The Spirodela polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle.

The subfamily of the Lemnoideae belongs to a different order than other monocotyledonous species that have been sequenced and comprises aquatic plants that grow rapidly on the water surface. Here we select Spirodela polyrhiza for whole-genome sequencing. We show that Spirodela has a genome with no signs of recent retrotranspositions but signatures of two ancient whole-genome duplications, possibly 95 million years ago (mya), older than those in Arabidopsis and rice. Its genome has only 19,623 predicted protein-coding genes, which is 28% less than the dicotyledonous Arabidopsis thaliana and 50% less than monocotyledonous rice. We propose that at least in part, the neotenous reduction of these aquatic plants is based on readjusted copy numbers of promoters and repressors of the juvenile-to-adult transition. The Spirodela genome, along with its unique biology and physiology, will stimulate new insights into environmental adaptation, ecology, evolution and plant development, and will be instrumental for future bioenergy applications.

KANADI regulates organ polarity in Arabidopsis.

Leaves and floral organs are polarized along their adaxial-abaxial (dorsal-ventral) axis. In Arabidopsis, this difference is particularly obvious in the first two rosette leaves, which possess trichomes (leaf hairs) on their adaxial surface but not their abaxial surface. Mutant alleles of KANADI (KAN) were identified in a screen for mutants that produce abaxial trichomes on these first two leaves. kan mutations were originally identified as enhancers of the mutant floral phenotype of crabs claw (crc), a gene that specifies abaxial identity in carpels. Here we show that KAN is required for abaxial identity in both leaves and carpels, and encodes a nuclear-localized protein in the GARP family of putative transcription factors. The expression pattern of KAN messenger RNA and the effect of ectopically expressing KAN under the regulation of the cauliflower mosaic virus (CAMV) 35S promoter indicate that KAN may also specify peripheral identity in the developing embryo.

The specification of leaf identity during shoot development.

A single plant produces several different types of leaves or leaf-like organs during its life span. This phenomenon, which is termed heteroblasty, is an invariant feature of shoot development but is also regulated by environmental factors that affect the physiology of the plant. Invariant patterns of heteroblastic development reflect global changes in the developmental status of the shoot, such as the progression from embryogenesis through juvenile and adult phases of vegetative development, culminating in the production of reproductive structures. Genes that regulate these phase-specific aspects of leaf identity have been identified by mutational analysis in both maize and Arabidopsis. These mutations have revealed that leaf production is regulated independently of leaf identity, implying that the identity of a leaf at a particular position on the shoot may depend on when the leaf was initiated in relation to a temporal program of shoot development.

Loss-of-function mutations in the maize homeobox gene, knotted1, are defective in shoot meristem maintenance.

The product of the maize homeobox gene, knotted1 (kn1), localizes to the nuclei of cells in shoot meristems, but is absent from portions of the meristem where leaf primordia or floral organs initiate. Recessive mutant alleles of kn1 were obtained by screening for loss of the dominant leaf phenotype in maize. Mutant kn1 alleles carrying nonsense, splicing and frame shift mutations cause severe inflorescence and floral defects. Mutant tassels produce fewer branches and spikelets. Ears are often absent, and when present, are small with few spikelets. In addition, extra carpels form in female florets and ovule tissue proliferates abnormally. Less frequently, extra leaves form in the axils of vegetative leaves. These mutations reveal a role for kn1 in meristem maintenance, particularly as it affects branching and lateral organ formation.

Nucleotide sequence and analysis of the plant-inducible locus pinF from Agrobacterium tumefaciens.

Several loci on the tumor-inducing plasmid from Agrobacterium tumefaciens were transcriptionally activated in the presence of wounded plant tissue or extracts. The inducible virulence loci were required for efficient tumor formation. In contrast, the plant-inducible locus pinF was not observed to be absolutely essential for virulence. Mutants in pinF showed an attenuated virulence on a variety of dicotyledonous hosts, and this attenuation became more pronounced with decreasing numbers of bacterial cells in the inoculum. The DNA sequence of a 5.5-kilobase region which included the pinF locus from the octopine-type tumor-inducing plasmid A6 was determined. Four open reading frames consistent with the observed transcription of pinF were observed. Two of the open reading frames, pinF1 and pinF2, coded for polypeptides with relative molecular weights of 47,519 (pinF1) and 46,740 (pinF2). A comparison of the amino acid sequences of pinF1 and pinF2 indicated that they were similar to each other and to known polypeptide sequences for cytochrome P-450 enzymes.

A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane.

The VirA protein is one of two proteins required for transcriptional activation of Agrobacterium tumefaciens virulence genes in response to phenolic compounds released by plants during infection. We describe two experimental approaches which indicate that this protein has a transmembrane topology. First, spheroplasts of Escherichia coli or wild-type A. tumefaciens expressing the VirA protein were treated with proteinase K to digest periplasmic proteins, and the remaining proteins were immunologically stained on Western blots (immunoblots) by using anti-VirA antibody. Second, transposon TnphoA was used to generate translational fusions between virA and phoA, the latter of which is the structural gene for alkaline phosphatase. Both techniques indicated that VirA spans the cytoplasmic membrane, with approximately 275 amino acids near the amino terminus being localized in the periplasmic space and the rest of the protein being localized in the cytoplasm. We also show that overexpression of VirA in E. coli is deleterious to cell growth and that this phenomenon depends on the synthesis of either the second hydrophobic core or some nearby portion of the VirA protein.

Transcriptional regulation of the virA and virG genes of Agrobacterium tumefaciens.

We have used transcriptional and translational fusions between various vir gene promoters and the lacZ gene to study the regulation of vir genes. Like other vir promoters, the virA promoter was induced by acetosyringone in a virA virG-dependent fashion. In addition to being induced by acetosyringone, the virG promoter was partially induced by acidic growth conditions and by starvation for inorganic phosphate. These two conditions appeared to act synergistically. The response to low pH and to phosphate starvation occurred in the absence of the Ti plasmid and must therefore have been mediated by chromosomal genes. Two transposon-generated mutations were obtained which attenuated induction by low pH. One of these transposons was cloned along with flanking DNA; the flanking DNA was sequenced (858 base pairs total), and the predicted amino acid sequence showed homology with a family of proteins including the Rhizobium leguminosarum nodI gene, many of whose members bind ATP and have been implicated in active transport systems. These results are discussed as possible explanations for previous observations that the induction of the octopine vir regulon (i) occurs only in acidic media and (ii) shows hyperbolic kinetics after a long lag phase.