Functional divergence of microtubule-associated TPX2 family members in Arabidopsis thaliana

TPX2 (Targeting Protein for Xklp2) is an evolutionary conserved microtubule-associated protein important for microtubule nucleation and mitotic spindle assembly. The protein was described as an activator of the mitotic kinase Aurora A in humans and the Arabidopsis AURORA1 (AUR1) kinase. In contrast to animal genomes that encode only one TPX2 gene, higher plant genomes encode a family with several TPX2-LIKE gene members (TPXL). TPXL genes of Arabidopsis can be divided into two groups. Group A proteins (TPXL2, 3, 4, and 8) contain Aurora binding and TPX2_importin domains, while group B proteins (TPXL1, 5, 6, and 7) harbor an Xklp2 domain. Canonical TPX2 contains all the above-mentioned domains. We confirmed using in vitro kinase assays that the group A proteins contain a functional Aurora kinase binding domain. Transient expression of Arabidopsis TPX2-like proteins in Nicotiana benthamiana revealed preferential localization to microtubules and nuclei. Co-expression of AUR1 together with TPX2-like proteins changed the localization of AUR1, indicating that these proteins serve as targeting factors for Aurora kinases. Taken together, we visualize the various localizations of the TPX2-LIKE family in Arabidopsis as a proxy to their functional divergence and provide evidence of their role in the targeted regulation of AUR1 kinase activity

Interaction specificity of Arabidopsis 14-3-3 proteins with phototropin receptor kinases

Phototropin receptor kinases play an important roles in optimising plant growth in response to blue light. Much is known regarding their photochemical reactivity, yet little progress has been made to identify downstream signalling components. Here, we isolated several interacting proteins for Arabidopsis phototropin 1 (phot1) by yeast two-hybrid screening. These include members of the NPH3/RPT2 (NRL) protein family, proteins associated with vesicle trafficking, and the 14-3-3 lambda (?) isoform from Arabidopsis . 14-3-3? and phot1 were found to colocalise and interact in vivo. Moreover, 14-3-3 binding to phot1 was limited to non-epsilon 14-3-3 isoforms and was dependent on key sites of receptor autophosphorylation. No 14-3-3 binding was detected for Arabidopsis phot2, suggesting that 14-3-3 proteins represent specific mode of phot1 signalling

Transcriptional Regulation: a Genomic Overview

The availability of the Arabidopsis thaliana genome sequence allows a comprehensive analysis of transcriptional regulation in plants using novel genomic approaches and methodologies. Such a genomic view of transcription first necessitates the compilation of lists of elements. Transcription factors are the most numerous of the different types of proteins involved in transcription in eukaryotes, and the Arabidopsis genome codes for more than 1,500 of them, or approximately 6% of its total number of genes. A genome-wide comparison of transcription factors across the three eukaryotic kingdoms reveals the evolutionary generation of diversity in the components of the regulatory machinery of transcription. However, as illustrated by Arabidopsis, transcription in plants follows similar basic principles and logic to those in animals and fungi. A global view and understanding of transcription at a cellular and organismal level requires the characterization of the Arabidopsis transcriptome and promoterome, as well as of the interactome, the localizome, and the phenome of the proteins involved in transcription

