Formation of corymb-like inflorescences due to delay in bolting and flower development in the corymbosa2 mutant of Arabidopsis

Among the wild-type ecotypes of Arabidopsis thaliana whose shape of inflorescence is categorized as raceme, the ecotype Landsberg harboring the erecta (er) mutation shows a corymb-like inflorescence, namely, a compact inflorescence with a flattened arrangement of flower buds at the tip. The fact that the ER gene encodes a receptor-like protein kinase implies the presence of a signaling cascade responsible for the inflorescence morphology of flowering plants. We report here the characterization of another mutant with a corymb-like inflorescence, named corymbosa2 (crm2), and the isolation of the CRM2 gene. While the er mutation causes a severe reduction in the length of pedicels, the crm2 mutation results in a significant delay in the initiation of internode elongation and in the development of flowers, despite having little effect on the timing of floral induction. Consequently, the number of flower buds is apparently increased at the tip of crm2 inflorescence. The crm2 er double mutant shows an additive phenotype. These results suggest that CRM2 and ER may act in different ways to generate wild-type inflorescence. The CRM2 gene was isolated by positional cloning and appears to encode a polypeptide with no significant homology to known sequences.     

Mutants of Arabidopsis thaliana with pleiotropic effects on the expression of the gene for β-amylase and on the accumulation of anthocyanin that are inducible by sugars

We identified a mutant of Arabidopsis thaliana ecotype Col-0 in which significantly reduced levels of expression of the gene for β-amylase ( ATβ-Amy ) were detected in leaves in response to high concentrations of sucrose, glucose or fructose. Genetic studies, including a cross with transgenic plants that harbored the ATβ-Amy:GUS transgene with the promoter of ATβ-Amy , indicated that this phenotype was caused by a recessive mutation, Iba1 , that affected expression of ATβ-Amy in trans . We also found a reduced level of sugar-induced expression of ATβ-Amy in the Landsberg erecta (L er ) ecotype compared with other ecotypes. This phenotype seemed to be due to a recessive trait, provisionally designated Iba2 , that was linked to neither erecta nor Iba1 . The Iba2 mutation also affected expression of ATβ-Amy:GUS transgene. Accumulation of starch and sugars after treatment of leaves with sucrose was not affected in the Iba1 mutant and L er plants. However, both Iba1 mutant and L er plants accumulated low levels of anthocyanin in response to sucrose, results that suggested the existence of some genetic linkage between regulation of the expression of ATβ-Amy and regulation of the accumulation of anthocyanin. Although the Iba1 and Iba2 mutations did not affect sugar-inducible gene expression in general, the expression of sugar-regulated genes other than the gene for β-amylase was differentially affected in the Iba1 mutant and L er plants. These results suggest that the sugar-regulated expression of many genes in plants might be mediated by multiple signal-transduction pathways.     

The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase.

The CLAVATA2 (CLV2) gene regulates both meristem and organ development in Arabidopsis. We isolated the CLV2 gene and found that it encodes a receptor-like protein (RLP), with a presumed extracellular domain composed of leucine-rich repeats similar to those found in plant and animal receptors, but with a very short predicted cytoplasmic tail. RLPs lacking cytoplasmic signaling domains have not been previously shown to regulate development in plants. Our prior work has demonstrated that the CLV1 receptor-like kinase (RLK) is present as a disulfide-linked multimer in vivo. We report that CLV2 is required for the normal accumulation of CLV1 protein and its assembly into protein complexes, indicating that CLV2 may form a heterodimer with CLV1 to transduce extracellular signals. Sequence analysis suggests that the charged residue in the predicted transmembrane domain of CLV2 may be a common feature of plant RLPs and RLKs. In addition, the chromosomal region in which CLV2 is located contains an extremely high rate of polymorphism, with 50 nucleotide and 15 amino acid differences between Landsberg erecta and Columbia ecotypes within the CLV2 coding sequence.     

