Sung Aeong Oh

ORE9, an F-Box Protein That Regulates Leaf Senescence in Arabidopsis

Senescence is a sequence of biochemical and physiological events that constitute the final stage of development. The identification of genes that alter senescence has practical value and is helpful in revealing pathways that influence senescence. However, the genetic mechanisms of senescence are largely unknown. The leaf of the oresara9 (ore9) mutant of Arabidopsis exhibits increased longevity during age-dependent natural senescence by delaying the onset of various senescence symptoms. It also displays delayed senescence symptoms during hormone-modulated senescence. Map-based cloning of ORE9 identified a 693–amino acid polypeptide containing an F-box motif and 18 leucine-rich repeats. The F-box motif of ORE9 interacts with ASK1 (Arabidopsis Skp1-like 1), a component of the plant SCF complex. These results suggest that ORE9 functions to limit leaf longevity by removing, through ubiquitin-dependent proteolysis, target proteins that are required to delay the leaf senescence program in Arabidopsis.

A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence

We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5′-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2–3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1 mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl2 which delayed loss of chlorophyll but not that of photochemical efficiency.

Differential expression of senescence-associated mRNAs during leaf senescence induced by different senescence-inducing factors in Arabidopsis

Four cDNA clones, named pSEN2, pSEN3, pSEN4, and pSEN5, for mRNAs induced during leaf senescence in Arabidopsis thaliana were characterized. The clones were isolated from a cDNA library of detached leaves incubated in darkness for 2 days to accelerate senescence, first by differential screening and then by examining expression of the primarily screened clones during age-dependent leaf senescence. Transcript levels detected by these cDNA clones, thus, were up-regulated in an age-dependent manner and during dark-induced leaf senescence. In contrast, when leaf senescence was induced by ethylene, ABA or methyl jasmonate, the transcript level detected by the clones was differentially regulated depending on the senescence-inducing hormones. The transcript level for pSEN4 increased during senescence induced by all three hormones, while the transcript detected by the pSEN2 clone did not increase during senescence induced by ethylene. The transcript level for pSEN5 was increased upon ABA-induced senescence but decreased during ethylene-induced senescence. The pSEN3 clone detected multiple transcripts that are differentially regulated by these factors. The results show that, although the apparent senescence symptoms of Arabidopsis leaf appear similar regardless of the senescence-inducing factors, the detailed molecular state of leaf cells during senescence induced by different senescence-inducing factors is different. The pSEN3 clone encodes a polyubiquitin and the pSEN4 clone encodes a peptide related to endoxyloglucan transferase. This result is consistent with the expected roles of senescence-induced genes during leaf senescence.

Control of plant germline proliferation by SCF FBL17 degradation of cell cycle inhibitors

Flowering plants possess a unique reproductive strategy, involving double fertilization by twin sperm cells. Unlike animal germ lines, the male germ cell lineage in plants only forms after meiosis and involves asymmetric division of haploid microspores, to produce a large, non-germline vegetative cell and a germ cell that undergoes one further division to produce the twin sperm cells. Although this switch in cell cycle control is critical for sperm cell production and delivery, the underlying molecular mechanisms are unknown. Here we identify a novel F-box protein of Arabidopsis thaliana, designated FBL17 (F-box-like 17), that enables this switch by targeting the degradation of cyclin-dependent kinase A;1 inhibitors specifically in male germ cells. We show that FBL17 is transiently expressed in the male germ line after asymmetric division and forms an SKP1–Cullin1–F-box protein (SCF) E3 ubiquitin ligase complex (SCFFBL17) that targets the cyclin-dependent kinase inhibitors KRP6 and KRP7 for proteasome-dependent degradation. Accordingly, the loss of FBL17 function leads to the stabilization of KRP6 and inhibition of germ cell cycle progression. Our results identify SCFFBL17 as an essential male germ cell proliferation complex that promotes twin sperm cell production and double fertilization in flowering plants.

A divergent cellular role for the FUSED kinase family in the plant-specific cytokinetic phragmoplast.

The FUSED (FU) Ser/Thr protein kinase family has a key role in the hedgehog signaling pathway known to control cell proliferation and patterning in fruit flies and humans . The genomes of Arabidopsis thaliana and rice each encode a single Fu ortholog, but their role is unknown. Here, we show that cytokinesis-defective mutants, which we named two-in-one (tio), result from mutations in Arabidopsis Fu. Phenotypic analysis of tio mutants reveals an essential role for TIO in conventional modes of cytokinesis in plant meristems and during male gametogenesis. TIO also has a key role in nonconventional modes of cytokinesis (cellularization) during female gametogenesis. We demonstrate that TIO is tightly localized to the midline of the nascent phragmoplast and remains associated with the expanding phragmoplast ring. These data reveal the evolution of a divergent role for the Fu kinase family as an essential phragmoplast-associated protein that functions in different cell type-specific modes of cytokinesis in plants.

