones, and rms1 mutant plants have approximately 40% fewer nodules than wild-type plants. Treatment with the synthetic strigolactone GR24 elevated nodule number in wild-type pea plants and also elevated nodule number in rms1 mutant plants to a level similar to that seen in untreated wild-type plants. Grafting studies revealed that nodule number and strigolactone levels in root tissue of rms1 roots were unaffected by grafting to wild-type scions indicating that strigolactones in the root, but not shoot-derived factors, regulate nodule number and provide the first direct evidence that the shoot does not make a major contribution to root strigolactone levels.     

The autoregulation gene SUNN mediates changes in root organ formation in response to nitrogen through alteration of shoot-to-root auxin transport

We tested whether a gene regulating nodule number in Medicago truncatula, Super Numeric Nodules (SUNN ), is involved in root architecture responses to carbon (C) and nitrogen (N) and whether this is mediated by changes in shoot-to-root auxin transport. Nodules and lateral roots are root organs that are under the control of nutrient supply, but how their architecture is regulated in response to nutrients is unclear. We treated wild-type and sunn-1 seedlings with four combinations of low or increased N (as nitrate) and C (as CO(2)) and determined responses in C/N partitioning, plant growth, root and nodule density, and changes in auxin transport. In both genotypes, nodule density was negatively correlated with tissue N concentration, while only the wild type showed significant correlations between N concentration and lateral root density. Shoot-to-root auxin transport was negatively correlated with shoot N concentration in the wild type but not in the sunn-1 mutant. In addition, the ability of rhizobia to alter auxin transport depended on N and C treatment as well as the SUNN gene. Nodule and lateral root densities were negatively correlated with auxin transport in the wild type but not in the sunn-1 mutant. Our results suggest that SUNN is required for the modulation of shoot-to-root auxin transport in response to altered N tissue concentrations in the absence of rhizobia and that this controls lateral root density in response to N. The control of nodule density in response to N is more likely to occur locally in the root.     

Characterization of the Lotus japonicus symbiotic mutant lot1 that shows a reduced nodule number and distorted trichomes

We isolated a recessive symbiotic mutant of Lotus japonicus that defines a genetic locus, LOT1 (for low nodulation and trichome distortion). The nodule number per plant of the mutant was about one-fifth of that of the wild type. The lot1 mutant showed a moderate dwarf phenotype and distorted trichomes, but its root hairs showed no apparent differences to those of the wild type. Infection thread formation after inoculation of Mesorhizobium loti was repressed in lot1 compared to that in the wild type. The nodule primordia of lot1 did not result in any aborted nodule-like structure, all nodules becoming mature and exhibiting high nitrogen fixation activity. The mutant was normally colonized by mycorrhizal fungi. lot1 also showed higher sensitivity to nitrate than the wild type. The grown-up seedlings of lot1 were insensitive to any ethylene treatments with regard to nodulation, although the mutant showed normal triple response on germination. It is conceivable that a nodulation-specific ethylene signaling pathway is constitutively activated in the mutant. Grafting experiments with lot1 and wild-type seedlings suggested that the root genotype mainly determines the low nodulation phenotype of the mutant, while the trichome distortion is regulated by the shoot genotype. Grafting of har1-4 shoots to lot1 roots resulted in an intermediate nodule number, i.e. more than that of lot1 and less than that of har1-4. Putative double mutants of lot1 and har1 also showed intermediate nodulation. Thus, it was indicated that LOT1 is involved in a distinct signal transduction pathway independent of HAR1.     