Dominant-negative receptor uncovers redundancy in the Arabidopsis ERECTA Leucine-rich repeat receptor-like kinase signaling pathway that regulates organ shape

Arabidopsis ERECTA, a Leu-rich repeat receptor-like Ser/Thr kinase (LRR-RLK), regulates organ shape and inflorescence architecture. Here, we show that a truncated ERECTA protein that lacks the cytoplasmic kinase domain (DeltaKinase) confers dominant-negative effects when expressed under the control of the native ERECTA promoter and terminator. Transgenic plants expressing DeltaKinase displayed phenotypes, including compact inflorescence and short, blunt siliques, that are characteristic of loss-of-function erecta mutant plants. The DeltaKinase fragment migrated as a stable approximately 400-kD protein complex in the complete absence of the endogenous ERECTA protein and significantly exaggerated the growth defects of the null erecta plants. A functional LRR domain of DeltaKinase was required for dominant-negative effects. Accumulation of DeltaKinase did not interfere with another LRR-RLK signaling pathway (CLAVATA1), which operates in the same cells as ERECTA but has a distinct biological function. Both the erecta mutation and DeltaKinase expression conferred a lesser number of large, disorganized, and expanded cortex cells, which are associated with an increased level of somatic endoploidy. These findings suggest that functionally redundant RLK signaling pathways, including ERECTA, are required to fine-tune the proliferation and growth of cells in the same tissue type during Arabidopsis organogenesis.     

Both the extracellular leucine-rich repeat domain and the kinase activity of FSL2 are required for flagellin binding and signaling in Arabidopsis

In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17, which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli. This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex.     

The KDEL receptor modulates the endoplasmic reticulum stress response through mitogen-activated protein kinase signaling cascades

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) evokes the ER stress response. The resultant outcomes are cytoprotective but also proapoptotic. ER chaperones and misfolded proteins exit to the secretory pathway and are retrieved to the ER, during which process the KDEL receptor plays a significant role. Using an expression of a mutant KDEL receptor that lacks the ability for ligand recognition, we show that the impairment of retrieval by the KDEL receptor led to a mis-sorting of the immunoglobulin-binding protein BiP, an ER chaperone that has a retrieval signal from the early secretory pathway, which induced intense ER stress response and an increase in susceptibility to ER stress in HeLa cells. Furthermore, we show that the ER stress response accompanied the activation of p38 mitogen-activated protein (MAP) kinases and c-Jun amino-terminal kinases (JNKs) and that the expression of the mutant KDEL receptor suppressed the activation of p38 and JNK1 but not JNK2. The effect of the expression of the mutant KDEL receptor was consistent with the effect of a specific inhibitor for p38 MAP kinases, because the inhibitor sensitized HeLa cells to ER stress. We also found that activation of the KDEL receptor by the ligand induced the phosphorylation of p38 MAP kinases. These results indicate that the KDEL receptor participates in the ER stress response not only by its retrieval ability but also by modulating MAP kinase signaling, which may affect the outcomes of the mammalian ER stress response.     

Study of the effect of the gene ERECTA2 on the development of Arabidopsis thaliana shoot

The role of the gene ER2 in plant development has been studied by the analysis of the erecta2 (er2) mutant of Arabidopsis thaliana (L.) Heynh. It was shown that the mutation er2 provides pleiotropic effect on the development of all aboveground organs. It induces shortening and thickening of the stem, leaves and all flower organs, though it does not change the sensitivity to gibberellin. Changes in the morphology of the shoot organs are due to the changes in cell polarity. The cells get wider and shorter compared to the wild type. It was found that the gene ER2 is located in the lower arm of the chromosome 1. It complementarily interacts with the gene ER that plays an important role in the control of intercellular interactions.     

