RESUMO
The AUX1 and PIN auxin influx and efflux facilitators are key regulators of root growth and development. For root gravitropism to occur, AUX1 and PIN2 must transport auxin via the lateral root cap to elongating epidermal cells. Genetic studies suggest that AXR4 functions in the same pathway as AUX1. Here we show that AXR4 is a previously unidentified accessory protein of the endoplasmic reticulum (ER) that regulates localization of AUX1 but not of PIN proteins. Loss of AXR4 resulted in abnormal accumulation of AUX1 in the ER of epidermal cells, indicating that the axr4 agravitropic phenotype is caused by defective AUX1 trafficking in the root epidermis.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Raízes de Plantas/metabolismo , Ácido 2,4-Diclorofenoxiacético/farmacologia , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/química , Transporte Biológico , Proteínas de Transporte/química , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Gravitropismo , Herbicidas/farmacologia , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Coifa/citologia , Coifa/metabolismo , Plantas Geneticamente Modificadas , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. Although expression of GFP in plants has been widely reported, research on the use of GFP in plant cell cultures for bioprocess applications has been limited. In this study, the suitability of GFP as a secretory reporter and a useful tool in plant cell bioprocess optimization was demonstrated. GFP was produced and secreted from suspension cells derived from tobacco that was transformed with a binary vector containing mgfp5-ER cDNA, a modified GFP for efficient sorting to the endoplasmic reticulum, under control of the CaMV 35S promoter. For cell line gfp-13, extracellular and intracellular GFP accumulated to 15.4 and 29.4 mg x 1(-1), respectively. Extracellular GFP accounted for 30.9% of the total extracellular protein. The molecular mass of extracellular GFP was nearly identical to that of a recombinant GFP standard, indicating cleavage of the signal sequence. Neomycin phosphotransferase II, a cytosolic selection marker, was found almost exclusively in cellular extracts with less than 2% in the extracellular medium. These results suggest that extracellular GFP is most likely the result of secretion rather than nonspecific leakage from cells. Furthermore, medium fluorescence intensity correlated nicely with extracellular GFP concentration supporting the use of GFP as a quantitative secretory reporter. During the batch cultivation, culture GFP fluorescence also followed closely with cell growth. A medium feeding strategy was then developed based on culture GFP fluorescence that resulted in improved biomass as well as GFP production in a fed-batch culture.