Bidirectional promoters in seed development and related hormone/stress responses.

BACKGROUND: Bidirectional promoters are common in genomes but under-studied experimentally, particularly in plants. We describe a targeted identification and selection of a subset of putative bidirectional promoters to identify genes involved in seed development and to investigate possible coordinated responses of gene pairs to conditions important in seed maturation such as desiccation and ABA-regulation. RESULTS: We combined a search for 100-600 bp intergenic regions in the Arabidopsis genome with a cis-element based selection for those containing multiple copies of the G-box motif, CACGTG. One of the putative bidirectional promoters identified also contained a CE3 coupling element 5 bp downstream of one G-box and is identical to that characterized previously in the HVA1 promoter of barley. CE3 elements are significantly under-represented and under-studied in Arabidopsis. We further characterized the pair of genes associated with this promoter and uncovered roles for two small, previously uncharacterized, plant-specific proteins in Arabidopsis seed development and stress responses. CONCLUSIONS: Using bioinformatics we identified putative bidirectional promoters involved in seed development and analysed expression patterns for a pair of plant-specific genes in various tissues and in response to hormones/stress. We also present preliminary functional analysis of these genes that is suggestive of roles in seed development.

AtTRB1, a telomeric DNA-binding protein from Arabidopsis, is concentrated in the nucleolus and shows highly dynamic association with chromatin.

AtTRB1, 2 and 3 are members of the SMH (single Myb histone) protein family, which comprises double-stranded DNA-binding proteins that are specific to higher plants. They are structurally conserved, containing a Myb domain at the N-terminus, a central H1/H5-like domain and a C-terminally located coiled-coil domain. AtTRB1, 2 and 3 interact through their Myb domain specifically with telomeric double-stranded DNA in vitro, while the central H1/H5-like domain interacts non-specifically with DNA sequences and mediates protein-protein interactions. Here we show that AtTRB1, 2 and 3 preferentially localize to the nucleus and nucleolus during interphase. Both the central H1/H5-like domain and the Myb domain from AtTRB1 can direct a GFP fusion protein to the nucleus and nucleolus. AtTRB1-GFP localization is cell cycle-regulated, as the level of nuclear-associated GFP diminishes during mitotic entry and GFP progressively re-associates with chromatin during anaphase/telophase. Using fluorescence recovery after photobleaching and fluorescence loss in photobleaching, we determined the dynamics of AtTRB1 interactions in vivo. The results reveal that AtTRB1 interaction with chromatin is regulated at two levels at least, one of which is coupled with cell-cycle progression, with the other involving rapid exchange.

Reprogramming of Stem Cell Activity to Convert Thorns into Branches.

Thorns arise from axillary shoot apical meristems that proliferate for a time and then terminally differentiate into a sharp tip. Like other meristems, thorn meristems contain stem cells but, in the case of thorns, these stem cells undergo a programmed cessation of proliferative activity. Using Citrus, we characterize a gene network necessary for thorn development. We identify two Citrus genes, THORN IDENTITY1 (TI1) and THORN IDENTITY2 (TI2), encoding TCP transcription factors, as necessary for stem cell quiescence and thorn identity. Disruption of TI1 and TI2 function results in reactivation of stem cells and concomitant conversion of thorns to branches. Expression of WUSCHEL (WUS) defines the shoot stem cell niche in the apical meristems of many angiosperm species; we show that TI1 binds to the Citrus WUS promoter and negatively regulates its expression to terminate stem cell proliferation. We propose that shifts in the timing and function of components of this gene network can account for the evolution of Citrus thorn identity. Modulating this pathway can significantly alter plant architecture and could be leveraged to improve crop yields.

The ULT1 and ULT2 trxG genes play overlapping roles in Arabidopsis development and gene regulation.

