The har1-1 mutant of Lotus japonicus line Gifu is characterised by increased nodulation and significantly inhibited root growth in the presence of its microsymbiont Mesorhizoboium loti (for example strain NZP2235). A sexual cross between the mutant and another L. japonicus genotype Funakura (with wild-type root and nodule morphology) demonstrated Mendelian recessive segregation of both phenotypes (for root and nodule) in 242 F2 individuals. No separation of phenotypes was observed, suggesting a single mutation with pleiotropic effects. Reciprocal grafting showed that the har1-1 controlled phenotype is governed by the shoot. Using a skeletal genetic map of arbitrary molecular markers produced from a Gifu x Funakura cross, the har1-1 locus was positioned between two markers at about 7 and 13 cM distance. Single nucleotide polymorphisms (SNPs) and transgene sequences were detected by allele-specific PCR in DNA isolated from small (1 mg mass) individual seeds and half-cotyledon of the model legume Lotus japonicus, allowing fast determination of a seedling’s genomic status. This permitted a shortening of the breeding cycle for multi-trait seed lines. Fast neutron mutagenesis of Lotus japonicus wild-type genotype Gifu resulted in the first time isolation of a stable mutant (FNN5-2) unable to form nitrogen-fixing nodules in symbiosis with Mesorhizobium loti, though being infected by mycorrhizal fungi. The mutation behaves as a loss-of-function recessive, and has no other apparent phenotypic effects. Molecular characterization indicates a partial loss of the LjNFR1 LysM type receptor kinase gene. Additionally part of the LjNIN gene (encoding a putative transcription factor needed for nodulation) is also missing. Transcript levels for both genes are severely reduced. As LjNIN and LjNFR1 are in the same chromosomal region we tested whether this terminal portion is lacking. PCR analysis confirms that genes within the relevant interval (such as LjPAL1 (encoding phenylalanine ammonia lyase) and LjEIL2 (encoding an ethylene insensitive-like response regulator)) are present, suggesting that the mutational event induced by the fast neutrons was either a double hit coincidently involving two nodulation-related genes, a major genome rearrangement, or a major segmental inversion. To develop an integrated nodule developmental model based on gene interactions in autoregulation, nodulation and plant hormone response deficient lines, HE double mutants have been built using the har1-1 mutant (hypernodulation and aberrant root) and the ethylene insensitive transgenic line Etr1-1. The homozygous loss-of-function mutant har1-1 has increased nodulation and decreased root growth. Ethylene insensitivity mediated by the transgene 35S::AtETR1-1 restores the normal root growth. The HE double mutants were confirmed by triple response test and allele- or gene-specific PCR. The current results in this study indicate that a) HE double mutants shown the same nodulation pattern as har1-1 and normal root formation as Etr1-1, suggesting that nodule and root control diverge at some stage with root control being ethylene-mediated and the Har1 gene, the orthologue of GmNARK is involved in nodulation. b) Grafting demonstrated that the shoot is the source of ethylene suppression of the har1-1 induced inhibition of root growth. c) The mutated Etr1-1 gene was able to replace AVG in BAP root inhibition; d) IPT-dependent cytokinin overproduction led to aberrant root architecture in har1-1; e) Crosstalk between ethylene and cytokinin in HE double mutant by qRT-PCR.

