A phylogenetically conserved APETALA2/ETHYLENE RESPONSE FACTOR, ERF12, regulates Arabidopsis floral development.

KEY MESSAGE: Arabidopsis ETHYLENE RESPONSE FACTOR12 (ERF12), the rice MULTIFLORET SPIKELET1 orthologue pleiotropically affects meristem identity, floral phyllotaxy and organ initiation and is conserved among angiosperms. Reproductive development necessitates the coordinated regulation of meristem identity and maturation and lateral organ initiation via positive and negative regulators and network integrators. We have identified ETHYLENE RESPONSE FACTOR12 (ERF12) as the Arabidopsis orthologue of MULTIFLORET SPIKELET1 (MFS1) in rice. Loss of ERF12 function pleiotropically affects reproductive development, including defective floral phyllotaxy and increased floral organ merosity, especially supernumerary sepals, at incomplete penetrance in the first-formed flowers. Wildtype floral organ number in early formed flowers is labile, demonstrating that floral meristem maturation involves the stabilisation of positional information for organogenesis, as well as appropriate identity. A subset of erf12 phenotypes partly defines a narrow developmental time window, suggesting that ERF12 functions heterochronically to fine-tune stochastic variation in wild type floral number and similar to MFS1, promotes meristem identity. ERF12 expression encircles incipient floral primordia in the inflorescence meristem periphery and is strong throughout the floral meristem and intersepal regions. ERF12 is a putative transcriptional repressor and genetically opposes the function of its relatives DORNRÖSCHEN, DORNRÖSCHEN-LIKE and PUCHI and converges with the APETALA2 pathway. Phylogenetic analysis suggests that ERF12 is conserved among all eudicots and appeared in angiosperm evolution concomitant with the generation of floral diversity.

Alternative splicing of two leading exons partitions promoter activity between the coding regions of the maize homeobox gene Zmhox1a and Trap (transposon-associated protein).

Elucidation of the exon/intron structure of the maize Zmhox1a homeobox gene revealed two small introns in the homeodomain. Both intron positions are conserved in animal counterparts encoded in the metazoan homeobox gene clusters and thus may indicate a common ancestor. The transcription start of the Zmhox1a gene has been localized far from the protein-coding region. Two distal untranslated leading exons are alternatively spliced to either the Zmhox1a coding exons or an unrelated open reading frame comprising two exons located internally of the large second Zmhox1a intron. Due to significant homology to the C-terminus of the Mutator transposase this alternative gene product was named Trap (transposon-associated protein). Splice site selection may involve two sequence elements conserved at the splice acceptor sites in front of the Zmhox1a and Trap protein-coding regions. The translation of a mRNA species devoid of exon 3 which encodes the Zmhox1a transcription start codon may give rise to an N-terminal deletion polypeptide, deltaZmhox1a. Ectopic expression experiments in transgenic tobacco indicate a putative function distinct from the full-length Zmhox1a protein.

The IBP genes of maize are expressed in non-meristematic, elongating cells of the seedling and in abortive floral organs.

The transcription start site of the maize Shrunken-1 (Sh-1) gene is sufficient for transcriptional initiation in the absence of other promoter elements and is recognized in vitro by the Initiator Binding Protein (IBP). We describe here in situ hybridization experiments performed on various maize tissues to quantify IBP transcription at the cellular level. IBP transcripts are found in the endosperm and in differentiating, enlarging cells of the shoot and the root of the maize seedling. This expression pattern overlaps with that of the Sh-1 gene and is therefore compatible with the hypothesis that the Sh-1 transcription start site is a target for IBP. In the developing spikelets of male and female inflorescences IBP transcript levels are very high in those organs that are later aborted when flowers become unisexual. Overexpression of the maize IBP1 gene product in transgenic tobacco causes a reduction in internodal elongation and effects gibberellin hormonal balance. The cellular expression pattern described here establishes IBP transcripts as an interesting molecular marker for enlarging, and presumably differentiating, cells released from the root or shoot apex.

Ectopic expression of the maize homeobox genes ZmHox1a or ZmHox1b causes pleiotropic alterations in the vegetative and floral development of transgenic tobacco.