LjABCB1, an ATP-binding cassette protein specifically induced in uninfected cells of Lotus japonicus nodules

Legume plants develop root nodules through symbiosis with rhizobia, and fix atmospheric nitrogen in this symbiotic organ. Development of root nodules is regulated by many metabolites including phytohormones. Previously, we reported that auxin is strongly involved in the development of the nodule vascular bundle and lenticel formation on the nodules of Lotus japonicus. Here we show that an ATP-binding cassette (ABC) protein, LjABCB1, which is a homologue of Arabidopsis auxin transporter AtABCB4, is specifically expressed during nodulation of L. japonicus. A reporter gene analysis indicated that the expression of LjABCB1 was restricted to uninfected cells adjacent to infected cells in the nodule, while no expression was observed in shoot apical meristems or root tips, in which most auxin transporter genes are expressed. The auxin transport activity of LjABCB1 was confirmed using a heterologous expression system.     

Gibberellin controls the nodulation signaling pathway in Lotus japonicus

Root nodule formation is regulated by several plant hormones, but the details of the regulation of the nodulation signaling pathway are largely unknown. In this study, the role of gibberellin (GA) in the control of root nodule symbiosis was investigated at the physiological and genetic levels in Lotus japonicus . Exogenous application of biologically active GA, GA₃, inhibited the formation of infection threads and nodules, which was counteracted by the application of a biosynthesis inhibitor of GA, Uniconazole P. Nod factor-induced root hair deformation was severely blocked in the presence of GA, which was phenocopied by nsp2 mutants. The number of spontaneous nodules triggered by the gain-of-function mutation of calcium/calmodulin-dependent kinase (CCaMK) or the lotus histidine kinase 1 (LHK1) was decreased upon the addition of GA; moreover, the overexpression of the gain-of-function mutation of L. japonicus , SLEEPY1, a positive regulator of GA signaling, resulted in a reduced nodule number, without other aspects of root development being affected. These results indicate that higher GA signaling levels specifically inhibit the nodulation signaling pathway. Nod factor-dependent induction of NSP2 and NIN was inhibited by exogenous GA. Furthermore, the cytokinin-dependent induction of NIN was suppressed by GA. From these results, we conclude that GA inhibits the nodulation signaling pathway downstream of cytokinin, possibly at NSP2, which is required for Nod factor-dependent NIN expression. These results clarify the roles of GA in the nodulation signaling pathway, and in relation to the cytokinin signaling pathway for nodulation in L. japonicus .     

Shoot apex removal does not alter autoregulation of Modulation in soybean

The suppression of new nodule development in soybean (Glycine max (L.) Merr.) has been previously demonstrated to involve the shoot through reciprocal grafts between the wild-type cultivar Bragg and its supernodulating mutant nts382. Using the same grafting technique, but modified through the excision of the shoot apex region and emerging lateral shoots, we show here that autoregulation of nodule number still existed despite apex removal. This radical treatment lowered total nodule number per plant as well as root, shoot and nodule dry weight. Bragg shoots grafted onto nts382 roots gave wild-type nodulation (26 nodules, 15mg total nodule mass) as compared to nts382 shoots grafted onto Bragg roots (340 nodules, 277 mg total nodule mass). Specific nodule mass differed between supernodulating (about 0·5-1·0mg per nodule) and wild-type nodulating (2·3 mg per nodule) plants. In contrast to other growth characteristics, apex removal did not affect specific nodule size, except in plants with wild-type shoots and nts382 (supernodulation) roots. Apex removal only slightly affected the percentage of nodule weight per total root weight in nts382, but had a severe effect in wild type. Growth reductions varied between the normal and supernodulating plants. The fact that autoregulation of nodulation still functions in plants devoid of functional shoot apices suggests that the autoregulation signal may not be derived from the apex regions and that the leaf may be a likely source.     