A regeneração in vitro é bastante utilizada em processos biotecnológicos. No entanto, pouco se conhece sobre os mecanismos envolvidos na aquisição de competência para formação de novos órgãos. Em tomateiro (S. lycopersicum), a alta capacidade de regeneração in vitro é atribuída ao alelo Rg1, vinda de Solanum peruvianum, e que está presente na cv MsK (S. lycopersicum x S. peruvianum). Os genes de nanismo da cv Micro-Tom (MT) foram passados para MsK, obtendo-se, após 8 gerações de autofecundação (F8), a cv Micro-MsK. No presente estudo, após 6 gerações de retrocruzamentos (BC6Fn), criou-se MT-Rg1, com Rg1 isogênico a MT. Testes de regeneração in vitro mostraram que a formação de gemas caulinares adventícias para MT-Rg1 e Micro-MsK são equivalentes, evidenciando que a alta capacidade de regeneração de Micro-MsK é basicamente devido ao Rg1. Plantas MTRg1 apresentam freqüente formação de 3 cotilédones, excesso de ramificações caulinares e senescência atrasada, características ausentes nos parentais. Esses efeitos pleiotrópicos de Rg1 coincidem com aqueles descritos para alterações no hormônio citocinina. Testes de sensibilidade à citocinina e de tempo de senescência realizados por nós, bem como dosagem desse hormônio feita por outras equipes, descartaram essa hipótese. Enxertias recíprocas evidenciaram que a promoção da ramificação caulinar por Rg1 não é um sinal translocável, mas parece ser uma característica intrínseca do tecido. Rg1 também promoveu uma maior formação de raízes adventícias em estacas ex vitro, sugerindo que ele afeta o processo de competência para formação de órgãos em geral, e não somente a indução específica de gemas caulinares. Duplos mutantes entre Rg1 e mutantes com alteração na sensibilidade/metabolismo hormonal (dgt, brt e pro), ausência de ramificações caulinares (ls) e senescência acelerada (l) foram obtidos e testados quanto ao padrão de ramificações e à capacidade de regeneração in vitro. Além desses parâmetros, a presença de Rg1 suprimiu o fenótipo de folhas pouco recortadas do mutante hipersensível a giberelina (pro) e recuperou o sistema radicular pouco desenvolvido do mutante com baixa sensibilidade à auxina (dgt). Rg1 também foi capaz de reverter a ausência de ramificações laterais de ls, sendo que a mutação Me, o qual representa uma superexpressão de um gene do tipo KNOX, não foi capaz. Esse resultado sugere que Rg1 não é um gene do tipo KNOX, embora esses genes sejam considerados os principais controladores da competência. Analisando diferentes tipos de explantes em experimentos independentes, contatou-se que, em geral, as mutações brt (baixa sensibilidade a citocinina), dgt e ls diminuíram a capacidade de regeneração de Rg1, enquanto l aumentou. Rg1 mostrou-se particularmente epistático à mutação pro, revertendo o fenótipo de baixa formação de gemas caulinares desse mutante. Surpreendentemente, Rg1 provou ser mais sensível à auxina em testes de alongamento de segmentos de hipocótilos, sendo capaz de reverter o fenótipo do mutante dgt nesse mesmo teste. Em conjunto, esses resultados confirmam o papel de Rg1 na fase de aquisição da competência e sugerem uma interação dos hormônios giberelina e auxina nesse processo. / The In vitro regeneration process is widely used in plant biotechnology. However, the mechanisms involved in the acquisition of competence for organ formation are hitherto unknown. In tomato (S. lycopersicum), the high capacity for in vitro regeneration is attributed to the Rg1 allele from S. peruvianum, which is present in the cv MsK (S. lycopersicum x S. peruvianum). The dwarfism genes of the cv Micro-Tom (MT) were transferred to MsK, and, after 8 generations of selfing (F8), the cv Micro-Msk was obtained. Here, after 6 generations of backcrosses (BC6Fn), we created the MT-Rg1, which has Rg1 isogenic to MT. Tests of in vitro regeneration showed that shoot formation in MT-Rg1 and Micro-MsK are equivalent, suggesting that the high capacity of regeneration of Micro-MsK is basically due to Rg1. Comparing MT-Rg1 with the control MT, we noticed a high frequency of 3 cotyledon formation, increased shoot branching and late senescence, which are absent in the parentals. These pleiotropic effects