PLANT PHYSIOLOGY CELL BIOLOGY AND SIGNAL TRANSDUCTION

Comparative Analyses of Arabidopsis complex glycan1 Mutants and Genetic Interaction with staurosporin and temperature sensitive3a

Frank, J., Kaulfurst-Soboll, H., Rips, S., Koiwa, H., von Schaewen, A. — 2008-11-06

We compare three Arabidopsis (Arabidopsis thaliana) complex glycan1 (cgl1) alleles and report on genetic interaction with staurosporin and temperature sensitive3a (stt3a). STT3a encodes a subunit of oligosaccharyltransferase that affects efficiency of N-glycan transfer to nascent secretory proteins in the endoplasmic reticulum; cgl1 mutants lack N-acetyl-glucosaminyltransferase I activity and are unable to form complex N-glycans in the Golgi apparatus. By studying CGL1-green fluorescent protein fusions in transient assays, we show that the extra N-glycosylation site created by a point mutation in cgl1 C5 is used in planta and interferes with folding of full-length membrane-anchored polypeptides in the endoplasmic reticulum. Tunicamycin treatment or expression in the stt3a-2 mutant relieved the folding block, and migration to Golgi stacks resumed. Complementation tests with C5-green fluorescent protein and other N-glycosylation variants of CGL1 demonstrated that suppression of aberrant N-glycosylation restores activity. Interestingly, CGL1 seems to be functional also as nonglycosylated enzyme. Two other cgl1 alleles showed splicing defects of their transcripts. In cgl1 C6, a point mutation affects the 3' splice site of intron 14, resulting in frame shifts; in cgl1-T, intron 11 fails to splice due to insertion of a T-DNA copy. Introgression of stt3a-2 did not restore complex glycan formation in cgl1 C6 or cgl1-T but suppressed the N-acetyl-glucosaminyltransferase I defect in cgl1 C5. Root growth assays revealed synergistic effects in double mutants cgl1 C6 stt3a-2 and cgl1-T stt3a-2 only. Besides demonstrating the conditional nature of cgl1 C5 in planta, our observations with loss-of-function alleles cgl1 C6 and cgl1-T in the stt3a-2 underglycosylation background prove that correct N-glycosylation is important for normal root growth and morphology in Arabidopsis.

The Pollen Receptor Kinase LePRK2 Mediates Growth-Promoting Signals and Positively Regulates Pollen Germination and Tube Growth

2008-11-06

In flowering plants, the process of pollen germination and tube growth is required for successful fertilization. A pollen receptor kinase from tomato (Solanum lycopersicum), LePRK2, has been implicated in signaling during pollen germination and tube growth as well as in mediating pollen (tube)-pistil communication. Here we show that reduced expression of LePRK2 affects four aspects of pollen germination and tube growth. First, the percentage of pollen that germinates is reduced, and the time window for competence to germinate is also shorter. Second, the pollen tube growth rate is reduced both in vitro and in the pistil. Third, tip-localized superoxide production by pollen tubes cannot be increased by exogenous calcium ions. Fourth, pollen tubes have defects in responses to style extract component (STIL), an extracellular growth-promoting signal from the pistil. Pollen tubes transiently overexpressing LePRK2-fluorescent protein fusions had slightly wider tips, whereas pollen tubes coexpressing LePRK2 and its cytoplasmic partner protein KPP (a Rop-GEF) had much wider tips. Together these results show that LePRK2 positively regulates pollen germination and tube growth and is involved in transducing responses to extracellular growth-promoting signals.

The Mitochondrial Cycle of Arabidopsis Shoot Apical Meristem and Leaf Primordium Meristematic Cells Is Defined by a Perinuclear Tentaculate/Cage-Like Mitochondrion

