AT-hook Motif Nuclear Localized Protein Research Articles

Dissection of Functional Modules of AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 4 in the Development of the Root Xylem

Xylem development in the Arabidopsis root apical meristem requires a complex cross talk between plant hormone signaling and transcriptional factors (TFs). The key processes involve fine-tuning between neighboring cells, mediated via the intercellular movement of signaling molecules. As an example, we previously reported that AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN (AHL) 4 (AHL4), a member of the 29 AT-hook family TFs in Arabidopsis, moves into xylem precursors from their neighbors to determine xylem differentiation. As part of the effort to understand the molecular functions of AHL4, we performed domain swapping analyses using AHL1 as a counterpart, finding that AHL4 has three functionally distinctive protein modules. The plant and prokaryotes conserved (PPC) domain of AHL4 acts as a mediator of protein–protein interactions with AHL members. The N-terminus of AHL4 is required for the regulation of xylem development likely via its unique DNA-binding activity. The C-terminus of AHL4 confers intercellular mobility. Our characterization of modules in the AHL4 protein will augment our understanding of the complexity of regulation and the evolution of intercellular mobility in AHL4 and its relatives.

Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered immunity

In many eukaryotic systems during immune responses, mitogen-activated protein kinases (MAPKs) link cytoplasmic signaling to chromatin events by targeting transcription factors, chromatin remodeling complexes, and the RNA polymerase machinery. So far, knowledge on these events is scarce in plants and no attempts have been made to focus on phosphorylation events of chromatin-associated proteins. Here we carried out chromatin phosphoproteomics upon elicitor-induced activation of Arabidopsis The events in WT were compared with those in mpk3, mpk4, and mpk6 mutant plants to decipher specific MAPK targets. Our study highlights distinct signaling networks involving MPK3, MPK4, and MPK6 in chromatin organization and modification, as well as in RNA transcription and processing. Among the chromatin targets, we characterized the AT-hook motif containing nuclear localized (AHL) DNA-binding protein AHL13 as a substrate of immune MAPKs. AHL13 knockout mutant plants are compromised in pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species production, expression of defense genes, and PAMP-triggered immunity. Transcriptome analysis revealed that AHL13 regulates key factors of jasmonic acid biosynthesis and signaling and affects immunity toward Pseudomonas syringae and Botrytis cinerea pathogens. Mutational analysis of the phosphorylation sites of AHL13 demonstrated that phosphorylation regulates AHL13 protein stability and thereby its immune functions.

At-Hook Motif Nuclear Localised Protein 18 as a Novel Modulator of Root System Architecture.

The At-Hook Motif Nuclear Localized Protein (AHL) gene family encodes embryophyte-specific nuclear proteins with DNA binding activity. They modulate gene expression and affect various developmental processes in plants. We identify AHL18 (At3G60870) as a developmental modulator of root system architecture and growth. AHL18 is involved in regulation of the length of the proliferation domain and number of dividing cells in the root apical meristem and thereby, cell production. Both primary root growth and lateral root development respond according to AHL18 transcription level. The ahl18 knock-out plants show reduced root systems due to a shorter primary root and a lower number of lateral roots. This change results from a higher number of arrested and non-developing lateral root primordia (LRP) rather than from a decreased LRP initiation. The over-expression of AHL18 results in a more extensive root system, longer primary roots, and increased density of lateral root initiation events. AHL18 is thus involved in the formation of lateral roots at both LRP initiation and their later development. We conclude that AHL18 participates in modulation of root system architecture through regulation of root apical meristem activity, lateral root initiation and emergence; these correspond well with expression pattern of AHL18.

Efficient use of artificial micro-RNA to downregulate the expression of genes at the post-transcriptional level in Arabidopsis thaliana.

Micro-RNAs are cellular components regulating gene expression at the post-transcription level. In the present study, artificial micro-RNAs were used to decrease the transcript level of two genes, AtExpA8 (encoding an expansin) and AHL25 (encoding an AT-hook motif nuclear localized protein) in Arabidopsis thaliana. The backbone of the Arabidopsis endogenous MIR319a micro-RNA was used in a site-directed mutagenesis approach for the generation of artificial micro-RNAs targeting two genes. The recombinant cassettes were expressed under the control of the CaMV 35S promoter in individual A. thaliana plants. Transgenic lines of the third generation were tested by isolating total RNA and by subsequent cDNA synthesis using oligo-dT18 primers and mRNAs as templates. The expression of the two target genes was checked through quantitative real-time polymerase chain reaction to confirm reduced transcript levels for AtExpA8 and AHL25. Downregulation of AtExpA8 resulted in the formation of short hypocotyls compared with those of the wild-type control in response to low pH and high salt concentration. This technology could be used to prevent the expression of exogenous and invading genes posing a threat to the normal cellular physiology of the host plant.