The engagement of Sec61p in the ER dislocation process

Sec61p comprises the endoplasmic reticulum (ER) channel through which nascent polypeptides are imported and from which malfolded proteins have been suggested to be exported, or dislocated, back to the cytoplasm. We have devised a genetic screen for dislocation-specific mutant alleles of SEC61 from S. cerevisiae by employing the unfolded protein response to report on the accumulation of misfolded proteins in the ER. Three of the isolated sec61 alleles are fully proficient in protein translocation into the ER, but defective in the elimination of a misfolded ER luminal substrate and a short-lived ER membrane-spanning model protein, which are otherwise rapidly degraded by cytoplasmic proteolysis in wild-type cells. Our results point to the fourth luminal loop and third transmembrane domain of Sec61p that markedly influence dislocation. We suggest that distinct features of the Sec61-translocon direct the two-way translocation processes.     

Both the Extracellular Leucine-Rich Repeat Domain and the Kinase Activity of FLS2 Are Required for Flagellin Binding and Signaling in Arabidopsis

In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17, which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli. This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex.     

Promastigote to amastigote differentiation of Leishmania is markedly delayed in the absence of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation

The parasitic protozoan Leishmania is the etiological agent of human leishmaniasis worldwide. It undergoes cellular differentiation from the sandfly promastigote form into amastigotes within mammalian macrophages, a process that is essential for its intracellular survival. Here, we characterized the Leishmania infantum PERK eIF2alpha kinase homologue and addressed its role in the parasite's cytodifferentiation. We show that Leishmania PERK is an endoplasmic reticulum (ER) transmembrane protein that largely colocalizes with the ER BiP chaperone. The Leishmania PERK catalytic kinase domain undergoes autohyperphosphorylation and phosphorylates the translation initiation factor 2-alpha subunit (eIF2alpha) in vitro at threonine 166. We also report that PERK is post-translationally regulated specifically in the intracellular stage of the parasite or under ER stress, most likely through extensive autohyperphosphorylation. We have generated a PERK dominant negative mutant overexpressing a truncated PERK protein lacking the N-terminal luminal domain and showed that this mutant is impaired in eIF2alpha phosphorylation in response to ER stress or during amastigote differentiation. Most importantly, we showed that lack of eIF2alpha phosphorylation markedly delays the Leishmania differentiation process towards amastigote forms both in parasites grown axenically or within macrophages. These data highlight the importance of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation in the intracellular development of Leishmania.     

Cloning and characterization of the histidine kinase gene<Emphasis Type="Italic"> Dic1</Emphasis> from<Emphasis Type="Italic"> Cochliobolus heterostrophus</Emphasis> that confers dicarboximide resistance and osmotic adaptation

A gene for a putative two-component histidine kinase, which is homologous to os-1 from Neurospora crassa, was cloned and sequenced from the plant-pathogenic fungus Cochliobolus heterostrophus. The predicted protein possessed the conserved histidine kinase domain, the response regulator domain, and six tandem repeats of 92-amino-acids at the N-terminal end that are found in histidine kinases from other filamentous fungi. Introduction of the histidine kinase gene complemented the deficiency of the C. heterostrophus dic1 mutant, suggesting that the Dic1 gene product is a histidine kinase. Dic1 mutants are resistant to dicarboximide and phenylpyrrole fungicides, and they are sensitive to osmotic stress. We previously classified dic1 alleles into three types, based on their phenotypes. To explain the phenotypic differences among the dic1 mutant alleles, we cloned and sequenced the mutant dic1 genes and compared their sequences with that of the wild-type strain. Null mutants for Dic1, and mutants with a deletion or point mutation in the N-terminal repeat region, were highly sensitive to osmotic stress and highly resistant to both fungicides. A single amino acid change within the kinase domain or the regulator domain altered the sensitivity to osmotic stress and conferred moderate resistance to the fungicides. These results suggest that this predicted protein, especially its repeat region, has an important function in osmotic adaptation and fungicide resistance.Communicated by C. A. M. J. J. van den Hondel     

