RESUMO
Plant membrane transporters controlling metabolite distribution contribute key agronomic traits1-6. To eliminate anti-nutritional factors in edible parts of crops, the mutation of importers can block the accumulation of these factors in sink tissues7. However, this often results in a substantially altered distribution pattern within the plant8-12, whereas engineering of exporters may prevent such changes in distribution. In brassicaceous oilseed crops, anti-nutritional glucosinolate defence compounds are translocated to the seeds. However, the molecular targets for export engineering of glucosinolates remain unclear. Here we identify and characterize members of the USUALLY MULTIPLE AMINO ACIDS MOVE IN AND OUT TRANSPORTER (UMAMIT) family-UMAMIT29, UMAMIT30 and UMAMIT31-in Arabidopsis thaliana as glucosinolate exporters with a uniport mechanism. Loss-of-function umamit29 umamit30 umamit31 triple mutants have a very low level of seed glucosinolates, demonstrating a key role for these transporters in translocating glucosinolates into seeds. We propose a model in which the UMAMIT uniporters facilitate glucosinolate efflux from biosynthetic cells along the electrochemical gradient into the apoplast, where the high-affinity H+-coupled glucosinolate importers GLUCOSINOLATE TRANSPORTERS (GTRs) load them into the phloem for translocation to the seeds. Our findings validate the theory that two differently energized transporter types are required for cellular nutrient homeostasis13. The UMAMIT exporters are new molecular targets to improve nutritional value of seeds of brassicaceous oilseed crops without altering the distribution of the defence compounds in the whole plant.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Glucosinolatos , Proteínas de Membrana Transportadoras , Sementes , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Glucosinolatos/metabolismo , Homeostase , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Floema/metabolismo , Reprodutibilidade dos Testes , Sementes/metabolismoRESUMO
In plants, transport processes are important for the reallocation of defence compounds to protect tissues of high value, as demonstrated in the plant model Arabidopsis, in which the major defence compounds, glucosinolates, are translocated to seeds on maturation. The molecular basis for long-distance transport of glucosinolates and other defence compounds, however, remains unknown. Here we identify and characterize two members of the nitrate/peptide transporter family, GTR1 and GTR2, as high-affinity, proton-dependent glucosinolate-specific transporters. The gtr1 gtr2 double mutant did not accumulate glucosinolates in seeds and had more than tenfold over-accumulation in source tissues such as leaves and silique walls, indicating that both plasma membrane-localized transporters are essential for long-distance transport of glucosinolates. We propose that GTR1 and GTR2 control the loading of glucosinolates from the apoplasm into the phloem. Identification of the glucosinolate transporters has agricultural potential as a means to control allocation of defence compounds in a tissue-specific manner.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glucosinolatos/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Sementes/metabolismo , Animais , Arabidopsis/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/efeitos dos fármacos , Extratos Celulares/química , Evolução Molecular , Deleção de Genes , Biblioteca Gênica , Genes de Plantas/genética , Glucosinolatos/farmacologia , Proteínas de Transporte de Monossacarídeos/deficiência , Proteínas de Transporte de Monossacarídeos/genética , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Especificidade de Órgãos , Floema/metabolismo , Prótons , Xenopus laevisRESUMO
In Arabidopsis (Arabidopsis thaliana), a strategy to defend its leaves against herbivores is to accumulate glucosinolates along the midrib and at the margin. Although it is generally assumed that glucosinolates are synthesized along the vasculature in an Arabidopsis leaf, thereby suggesting that the margin accumulation is established through transport, little is known about these transport processes. Here, we show through leaf apoplastic fluid analysis and glucosinolate feeding experiments that two glucosinolate transporters, GTR1 and GTR2, essential for long-distance transport of glucosinolates in Arabidopsis, also play key roles in glucosinolate allocation within a mature leaf by effectively importing apoplastically localized glucosinolates into appropriate cells. Detection of glucosinolates in root xylem sap unambiguously shows that this transport route is involved in root-to-shoot glucosinolate allocation. Detailed leaf dissections show that in the absence of GTR1 and GTR2 transport activity, glucosinolates accumulate predominantly in leaf margins and leaf tips. Furthermore, we show that glucosinolates accumulate in the leaf abaxial epidermis in a GTR-independent manner. Based on our results, we propose a model for how glucosinolates accumulate in the leaf margin and epidermis, which includes symplasmic movement through plasmodesmata, coupled with the activity of putative vacuolar glucosinolate importers in these peripheral cell layers.