The hpa1 mutant of Arabidopsis reveals a crucial role of histidine homeostasis in root meristem maintenance

Histidine (His) is an essential ingredient for protein synthesis and is required by all living organisms. In higher plants, although there is considerable evidence that His is essential for plant growth and survival, there is very little information as to whether it plays any specific role in plant development. Here, we present evidence for such a role of this amino acid in root development in Arabidopsis (Arabidopsis thaliana) from the characterization of a novel Arabidopsis mutant, hpa1, which has a very short root system and carries a mutation in one of the two Arabidopsis histidinol-phosphate aminotransferase (HPA) genes, AtHPA1. We have established that AtHPA1 encodes a functional HPA and that its complete knockout is embryo lethal. Biochemical analysis shows that the mutation in hpa1 only resulted in a 30% reduction in free His content and had no significant impact on the total His content. It did not cause any known symptoms of His starvation. However, the mutant displayed a specific developmental defect in root meristem maintenance and was unable to sustain primary root growth 2 d after germination. We have demonstrated that the root meristem failure in the mutant is tightly linked to the reduction in free His content and could be rescued by either exogenous His supplementation or AtHPA1 overexpression. Our results therefore reveal an important role of His homeostasis in plant development.     

The Arabidopsis ARCP protein, CSI1, which is required for microtubule stability, is necessary for root and anther development

Armadillo repeat-containing proteins (ARCPs) are conserved across eukaryotic kingdoms and function in various processes. Regulation of microtubule stability by ARCPs exists widely in mammals and algae, but little is known in plants. Here, we present the functional characterization of an Arabidopsis thaliana ARCP, which was previously identified as Cellulose synthase-interactive protein1 (CSI1), and prove its crucial role in anther and root development. CSI1 is highly expressed in floral tissues, and knockout mutants of CSI1 (three allelic lines) accordingly exhibit defective anther dehiscence, which can be partially rescued by mammalian microtubule-stabilizer MAP4, suggesting that CSI1 functions by stabilizing the microtubular cytoskeleton. CSI1 binds microtubules in vitro, and immunofluorescence and coimmunoprecipitation studies confirmed the physical interactions between CSI1 and microtubules in vivo. Analysis using oryzalin, a microtubule-disrupting drug, further revealed the destabilized microtubules under CSI1 deficiency and confirmed the crucial role of CSI1 in microtubule stability. The dynamic change of CSI1 in response to dehydration strongly suggests the important function of CSI1 in dehydration-induced microtubule depolymerization and reorganization, which is crucial for anther development. These results indicate the pivotal role of CSI1 in anther development by regulating microtubule stability and hence cell morphogenesis.     

Root-specific CLE19 overexpression and the sol1/2 suppressors implicate a CLV-like pathway in the control of Arabidopsis root meristem maintenance

In the Arabidopsis shoot apical meristem, an organizing center signals in a non-cell-autonomous manner to specify the overlying stem cells. Stem cells express the small, secreted protein CLAVATA3 (CLV3; ) that activates the CLV1-CLV2 receptor complex, which negatively controls the size of the organizing center. Consistently, CLV3 overexpression restricts shoot meristem size. The root meristem also contains a stem cell organizer, and here we show that localized overexpression in roots of CLE19, encoding a CLV3 homolog, restricts the size of the root meristem. This suggests that CLE19 acts by overactivating an endogenous CLV-like pathway involved in root meristem maintenance. Surprisingly, CLE19 restricts meristem size without directly interfering with organizer and stem cell specification. We isolated mutations in two loci, SOL1 and SOL2, which suppress the CLE19 overexpression phenotype. sol2 plants display floral phenotypes reminiscent of clv weak alleles; these phenotypes suggest that components of a CLV pathway are shared in roots and shoots. SOL1 encodes a putative Zn(2+)-carboxypeptidase, which may be involved in ligand processing.     

An Arabidopsis protein with a novel calcium-binding repeat sequence interacts with TONSOKU/MGOUN3/BRUSHY1 involved in meristem maintenance

TONSOKU(TSK)/MGOUN3/BRUSHY1 from Arabidopsis thaliana, which plays an important role in the maintenance of meristem organization, contains an LGN repeat motif similar to that found in animal proteins involved in asymmetric cell division. One protein that interacts with the LGN motif of TSK in a yeast two-hybrid screen, TSK-associating protein 1 (TSA1), contains a 10-fold repeat of a unique 41 amino acid sequence. The repeat sequence, with a glutamic acid-phenylalanine-glutamic acid (EFE) conserved core sequence, is enriched with acidic amino acids. TSA1 also contains an N-terminal putative signal peptide and it interacts with the LGN motif of TSK through a C-terminal region separated from the EFE repeats by a putative membrane-spanning region. The recombinant protein consisting of EFE repeats was rich in alpha-helical structure and possessed Ca2+-binding activity. Unlike nuclear localization of TSK, the TSA1 fused with green fluorescent protein (GFP) expressed in tobacco BY-2 cells was localized in small cytoplasmic vesicles during interphase. However, cellular localization of both TSA1-GFP and GFP-TSK changed dynamically during mitosis. In particular, both GFP-TSK and TSA1-GFP were concentrated in limited areas that are close to the ends of spindle microtubules ahead of separating chromatids. These results are discussed in terms of the possible involvement of TSK and TSA1 in mitosis.     

