RESUMO
Plants have evolved different mechanisms to increase their tolerance to aluminum (Al) toxicity and low pH in the soil. The Zn finger transcription factor SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) plays an essential role in the adaptation of plants to Al and low pH stresses. In this work, we isolated the ScSTOP1 gene from rye (Secale cereale L.), which is located on chromosome 3RS. The ectopic expression of ScSTOP1 complements the Arabidopsis stop1 mutation in terms of root growth inhibition due to Al and pH stress, as well as phosphate starvation tolerance, suggesting that rye ScSTOP1 is a functional ortholog of AtSTOP1. A putative STOP1 binding motif was identified in the promoter of a well-known STOP1 target from rye and Arabidopsis and was later corroborated by genomic DAP-seq analyses. Coexpression analyses verified that ScSTOP1 activated the promoter of ScALMT1. We have also identified a putative phosphorylatable serine in STOP1 that is phylogenetically conserved and critical for such activation. Our data indicated that ScSTOP1 also regulated Al and pH tolerance in rye.
Assuntos
Alumínio/toxicidade , Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Secale/metabolismo , Proteínas de Arabidopsis/genética , Cromossomos de Plantas/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Concentração de Íons de Hidrogênio , Mutação/genética , Transportadores de Ânions Orgânicos/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Secale/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Plant roots have the potential capacity to grow almost indefinitely if meristematic and lateral branching is sustained. In a genetic screen we identified an Arabidopsis mutant showing limited root growth (lrg1) due to defects in cell division and elongation in the root meristem. Positional cloning determined that lrg1 affects an alpha-1,2-mannosyltransferase gene, LEW3, involved in protein N-glycosylation. The lrg1 mutation causes a synonymous substitution that alters the correct splicing of the fourth intron in LEW3, causing a mix of wild-type and truncated protein. LRG1 RNA missplicing in roots and short root phenotypes in lrg1 are light-intensity dependent. This mutation disrupts a GC-base pair in a three-base-pair stem with a four-nucleotide loop, which seems to be necessary for correct LEW3 RNA splicing. We found that the lrg1 short root phenotype correlates with high levels of reactive oxygen species and low pH in the apoplast. Proteomic analyses of N-glycosylated proteins identified GLU23/PYK10 and PRX34 as N-glycosylation targets of LRG1 activity. The lrg1 mutation reduces the positive interaction between Arabidopsis and Serendipita indica. A prx34 mutant showed a significant reduction in root growth, which is additive to lrg1. Taken together our work highlights the important role of N-glycosylation in root growth and development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Basidiomycota/fisiologia , Manosiltransferases/metabolismo , Peroxidases/metabolismo , beta-Glucosidase/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Divisão Celular , Glicosilação , Concentração de Íons de Hidrogênio , Íntrons/genética , Manosiltransferases/genética , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/efeitos da radiação , Mutação , Peroxidases/genética , Fenótipo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/efeitos da radiação , Proteômica , Splicing de RNA , Espécies Reativas de Oxigênio/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/efeitos da radiação , beta-Glucosidase/genéticaRESUMO
Plants have developed several external and internal aluminium (Al) tolerance mechanisms. The external mechanism best characterised is the exudation of organic acids induced by Al. Rye (Secale cereale L.), one of the most Al-tolerant cereal crops, secretes both citrate and malate from its roots in response to Al. However, the role of malate dehydrogenase (MDH) genes in Al-induced stress has not been studied in rye. We have isolated the ScMDH1 and ScMDH2 genes, encoding two different mitochondrial MDH isozymes, in three Al-tolerant rye cultivars (Ailés, Imperial and Petkus) and one sensitive inbred rye line (Riodeva). These genes, which have seven exons and six introns, were located on the 1R (ScMDH1) and 3RL (ScMDH2) chromosomes. Exon 1 of ScMDH1 and exon 7 of ScMDH2 were the most variable among the different ryes. The hypothetical proteins encoded by these genes were classified as putative mitochondrial MDH isoforms. The phylogenetic relationships obtained using both cDNA and protein sequences indicated that the ScMDH1 and ScMDH2 proteins are orthologous to mitochondrial MDH1 and MDH2 proteins of different Poaceae species. The expression studies of the ScMDH1 and ScMDH2 genes indicate that it is more intense in roots than in leaves. Moreover, the amount of their corresponding mRNAs in roots from plants treated and not treated with Al was higher in the tolerant cultivar Petkus than in the sensitive inbred line Riodeva. In addition, ScMDH1 and ScMDH2 mRNA levels decreased in response to Al stress (repressive behaviour) in the roots of both the tolerant Petkus and the sensitive line Riodeva.
Assuntos
Alumínio/metabolismo , Malato Desidrogenase/genética , Secale/fisiologia , Sequência de Bases , Mapeamento Cromossômico , Peroxidação de Lipídeos , Malatos/metabolismo , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , RNA Mensageiro/genética , RNA de Plantas/genética , Secale/enzimologia , Secale/genética , Plântula/enzimologia , Plântula/genética , Plântula/fisiologia , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Among cereal crops, rye is one of the most tolerant species to aluminum. A candidate gene approach was used to determine the likely molecular identity of an Al tolerance locus (Alt4). Using PCR primers designed from a wheat aluminum tolerance gene encoding an aluminum-activated malate transporter (TaALMT1), a rye gene (ScALMT1) was amplified, cloned and sequenced. Subsequently, the ScALMT1 gene of rye was found to be located on 7RS by PCR amplification using the wheat-rye addition lines. SNP polymorphisms for this gene were detected among the parents of three F(2) populations that segregate for the Alt4 locus. A map of the rye chromosome 7R, including the Alt4 locus ScALMT1 and several molecular markers, was constructed showing a complete co-segregation between Alt4 and ScALMT1. Furthermore, expression experiments were carried out to clarify the function of this candidate gene. Briefly, the ScALMT1 gene was found to be primarily expressed in the root apex and upregulated when aluminum was present in the medium. Five-fold differences in the expression were found between the Al tolerant and the Al non-tolerant genotypes. Additionally, much higher expression was detected in the rye genotypes than the moderately tolerant "Chinese Spring" wheat cultivar. These results suggest that the Alt4 locus encodes an aluminum-activated organic acid transporter gene that could be utilized to increase Al tolerance in Al sensitive plant species. Finally, TaALMT1 homologous sequences were identified in different grasses and in the dicotyledonous plant Phaseolus vulgaris. Our data support the hypothesis of the existence of a common mechanism of Al tolerance encoded by a gene located in the homoeologous group four of cereals.
