scholarly journals Unfolded protein-independent IRE1 activation contributes to multifaceted developmental processes in Arabidopsis

2019 ◽  
Vol 2 (5) ◽  
pp. e201900459 ◽  
Author(s):  
Kei-ichiro Mishiba ◽  
Yuji Iwata ◽  
Tomofumi Mochizuki ◽  
Atsushi Matsumura ◽  
Nanami Nishioka ◽  
...  
Keyword(s):  
Male Gametophyte ◽  
Pollen Grains ◽  
Activation Mechanism ◽  
Alternative Activation ◽  
Developmental Defect ◽  
Mutant Form ◽  
Triple Mutant ◽  
Developmental Defects ◽  
Unfolded Protein

In Arabidopsis, the IRE1A and IRE1B double mutant (ire1a/b) is unable to activate cytoplasmic splicing of bZIP60 mRNA and regulated IRE1-dependent decay under ER stress, whereas the mutant does not exhibit severe developmental defects under normal conditions. In this study, we focused on the Arabidopsis IRE1C gene, whose product lacks a sensor domain. We found that the ire1a/b/c triple mutant is lethal, and heterozygous IRE1C (ire1c/+) mutation in the ire1a/b mutants resulted in growth defects and reduction of the number of pollen grains. Genetic analysis revealed that IRE1C is required for male gametophyte development in the ire1a/b mutant background. Expression of a mutant form of IRE1B that lacks the luminal sensor domain (ΔLD) complemented a developmental defect in the male gametophyte in ire1a/b/c haplotype. In vivo, the ΔLD protein was activated by glycerol treatment that increases the composition of saturated lipid and was able to activate regulated IRE1-dependent decay but not bZIP60 splicing. These observations suggest that IRE1 contributes to plant development, especially male gametogenesis, using an alternative activation mechanism that bypasses the unfolded protein-sensing luminal domain.

scholarly journals Unfolded protein-independent IRE1 activation contributes to multifaceted developmental processes in Arabidopsis

2019 ◽  
Author(s):  
Kei-ichiro Mishiba ◽  
Yuji Iwata ◽  
Tomofumi Mochizuki ◽  
Atsushi Matsumura ◽  
Nanami Nishioka ◽  
...  
Keyword(s):  
Er Stress ◽  
Male Gametophyte ◽  
Activation Mechanism ◽  
Alternative Activation ◽  
Developmental Defect ◽  
Mutant Form ◽  
Triple Mutant ◽  
Unfolded Protein ◽  
Stress Dependent

AbstractAs an initial step for the unfolded protein response (UPR) pathway, the luminal domain of inositol requiring enzyme 1 (IRE1) senses unfolded proteins in the endoplasmic reticulum (ER). Recent findings in yeast and metazoans suggest alternative IRE1 activation without the sensor domain, although its mechanism and physiological significance remain to be elucidated. In Arabidopsis, the IRE1A and IRE1B double mutant (ire1a/b) is unable to activate cytoplasmic splicing of bZIP60 mRNA and regulated IRE1-dependent decay (RIDD) under ER stress, while the mutant does not exhibit severe developmental defects and is fertile under non-stress conditions. In this study, we focused on a third Arabidopsis IRE1 gene, designated as IRE1C, whose product lacks a sensor domain. We found that even though ire1c and ire1a/c mutants did not exhibit defective bZIP60 splicing and RIDD under ER stress, the ire1a/b/c triple mutant is lethal. Heterozygous IRE1C (ire1c/+) mutation in the ire1a/b mutants resulted in growth defects and reduction of the number of pollen grains. Genetic analysis revealed that IRE1C is required for male gametophyte development in the ire1a/b mutant background. Expression of a mutant form of IRE1B that lacks the luminal sensor domain (ΔLD) in the ire1a/b mutant did not complement defects in ER stress-dependent bZIP60 splicing and RIDD. Nevertheless, expression of ΔLD complemented a developmental defect in the male gametophyte in ire1a/b/c haplotype. In vivo, the ΔLD protein was activated by glycerol treatment that increases the composition of saturated lipid and was able to activate RIDD but not bZIP60 splicing. Phenotypes of IRE1B mutants lacking the sensor domain produced by CRISPR/Cas9-mediated gene editing in the ire1a/c mutant background were essentially same as those of ΔLD-expressing ire1a/b mutant. These observations suggest that IRE1 contributes to plant development, especially male gametogenesis, using an alternative activation mechanism that bypasses the unfolded protein-sensing luminal domain.

