Peter Huijser

AtPIN2 defines a locus of Arabidopsis for root gravitropism control

The molecular mechanisms underlying gravity perception and signal transduction which control asymmetric plant growth responses are as yet unknown, but are likely to depend on the directional flux of the plant hormone auxin. We have isolated an Arabidopsis mutant of the AtPIN2 gene using transposon mutagenesis. Roots of the Atpin2::En701 null‐mutant were agravitropic and showed altered auxin sensitivity, a phenotype characteristic of the agravitropic wav6‐52 mutant. The AtPIN2 gene was mapped to chromosome 5 (115.3 cM) corresponding to the WAV6 locus and subsequent genetic analysis indicated that wav6‐52 and Atpin2::En701 were allelic. The AtPIN2 gene consists of nine exons defining an open reading frame of 1944 bp which encodes a 69 kDa protein with 10 putative transmembrane domains interrupted by a central hydrophilic loop. The topology of AtPIN2p was found to be similar to members of the major facilitator superfamily of transport proteins. We have shown that the AtPIN2 gene was expressed in root tips. The AtPIN2 protein was localized in membranes of root cortical and epidermal cells in the meristematic and elongation zones revealing a polar localization. These results suggest that AtPIN2 plays an important role in control of gravitropism regulating the redistribution of auxin from the stele towards the elongation zone of roots.

Genetic Control of Flower Development by Homeotic Genes in Antirrhinum majus

Homeotic mutants have been useful for the study of animal development. Such mutants are also known in plants. The isolation and molecular analysis of several homeotic genes in Antirrhinum majus provide insights into the underlying molecular regulatory mechanisms of flower development. A model is presented of how the characteristic sequential pattern of developing organs, comprising the flower, is established in the process of morphogenesis.

Deficiens, a homeotic gene involved in the control of flower morphogenesis in Antirrhinum majus: the protein shows homology to transcription factors

Deficiens (defA+) is a homeotic gene involved in the genetic control of Antirrhinum majus flower development. Mutation of this gene (defA‐1) causes homeotic transformation of petals into sepals and of stamina into carpels in flowers displaying the ‘globifera’ phenotype, as shown by cross sections and scanning electronmicroscopy of developing flowers. A cDNA derived from the wild type defA+ gene has been cloned by differential screening of a subtracted ‘flower specific’ cDNA library. The identity of this cDNA with the defA+ gene product has been confirmed by utilizing the somatic and germinal instability of defA‐1 mutants. According to Northern blot analyses the defA+ gene is expressed in flowers but not in leaves, and its expression is nearly constant during all stages of flower development. The 1.1 kb long mRNA has a 681 bp long open reading frame that can code for a putative protein of 227 amino acids (mol. wt 26.2 kd). At its N‐terminus the DEF A protein reveals homology to a conserved domain of the regulatory proteins SRF (activating c‐fos) in mammals and GRM/PRTF (regulating mating type) in yeast. We discuss the structure and the possible function of the DEF A protein in the control of floral organogenesis.

Relationship between nucleosome positioning and DNA methylation

Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana using massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified 10-base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results indicate that nucleosome positioning influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA, indicating that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron–exon and exon–intron boundaries. RNA polymerase II (Pol II) was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is also enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition.

Bracteomania, an inflorescence anomaly, is caused by the loss of function of the MADS-box gene squamosa in Antirrhinum majus.

Anomalous flowering of the Antirrhinum majus mutant squamosa (squa) is characterized by excessive formation of bracts and the production of relatively few and often malformed or incomplete flowers. To study the function of squamosa in the commitment of an inflorescence lateral meristem to floral development, the gene was cloned and its genomic structure, a well as that of four mutant alleles, was determined. SQUA is a member of a family of transcription factors which contain the MADS‐box, a conserved DNA binding domain. In addition, we analysed the temporal and spatial expression pattern of the squa gene. Low transcriptional activity of squa is detectable in bracts and in the leaves immediately below the inflorescence. High squa transcript levels are seen in the inflorescence lateral meristems as soon as they are formed in the axils of bracts. Squa transcriptional activity persists through later stages of floral morphogenesis, with the exception of stamen differentiation. Although necessary for shaping a normal racemose inflorescence, the squa function is not absolutely essential for flower development. We discuss the function of the gene during flowering, its likely functional redundancy and its possible interaction with other genes participating in the genetic control of flower formation in Antirrhinum.

Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development.