Segui-Simarro, J. M., Coronado, M. J., Staehelin, L. A. — 2008-11-06

Plant cells exhibit a high rate of mitochondrial DNA (mtDNA) recombination. This implies that before cytokinesis, the different mitochondrial compartments must fuse to allow for mtDNA intermixing. When and how the conditions for mtDNA intermixing are established are largely unknown. We have investigated the cell cycle-dependent changes in mitochondrial architecture in different Arabidopsis (Arabidopsis thaliana) cell types using confocal microscopy, conventional, and three-dimensional electron microscopy techniques. Whereas mitochondria of cells from most plant organs are always small and dispersed, shoot apical and leaf primordial meristematic cells contain small, discrete mitochondria in the cell periphery and one large, mitochondrial mass in the perinuclear region. Serial thin-section reconstructions of high-pressure-frozen shoot apical meristem cells demonstrate that during G1 through S phase, the large, central mitochondrion has a tentaculate morphology and wraps around one nuclear pole. In G2, both types of mitochondria double their volume, and the large mitochondrion extends around the nucleus to establish a second sheet-like domain at the opposite nuclear pole. During mitosis, approximately 60% of the smaller mitochondria fuse with the large mitochondrion, whose volume increases to 80% of the total mitochondrial volume, and reorganizes into a cage-like structure encompassing first the mitotic spindle and then the entire cytokinetic apparatus. During cytokinesis, the cage-like mitochondrion divides into two independent tentacular mitochondria from which new, small mitochondria arise by fission. These cell cycle-dependent changes in mitochondrial architecture explain how these meristematic cells can achieve a high rate of mtDNA recombination and ensure the even partitioning of mitochondria between daughter cells.

Physiological and Transcriptomic Evidence for a Close Coupling between Chloroplast Ontogeny and Cell Cycle Progression in the Pennate Diatom Seminavis robusta

2008-11-06

Despite the growing interest in diatom genomics, detailed time series of gene expression in relation to key cellular processes are still lacking. Here, we investigated the relationships between the cell cycle and chloroplast development in the pennate diatom Seminavis robusta. This diatom possesses two chloroplasts with a well-orchestrated developmental cycle, common to many pennate diatoms. By assessing the effects of induced cell cycle arrest with microscopy and flow cytometry, we found that division and reorganization of the chloroplasts are initiated only after S-phase progression. Next, we quantified the expression of the S. robusta FtsZ homolog to address the division status of chloroplasts during synchronized growth and monitored microscopically their dynamics in relation to nuclear division and silicon deposition. We show that chloroplasts divide and relocate during the S/G2 phase, after which a girdle band is deposited to accommodate cell growth. Synchronized cultures of two genotypes were subsequently used for a cDNA-amplified fragment length polymorphism-based genome-wide transcript profiling, in which 917 reproducibly modulated transcripts were identified. We observed that genes involved in pigment biosynthesis and coding for light-harvesting proteins were up-regulated during G2/M phase and cell separation. Light and cell cycle progression were both found to affect fucoxanthin-chlorophyll a/c-binding protein expression and accumulation of fucoxanthin cell content. Because chloroplasts elongate at the stage of cytokinesis, cell cycle-modulated photosynthetic gene expression and synthesis of pigments in concert with cell division might balance chloroplast growth, which confirms that chloroplast biogenesis in S. robusta is tightly regulated.

Molecular Characterization of Organelle-Type Nudix Hydrolases in Arabidopsis

2008-11-06

Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases act to hydrolyze ribonucleoside and deoxyribonucleoside triphosphates, nucleotide sugars, coenzymes, or dinucleoside polyphosphates. Arabidopsis (Arabidopsis thaliana) contains 27 genes encoding Nudix hydrolase homologues (AtNUDX1 to -27) with a predicted distribution in the cytosol, mitochondria, and chloroplasts. Previously, cytosolic Nudix hydrolases (AtNUDX1 to -11 and -25) were characterized. Here, we conducted a characterization of organelle-type AtNUDX proteins (AtNUDX12 to -24, -26, and -27). AtNUDX14 showed pyrophosphohydrolase activity toward both ADP-ribose and ADP-glucose, although its K_m value was approximately 100-fold lower for ADP-ribose (13.0 ± 0.7 µm) than for ADP-glucose (1,235 ± 65 µm). AtNUDX15 hydrolyzed not only reduced coenzyme A (118.7 ± 3.4 µm) but also a wide range of its derivatives. AtNUDX19 showed pyrophosphohydrolase activity toward both NADH (335.3 ± 5.4 µm) and NADPH (36.9 ± 3.5 µm). AtNUDX23 had flavin adenine dinucleotide pyrophosphohydrolase activity (9.1 ± 0.9 µm). Both AtNUDX26 and AtNUDX27 hydrolyzed diadenosine polyphosphates (n = 4–5). A confocal microscopic analysis using a green fluorescent protein fusion protein showed that AtNUDX15 is distributed in mitochondria and AtNUDX14 -19, -23, -26, and -27 are distributed in chloroplasts. These AtNUDX mRNAs were detected ubiquitously in various Arabidopsis tissues. The T-DNA insertion mutants of AtNUDX13, -14, -15, -19, -20, -21, -25, -26, and -27 did not exhibit any phenotypical differences under normal growth conditions. These results suggest that Nudix hydrolases in Arabidopsis control a variety of metabolites and are pertinent to a wide range of physiological processes.