Overlap of proteome changes in Medicago truncatula in response to auxin and Sinorhizobium meliloti

We used proteome analysis to identify proteins induced during nodule initiation and in response to auxin in Medicago truncatula. From previous experiments, which found a positive correlation between auxin levels and nodule numbers in the M. truncatula supernodulation mutant sunn (supernumerary nodules), we hypothesized (1) that auxin mediates protein changes during nodulation and (2) that auxin responses might differ between the wild type and the supernodulating sunn mutant during nodule initiation. Increased expression of the auxin response gene GH3:beta-glucuronidase was found during nodule initiation in M. truncatula, similar to treatment of roots with auxin. We then used difference gel electrophoresis and tandem mass spectrometry to compare proteomes of wild-type and sunn mutant roots after 24 h of treatment with Sinorhizobium meliloti, auxin, or a control. We identified 131 of 270 proteins responding to treatment with S. meliloti and/or auxin, and 39 of 89 proteins differentially displayed between the wild type and sunn. The majority of proteins changed similarly in response to auxin and S. meliloti after 24 h in both genotypes, supporting hypothesis 1. Proteins differentially accumulated between untreated wild-type and sunn roots also showed changes in auxin response, consistent with altered auxin levels in sunn. However, differences between the genotypes after S. meliloti inoculation were largely not due to differential auxin responses. The role of the identified candidate proteins in nodule initiation and the requirement for their induction by auxin could be tested in future functional studies.     

Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid

The mechanisms imposing a gibberellin (GA) requirement to promote the germination of dormant and non-dormant Arabidopsis seeds were analyzed using the GA-deficient mutant ga1, several seed coat pigmentation and structure mutants, and the abscisic acid (ABA)-deficient mutant aba1. Testa mutants, which exhibit reduced seed dormancy, were not resistant to GA biosynthesis inhibitors such as tetcyclacis and paclobutrazol, contrarily to what was found before for other non-dormant mutants in Arabidopsis. However, testa mutants were more sensitive to exogenous GAs than the wild-types in the presence of the inhibitors or when transferred to a GA-deficient background. The germination capacity of the ga1-1 mutant could be integrally restored, without the help of exogenous GAs, by removing the envelopes or by transferring the mutation to a tt background (tt4 and ttg1). The double mutants still required light and chilling for dormancy breaking, which may indicate that both agents can have an effect independently of GA biosynthesis. The ABA biosynthesis inhibitor norflurazon was partially efficient in releasing the dormancy of wild-type and mutant seeds. These results suggest that GAs are required to overcome the germination constraints imposed both by the seed coat and ABA-related embryo dormancy.     

Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity

Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules.     

Root Isoflavonoid Response to Grafting between Wild-Type and Nodulation-Mutant Soybean Plants

It was previously reported that the hypernodulating soybean (Glycine max [L.] Merr.) mutants, derived from the cultivar Williams, had higher root concentration of isoflavonoid compounds (daidzein, genistein, and coumestrol) than did Williams at 9 to 12 days after inoculation with Bradyrhizobium japonicum. These compounds are known inducers of nod genes in B. japonicum and may be involved in subsequent nodule development. The current study involving reciprocal grafts between NOD1-3 (hypernodulating mutant) and Williams showed that root isoflavonoid concentration and content was more than twofold greater when the shoot genotype was NOD1-3. When grafted, NOD1-3 shoots also induced hypernodulation on roots of both Williams and NOD1-3, while Williams shoots induced normal nodulation on both root genotypes. This shoot control of hypernodulation may be causally related to differential root isoflavonoid levels, which are also controlled by the shoot. In contrast, the nonnodulating characteristic of the NN5 mutant was strictly root controlled, based on reciprocal grafts. Delayed inoculation (7 days after planting) resulted in greater nodule numbers on both NOD1-3 and Williams, compared with a seed inoculation treatment. The nodulation pattern of grafted plants was independent of whether the shoot portion was derived from inoculated seed or uninoculated seed, when grafted at day 7 onto seedling roots derived from inoculated seed. This observation, coupled with the fact that no difference existed in nodule number of NOD1-3 and Williams until after 9 days from seed inoculation, indicated that if isoflavonoids play a role in differential nodulation of the hypernodulating mutant and the wild type, the effect is on advanced stages of nodule ontogeny, possibly related to autoregulation, rather than on initial infection stages.     