The ZmHox1a and ZmHox1b (for Zea mays homeobox) genes map on chromosomes 8 and 6, respectively. Both homeobox genes encode proteins that show 91% similarity and are transcribed simultaneously in meristematic and proliferating cells of the maize plant. To gain insight into the biological function of these genes, both open reading frames were expressed in tobacco, under the control of the cauliflower mosaic virus 35S promoter. The resulting transgenic ZmHox1a or ZmHox1b plants showed identical phenotypic alterations that fall into three classes: size reduction, formation of adventitious shoots, and homeotic floral transformations. Approximately 30% of the ZmHox1-expressing plants grew to only one-third of the wild-type size, and most axillary buds gave rise to lateral shoots. Flower abnormalities included formation of petaloid stamens and development of secondary flowers within the primary gynoecium. Therefore, the ectopic expression of the maize ZmHox1 homeobox gene products affects the vegetative as well as the reproductive phase of tobacco plants. All phenotypic alterations were transmitted to the next generation.

A novel DNA-binding domain in the Shrunken initiator-binding protein (IBP1).

South-western screening of lambda gt11 expression library with a fragment of the Shrunken promoter containing the initiator element resulted in cloning of a novel maize gene. The encoded initiator-binding protein (IBP1) interacts at the transcription start site of the Shrunken promoter. Analysis of the 680 amino acid (aa) long polypeptide revealed a novel bipartite DNA-binding domain at the carboxyl terminus. In its amino-terminal part, it is weakly related to Myb R-repeats but the following basic region is also essential for DNA binding. A region of similarity to the conserved 2.1 and 2.2 motifs in bacterial sigma-factors is located close to the IBP1 amino terminus. Two putative nuclear localization signals are compatible with the presence of antigenically related polypeptides in nuclear protein extracts. The IBP1 gene was mapped to the long arm of chromosome 9 (9L095); a second highly related gene IBP2 is located on the short arm of chromosome 1 (1S014). Both genes encode proteins sharing 93% similarity and are transcribed with similar activity in different plant organs. A small 82 nucleotide intron in the IBP2 transcript is found unspliced to a variable degree in different tissues. Translation of this incompletely processed transcript would result in a truncated amino-terminal polypeptide lacking the DNA-binding domain.

Zmhox1a, the product of a novel maize homeobox gene, interacts with the Shrunken 26 bp feedback control element.

A new maize homeobox gene was isolated by screening a lambda gt11 expression library with the 26 bp Shrunken feedback control element. Zmhox1a (Zea mays homeobox) is an unidentified maize gene mapping to the long arm of chromosome 8. It is a member of a new class of maize homeobox genes only distantly related to the Knotted class. The 3.1 kb Zmhox1a transcript can be detected in different maize tissues and encodes a polypeptide of 719 amino acids. Western blotting experiments detect the native 112 or 115 kDa protein in nuclear protein extracts, the nuclear localization being compatible with a function in transcriptional control. No Zmhox1a protein is detected in maize roots despite the presence of the Zmhox1a transcript; this may indicate a post-transcriptional control mechanism. A highly acidic central region of the Zmhox1a polypeptide implies a transcriptional activator function. The carboxy-terminal part of the maize homeodomain protein is related to the human Oct2 transcription factor, but homology to the POU specific domain is restricted to the POU-B subdomain. It was confirmed by DNase I footprinting experiments that DNA binding of the Zmhox1a homeodomain was at three sites flanking the TATA-box of the Shrunken promoter.

The combination of a novel stimulatory element in the first exon of the maize Shrunken-1 gene with the following intron 1 enhances reporter gene expression up to 1000-fold.

Both exon 1 and intron 1 of the maize Shrunken-1 (Sh1) gene individually stimulate expression of reporter genes in transient gene expression experiments if present within the transcription unit. The Sh1 exon 1 mediates a 10-fold increase in activity when inserted at the 5' end of the bacterial chloramphenicol transacetylase (CAT) marker gene in both monocot and dicot protoplasts. The Sh1 intron 1 enhances chimeric gene expression in rice and maize protoplasts approximately 100-fold but inhibits CAT expression in tobacco protoplasts. In combination, the stimulatory effects of Sh1 exon 1 and intron 1 are multiplicative in monocot protoplasts resulting in a final enhancement of up to 1000-fold compared to the unmodified CAT or luciferase marker genes.