Membrane proteins in the outer mebrane of plastids and mitochondria

Channels of the plastid and mitochondrial outer membranes facilitate the turnover of molecules and ions via these membranes. Although channels have been studied many questions pertaining to the whole diversity of plastid and mitochondrial channels in Arabidopsis thaliana and Pisum sativum remain unanswered. In this thesis I studied OEP16, OEP37 and VDAC families in two model plants, in Arabidopsis and pea. The Arabidopsis OEP16 family represents four channels of α-helical structure, similar to the pea OEP16 protein. These channels are suggested to transport amino acids and compounds with primary amino groups. Immunoblot analysis, GFP/RFP protein fusion expression, as well as proteomic analysis showed that AtOEP16.1, AtOEP16.2 and AtOEP16.4 are located in the outer envelope membrane of plastids, while AtOEP16.3 is in mitochondria. The gene expression and immunoblot analyses revealed that AtOEP16.1 and AtOEP16.3 proteins are highly abundant and ubiquitous; expression of AtOEP16.1 is regulated by light and cold. AtOEP16.2 is highly expressed in pollen, seeds and seedlings. AtOEP16.4 is a low expressed housekeeping protein. Single knockout mutants of AtOEP16.1, AtOEP16.2 and AtOEP16.4, and double mutants of AtOEP16 gene family did not show any remarkable phenotype. However, macroarray analysis of Atoep16.1-p T-DNA mutant revealed 10 down-regulated and 6 up-regulated genes. In contrast to the α-helical OEP16 proteins, the OEP37 and VDAC proteins are of β-barrel structure. The PsOEP37 and AtOEP37 channel proteins form a selective barrier in the outer envelope of chloroplasts. Electrophysiological studies in lipid bilayer membranes showed that the PsOEP37 channel is permeable for cations. Specific expression profiles showed that AtOEP37 and PsOEP37 are highly expressed in the entire plant. The isolated PsVDAC gene encodes a protein, which is located in mitochondria. In Arabidopsis gene database, five Arabidopsis genes, which code for VDAC-like proteins were announced. One gene was not detected, whereas four of these genes expressed in leaves, roots, flower buds and pollen

Nuclear-localized subtype of end-binding 1 protein regulates spindle organization in Arabidopsis

End-binding 1 (EB1) proteins are evolutionarily conserved plus-end-tracking proteins that localize to growing microtubule plus ends where they regulate microtubule dynamics and interactions with intracellular targets. Animal EB1 proteins have acidic C-terminal tails that might induce an autoinhibitory conformation. Although EB1 proteins with the same structural features occur in plants (EB1a and EB1b in Arabidopsis thaliana), a variant form (EB1c) is present that lacks the characteristic tail. We show that in Arabidopsis the tail region of EB1b, but not of EB1c, inhibits microtubule assembly in vitro. EB1a and EB1b form heterodimers with each other, but not with EB1c. Furthermore, the EB1 genes are expressed in various cell types of Arabidopsis, but the expression of EB1c is particularly strong in the meristematic cells where it is targeted to the nucleus by a nuclear localization signal in the C-terminal tail. Reduced expression of EB1c compromised the alignment of spindle and phragmoplast microtubules and caused frequent lagging of separating chromosomes at anaphase. Roots of the eb1c mutant were hypersensitive to a microtubule-disrupting drug and complete rescue of the mutant phenotype required the tail region of EB1c. These results suggest that a plant-specific EB1 subtype has evolved to function preferentially on the spindle microtubules by accumulating in the prophase nucleus

Beyond the photocycle-how cryptochromes regulate photoresponses in plants?

Cryptochromes (CRYs) are blue light receptors that mediate light regulation of plant growth and development. Land plants possess various numbers of cryptochromes, CRY1 and CRY2, which serve overlapping and partially redundant functions in different plant species. Cryptochromes exist as physiologically inactive monomers in darkness; photoexcited cryptochromes undergo homodimerization to increase their affinity to the CRY-signaling proteins, such as CIBs (CRY2-interacting bHLH), PIFs (Phytochrome-Interacting Factors), AUX/IAA (Auxin/INDOLE-3-ACETIC ACID), and the COP1-SPAs (Constitutive Photomorphogenesis 1-Suppressors of Phytochrome A) complexes. These light-dependent protein-protein interactions alter the activity of the CRY-signaling proteins to change gene expression and developmental programs in response to light. In the meantime, photoexcitation also changes the affinity of cryptochromes to the CRY-regulatory proteins, such as BICs (Blue-light Inhibitors of CRYs) and PPKs (Photoregulatory Protein Kinases), to modulate the activity, modification, or abundance of cryptochromes and photosensitivity of plants in response to the changing light environment

ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing.