Protein Tyrosine Kinases and Protein Tyrosine Phosphatases Are Involved in Abscisic Acid-Dependent Processes in Arabidopsis Seeds and Suspension Cells

2008-11-06

Protein tyrosine (Tyr) phosphorylation plays a central role in many signaling pathways leading to cell growth and differentiation in animals. Tyr phosphorylated proteins have been detected in higher plants, and the roles of protein Tyr phosphatases and protein Tyr kinases in some physiological responses have been shown. We investigated the involvement of Tyr phosphorylation events in abscisic acid (ABA) signaling using a pharmacological approach. Phenylarsine oxide, a specific inhibitor of protein Tyr phosphatase activity, abolished the ABA-dependent accumulation of RAB18 (responsive to ABA 18) transcripts. Protein Tyr kinase inhibitors like genistein, tyrphostin A23, and erbstatin blocked the RAB18 expression induced by ABA in Arabidopsis (Arabidopsis thaliana). Stomatal closure induced by ABA was also inhibited by phenylarsine oxide and genistein. We studied the changes in the Tyr phosphorylation levels of proteins in Arabidopsis seeds after ABA treatment. Proteins were separated by two-dimensional gel electrophoresis, and those phosphorylated on Tyr residues were detected using an anti-phosphotyrosine antibody by western blot. Changes were detected in the Tyr phosphorylation levels of 19 proteins after ABA treatment. Genistein inhibited the ABA-dependent Tyr phosphorylation of proteins. The 19 proteins were analyzed by matrix-assisted laser-desorption ionization time-of-flight/time-of-flight mass spectrometry. Among the proteins identified were storage proteins like cruciferins, enzymes involved in the mobilization of lipid reserves like aconitase, enolase, aldolase, and a lipoprotein, and enzymes necessary for seedling development like the large subunit of Rubisco. Additionally, the identification of three putative signaling proteins, a peptidyl-prolyl isomerase, an RNA-binding protein, and a small ubiquitin-like modifier-conjugating enzyme, enlightens how Tyr phosphorylation might regulate ABA transduction pathways in plants.

Innate Immunity Signaling: Cytosolic Ca2+ Elevation Is Linked to Downstream Nitric Oxide Generation through the Action of Calmodulin or a Calmodulin-Like Protein

Ma, W., Smigel, A., Tsai, Y.-C., Braam, J., Berkowitz, G. A. — 2008-10-06

Ca²⁺ rise and nitric oxide (NO) generation are essential early steps in plant innate immunity and initiate the hypersensitive response (HR) to avirulent pathogens. Previous work from this laboratory has demonstrated that a loss-of-function mutation of an Arabidopsis (Arabidopsis thaliana) plasma membrane Ca²⁺-permeable inwardly conducting ion channel impairs HR and that this phenotype could be rescued by the application of a NO donor. At present, the mechanism linking cytosolic Ca²⁺ rise to NO generation during pathogen response signaling in plants is still unclear. Animal nitric oxide synthase (NOS) activation is Ca²⁺/calmodulin (CaM) dependent. Here, we present biochemical and genetic evidence consistent with a similar regulatory mechanism in plants: a pathogen-induced Ca²⁺ signal leads to CaM and/or a CaM-like protein (CML) activation of NOS. In wild-type Arabidopsis plants, the use of a CaM antagonist prevents NO generation and the HR. Application of a CaM antagonist does not prevent pathogen-induced cytosolic Ca²⁺ elevation, excluding the possibility of CaM acting upstream from Ca²⁺. The CaM antagonist and Ca²⁺ chelation abolish NO generation in wild-type Arabidopsis leaf protein extracts as well, suggesting that plant NOS activity is Ca²⁺/CaM dependent in vitro. The CaM-like protein CML24 has been previously associated with NO-related phenotypes in Arabidopsis. Here, we find that innate immune response phenotypes (HR and [avirulent] pathogen-induced NO elevation in leaves) are inhibited in loss-of-function cml24-4 mutant plants. Pathogen-associated molecular pattern-mediated NO generation in cells of cml24-4 mutants is impaired as well. Our work suggests that the initial pathogen recognition signal of Ca²⁺ influx into the cytosol activates CaM and/or a CML, which then acts to induce downstream NO synthesis as intermediary steps in a pathogen perception signaling cascade, leading to innate immune responses, including the HR.

