A molecular rheostat adjusts auxin flux to promote root protophloem differentiation.

Auxin influences plant development through several distinct concentration-dependent effects 1 . In the Arabidopsis root tip, polar auxin transport by PIN-FORMED (PIN) proteins creates a local auxin accumulation that is required for the maintenance of the stem-cell niche2-4. Proximally, stem-cell daughter cells divide repeatedly before they eventually differentiate. This developmental gradient is accompanied by a gradual decrease in auxin levels as cells divide, and subsequently by a gradual increase as the cells differentiate5,6. However, the timing of differentiation is not uniform across cell files. For instance, developing protophloem sieve elements (PPSEs) differentiate as neighbouring cells still divide. Here we show that PPSE differentiation involves local steepening of the post-meristematic auxin gradient. BREVIS RADIX (BRX) and PROTEIN KINASE ASSOCIATED WITH BRX (PAX) are interacting plasma-membrane-associated, polarly localized proteins that co-localize with PIN proteins at the rootward end of developing PPSEs. Both brx and pax mutants display impaired PPSE differentiation. Similar to other AGC-family kinases, PAX activates PIN-mediated auxin efflux, whereas BRX strongly dampens this stimulation. Efficient BRX plasma-membrane localization depends on PAX, but auxin negatively regulates BRX plasma-membrane association and promotes PAX activity. Thus, our data support a model in which BRX and PAX are elements of a molecular rheostat that modulates auxin flux through developing PPSEs, thereby timing PPSE differentiation.

Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis.

In planta Agrobacterium-mediated transformation combined with a soil-based herbicide selection for transgenic plants was used to recover large numbers of transgenic Arabidopsis plants for functional genomic studies. A tissue-culture-free system for generating transgenic plants was achieved by infiltrating Arabidopsis plants with Agrobacterium tumefaciens harboring a binary T-DNA vector containing the phosphinothricin acetyltransferase gene from Streptomyces hygroscopicus, and by selecting transgenic Arabidopsis growing in soil by foliar application of the herbicide Finale (phosphinothricin). Analysis of herbicide-resistant plants indicated that all were transgenic and that the T-DNA transformation process occurred late during flower development, resulting in a preponderance of independently derived T-DNA insertions. T-DNA insertions were usually integrated in a concatenated, rearranged form, and using linkage analysis, we estimated that T1 plants carried between one and five T-DNA loci. Using pooling strategies, both DNA and seed pools were generated from about 38,000 Arabidopsis plants representing over 115,000 independent T-DNA insertions. We show the utility of these transgenic lines for identifying insertion mutations using gene sequence and PCR-based screening.

PLANT TRANSCRIPTION FACTOR STUDIES.

Major advances have been made in understanding the role of transcription factors in gene expression in yeast, Drosophila, and man. Transcription factor modification, synergistic events, protein-protein interactions, and chromatin structure have been successfully integrated into transcription factor studies in these organisms. While many putative transcription factors have been isolated from plants, most of them are only poorly characterized. This review summarizes examples where molecular biological techniques have been successfully employed to study plant transcription factors. The functional analysis of transcription factors is described as well as techniques for studying the interactions of transcription factors with other proteins and with DNA.