scholarly journals Multiple autophosphorylation sites are dispensable for murine ATM activation in vivo

2008 ◽  
Vol 183 (5) ◽  
pp. 777-783 ◽  
Author(s):  
Jeremy A. Daniel ◽  
Manuela Pellegrini ◽  
Ji-Hoon Lee ◽  
Tanya T. Paull ◽  
Lionel Feigenbaum ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Cycle Checkpoints ◽  
Activation Mechanism ◽  
Mutant Form ◽  
Dna Double Strand Breaks ◽  
Atm Kinase ◽  
Strand Breaks ◽  
Physiological Importance ◽  
Evolutionarily Conserved

Cellular responses to both physiological and pathological DNA double-strand breaks are initiated through activation of the evolutionarily conserved ataxia telangiectasia mutated (ATM) kinase. Upon DNA damage, an activation mechanism involving autophosphorylation has been reported to allow ATM to phosphorylate downstream targets important for cell cycle checkpoints and DNA repair. In humans, serine residues 367, 1893, and 1981 have been shown to be autophosphorylation sites that are individually required for ATM activation. To test the physiological importance of these sites, we generated a transgenic mouse model in which all three conserved ATM serine autophosphorylation sites (S367/1899/1987) have been replaced with alanine. In this study, we show that ATM-dependent responses at both cellular and organismal levels are functional in mice that express a triple serine mutant form of ATM as their sole ATM species. These results lend further support to the notion that ATM autophosphorylation correlates with the DNA damage–induced activation of the kinase but is not required for ATM function in vivo.

scholarly journals Maize ROP2 GTPase Provides a Competitive Advantage to the Male Gametophyte

Genetics ◽  
2003 ◽  
Vol 165 (4) ◽  
pp. 2137-2151 ◽  
Author(s):  
K M Arthur ◽  
Z Vejlupkova ◽  
R B Meeley ◽  
J E Fowler
Keyword(s):  
Male Gametophyte ◽  
Transmission Rate ◽  
Pollen Grains ◽  
Duplicate Gene ◽  
Wild Type ◽  
Allelic Series ◽  
Plant Signal Transduction ◽  
Relative Transmission ◽  
Mutant Pollen

Abstract Rop GTPases have been implicated in the regulation of plant signal transduction and cell morphogenesis. To explore ROP2 function in maize, we isolated five Mutator transposon insertions (rop2::Mu alleles). Transmission frequency through the male gametophyte, but not the female, was lower than expected in three of the rop2::Mu mutants. These three alleles formed an allelic series on the basis of the relative transmission rate of each when crossed as trans-heterozygotes. A dramatic reduction in the level of ROP2-mRNA in pollen was associated with the three alleles causing a transmission defect, whereas a rop2::Mu allele that did not result in a defect had wild-type transcript levels, thus confirming that mutation of rop2 causes the mutant phenotype. These data strongly support a role for rop2 in male gametophyte function, perhaps surprisingly, given the expression in pollen of the nearly identical duplicate gene rop9. However, the transmission defect was apparent only when a rop2::Mu heterozygote was used as the pollen donor or when a mixture of wild-type and homozygous mutant pollen was used. Thus, mutant pollen is at a competitive disadvantage compared to wild-type pollen, although mutant pollen grains lacked an obvious cellular defect. Our data demonstrate the importance in vivo of a specific Rop, rop2, in the male gametophyte.

scholarly journals AtPIG-S, a predicted Glycosylphosphatidylinositol Transamidase Subunit, is critical for pollen tube growth in Arabidopsis

2020 ◽  
Author(s):  
Nick Desnoyer ◽  
Greg Howard ◽  
Emma Jong ◽  
Ravishankar Palanivelu
Keyword(s):  
Pollen Tube ◽  
Male Fertility ◽  
Male Gametophyte ◽  
Pollen Grains ◽  
Unexpected Finding ◽  
Gpi Anchor ◽  
Developmental Defects ◽  
Post Translational Modifications ◽  
Gpi Transamidase