Chloroplast Outer Envelope Protein CHUP1 Is Essential for Chloroplast Anchorage to the Plasma Membrane and Chloroplast Movement

2008-10-06

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.

Rice ROOT ARCHITECTURE ASSOCIATED1 Binds the Proteasome Subunit RPT4 and Is Degraded in a D-Box and Proteasome-Dependent Manner

2008-10-06

Root growth is mainly determined by cell division and subsequent elongation in the root apical area. Components regulating cell division in root meristematic cells are largely unknown. Previous studies have identified rice (Oryza sativa) ROOT ARCHITECTURE ASSOCIATED1 (OsRAA1) as a regulator in root development. Yet, the function of OsRAA1 at the cellular and molecular levels is unclear. Here, we show that OsRAA1-overexpressed transgenic rice showed reduced primary root growth, increased numbers of cells in metaphase, and reduced numbers of cells in anaphase, which suggests that OsRAA1 is responsible for limiting root growth by inhibiting the onset of anaphase. The expression of OsRAA1 in fission yeast also induced metaphase arrest, which is consistent with the fact that OsRAA1 functions through a conserved mechanism of cell cycle regulation. Moreover, a colocalization assay has shown that OsRAA1 is expressed predominantly at spindles during cell division. Yeast two-hybrid and pull-down assays, as well as a bimolecular fluorescence complementation assay, all have revealed that OsRAA1 interacts with a rice homolog of REGULATORY PARTICLE TRIPLE-A ATPASE4, a component that is involved in the ubiquitin pathway. Treating transgenic rice with specific inhibitors of the 26S proteasome blocked the degradation of OsRAA1 and increased the number of cells in metaphase. Mutation of a putative ubiquitination-targeting D-box (RGSLDLISL) in OsRAA1 interrupted the destruction of OsRAA1 in transgenic yeast. These results suggest that ubiquitination and proteasomic proteolysis are involved in OsRAA1 degradation, which is essential for the onset of anaphase, and that OsRAA1 may modulate root development mediated by the ubiquitin-proteasome pathway as a novel regulatory factor of the cell cycle.

AtPTR1 and AtPTR5 Transport Dipeptides in Planta

2008-10-06

Transporters for di- and tripeptides belong to the large and poorly characterized PTR/NRT1 (peptide transporter/nitrate transporter 1) family. A new member of this gene family, AtPTR5, was isolated from Arabidopsis (Arabidopsis thaliana). Expression of AtPTR5 was analyzed and compared with tissue specificity of the closely related AtPTR1 to discern their roles in planta. Both transporters facilitate transport of dipeptides with high affinity and are localized at the plasma membrane. Mutants, double mutants, and overexpressing lines were exposed to several dipeptides, including toxic peptides, to analyze how the modified transporter expression affects pollen germination, growth of pollen tubes, root, and shoot. Analysis of atptr5 mutants and AtPTR5-overexpressing lines showed that AtPTR5 facilitates peptide transport into germinating pollen and possibly into maturating pollen, ovules, and seeds. In contrast, AtPTR1 plays a role in uptake of peptides by roots indicated by reduced nitrogen (N) levels and reduced growth of atptr1 mutants on medium with dipeptides as the sole N source. Furthermore, overexpression of AtPTR5 resulted in enhanced shoot growth and increased N content. The function in peptide uptake was further confirmed with toxic peptides, which inhibited growth. The results show that closely related members of the PTR/NRT1 family have different functions in planta. This study also provides evidence that the use of organic N is not restricted to amino acids, but that dipeptides should be considered as a N source and transport form in plants.