The retrieval function of the KDEL receptor requires PKA phosphorylation of its C-terminus

The KDEL receptor is a Golgi/intermediate compartment-located integral membrane protein that carries out the retrieval of escaped ER proteins bearing a C-terminal KDEL sequence. This occurs throughout retrograde traffic mediated by COPI-coated transport carriers. The role of the C-terminal cytoplasmic domain of the KDEL receptor in this process has been investigated. Deletion of this domain did not affect receptor subcellular localization although cells expressing this truncated form of the receptor failed to retain KDEL ligands intracellularly. Permeabilized cells incubated with ATP and GTP exhibited tubular processes-mediated redistribution from the Golgi area to the ER of the wild-type receptor, whereas the truncated form lacking the C-terminal domain remained concentrated in the Golgi. As revealed with a peptide-binding assay, this domain did not interact with both coatomer and ARF-GAP unless serine 209 was mutated to aspartic acid. In contrast, alanine replacement of serine 209 inhibited coatomer/ARF-GAP recruitment, receptor redistribution into the ER, and intracellular retention of KDEL ligands. Serine 209 was phosphorylated by both cytosolic and recombinant protein kinase A (PKA) catalytic subunit. Inhibition of endogenous PKA activity with H89 blocked Golgi-ER transport of the native receptor but did not affect redistribution to the ER of a mutated form bearing aspartic acid at position 209. We conclude that PKA phosphorylation of serine 209 is required for the retrograde transport of the KDEL receptor from the Golgi complex to the ER from which the retrieval of proteins bearing the KDEL signal depends.     

In vitro phosphorylation and hormone binding activation of the synthetic wild type human estradiol receptor.

A tyrosine kinase purified from calf uterus activates the hormone binding of endogenous estradiol receptor (ER) predephosphorylated and preinactivated by a nuclear phosphotyrosine phosphatase. The kinase also activates and phosphorylates the human estradiol receptor HEO synthesized in vitro, which differs from the wild type receptor HEGO because a glycine is replaced by a valine at position 400. Moreover, the kinase activates and phosphorylates a deletion mutant of HEO which consists almost exclusively of the hormone binding domain. Using HEGO and HEO in parallel and measuring both binding activation and phosphorylation of ER we now observe that the wild type receptor is a good kinase substrate, slightly better than HEO. Furthermore, HEGO like the calf uterus receptor in the presence of estradiol, stimulates the kinase. From present findings it appears that ER and uterus tyrosine kinase are functionally associated and that this association is abolished by glycine to valine substitution at position 400 of ER.     

Gene expression profiling of yeasts overexpressing wild type or misfolded Pma1 variants reveals activation of the Hog1 MAPK pathway

Dominant negative PMA1 mutants render misfolded proteins that are retained in the endoplasmic reticulum (ER) and slowly degraded by ER-associated degradation. Accumulation of misfolded proteins in the ER activates an ER-to-nucleus signalling pathway termed the unfolded protein response (UPR). We have used a PMA1-D378T dominant negative mutant to analyse its impact on UPR induction. Our results show that overexpression of the misfolded mutant Pma1 does not lead to activation of the UPR. In addition, in mutants with a constitutively activated UPR the turnover rate of the mutant ATPase is not altered. To determine if the expression of the misfolded mutant protein induces some other kind of response we performed global gene expression analysis experiments in yeasts overexpressing either wild type or dominant lethal PMA1 alleles. The results suggest that the high osmolarity glycerol (Hog1) mitogen-activated protein kinase (MAPK) pathway is activated by both wild type and mutant ATPases. We show that expression of the PMA1 alleles induces phosphorylation of Hog1 and activation of the Hog1 MAPK cascade. This activation is mediated by the Sln1 branch of the stress-dependent Hog1 MAPK network. Finally, we show that at least two other plasma membrane proteins are also able to activate the Hog1 MAPK system.     