of Rg1 coincide with those described for plants with alterations in the hormone cytokinin. Tests of sensitivity for cytokinin and senescence behavior carried out by us, as well as dosage of that hormone made by other researches, discarded that hypothesis. Reciprocal grafting showed that the promotion of shoot branching by Rg1 is not a transmissible signal, but seems to be an inherent characteristic of the tissue. Rg1 also increases adventitious roots formation of ex vitro cuttings, suggesting that it affects the process of competence, which is common for shoots and roots, instead of the specific induction of shoots. Double mutants between Rg1 and mutants with alteration in the sensitivity/metabolism of plant hormones (dgt, brt and pro), as well as mutants with absence of shoot branching (ls) and accelerated senescence (l) were obtained and tested for the capacity of in vitro regeneration. The presence of Rg1 suppressed the phenotype of less dissected leaves of the mutant hypersensitive to gibberrellin (pro) and rescued the phenotype of poor developed root system of the mutant with low auxin sensitivity (dgt). Rg1 was also able to revert the absence of axillary shoot formation in ls, whereas the mutant Me, which represents an over expression of a KNOX gene was not. This result suggests that Rg1 is not a KNOX gene, although those genes are considered the main controllers of the competence. Analyzing different types of explants in independent experiments, it was verified that, in general, the mutations brt (low sensibility to cytokinin), dgt and ls decreased the regeneration capacity of Rg1, while l increased it. Rg1 was particularly epistatic to the pro mutation, reverting the phenotype of little shoot formation of that mutant. Surprisingly, Rg1 proved to be more sensitive to auxin in the hypocotyl segment elongation test, being able to revert the phenotype of the mutant dgt in the same test. Together, these results confirm the role of the Rg1 in the process of acquisition of the competence and suggest an interaction of the hormones gibberellin and auxin in this process.

A regeneração in vitro é bastante utilizada em processos biotecnológicos. No entanto, pouco se conhece sobre os mecanismos envolvidos na aquisição de competência para formação de novos órgãos. Em tomateiro (S. lycopersicum), a alta capacidade de regeneração in vitro é atribuída ao alelo Rg1, vinda de Solanum peruvianum, e que está presente na cv MsK (S. lycopersicum x S. peruvianum). Os genes de nanismo da cv Micro-Tom (MT) foram passados para MsK, obtendo-se, após 8 gerações de autofecundação (F8), a cv Micro-MsK. No presente estudo, após 6 gerações de retrocruzamentos (BC6Fn), criou-se MT-Rg1, com Rg1 isogênico a MT. Testes de regeneração in vitro mostraram que a formação de gemas caulinares adventícias para MT-Rg1 e Micro-MsK são equivalentes, evidenciando que a alta capacidade de regeneração de Micro-MsK é basicamente devido ao Rg1. Plantas MTRg1 apresentam freqüente formação de 3 cotilédones, excesso de ramificações caulinares e senescência atrasada, características ausentes nos parentais. Esses efeitos pleiotrópicos de Rg1 coincidem com aqueles descritos para alterações no hormônio citocinina. Testes de sensibilidade à citocinina e de tempo de senescência realizados por nós, bem como dosagem desse hormônio feita por outras equipes, descartaram essa hipótese. Enxertias recíprocas evidenciaram que a promoção da ramificação caulinar por Rg1 não é um sinal translocável, mas parece ser uma característica intrínseca do tecido. Rg1 também promoveu uma maior formação de raízes adventícias em estacas ex vitro, sugerindo que ele afeta o processo de competência para formação de órgãos em geral, e não somente a indução específica de gemas caulinares. Duplos mutantes entre Rg1 e mutantes com alteração na sensibilidade/metabolismo hormonal (dgt, brt e pro), ausência de ramificações caulinares (ls) e senescência acelerada (l) foram obtidos e testados quanto ao padrão de ramificações e à capacidade de regeneração in vitro. Além desses parâmetros, a presença de Rg1 suprimiu o fenótipo de folhas pouco recortadas do mutante hipersensível a giberelina (pro) e recuperou o sistema