Interactions of Two Transcriptional Repressors and Two Transcriptional Activators in Modulating Gibberellin Signaling in Aleurone Cells

Zou, X., Neuman, D., Shen, Q. J. — 2008-09-04

Gibberellins (GAs) regulate many aspects of plant development, such as germination, growth, and flowering. The barley (Hordeum vulgare) Amy32b -amylase promoter contains at least five cis-acting elements that govern its GA-induced expression. Our previous studies indicate that a barley WRKY gene, HvWRKY38, and its rice (Oryza sativa) ortholog, OsWRKY71, block GA-induced expression of Amy32b-GUS. In this work, we investigated the functional and physical interactions of HvWRKY38 with another repressor and two activators in barley. HvWRKY38 blocks the inductive activities of SAD (a DOF protein) and HvGAMYB (a R2R3 MYB protein) when either of these proteins is present individually. However, SAD and HvGAMYB together overcome the inhibitory effect of HvWRKY38. Yet, the combination of HvWRKY38 and BPBF (another DOF protein) almost diminishes the synergistic effect of SAD and HvGAMYB transcriptional activators. Electrophoretic mobility shift assays indicate that HvWRKY38 blocks the GA-induced expression of Amy32b by interfering with the binding of HvGAMYB to the cis-acting elements in the -amylase promoter. The physical interaction of HvWRKY38 and BPBF repressors is demonstrated via bimolecular fluorescence complementation assays. These data suggest that the expression of Amy32b is modulated by protein complexes that contain either activators (e.g. HvGAMYB and SAD) or repressors (e.g. HvWRKY38 and BPBF). The relative amounts of the repressor or activator complexes binding to the Amy32b promoter regulate its expression level in barley aleurone cells.

Immunolocalization of Solanaceous SUT1 Proteins in Companion Cells and Xylem Parenchyma: New Perspectives for Phloem Loading and Transport

Schmitt, B., Stadler, R., Sauer, N. — 2008-09-04

Leaf sucrose (Suc) transporters are essential for phloem loading and long-distance partitioning of assimilates in plants that load their phloem from the apoplast. Suc loading into the phloem is indispensable for the generation of the osmotic potential difference that drives phloem bulk flow and is central for the long-distance movement of phloem sap compounds, including hormones and signaling molecules. In previous analyses, solanaceous SUT1 Suc transporters from tobacco (Nicotiana tabacum), potato (Solanum tuberosum), and tomato (Solanum lycopersicum) were immunolocalized in plasma membranes of enucleate sieve elements. Here, we present data that identify solanaceous SUT1 proteins with high specificity in phloem companion cells. Moreover, comparisons of SUT1 localization in the abaxial and adaxial phloem revealed higher levels of SUT1 protein in the abaxial phloem of all three solanaceous species, suggesting different physiological roles for these two types of phloem. Finally, SUT1 proteins were identified in files of xylem parenchyma cells, mainly in the bicollateral veins. Together, our data provide new insight into the role of SUT1 proteins in solanaceous species.

Functional Characterization of the Arabidopsis AtSUC2 Sucrose/H+ Symporter by Tissue-Specific Complementation Reveals an Essential Role in Phloem Loading But Not in Long-Distance Transport