AbstractBackgroundGlycosylphosphatidylinositol (GPI) addition is one of the several post-translational modifications to proteins that increase their affinity for membranes. In eukaryotes, the GPI transamidase complex (GPI-T) catalyzes the attachment of pre-assembled GPI anchors to GPI-anchored proteins (GAPs) through a transamidation reaction. A mutation in AtGPI8 (gpi8-2), the putative catalytic subunit of GPI-T in Arabidopsis, is transmitted normally through the female gametophyte (FG), indicating the FG tolerates loss of GPI transamidation. In contrast, gpi8-2 almost completely abolishes male gametophyte (MG) function. Still, the unexpected finding that gpi8-2 FGs function normally requires further investigation. Additionally, specific developmental defects in the MG caused by loss of GPI transamidation remain poorly characterized.ResultsHere we investigated the effect of loss of AtPIG-S, another GPI-T subunit, in both gametophytes. Like gpi8-2, we showed that a mutation in AtPIG-S (pigs-1) disrupted synergid localization of LORELEI (LRE), a putative GAP critical for pollen tube reception by the FG, yet is transmitted normally through the FG. Conversely, pigs-1 severely impaired male gametophyte (MG) function during pollen tube emergence and growth in the pistil. A pPIGS:PIGS-GFP transgene complemented these MG defects and enabled generation of pigs-1/pigs-1 seedlings, but seemingly failed to rescue the function of AtPIG-S in the sporophyte, as pigs-1/pigs-1, pPIGS:PIGS-GFP seedlings died soon after germination.ConclusionsCharacterization of pigs-1 provided further evidence that the FG tolerates loss of GPI transamidation more than the MG and that the MG compared to the FG may be a better haploid system to study the role of GPI-anchoring. pigs-1 pollen develops normally and thus represent a tool in which GPI anchor biosynthesis and transamidation of GAPs have been uncoupled, offering a potential way to study free GPI in plant development. While previously reported male fertility defects of GPI biosynthesis mutants could have been due either to loss of GPI or GAPs lacking the GPI anchor, our results clarified that the loss of mature GAPs underlie male fertility defects of GPI-deficient pollen grains, as pigs-1 is defective only in the downstream transamidation step. Our study also provided further evidence that GPI transamidation is essential in seedling development.

Characterization of self-incompatibility in Campanula rapunculoides (Campanulaceae) through genetic analyses and microscopy

Botany ◽  
2008 ◽  
Vol 86 (1) ◽  
pp. 1-13 ◽  
Author(s):  
S. V. Good-Avila ◽  
D. Majumder ◽  
H. Amos ◽  
A. G. Stephenson
Keyword(s):  
Genetic Basis ◽  
Male Gametophyte ◽  
Single Gene ◽  
Pollen Grains ◽  
Diallel Analysis ◽  
Pollen Tubes ◽  
Self Incompatibility ◽  
The Family

In this paper, we seek to identify the genetic basis of self-incompatibility (SI) in Campanula rapunculoides L. through diallel analysis of full siblings; to characterize the growth of pollen tubes in vivo after incompatible and compatible pollination; and to determine whether the SI system is based on pistil S-RNases. Pollinations were performed among individuals from five diallel crosses and scored for both fruit set and pollen-tube growth to determine the genetic basis of SI. On a subset of these individuals with known cross-(in)compatibility relationships, additional crosses were performed and pistils collected 1, 3, 6, 12, and 24 h after pollination to assess both the percentage of pollen grains that had germinated on the stigma, and the number of pollen tubes that had grown 20%, 40% 60%, 80%, and 100% of the distance down the pistil over five time intervals. Finally, total pistillate proteins were extracted and subjected to isoelectric focusing and RNase activity staining to find evidence of a highly basic S-RNases associated with SI in the Solanaceae. We found evidence that the SI system was based on the haplotype of the male gametophyte, and was not sporophytic. Protein analyses showed that SI was not based on a pistillate S-RNase. The existence of modifiers of SI and possible polyploidy at the S-locus complicated the expression of SI in this species, and single-gene inheritance could not be determined. This represents the first published characterization of incompatibility in the family Campanulaceae.

scholarly journals Activation of AtMPK9 through autophosphorylation that makes it independent of the canonical MAPK cascades

2015 ◽  
Vol 467 (1) ◽  
pp. 167-175 ◽  
Author(s):  
Szilvia K. Nagy ◽  
Zsuzsanna Darula ◽  
Brigitta M. Kállai ◽  
László Bögre ◽  
Gábor Bánhegyi ◽  
...  
Keyword(s):  
Mitogen Activated Protein Kinase ◽  
Activation Mechanism ◽  
Mutant Form ◽  
Mapk Cascades ◽  
Dual Specificity ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Mapk Kinases