The epigenetic regulation of gene expression is critical for ensuring the proper deployment and stability of defined genome transcription programs at specific developmental stages. The cellular memory of stable gene expression states during animal and plant development is mediated by the opposing activities of Polycomb group (PcG) factors and trithorax group (trxG) factors. Yet, despite their importance, only a few trxG factors have been characterized in plants and their roles in regulating plant development are poorly defined. In this work, we report that the closely related Arabidopsis trxG genes ULTRAPETALA1 (ULT1) and ULT2 have overlapping functions in regulating shoot and floral stem cell accumulation, with ULT1 playing a major role but ULT2 also making a minor contribution. The two genes also have a novel, redundant activity in establishing the apical­basal polarity axis of the gynoecium, indicating that they function in differentiating tissues. Like ULT1 proteins, ULT2 proteins have a dual nuclear and cytoplasmic localization, and the two proteins physically associate in planta. Finally, we demonstrate that ULT1 and ULT2 have very similar overexpression phenotypes and regulate a common set of key development target genes, including floral MADS-box genes and class I KNOX genes. Our results reveal that chromatin remodeling mediated by the ULT1 and ULT2 proteins is necessary to control the development of meristems and reproductive organs. They also suggest that, like their animal counterparts, plant trxG proteins may function in multi-protein complexes to up-regulate the expression of key stage- and tissue-specific developmental regulatory genes.

Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins.

Leaves are determinate organs that arise from the flanks of the shoot apical meristem as polar structures with distinct adaxial (dorsal) and abaxial (ventral) sides. Opposing regulatory interactions between genes specifying adaxial or abaxial fates function to maintain dorsoventral polarity. One component of this regulatory network is the Myb-domain transcription factor gene ASYMMETRIC LEAVES1 (AS1). The contribution of AS1 to leaf polarity varies across different plant species; however, in Arabidopsis, as1 mutants have only mild defects in leaf polarity, suggesting that alternate pathways exist for leaf patterning. Here, we describe three genes, PIGGYBACK1 (PGY1), PGY2 and PGY3, which alter leaf patterning in the absence of AS1. All three pgy mutants develop dramatic ectopic lamina outgrowths on the adaxial side of the leaf in an as1 mutant background. This leaf-patterning defect is enhanced by mutations in the adaxial HD-ZIPIII gene REVOLUTA (REV), and is suppressed by mutations in abaxial KANADI genes. Thus, PGY genes influence leaf development via genetic interactions with the HD-ZIPIII-KANADI pathway. PGY1, PGY2 and PGY3 encode cytoplasmic large subunit ribosomal proteins, L10a, L9 and L5, respectively. Our results suggest a role for translation in leaf dorsoventral patterning and indicate that ribosomes are regulators of key patterning events in plant development.

Arabidopsis POT1A interacts with TERT-V(I8), an N-terminal splicing variant of telomerase.

Chromosome integrity is maintained via the actions of ribonucleoprotein complexes that can add telomeric repeats or can protect the chromosome end from being degraded. POT1 (protection of telomeres 1), a class of single-stranded-DNA-binding proteins, is a regulator of telomeric length. The Arabidopsis genome contains three POT1 homologues: POT1A, POT1B and POT1C. Using yeast two-hybrid assays to identify components of a potential POT1A complex, we retrieved three interactors: the N-terminus of the telomerase, a protein kinase and a plant-specific protein. Further analysis of the interaction of POT1 proteins with telomerase showed that this interaction is specific to POT1A, suggesting a specific role for this paralogue. The interaction is specific to the N-terminal region of the telomerase, which can be encoded by splicing variants. This interaction indicates possible mechanisms for telomerase regulation by alternative splicing and by POT1 proteins.

Two E2F sites in the Arabidopsis MCM3 promoter have different roles in cell cycle activation and meristematic expression.

The commitment to DNA replication is a key step in cell division control. The Arabidopsis MCM3 homologue forms part of the mini chromosome maintenance (MCM) complex involved in the initiation of DNA replication at the transition G(1)/S. Consistent with its role at the G(1)/S transition we show that the AtMCM3 gene is transcriptionally regulated at S phase. The 5' region of this gene contains several E2F consensus binding sites, two of which match the human consensus closely and whose roles have been studied here. The identity of the two sequences as E2F binding sites has been confirmed by electrophoretic mobility shift assay analyses. Furthermore the promoter is activated by AtE2F-a and AtDP-a factors in transient expression studies. One of the E2F binding sites is shown to be responsible for the G(2)-specific repression of the promoter in synchronized cell suspension cultures. In contrast, the second E2F binding site has a role in meristem-specific expression in planta as deletion of this site eliminates the expression of a reporter gene in root and apical meristems. Thus two highly similar E2F binding sites in the promoter of the MCM3 gene are responsible for different cell cycle regulation or developmental expression patterns depending on the cellular environment.