Cloning and characterization of MS5 from Arabidopsis : a gene critical in male meiosis

In this paper, we describe the cloning of the MS5 gene, a gene essential for male fertility in Arabidopsis . We previously defined the MS5 locus by characterizing an EMS-induced allele, ms5–1 . We identified a new allele of MS5 ( ms5–2 ) that was T-DNA-generated and used the T-DNA tag to clone the gene. Sequencing of mutant and wild-type alleles together with complementation of the ms5–1 mutant phenotype with a wild-type genomic clone confirmed the identity of the gene. Differences between the phenotypes of the two mutant alleles could be attributed to differences in mutant gene structure. The semi-dominant and dominant negative phenotypes of the ms5–2 mutant probably result from production of a truncated polypeptide. An unknown locus in Landsberg erecta can counteract the dominant negative phenotype of ms5–2 . Mutations in MS5 cause the formation of ‘polyads’– tetrads with more than four pools of chromosomes after male meiosis. Similarities between the MS5 sequence and that of a number of proteins were found; two that may be significant were with a synaptonemal complex protein and with a regulatory subunit of a cyclin-dependent kinase. The MS5 gene is a member of a small gene family highly conserved amongst plant species.     

The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmic reticulum retrieval in yeast

Sec22p is an endoplasmic reticulum (ER)-Golgi v-SNARE protein whose retrieval from the Golgi compartment to the endoplasmic reticulum (ER) is mediated by COPI vesicles. Whether Sec22p exhibits its primary role at the ER or the Golgi apparatus is still a matter of debate. To determine the role of Sec22p in intracellular transport more precisely, we performed a synthetic lethality screen. We isolated mutant yeast strains in which SEC22 gene function, which in a wild type strain background is non-essential for cell viability, has become essential. In this way a novel temperature-sensitive mutant allele, dsl1-22, of the essential gene DSL1 was obtained. The dsl1-22 mutation causes severe defects in Golgi-to-ER retrieval of ER-resident SNARE proteins and integral membrane proteins harboring a C-terminal KKXX retrieval motif, as well as of the soluble ER protein BiP/Kar2p, which utilizes the HDEL receptor, Erd2p, for its recycling to the ER. DSL1 interacts genetically with mutations that affect components of the Golgi-to-ER recycling machinery, namely sec20-1, tip20-5, and COPI-encoding genes. Furthermore, we demonstrate that Dsl1p is a peripheral membrane protein, which in vitro specifically binds to coatomer, the major component of the protein coat of COPI vesicles.     

A novel positive feedback loop mediated by the docking protein Gab1 and phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4, 5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR.     

Dsl1p, an essential protein required for membrane traffic at the endoplasmic reticulum/Golgi interface in yeast

To identify novel factors required for ER to Golgi transport in yeast we performed a screen for genes that, when mutated, confer a dependence on a dominant mutant form of the ER to Golgi vesicle docking factor Sly1p, termed Sly1-20p. DSL1 , a novel gene isolated in the screen, encodes an essential protein with a predicted molecular mass of 88 kDa. DSL1 is required for transport between the ER and the Golgi because strains bearing mutant alleles of this gene accumulate the pre-Golgi form of transported proteins at the restrictive temperature. Two strains bearing temperature-sensitive alleles of DSL1 display distinct phenotypes as observed by electron microscopy at the restrictive temperature; although both strains accumulate ER membrane, one also accumulates vesicles. Interestingly, the inviability of strains bearing several mutant alleles of DSL1 can be suppressed by expression of either Erv14p (a protein required for the movement of a specific protein from the ER to the Golgi), Sec21p (the γ-subunit of the COPI coat protein complex coatomer), or Sly1-20p. Because the strongest suppressor is SEC21 , we proposed that Dsl1p functions primarily in retrograde Golgi to ER traffic, although it is possible that Dsl1p functions in anterograde traffic as well, perhaps at the docking stage, as suggested by the suppression by SLY1-20 .