Srivastava, A. C., Ganesan, S., Ismail, I. O., Ayre, B. G. — 2008-09-04

AtSUC2 (At1g22710) encodes a phloem-localized sucrose (Suc)/H⁺ symporter necessary for efficient Suc transport from source tissues to sink tissues in Arabidopsis (Arabidopsis thaliana). AtSUC2 is highly expressed in the collection phloem of mature leaves, and its function in phloem loading is well established. AtSUC2, however, is also expressed strongly in the transport phloem, where its role is more ambiguous, and it has been implicated in mediating both efflux and retrieval to and from flanking tissues via the apoplast. To characterize the role of AtSUC2 in controlling carbon partitioning along the phloem path, AtSUC2 cDNA was expressed from tissue-specific promoters in an Atsuc2 mutant background. Suc transport in this mutant is highly compromised, as indicated by stunted growth and the accumulation of large quantities of sugar and starch in vegetative tissues. Expression of AtSUC2 cDNA from the 2-kb AtSUC2 promoter was sufficient to restore growth and carbon partitioning to nearly wild-type levels. The GALACTINOL SYNTHASE promoter of Cucumis melo (CmGAS1p) confers expression only in the minor veins of mature leaves, not in the transport phloem of larger leaf veins and stems. Mutant plants expressing AtSUC2 cDNA from CmGAS1p had intermediate growth and accumulated sugar and starch, but otherwise they had normal morphology. These characteristics support a role for AtSUC2 in retrieval but not efflux along the transport phloem and show that the only vital function of AtSUC2 in photoassimilate distribution is phloem loading. In addition, Atsuc2 mutant plants, although debilitated, do grow, and AtSUC2-independent modes of phloem transport are discussed, including an entirely symplastic pathway from mesophyll cells to sink tissues.

Arabidopsis Mitogen-Activated Protein Kinase Kinases MKK1 and MKK2 Have Overlapping Functions in Defense Signaling Mediated by MEKK1, MPK4, and MKS1

2008-09-04

The Arabidopsis (Arabidopsis thaliana) MKK1 and MKK2 mitogen-activated protein kinase kinases have been implicated in biotic and abiotic stress responses as part of a signaling cascade including MEKK1 and MPK4. Here, the double loss-of-function mutant (mkk1/2) of MKK1 and MKK2 is shown to have marked phenotypes in development and disease resistance similar to those of the single mekk1 and mpk4 mutants. Because mkk1 or mkk2 single mutants appear wild type, basal levels of MPK4 activity are not impaired in them, and MKK1 and MKK2 are in part functionally redundant in unchallenged plants. These findings are confirmed and extended by biochemical and molecular analyses implicating the kinases in jasmonate- and salicylate-dependent defense responses, mediated in part via the MPK4 substrate MKS1. In addition, transcriptome analyses delineate overlapping and specific effects of the kinases on global gene expression patterns demonstrating both redundant and unique functions for MKK1 and MKK2.

A Novel RNA-Binding Protein Associated with Cell Plate Formation

Ma, L., Xie, B., Hong, Z., Verma, D. P. S., Zhang, Z. — 2008-09-04

Building a cell plate during cytokinesis in plant cells requires the participation of a number of proteins in a multistep process. We previously identified phragmoplastin as a cell plate-specific protein involved in creating a tubulovesicular network at the cell plate. We report here the identification and characterization of a phragmoplastin-interacting protein, PHIP1, in Arabidopsis (Arabidopsis thaliana). It contains multiple functional motifs, including a lysine-rich domain, two RNA recognition motifs, and three CCHC-type zinc fingers. Polypeptides with similar motif structures were found only in plant protein databases, but not in the sequenced prokaryotic, fungal, and animal genomes, suggesting that PHIP1 represents a plant-specific RNA-binding protein. In addition to phragmoplastin, two Arabidopsis small GTP-binding proteins, Rop1 and Ran2, are also found to interact with PHIP1. The zinc fingers of PHIP1 were not required for its interaction with Rop1 and phragmoplastin, but they may participate in its binding with the Ran2 mRNA. Immunofluorescence, in situ RNA hybridization, and green fluorescent protein tagging experiments showed the association of PHIP1 with the forming cell plate during cytokinesis. Taken together, our data suggest that PHIP1 is a novel RNA-binding protein and may play a unique role in the polarized mRNA transport to the vicinity of the cell plate.

The Omp85-Related Chloroplast Outer Envelope Protein OEP80 Is Essential for Viability in Arabidopsis

