| Arabidopsis thaliana, a dicotyledonous model plant, plays an irreplaceable role in plant development research. Auxin is an important plant hormone, playing an essential role in the regulation of plant growth and development, especially in the root. IAA can be synthesized in the meristem and young primordia, in particular in the shoot apical meristem (SAM) and root apical meristem (RAM). Auxin is delivered to all parts of the plant by its unique mode of polar transport, and thus plays an important function. The polar transport of auxin from cell to cell is facilitated by auxin transport proteins, which are asymmetrically distributed on the plasma membrane (PM). These proteins mainly belong to PIN-FORMED (PIN) and P-GLYCOPROTEIN (PGP) families of auxin efflux transporters, and AUXIN1/LIKE AUX1(AUX/LAX) family of auxin influx transporters. Shoot-derived auxin transports through the central vascular tissue to the junction of shoot and root mediated via PIN1. In the root, PIN1mediates the transport of auxin to the root tip, assisted by PIN3, PIN4and PIN7. Whereas transport of auxin from the root cap region to the root elongation zone is mainly mediated by PIN2and facilitated by PIN3and PIN7, allowing the reflux of auxin to the central cylinder and the formation of ’auxin reflux’ loop. This phenomenon maintains QC function, the root auxin concentration gradient and tropism growth.Aimed at screening mutants associated with shoot-to-root auxin transport in Arabidopsis, and to explore the important role of long distance auxin transport in plants, especially during root development process, we generated Arabidopsis transgenic lines carrying CLV3::IAAH-DR5::GFP, which specifically induces auxin biosynthesis in the shoot apical meristem (SAM) in the presence of the auxin precursor IAM. The accumulation and distribution pattern of shoot-derived auxin in the root tip can be monitored in real-time by DR5::GFP expression. A mutant library was constructed by performing EMS mutagenesis in the background of CLV3::IAAH-DR5::GFP, and was screened to identify mutants that specifically affect auxin transport from shoot to root based on changes in root hair number, DR5::GFP fluorescence intensity, lateral root number, primary root length, hypocotyls length and root gravitropism. Mutants obtained from this study is expected to further improve our understanding on the modes of polar auxin transport, and on the role of polar auxin transport in plant growth and development. The main results are as follows:1) Through applying different concentrations of I AM to the CLV3::IAAH-DR5::GFP plants, we found that treatment with20nmol/L IAM for7days led to significant difference of root length between mock-treated and IAM-treated seedlings. We therefore used this concentration for the screening, and further define the time periods required for the screening of different phenotypic changes. We also examined the effects of different concentrations of exogenous auxin and auxin transport inhibitor NPA on the root and found that5nmol/L2,4-D,80nmol/L NAA and40nmol/L IAA could promote root elongation, and20nmol/L2,4-D,300nmol/L NAA and150nmol/L IAA inhibited the primary root length, and10μmol/L NPA treatment led to the loss of root gravitropism.2) EMS mutant library was successfully constructed and2124M2lines were harvested.3) Through screening and several rounds of rescreening of phenotypes related to root hair number, fluorescence intensity, lateral root number, primary root length, hypocotyl length and root geotropism, we identified64mutants which are genetically stable and likely involved in the transport of auxin from the shoot to the root. These mutants were further analyzed with high concentration of IAM (1000nmol/L), exogenous auxin or NPA, and were divided into13categories based on their phenotypic similarities and allelism test. We expect that these mutants will provide us with a better insight into the factors involved in shoot-to-root auxin transport. |
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