Cell-to-Cell Movement of Two Interacting AT-Hook Factors inArabidopsisRoot Vascular Tissue Patterning

The xylem and phloem, major conducting and supporting tissues in vascular plants, are established by cell division and cell-type specification in the procambium/cambium. The organization of the xylem, phloem, and procambium/cambium is tightly controlled. However, the underlying regulatory mechanisms remain largely unknown. In this study, we report the discovery of two transcription factors, AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 3 (AHL3) and AHL4, which regulate vascular tissue boundaries in Arabidopsis thaliana roots. In either of the knockout mutants of AHL3 and AHL4, encoding closely related AT-hook transcription factors, a misspecification of tissue boundaries between the xylem and procambium occurred and ectopic xylem developed in the procambium domain. In plants, specific types of transcription factors can serve as direct intercellular signals by moving from one cell to another, playing crucial roles in tissue patterning. Adding to this paradigm, AHL4 moves actively from the procambium to xylem in the root meristem to regulate the tissue boundaries. When the intercellular movement of AHL4 was impaired, AHL4 could not complement the xylem phenotype in the ahl4. Furthermore, AHL4 revealed unique characteristics in that it interacts with AHL3 in vivo and that this interaction facilitates their intercellular trafficking. Taken together, this study uncovered a novel mechanism in vascular tissue patterning that requires the intercellular trafficking of two interacting transcription factors.

Comprehensive analysis of AHL homologous genes encoding AT-hook motif nuclear localized protein in rice

The AT-hook motif is a small DNA-binding protein motif that has been found in the high mobility group of non-histone chromosomal proteins. The Arabidopsis genome contains 29 genes encoding the AT-hook motif DNA-binding protein (AHL). Recent studies of Arabidopsis genes (AtAHLs) have revealed that they might play diverse functional roles during plant growth and development. In this report, we mined 20 AHL genes (OsAHLs) from the rice genome database using AtAHL genes as queries and characterized their molecular features. A phylogenetic tree revealed that OsAHL proteins can be classified into 2 evolutionary clades. Tissue expression pattern analysis revealed that all of the OsAHL genes might be functionally expressed genes with 3 distinct expression patterns. Nuclear localization analysis using transgenic Arabidopsis showed that several OsAHL proteins are exclusively localized in the nucleus, indicating that they may act as architectural transcription factors to regulate expression of their target genes during plant growth and development.

Crystallization and preliminary X-ray crystallographic analysis of a conserved domain in plants and prokaryotes from Pyrococcus horikoshii OT3.

A plant- and prokaryote-conserved domain (PPC) has previously been found in AT-hook motif nuclear localized protein 1 (AHL1) localized in the nuclear matrix of Arabidopsis thaliana (AtAHL1). AtAHL1 has a DNA-binding function. Mutation analyses of AtAHL1 has previously revealed that the hydrophobic region of the PPC domain is essential for its nuclear localization. In this study, the PPC of the hyperthermophilic archaebacterium Pyrococcus horikoshii (PhPPC) was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 53.69, c = 159.2 A. Data were obtained at 100 K, with diffraction being observed to a resolution of 1.7 A. A complete data set from crystals of the SeMet-substituted protein was also obtained.

Identification of a novel plant MAR DNA binding protein localized on chromosomal surfaces

We identified a novel nucleoplasm localized protein in Arabidopsis called AT-hook motif nuclear localized protein 1 (AHL1), which was isolated by visual screening of transformants using random GFP::cDNA fusions. AHL1 contains an AT-hook motif and unknown conserved PPC (plants and prokaryotes conserved) domain that includes a hydrophobic region. Approximately 30 paralogues were identified in the Arabidopsis genome. Proteins with PPC-like domains are found in Bacteria, Archaea and the plant kingdom, but in Bacteria and Archaea the PPC containing proteins of do not have an AT-hook motif. Thus, the PPC domain is evolutionary conserved and has a new function such as AT-rich DNA binding. AHL1 was mainly localized in the nucleoplasm, but little in the nucleolus and heterochromatic region, and was concentrated in the boundary region between euchromatin and heterochromatin. Biochemically, AHL1 was also found in the nuclear matrix fraction. In the M phase, AHL1 was localized on the chromosomal surface. The AT-hook motif was essential for matrix attachment region (MAR) binding, and the hydrophobic region of the PPC was indispensable for nuclear localization. Our results suggest that AHL1 is a novel plant MAR binding protein, which is related to the positioning of chromatin fibers in the nucleus by the presence of an AT-hook motif and PPC domain. In addition, AHL1 is located on the surface of chromosomes during mitosis.