radicular pouco desenvolvido do mutante com baixa sensibilidade à auxina (dgt). Rg1 também foi capaz de reverter a ausência de ramificações laterais de ls, sendo que a mutação Me, o qual representa uma superexpressão de um gene do tipo KNOX, não foi capaz. Esse resultado sugere que Rg1 não é um gene do tipo KNOX, embora esses genes sejam considerados os principais controladores da competência. Analisando diferentes tipos de explantes em experimentos independentes, contatou-se que, em geral, as mutações brt (baixa sensibilidade a citocinina), dgt e ls diminuíram a capacidade de regeneração de Rg1, enquanto l aumentou. Rg1 mostrou-se particularmente epistático à mutação pro, revertendo o fenótipo de baixa formação de gemas caulinares desse mutante. Surpreendentemente, Rg1 provou ser mais sensível à auxina em testes de alongamento de segmentos de hipocótilos, sendo capaz de reverter o fenótipo do mutante dgt nesse mesmo teste. Em conjunto, esses resultados confirmam o papel de Rg1 na fase de aquisição da competência e sugerem uma interação dos hormônios giberelina e auxina nesse processo. / The In vitro regeneration process is widely used in plant biotechnology. However, the mechanisms involved in the acquisition of competence for organ formation are hitherto unknown. In tomato (S. lycopersicum), the high capacity for in vitro regeneration is attributed to the Rg1 allele from S. peruvianum, which is present in the cv MsK (S. lycopersicum x S. peruvianum). The dwarfism genes of the cv Micro-Tom (MT) were transferred to MsK, and, after 8 generations of selfing (F8), the cv Micro-Msk was obtained. Here, after 6 generations of backcrosses (BC6Fn), we created the MT-Rg1, which has Rg1 isogenic to MT. Tests of in vitro regeneration showed that shoot formation in MT-Rg1 and Micro-MsK are equivalent, suggesting that the high capacity of regeneration of Micro-MsK is basically due to Rg1. Comparing MT-Rg1 with the control MT, we noticed a high frequency of 3 cotyledon formation, increased shoot branching and late senescence, which are absent in the parentals. These pleiotropic effects of Rg1 coincide with those described for plants with alterations in the hormone cytokinin. Tests of sensitivity for cytokinin and senescence behavior carried out by us, as well as dosage of that hormone made by other researches, discarded that hypothesis. Reciprocal grafting showed that the promotion of shoot branching by Rg1 is not a transmissible signal, but seems to be an inherent characteristic of the tissue. Rg1 also increases adventitious roots formation of ex vitro cuttings, suggesting that it affects the process of competence, which is common for shoots and roots, instead of the specific induction of shoots. Double mutants between Rg1 and mutants with alteration in the sensitivity/metabolism of plant hormones (dgt, brt and pro), as well as mutants with absence of shoot branching (ls) and accelerated senescence (l) were obtained and tested for the capacity of in vitro regeneration. The presence of Rg1 suppressed the phenotype of less dissected leaves of the mutant hypersensitive to gibberrellin (pro) and rescued the phenotype of poor developed root system of the mutant with low auxin sensitivity (dgt). Rg1 was also able to revert the absence of axillary shoot formation in ls, whereas the mutant Me, which represents an over expression of a KNOX gene was not. This result suggests that Rg1 is not a KNOX gene, although those genes are considered the main controllers of the competence. Analyzing different types of explants in independent experiments, it was verified that, in general, the mutations brt (low sensibility to cytokinin), dgt and ls decreased the regeneration capacity of Rg1, while l increased it. Rg1 was particularly epistatic to the pro mutation, reverting the phenotype of little shoot formation of that mutant. Surprisingly, Rg1 proved to be more sensitive to auxin in the hypocotyl segment elongation test, being able to revert the phenotype of the mutant dgt in the same test. Together, these results confirm the role of the Rg1 in the process of acquisition of the competence and suggest an interaction of the hormones gibberellin and auxin in this process.