Patel, R., Hsu, S.-C., Bedard, J., Inoue, K., Jarvis, P. — 2008-09-04

β-Barrel proteins of the Omp85 (Outer membrane protein, 85 kD) superfamily exist in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Prominent Omp85 proteins in bacteria and mitochondria mediate biogenesis of other β-barrel proteins and are indispensable for viability. In Arabidopsis (Arabidopsis thaliana) chloroplasts, there are two distinct types of Omp85-related protein: Toc75 (Translocon at the outer envelope membrane of chloroplasts, 75 kD) and OEP80 (Outer Envelope Protein, 80 kD). Toc75 functions as a preprotein translocation channel during chloroplast import, but the role of OEP80 remains elusive. We characterized three T-DNA mutants of the Arabidopsis OEP80 (AtOEP80) gene. Selectable markers associated with the oep80-1 and oep80-2 insertions segregated abnormally, suggesting embryo lethality of the homozygous genotypes. Indeed, no homozygotes were identified among >100 individuals, and heterozygotes of both mutants produced approximately 25% aborted seeds upon self-pollination. Embryo arrest occurred at a relatively late stage (globular embryo proper) as revealed by analysis using Nomarski optics microscopy. This is substantially later than arrest caused by loss of the principal Toc75 isoform, atToc75-III (two-cell stage), suggesting a more specialized role for AtOEP80. Surprisingly, the oep80-3 T-DNA (located in exon 1 between the first and second ATG codons of the open reading frame) did not cause any detectable developmental defects or affect the size of the AtOEP80 protein in chloroplasts. This indicates that the N-terminal region of AtOEP80 is not essential for the targeting, biogenesis, or functionality of the protein, in contrast with atToc75-III, which requires a bipartite targeting sequence.

In Planta Analysis of the Cell Cycle-Dependent Localization of AtCDC48A and Its Critical Roles in Cell Division, Expansion, and Differentiation

Park, S., Rancour, D. M., Bednarek, S. Y. — 2008-09-04

CDC48/p97 is a conserved homohexameric AAA-ATPase chaperone required for a variety of cellular processes but whose role in the development of a multicellular model system has not been examined. Here, we have used reverse genetics, visualization of a functional Arabidopsis (Arabidopsis thaliana) CDC48 fluorescent fusion protein, and morphological analysis to examine the subcellular distribution and requirements for AtCDC48A in planta. Homozygous Atcdc48A T-DNA insertion mutants arrest during seedling development, exhibiting decreased cell expansion and displaying pleiotropic defects in pollen and embryo development. Atcdc48A insertion alleles show significantly reduced male transmission efficiency due to defects in pollen tube growth. Yellow fluorescent protein-AtCDC48A, a fusion protein that functionally complements the insertion mutant defects, localizes in the nucleus and cytoplasm and is recruited to the division mid-zone during cytokinesis. The pattern of nuclear localization differs according to the stage of the cell cycle and differentiation state. Inducible expression of an Atcdc48A Walker A ATPase mutant in planta results in cytokinesis abnormalities, aberrant cell divisions, and root trichoblast differentiation defects apparent in excessive root hair emergence. At the biochemical level, our data suggest that the endogenous steady-state protein level of AtCDC48A is dependent upon the presence of ATPase-active AtCDC48A. These results demonstrate that CDC48A/p97 is critical for cytokinesis, cell expansion, and differentiation in plants.

Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE Proteins Serve Brassinosteroid-Dependent and -Independent Signaling Pathways

Albrecht, C., Russinova, E., Kemmerling, B., Kwaaitaal, M., de Vries, S. C. — 2008-09-04

The Arabidopsis (Arabidopsis thaliana) SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes belong to a small family of five plant receptor kinases that are involved in at least five different signaling pathways. One member of this family, BRASSINOSTEROID INSENSITIVE1 (BRI1)-ASSOCIATED KINASE1 (BAK1), also known as SERK3, is the coreceptor of the brassinolide (BR)-perceiving receptor BRI1, a function that is BR dependent and partially redundant with SERK1. BAK1 (SERK3) alone controls plant innate immunity, is also the coreceptor of the flagellin receptor FLS2, and, together with SERK4, can mediate cell death control, all three in a BR-independent fashion. SERK1 and SERK2 are essential for male microsporogenesis, again independent from BR. SERK5 does not appear to have any function under the conditions tested. Here, we show that the different SERK members are only redundant in pairs, whereas higher order mutant combinations only show additive phenotypes. Surprisingly, SERK members that are redundant within one are not redundant in another pathway. We also show that this evolution of functional pairs occurred by a change in protein function and not by differences in spatial expression. We propose that, in plants, closely related receptor kinases have a minimal homo- or heterodimeric configuration to achieve specificity.