We have determined the structure of the floral homeotic deficiens (defA) gene whose mutants display sepaloid petals and carpelloid stamens, and have analysed its spatial and temporal expression pattern. In addition, several mutant alleles (morphoalleles) were studied. The results of these analyses define three functional domains of the DEF A protein and identify in the deficiens promoter a possible cis‐acting binding site for a transcription factor which specifically upregulates expression of deficiens in petals and stamens. In vitro DNA binding studies show that DEF A binds to specific DNA motifs as a heterodimer, together with the protein product of the floral homeotic globosa gene, thus demonstrating that the protein encoded by deficiens is a DNA binding protein. Furthermore, Northern analysis of a temperature sensitive allele at permissive and non‐permissive temperatures provides evidence for autoregulation of the persistent expression of deficiens throughout flower development. A possible mechanism of autoregulation is discussed.

The control of developmental phase transitions in plants

Plant development progresses through distinct phases: vegetative growth, followed by a reproductive phase and eventually seed set and senescence. The transitions between these phases are controlled by distinct genetic circuits that integrate endogenous and environmental cues. In recent years, however, it has become evident that the genetic networks that underlie these phase transitions share some common factors. Here, we review recent advances in the field of plant phase transitions, highlighting the role of two microRNAs – miR156 and miR172 – and their respective targets during these transitions. In addition, we discuss the evolutionary conservation of the functions of these miRNAs in regulating the control of plant developmental phase transitions.

The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis.

Throughout development the Arabidopsis shoot apical meristem successively undergoes several major phase transitions such as the juvenile-to-adult and floral transitions until, finally, it will produce flowers instead of leaves and shoots. Members of the Arabidopsis SBP-box gene family of transcription factors have been implicated in promoting the floral transition in dependence of miR156 and, accordingly, transgenics constitutively over-expressing this microRNA are delayed in flowering. To elaborate their roles in Arabidopsis shoot development, we analysed two of the 11 miR156 regulated Arabidopsis SBP-box genes, i.e. the likely paralogous genes SPL9 and SPL15. Single and double mutant phenotype analysis showed these genes to act redundantly in controlling the juvenile-to-adult phase transition. In addition, their loss-of-function results in a shortened plastochron during vegetative growth, altered inflorescence architecture and enhanced branching. In these aspects, the double mutant partly phenocopies constitutive MIR156b over-expressing transgenic plants and thus a major contribution to the phenotype of these transgenics as a result of the repression of SPL9 and SPL15 is strongly suggested.

Molecular characterisation of the Arabidopsis SBP-box genes

The Arabidopsis thaliana SPL gene family represents a group of structurally diverse genes encoding putative transcription factors found apparently only in plants. The distinguishing characteristic of the SPL gene family is the SBP-box encoding a conserved protein domain of 76 amino acids in length, the SBP-domain, which is responsible for the interaction with DNA. We present here characterisation of 12 members of the SPL gene family. These genes show highly diverse genomic organisations and are found scattered over the Arabidopsis genome. Some SPL genes are constitutively expressed, while transcriptional activity of others is under developmental control. Based on phylogenetic reconstruction, gene structure and expression patterns, they can be divided into subfamilies. In addition to the Arabidopsis SPL genes, we isolated and determined the sequences of three SBP-box genes from Antirrhinum majus and seven from Zea mays.

A new family of DNA binding proteins includes putative transcriptional regulators of the Antirrhinum majus floral meristem identity gene SQUAMOSA

Several sites of nuclear protein interaction within the promoter region of theAntirrhinum majus floral meristem identity geneSQUAMOSA were detected using an electrophoretic mobility shift assay. One of these sites displayed a particularly clear interaction with nuclear protein extracted from inflorescences but not with nuclear protein extracted from young, non-flowering plants. This site could thus represent a binding motif for a transcriptional activator. A South-western screen of an inflorescence cDNA expression library resulted in the isolation of several cDNAs representing two different genes namedSBP1 andSBP2 (forSQUAMOSA-pROMOTERBINDINGPROTEIN gene 1 and 2). Both genes encode highly similar protein domains which were found to be necessary and sufficient for binding DNA in a sequence-specific manner. This DNA-binding domain showed no similarity to known proteins in the databases. However, it is characteristic for a small family of gene products inA. majus and other plant species. Expression ofSBP1 and2 is developmentally regulated and their transcriptional activation precedes that ofSQUAMOSA. The data presented support the idea that members of the newly identifiedSBP gene family function as transcription factors involved in the control of early flower development.