The plant-specific VIMAG domain of Glutamine Dumper1 is necessary for the function of the protein in Arabidopsis

The over-expression of the arabidopsis GLUTAMINE DUMPER1 gene (GDU1) leads to increased amino acid content and transport. In a screening for mutations suppressing this phenotype, a mutant was isolated. The mutation leads to a glycine to arginine substitution in one of the two conserved domains of the protein, the VIMAG domain. More detailed structure function relationship analyses showed that the presence of this domain and the membrane localisation are both necessary for the function of the GDU1 protein. These results shed light on the function of the GDU1 protein whose family is specific to plants.     

Requirement of homeobox gene STIMPY/WOX9 for Arabidopsis meristem growth and maintenance

Most organs of flowering plants develop postembryonically from groups of pluripotent cells called meristems [1]. The shoot apical meristem (SAM) is specified by two complementary pathways [2-4]. SHOOT MERISTEMLESS (STM; [5]) defines the entire SAM region [6]. WUSCHEL (WUS), on the other hand, functions in a more restricted set of cells to promote stem-cell fate and is regulated by the CLAVATA genes in a negative feedback loop [7-10]. In contrast, little is known about how the growth of the SAM, which increases in size during vegetative development [11], is regulated. We have characterized STIMPY (STIP; also called WOX9 [12]), a homeobox gene required for the growth of the vegetative SAM, in part by positively regulating WUS expression. In addition, STIP is required in several other aerial organs and the root. What sets STIP apart from STM and WUS is that stip mutants can be fully rescued by stimulating the entry into the cell cycle with sucrose. Therefore, STIP is likely to act in all these tissues by maintaining cell division and preventing premature differentiation. Taken together, our findings suggest that STIP identifies a new genetic pathway integrating developmental signals with cell-cycle control.     

Molecular chaperone function of Arabidopsis thaliana phloem protein 2-A1, encodes a protein similar to phloem lectin

Although several phloem sap proteins have been identified from protein extracts of heat-treated Arabidopsis seedlings using FPLC gel filtration columns, many of the physiological roles played by these proteins remain to be elucidated. We functionally characterized a phloem protein 2-A1, which encodes a protein similar to phloem lectin. Using a bacterially expressed recombinant protein of AtPP2-A1, we found that it performs dual functions, showing both molecular chaperone activity and antifungal activity. mRNA expression of the AtPP2-1 gene was induced by diverse external stresses such as pathogens, and other signaling molecules, such as ethylene. These results suggest that the AtPP2-A1 molecular chaperone protein plays a critical role in the Arabidopsis defense system against diverse external stresses including fungal pathogenic attack and heat shock.     

The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana

The mutants irt1-1 and irt1-2 of Arabidopsis thaliana were identified among a collection of T-DNA-tagged lines on the basis of a decrease in the effective quantum yield of photosystem II. The mutations responsible interfere with expression of IRT1, a nuclear gene that encodes the metal ion transporter IRT1. In irt1 mutants, photosensitivity and chlorophyll fluorescence parameters, as well as abundance and composition of the photosynthetic apparatus, are significantly altered. Additional effects of the mutation under greenhouse conditions, including chlorosis and a drastic reduction in growth rate and fertility, are compatible with a deficiency in iron transport. Propagation of irt1 plants on media supplemented with additional quantities of iron salts restores almost all aspects of wild-type behaviour. The irt2-1 mutant, which carries an En insertion in the highly homologous IRT2 gene of Arabidopsis thaliana, was identified by reverse genetics and shows no symptoms of iron deficiency. This, together with the finding that irt1-1 can be complemented by 35S::IRT1 but not by 35S::IRT2, demonstrates that, although the products of the two genes are closely related, only AtIRT1 is required for iron homeostasis under physiological conditions.     

SeqA protein aggregation is necessary for SeqA function.

The binding of SeqA protein to hemimethylated GATC sequences is important in the negative modulation of chromosomal initiation at oriC, and in the formation of SeqA foci necessary for Escherichia coli chromosome segregation. Using gel-filtration chromotography and glycerol gradient sedimentation, we demonstrate that SeqA exists as a homotetramer. SeqA tetramers are able to aggregate or multimerize in a reversible, concentration-dependent manner. Using a bacterial two-hybrid system, we demonstrate that the N-terminal region of SeqA, especifically the 9th amino acid residue, glutamic acid, is required for functional SeqA-SeqA interaction. Although the SeqA(E9K) mutant protein, containing lysine rather than glutamic acid at the 9th amino acid residue, exists as a tetramer, the mutant protein binds to hemimethylated DNA with altered binding patterns as compared with wild-type SeqA. Aggregates of SeqA(E9K) are defective in hemimethylated DNA binding. Here we demonstrate that proper interaction between SeqA tetramers is required for both hemimethylated DNA binding and formation of active aggregates. SeqA tetramers and aggregates might be involved in the formation of SeqA foci required for the segregation of chromosomal DNA as well as the regulation of chromosomal initiation.     