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glucosinolatos/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Folhas de Planta/metabolismo , Proteínas de Arabidopsis/genética , Transporte Biológico , Técnicas de Inativação de Genes , Proteínas de Transporte de Monossacarídeos/genética , Epiderme Vegetal/metabolismo , Folhas de Planta/citologia , Raízes de Plantas/metabolismo , Plasmodesmos/metabolismo , Xilema/metabolismoRESUMO
As aphids are a pest on various crops worldwide, a better understanding of the interaction between aphids and plant host defenses is required. The green peach aphid (Myzus persicae) feeds on a variety of plant species, including the model plant Arabidopsis thaliana (Arabidopsis), in which glucosinolates function as a major part of the chemical defense. Several studies have shown that glucosinolates play a role in interactions between Arabidopsis and the green peach aphid. In this work, we used a recently identified Arabidopsis glucosinolate transporter mutant (gtr1gtr2 dKO), with altered glucosinolate content in the vasculature, to investigate the role of defense compound transport in aphid infestation. By monitoring aphid performance on caged leaves and analyzing glucosinolates in leaf tissue and phloem sap, as well as inside aphids, we examined if a change in spatial distribution of glucosinolates within a leaf influences aphid performance. Based on reduced glucosinolate content in the phloem sap of the transporter mutant, we hypothesized that aphids would perform better on gtr1gtr2 dKO leaves compared to WT. Unexpectedly, aphids performed poorly on gtr1gtr2 dKO leaves. Our data suggest that higher glucosinolate content in tissues surrounding the phloem of the double transporter mutant may play a role in reducing aphid performance on this genotype.
Assuntos
Afídeos/fisiologia , Aptidão Genética , Glucosinolatos/metabolismo , Herbivoria , Animais , Afídeos/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação , Floema/metabolismo , Folhas de Planta/metabolismoRESUMO
The nutritional value of Brassica seed meals is reduced by the presence of glucosinolates, which are toxic compounds involved in plant defense. Mutation of the genes encoding two glucosinolate transporters (GTRs) eliminated glucosinolates from Arabidopsis thaliana seeds, but translation of loss-of-function phenotypes into Brassica crops is challenging because Brassica is polyploid. We mutated one of seven and four of 12 GTR orthologs and reduced glucosinolate levels in seeds by 60-70% in two different Brassica species (Brassica rapa and Brassica juncea). Reduction in seed glucosinolates was stably inherited over multiple generations and maintained in field trials of two mutant populations at three locations. Successful translation of the gtr loss-of-function phenotype from model plant to two Brassica crops suggests that our transport engineering approach could be broadly applied to reduce seed glucosinolate content in other oilseed crops, such as Camelina sativa or Crambe abyssinica.
Assuntos
Brassica/genética , Melhoramento Genético/métodos , Glucosinolatos/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Óleos de Plantas/química , Sementes/genética , Glucosinolatos/análise , Mutação , Óleos de Plantas/análise , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/químicaRESUMO
The leaf apoplast comprises the extracellular continuum outside cell membranes. A broad range of processes take place in the apoplast, including intercellular signaling, metabolite transport, and plant-microbe interactions. To study these processes, it is essential to analyze the metabolite content in apoplastic fluids. Due to the fragile nature of leaf tissues, it is a challenge to obtain apoplastic fluids from leaves. Here, methods to collect apoplastic washing fluid and guttation fluid from Arabidopsis thaliana leaves are described.