Constitutive heterochromatin in Arabidopsis thaliana is marked by repressive chromatin modifications, including DNA methylation, histone H3 dimethylation at Lys9 (H3K9me2) and monomethylation at Lys27 (H3K27me1). The enzymes catalyzing DNA methylation and H3K9me2 have been identified; alterations in these proteins lead to reactivation of silenced heterochromatic elements. The enzymes responsible for heterochromatic H3K27me1, in contrast, remain unknown. Here we show that the divergent SET-domain proteins ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6 have H3K27 monomethyltransferase activity, and atxr5 atxr6 double mutants have reduced H3K27me1 in vivo and show partial heterochromatin decondensation. Mutations in atxr5 and atxr6 also lead to transcriptional activation of repressed heterochromatic elements. Notably, H3K9me2 and DNA methylation are unaffected in double mutants. These results indicate that ATXR5 and ATXR6 form a new class of H3K27 methyltransferases and that H3K27me1 represents a previously uncharacterized pathway required for transcriptional repression in Arabidopsis

Exploring plant tolerance to biotic and abiotic stresses

Plants are exposed to many stress factors, such as drought, high salinity or pathogens, which reduce the yield of the cultivated plants or affect the quality of the harvested products. Arabidopsis thaliana was used as a model plant to study the responses of plants to different sources of stress. With Agrobacterium T-DNA mediated promoter tagging, a novel di-/tripeptide transporter gene AtPTR3 was identified as a wound-induced gene. This gene was found to be induced by mechanical wounding, high salt concentrations, bacterial infection and senescence, and also in response to several plant hormones and signalling compounds, such as salicylic acid, jasmonic acid, ethylene and abscisic acid. Atptr3 mutants of two Arabidopsis ecotypes, C24 and Col-0, were impaired in germination on media containing a high salt concentration, which indicates that AtPTR3 is involved in seed germination under salt stress. Wounding caused local expression of the AtPTR3 gene, whereas inoculation with the plant pathogenic bacterium Erwinia carotovora subsp. carotovora caused both local and systemic expression of the gene. Atptr3 mutants showed increased susceptibility to infection caused by bacterial phytopathogens, E carotovora and Pseudomonas syringae pv. tomato, and the P. syringae type III secretion system was shown to be involved in suppression of the AtPTR3 expression in inoculated plants. Moreover, the Atptr3 mutation was found to reduce the expression of the marker gene for systemic acquired resistance, PR1 and the mutants accumulated reactive oxygen species (ROS) following the treatment of the plants with ROS generating substances. Overall results and observations suggest that the AtPTR3 is a novel and versatile stress responsive gene needed for defence reactions against many stresses. In a second part of the study, the yeast (Saccharomyces cerevisiae) trehalose-6-phosphate synthase gene (ScTPS1) was utilized to improve the drought tolerance of Arabidopsis. This gene codes for the first enzyme in the trehalose biosynthesis pathway of yeast, and expression in plants leads to improved drought tolerance but also growth aberrations. In this study, the ScTps1 protein was expressed in Arabidopsis using the constructs containing chloroplast targeting transit peptide sequence that facilitated the import of the ScTps1 into the chloroplast. The drought tolerance and growth phenotypes of Arabidopsis transgenics transformed with ScTPS1 with or without transit peptide, were characterized. The plants with cytosolic localization of the ScTps1 protein showed aberrant root phenotype, but the plants with the chloroplast targeted ScTps1 protein caused no aberration in root morphology. Even though both the transgenic lines showed enhanced drought tolerance, the relative water content of the lines was found to be similar to the wild type control. Moreover, both the transgenic lines showed slightly better water holding capacity or reduced water loss over time compared to wild type plants. The overall results indicated that the growth aberrations caused by cytosolic localization of ScTps1 could be uncoupled from the enhanced drought tolerance in the transgenic plants when the ScTps1 was targeted to chloroplast