Arabidopsis acyl-CoA-binding protein ACBP6 localizes in the phloem and affects jasmonate composition

Arabidopsis thaliana ACYL-COA-BINDING PROTEIN6 (AtACBP6) encodes a cytosolic 10-kDa AtACBP. It confers freezing tolerance in transgenic Arabidopsis, possibly by its interaction with lipids as indicated by the binding of acyl-CoA esters and phosphatidylcholine to recombinant AtACBP6. Herein, transgenic Arabidopsis transformed with an AtACBP6 promoter-driven β-glucuronidase (GUS) construct exhibited strong GUS activity in the vascular tissues. Immunoelectron microscopy using anti-AtACBP6 antibodies showed AtACBP6 localization in the phloem especially in the companion cells and sieve elements. Also, the presence of gold grains in the plasmodesmata indicated its potential role in systemic trafficking. The AtACBP6 protein, but not its mRNA, was found in phloem exudate of wild-type Arabidopsis. Fatty acid profiling using gas chromatography-mass spectrometry revealed an increase in the jasmonic acid (JA) precursor, 12-oxo-cis,cis-10,15-phytodienoic acid (cis-OPDA), and a reduction in JA and/or its derivatives in acbp6 phloem exudates in comparison to the wild type. Quantitative real-time PCR showed down-regulation of COMATOSE (CTS) in acbp6 rosettes suggesting that AtACBP6 affects CTS function. AtACBP6 appeared to affect the content of JA and/or its derivatives in the sieve tubes, which is consistent with its role in pathogen-defense and in its wound-inducibility of AtACBP6pro::GUS. Taken together, our results suggest the involvement of AtACBP6 in JA-biosynthesis in Arabidopsis phloem tissues.     

Arabidopsis glyoxalase II contains a zinc/iron binuclear metal center that is essential for substrate binding and catalysis

Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or anti-protozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors.     

Integrin-associated protein immunoglobulin domain is necessary for efficient vitronectin bead binding

Integrin-associated protein (IAP/CD47) is physically associated with the alpha v beta 3 vitronectin (Vn) receptor and a functionally and immunologically related integrin on neutrophils (PMN) and monocytes. Anti-IAP antibodies inhibit multiple phagocyte functions, including Arg- Gly-Asp (RGD)-initiated activation of phagocytosis, chemotaxis, and respiratory burst; PMN adhesion to entactin; and PMN transendothelial and transepithelial migration at a step subsequent to tight intercellular adhesion. Anti-IAP antibodies also inhibit binding of Vn- coated particles to many cells expressing alpha v beta 3. However, prior studies with anti-IAP did not directly address IAP function because they could not distinguish between IAP blockade and antibody- induced signaling effects on cells. To better determine the function of IAP, we have characterized and used an IAP-deficient human cell line. Despite expressing alpha v integrins, these cells do not bind Vn-coated particles unless transfected with IAP expression constructs. Increasing the level of alpha v beta 3 expression or increasing Vn density on the particle does not overcome the requirement for IAP. All known splice variants of IAP restore Vn particle binding equivalently. Indeed, the membrane-anchored IAP Ig variable domain suffices to mediate Vn particle binding in this system, while the multiply membrane-spanning and cytoplasmic domains are dispensable. In all cases, adhesion to a Vn- coated surface and fibronectin particle binding through alpha 5 beta 1 fibronectin receptors are independent of IAP expression. These data demonstrate that some alpha v integrin ligand-binding functions are IAP independent, whereas others require IAP, presumably through direct physical interaction between its Ig domain and the integrin.     

Tricellulin is a tight-junction protein necessary for hearing

The inner ear has fluid-filled compartments of different ionic compositions, including the endolymphatic and perilymphatic spaces of the organ of Corti; the separation from one another by epithelial barriers is required for normal hearing. TRIC encodes tricellulin, a recently discovered tight-junction (TJ) protein that contributes to the structure and function of tricellular contacts of neighboring cells in many epithelial tissues. We show that, in humans, four different recessive mutations of TRIC cause nonsyndromic deafness (DFNB49), a surprisingly limited phenotype, given the widespread tissue distribution of tricellulin in epithelial cells. In the inner ear, tricellulin is concentrated at the tricellular TJs in cochlear and vestibular epithelia, including the structurally complex and extensive junctions between supporting and hair cells. We also demonstrate that there are multiple alternatively spliced isoforms of TRIC in various tissues and that mutations of TRIC associated with hearing loss remove all or most of a conserved region in the cytosolic domain that binds to the cytosolic scaffolding protein ZO-1. A wild-type isoform of tricellulin, which lacks this conserved region, is unaffected by the mutant alleles and is hypothesized to be sufficient for structural and functional integrity of epithelial barriers outside the inner ear.