Cochleata: getting to the root of legume nodules

The homeotic mutant of Pisum sativum, cochleata, has stipules replaced by alternative leaf components, abnormal flowers and reduced fertility. Although the root system dry weight, root lengths and nodule numbers of cochleata are similar to those of its wild type, the nodulation phenotype of the mutant is unique. The nodules typically dichotomously branch and multiple callus and root structures emerge from their meristems. These nodule-roots incorporate a peripheral vascular bundle of the nodule into their own central vascular cylinder. Both the nodules and roots of the hybrid structures appear functional. Roles for COCHLEATA in development are discussed.     

Regulation of ovule initiation by gibberellins and brassinosteroids in tomato and Arabidopsis: two plant species,two molecular mechanisms

Ovule primordia formation is a complex developmental process with a strong impact on the production of seeds. In Arabidopsis this process is controlled by a gene network, including components of the signalling pathways of auxin, brassinosteroids (BRs) and cytokinins. Recently, we have shown that gibberellins (GAs) also play an important role in ovule primordia initiation, inhibiting ovule formation in both Arabidopsis and tomato. Here we reveal that BRs also participate in the control of ovule initiation in tomato, by promoting an increase on ovule primordia formation. Moreover, molecular and genetic analyses of the co‐regulation by GAs and BRs of the control of ovule initiation indicate that two different mechanisms occur in tomato and Arabidopsis. In tomato, GAs act downstream of BRs. BRs regulate ovule number through the downregulation of GA biosynthesis, which provokes stabilization of DELLA proteins that will finally promote ovule primordia initiation. In contrast, in Arabidopsis both GAs and BRs regulate ovule number independently of the activity levels of the other hormone. Taken together, our data strongly suggest that different molecular mechanisms could operate in different plant species to regulate identical developmental processes even, as for ovule primordia initiation, if the same set of hormones trigger similar responses, adding a new level of complexity.     

Autoregulation of soybean–Bradyrhizobium nodule symbiosis is controlled by shoot or/and root factors

Two strains of Bradyrhizobium japonicum were evaluated with five commercial cultivars of soybean (Clark, Crauford, Davis, Centaur, and Nessen) and one hypernodulating mutant NOD1-3. The hypernodulating NOD1-3 produced 30–50 times the number of nodules of commercial cultivars either inoculated with B. japonicum strain USDA 123 or RCR 3409. Grafting of NOD1-3 shoots to Clark and Davis roots induced hypernodulation on roots of Clark and Davis but did not enhance nodulation when grafted onto the roots of Crauford, Centaur, and Nessen. In contrast, the shoots of Clark, Davis, Centaur and Nessen significantly inhibited nodule formation on the root of NOD1-3. However, Crauford shoots did not alter nodule formation on the roots of NOD1-3 as compared with self-grafts of NOD1-3. It appears that the shoot of NOD1-3 has the ability to alter autoregulatory control of nodulation of Clark and Davis cultivars, but not of Crauford, Centaur and Nessen. The results suggest that the regulation of nodulation in soybean cultivars Clark and Davis is controlled by the shoot factors, while the Crauford was root controlled. Reciprocal grafts between NOD1-3 and Centaur or Nessen indicate that both shoot and root factors are involved in regulation of nodulation. The results suggested that the regulation of nodulation did not depend on bradyrhizobial strains. The shoot control of hypernodulation may be causally related to differential root isoflavonoid levels, which are also controlled by shoot. Application of daidzein significantly enhanced the nodulation and nitrogenase activity of soybean cv. Clark. Root control of restricted nodulation of soybean cv. Centaur did not respond to the addition of daidzein in nutrient solution indicating that this character is not related to isoflavonoids. Therefore, autoregulation in Clark and Centaur plants may be separate events in legume–rhizobia symbiosis and regulated by different kinds of signals.     

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.