Faculty Bibliography

Homeodomain proteins are key regulators of patterning during the development of animal and plant body plans. Knotted1-like TALE homeodomain proteins have been found to play important roles in the development of the Arabidopsis shoot apical meristem and are part of a complex regulatory network of protein interactions. We have investigated the possible role of the knotted1-like genes KNAT1, KNAT3, KNAT4, and KNAT5 in Arabidopsis root development. Root growth is indeterminate, and the organ shows distinct zones of cell proliferation, elongation and differentiation along its longitudinal axis. Here we show that KNAT1, KNAT3, KNAT4 and KNAT5 show cell type specific expression patterns in the Arabidopsis root. Moreover, they are expressed in different spatially restricted patterns along the longitudinal root axis and in lateral root primordia. Hormones play an important role in maintenance of root growth, and we have studied their effect on KNAT gene expression. We show that KNAT3 expression is repressed by moderate levels of cytokinin. In addition, we show that the subcellular localization of KNAT3 and KNAT4 is regulated, indicating post-translational control of the activities of these transcription factors. The regulated expression of KNAT1, KNAT3, KNAT4 and KNAT5 within the Arabidopsis root suggests a role for these genes in root development. Our data provide the first systematic survey of KNAT gene expression in the Arabidopsis root.

Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.

Lateral root development occurs throughout the life of the plant and is responsible for the plasticity of the root system. In Arabidopsis thaliana, lateral root founder cells originate from pericycle cells adjacent to xylem poles. In order to study the mechanisms of lateral root development, a population of Arabidopsis GAL4-GFP enhancer trap lines were screened and two lines were isolated with GAL4 expression in root xylem-pole pericycle cells (J0121), i.e. in cells competent to become lateral root founder cells, and in young lateral root primordia (J0192). These two enhancer trap lines are very useful tools with which to study the molecular and cellular bases of lateral root development using targeted gene expression. These lines were used for genetic ablation experiments by targeting the expression of a toxin-encoding gene. Moreover, the molecular bases of the enhancer trap expression pattern were characterized. These results suggest that the lateral-root-specific GAL4 expression pattern in J0192 is due to a strong enhancer in the promoter of the LOB-domain protein gene LBD16.

Polycomb (PcG) group proteins form modular complexes, which maintain repressed transcriptional states of target genes across cell divisions. As PcG complexes provide a memory of cell fate, such proteins might control temporal aspects of development. Loss-of-function of any of the FERTILIZATION INDEPENDENT SEED (FIS) PcG genes perturbs endosperm development. In this report we provide a detailed analysis of the phenotype of fis endosperm development using molecular and cellular markers. Wild type (WT) endosperm development undergoes a series of four major developmental phases timed by successive synchronous nuclei division. In fis endosperm the transition from phase 1, marked by a synchronous mode of nuclei divisions to phase 2, corresponding to the establishment of three mitotic domains, is absent. Accordingly, the expression of seven markers of phase 1 and phase 2 is temporally perturbed. In spite of such changes, specific sequences of developmental events still take place as in the WT. Overall, fis mutations are heterochronic mutations that cause a temporal deregulation in the ontogenic sequence of endosperm development.

We have generated a novel genetic system to visualize cell lineages in living tissues at high resolution. Heat shock was used to trigger the excision of a specific transposon and activation of a fluorescent marker gene. A histone-YFP marker was used to allow identification of cell lineages and easy counting of cells. Constitutive expression of a green fluorescent membrane protein was used to provide a precise outline of all surrounding cells. Marked lineages can be induced from specific cells within the organism by targeted laser irradiation, and the fate of the marked cells can be followed non-invasively. We have used the system to map cell lineages originating from the initials of primary and lateral roots in Arabidopsis. The lineage marking technique enabled us to measure the differential contribution of primary root pericycle cell files to developing lateral root primordia. The majority of cells in an emerging lateral root primordium derive from the central file of pericycle founder cells while off-centre founder cells contribute only a minor proliferation of tissue near the base of the root. The system shows great promise for the detailed study of cell division during morphogenesis.

We used enhancer trapping with the GAL4 transcriptional activator from yeast to obtain spatial control of transgene expression in all organs of the model monocotyledonous species rice (Oryza sativa L. cv. Nipponbare). Our T-DNA enhancer trapping cassette consisted of two principle components: (1) the minimal promoter-equipped gal4 gene placed adjacent to the right border, and (2) the green fluorescent protein gene (gfp) fused to the upstream activation sequence element (UAS) to which GAL4 binds and activates expression, so that gfp expression corresponds to gal4 expression. Agrobacterium-mediated integration of the cassette into the rice genome often brings the gal4 gene under transcriptional control of local genomic enhancers and promoters, resulting in gal4/gfp expression patterns ranging in specificity from single-cell types to constitutive expression. We produced more than 13 000 enhancer trap lines with this cassette and screened T(0) adult plants (1982 lines), T(1) seed (2684 lines) and T(1) seedlings (2667 lines) for gfp expression. Approximately 30% of the lines produced GFP, and we identified lines with gfp expression in specific cell types of all major organs of the rice plant. Subsequently, using the GUS reporter gene (uidA), we demonstrated that UAS:geneX constructs can be transactivated in specific cell types where gal4 and gfp are expressed, thus providing an excellent system for the manipulation of gene expression and physiological function in specific cell types of rice.

cg12 is an early actinorhizal nodulin gene from Casuarina glauca encoding a subtilisin-like serine protease. Using transgenic Casuarinaceae plants carrying cg12-gus and cg12-gfp fusions, we have studied the expression pattern conferred by the cg12 promoter region after inoculation with Frankia. cg12 was found to be expressed in root hairs and in root and nodule cortical cells containing Frankia infection threads. cg12 expression was also monitored after inoculation with ineffective Frankia strains, during mycorrhizae formation, and after diverse hormonal treatments. None of these treatments was able to induce its expression, therefore suggesting that cg12 expression is linked to plant cell infection by Frankia strains. Possible roles of cg12 in actinorhizal symbiosis are discussed.

Trans-splicing ribozymes are RNA-based catalysts capable of splicing RNA sequences from one transcript specifically into a separate target transcript. In doing so, a chimeric mRNA can be produced, and new gene activities triggered in living cells dependent on the presence of the target mRNA. Based on this ability of trans-splicing ribozymes to deliver new gene activities, a simple and versatile plating assay was developed in Saccharomyces cerevisiae for assessing and optimizing constructs in vivo. Trans-splicing ribozymes were used to splice sequences encoding a GAL4-derived transcription activator into a target transcript from a prevalent viral pathogen. The transcription activator translated from this new mRNA in turn triggered the expression of genes under the regulatory control of GAL4 upstream-activating sequences. Two of the activated genes complemented metabolic deficiencies in the host strain, and allowed growth on selective media. A simple genetic assay based on phenotypic conversion from auxotrophy to prototrophy was established to select efficient and specific trans-splicing ribozymes from a ribozyme library. This simple assay may prove valuable for selecting optimal target sites for therapeutic agents such as ribozymes, antisense RNA and antisense oligodeoxyribonucleotides, and for optimizing the design of the therapeutic agents themselves, in higher eukaryotes.