Mitogen-activated protein kinases (MAPKs) are part of conserved signal transduction modules in eukaryotes that are typically organized into three-tiered kinase cascades. The activation of MAPKs in these pathways is fully dependent on the bisphosphorylation of the TXY motif in the T-loop by the pertinent dual-specificity MAPK kinases (MAPKKs). The Arabidopsis mitogen-activated protein kinase 9 (AtMPK9) is a member of an atypical class of MAPKs. Representatives of this MAPK family have a TDY phosphoacceptor site, a long C-terminal extension and lack the common MAPKK-binding docking motif. In the present paper, we describe multiple in vitro and in vivo data showing that AtMPK9 is activated independently of any upstream MAPKKs but rather is activated through autophosphorylation. We mapped the autophosphorylation sites by MS to the TDY motif and to the C-terminal regulatory extension. We mutated the phosphoacceptor sites on the TDY, which confirmed the requirement for bisphorylation at this site for full kinase activity. Next, we demonstrated that the kinase-inactive mutant form of AtMPK9 is not trans-phosphorylated on the TDY site when mixed with an active AtMPK9, implying that the mechanism of the autocatalytic phosphorylation is intramolecular. Furthermore, we show that in vivo AtMPK9 is activated by salt and is regulated by okadaic acid-sensitive phosphatases. We conclude that the plant AtMPK9 shows similarities to the mammalian atypical MAPKs, such as extracellular-signal-regulated kinase (ERK) 7/8, in terms of an MAPKK-independent activation mechanism.

Pollen germination of Tsuga heterophylla in vitro

1971 ◽  
Vol 49 (1) ◽  
pp. 117-119 ◽  
Author(s):  
RongHui Ho ◽  
Oscar Sziklai
Keyword(s):  
Male Gametophyte ◽  
Generative Cell ◽  
Calcium Nitrate ◽  
Pollen Grains ◽  
Potassium Nitrate ◽  
Tsuga Heterophylla ◽  
The Body ◽  
Stalk Cell

The development of the male gametophyte from western hemlock (Tsuga heterophylla (Raf.) Sarg.) pollen was complete after 5 days of incubation. This normally takes at least 6 weeks in vivo. The pollen was cultured in a solution containing boron, calcium nitrate, magnesium sulfate, and potassium nitrate. In 2 days the generative cell divided into the body cell and the stalk cell and after a further 3 days the body nucleus divided into two sperm nuclei. Morphological descriptions and measurements of the germinating pollen grains were made.

scholarly journals Polymorphisms in Plasmodium falciparum dihydropteroate synthetase and dihydrofolate reductase genes in Nigerian children with uncomplicated malaria using high-resolution melting technique

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Adeyemi T. Kayode ◽  
Fehintola V. Ajogbasile ◽  
Kazeem Akano ◽  
Jessica N. Uwanibe ◽  
Paul E. Oluniyi ◽  
...  
Keyword(s):  
High Resolution ◽  
Dihydrofolate Reductase ◽  
Uncomplicated Malaria ◽  
High Resolution Melting ◽  
Intermittent Preventive Treatment ◽  
Preventive Treatment ◽  
Triple Mutant ◽  
Seasonal Malaria Chemoprevention

AbstractIn 2005, the Nigerian Federal Ministry of Health revised the treatment policy for uncomplicated malaria with the introduction of artemisinin-based combination therapies (ACTs). This policy change discouraged the use of Sulphadoxine-pyrimethamine (SP) as the second-line treatment of uncomplicated falciparum malaria. However, SP is used as an intermittent preventive treatment of malaria in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children aged 3–59 months. There have been increasing reports of SP resistance especially in the non-pregnant population in Nigeria, thus, the need to continually monitor the efficacy of SP as IPTp and SMC by estimating polymorphisms in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes associated with SP resistance. The high resolution-melting (HRM) assay was used to investigate polymorphisms in codons 51, 59, 108 and 164 of the dhfr gene and codons 437, 540, 581 and 613 of the dhps gene. DNA was extracted from 271 dried bloodspot filter paper samples obtained from children (< 5 years old) with uncomplicated malaria. The dhfr triple mutant I51R59N108, dhps double mutant G437G581 and quadruple dhfr I51R59N108 + dhps G437 mutant haplotypes were observed in 80.8%, 13.7% and 52.8% parasites, respectively. Although the quintuple dhfr I51R59N108 + dhps G437E540 and sextuple dhfr I51R59N108 + dhps G437E540G581 mutant haplotypes linked with in-vivo and in-vitro SP resistance were not detected, constant surveillance of these haplotypes should be done in the country to detect any change in prevalence.

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