Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana.

Controlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene-expression system, derived from the filamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to beta-glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP) genes, we demonstrate that alcR-mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose-dependent, with negligible activity in the absence of the exogenous inducer for soil-grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil-grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana.

PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem.

The process of organ positioning has been addressed, using the pin-formed 1 (pin1) mutant as a tool. PIN1 is a transmembrane protein involved in auxin transport in Arabidopsis. Loss of function severely affects organ initiation, and pin1 mutants are characterised by an inflorescence meristem that does not initiate any flowers, resulting in the formation of a naked inflorescence stem. This phenotype, combined with the proposed role of PIN1 in hormone transport, makes the mutant an ideal tool to study organ formation and phyllotaxis, and here we present a detailed analysis of the molecular modifications at the shoot apex caused by the mutation. We show that meristem structure and function are not severely affected in the mutant. Major alterations, however, are observed at the periphery of the pin1 meristem, where organ initiation should occur. Although two very early markers of organ initiation, LEAFY and AINTEGUMENTA, are expressed at the periphery of the mutant meristem, the cells are not recruited into distinct primordia. Instead a ring-like domain expressing those primordium specific genes is observed around the meristem. This ring-like domain also expresses a boundary marker, CUP-SHAPED COTYLEDON 2, involved in organ separation, showing that the zone at the meristem periphery has a hybrid identity. This implies that PIN1 is not only involved in organ outgrowth, but that it is also necessary for organ separation and positioning. A model is presented in which PIN1 and the local distribution of auxin control phyllotaxis.

A chromosomal paracentric inversion associated with T-DNA integration in Arabidopsis.

T-DNA integration in the nuclear plant genome may lead to rearrangements of the plant target site. Here we present evidence for a chromosomal inversion of 26 cM bordered by two T-DNAs in direct orientation, which is linked to the mgoun2 mutation. The integration sites of the T-DNAs map at positions 80 and 106 of chromosome I and we show that each T-DNA is bordered by plant sequences from positions 80 and 106, respectively. Although the T-DNAs are physically distant, they are genetically closely linked. In addition, three markers located on the chromosome segment between the two T-DNA integration sites show no recombination with the mgo2 mutation. We show that the inversion cannot be a consequence of a recombination event between the two T-DNAs, but that the integration of the T-DNAs and the inversion were two temporally linked events. T-DNA integration mechanisms that could have led to this inversion are discussed.

Cellular parameters of the shoot apical meristem in Arabidopsis.

The shoot apical meristem (SAM) is a small group of dividing cells that generate all of the aerial parts of the plant. With the goal of providing a framework for the analysis of Arabidopsis meristems at the cellular level, we performed a detailed morphometric study of actively growing inflorescence apices of the Landsberg erecta and Wassilewskija ecotypes. For this purpose, cell size, spatial distribution of mitotic cells, and the mitotic index were determined in a series of optical sections made with a confocal laser scanning microscope. The results allowed us to identify zones within the inflorescence SAM with different cell proliferation rates. In particular, we were able to define a central area that was four to six cells wide and had a low mitotic index. We used this technique to compare the meristem of the wild type with the enlarged meristems of two mutants, clavata3-1 (clv3-1) and mgoun2 (mgo2). One of the proposed functions of the CLV genes is to limit cell division rates in the center of the meristem. Our data allowed us to reject this hypothesis, because the mitotic index was reduced in the inflorescence meristem of the clv3-1 mutant. We also observed a large zone of slowly dividing cells in meristems of clv3-1 seedlings. This zone was not detectable in the wild type. These results suggest that the central area is increased in size in the mutant meristem, which is in line with the hypothesis that the CLV3 gene is necessary for the transition of cells from the central to the peripheral zone. Genetic and microscopic analyses suggest that mgo2 is impaired in the production of primordia, and we previously proposed that the increased size of the mgo2 meristem could be due to an accumulation of cells at the periphery. Our morphometric analysis showed that mgo2 meristems, in contrast to those of clv3-1, have an enlarged periphery with high cell proliferation rates. This confirms that clv3-1 and mgo2 lead to meristem overgrowth by affecting different aspects of meristem function.

MGOUN1 and MGOUN2: two genes required for primordium initiation at the shoot apical and floral meristems in Arabidopsis thaliana.

We report two new recessive mutations in Arabidopsis, mgoun1 and mgoun2 which cause a reduction in the number of leaves and floral organs, larger meristems and fasciation of the inflorescence stem. Although meristem structure is affected in the mutants, we provide evidence that its overall organisation is normal, as shown by the expression patterns of two meristem markers. Microscopical analyses suggest that both mutations affect organ primordia production. mgo1 strongly inhibits leaf production in a weak allele of shoot meristemless, stm-2. In addition, mgo1 and 2 severely reduce the ability of the fasciata1 and 2 mutants to initiate organs, although meristem formation per se was not inhibited. The strong allele, stm-5, is epistatic to mgo1, showing that the presence of meristematic cells is essential for MGO1 function. These results suggest a role for the MGO genes in primordia initiation although a more general role in meristem function can not be excluded. We describe a form of fasciation which is radically different from that described for clavata, which is thought to have an increased size of the meristem centre. Instead of one enlarged central meristem mgo1 and 2 show a continuous fragmentation of the shoot apex into multiple meristems, which leads to the formation of many extra branches. The phenotype of mgo1 clv3 and mgo2 clv3 double mutants suggest that the MGO and CLV genes are involved in different events. In conclusion, our results reveal two new components of the regulatory network controlling meristem function and primordia formation. A model for MGO genes is discussed.