Endoplasmic Reticulum– and Golgi-Localized Phospholipase A2 Plays Critical Roles in Arabidopsis Pollen Development and Germination

This study shows that Arabidopsis PLA2-γ and -δ, which are specifically expressed in pollen, localize to the endoplasmic reticulum and/or Golgi and that the suppression of PLA2s disrupts the endomembrane and induces pollen collapse. The PLA2 product, 18-1:LPE, was found to be required for pollen tube germination. The phospholipase A2 (PLA2) superfamily of lipolytic enzymes is involved in a number of essential biological processes, such as inflammation, development, host defense, and signal transduction. Despite the proven involvement of plant PLA2s in many biological functions, including senescence, wounding, elicitor and stress responses, and pathogen defense, relatively little is known about plant PLA2s, and their genes essentially remain uncharacterized. We characterized three of four Arabidopsis thaliana PLA2 paralogs (PLA2-β, -γ, and -δ) and found that they (1) are expressed during pollen development, (2) localize to the endoplasmic reticulum and/or Golgi, and (3) play critical roles in pollen development and germination and tube growth. The suppression of PLA2 using the RNA interference approach resulted in pollen lethality. The inhibition of pollen germination by pharmacological PLA2 inhibitors was rescued by a lipid signal molecule, lysophosphatidyl ethanolamine. Based on these results, we propose that plant reproduction, in particular, male gametophyte development, requires the activities of the lipid-modifying PLA2s that are conserved in other organisms.

The tobacco MAP215/Dis1-family protein TMBP200 is required for the functional organization of microtubule arrays during male germline establishment

The haploid microspore division during pollen development in flowering plants is an intrinsically asymmetric division which establishes the male germline for sexual reproduction. Arabidopsis gem1 mutants lack the male germline as a result of disturbed microspore polarity, division asymmetry, and cytokinesis and represent loss-of-function mutants in MOR1/GEM1, a plant orthologue of the conserved MAP215/Dis1 microtubule associated protein (MAP) family. This provides genetic evidence for the role of MAP215/Dis1 in the organization of gametophytic microtubule arrays, but it has remained unknown how microtubule arrays are affected in gem1 mutant microspores. Here, novel male gametophytic microtubule-reporter Nicotiana tabacum plants were constructed, expressing a green fluorescent protein-α-TUBULIN fusion protein (GFP-TUA6) under the control of a microspore-specific promoter. These plants allow effective visualization of all major male gametophytic microtubule arrays and provide useful tools to study the regulation of microtubule arrays by MAPs and other effectors. Depletion of TMBP200, a tobacco homologue of MOR1/GEM1 in gametophytic microtubule-reporter plants using microspore-targeted RNA interference, induced defects in microspore polarity, division asymmetry and cytokinesis that were associated with striking defects in phragmoplast position, orientation, and structure. Our observations further reveal a requirement for TMBP200 in gametophytic spindle organization and a novel role in spindle position and orientation in polarized microspores. These results provide direct evidence for the function of MAP215/Dis1 family protein TMBP200 in the organization of microtubule arrays critical for male germline formation in plants.

Arabidopsis Fused kinase and the Kinesin-12 subfamily constitute a signalling module required for phragmoplast expansion

The conserved Fused kinase plays vital but divergent roles in many organisms from Hedgehog signalling in Drosophila to polarization and chemotaxis in Dictyostelium. Previously we have shown that Arabidopsis Fused kinase termed TWO-IN-ONE (TIO) is essential for cytokinesis in both sporophytic and gametophytic cell types. Here using in vivo imaging of GFP-tagged microtubules in dividing microspores we show that TIO is required for expansion of the phragmoplast. We identify the phragmoplast-associated kinesins, PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B, as TIO-interacting proteins and determine TIO-Kinesin-12 interaction domains and their requirement in male gametophytic cytokinesis. Our results support the role of TIO as a functional protein kinase that interacts with Kinesin-12 subfamily members mainly through the C-terminal ARM repeat domain, but with a contribution from the N-terminal kinase domain. The interaction of TIO with Kinesin proteins and the functional requirement of their interaction domains support the operation of a Fused kinase signalling module in phragmoplast expansion that depends upon conserved structural features in diverse Fused kinases.

Insulin-induced maturation of Xenopus oocytes is inhibited by microinjection of a Brassica napus cDNA clone with high similarity to a mammalian receptor for activated protein kinase C

A cDNA clone encoding a WD-40 repeat protein (BGB1) was characterized in Brassica napus L. The clone contained an open reading frame of 327 amino acid residues almost entirely composed of seven segments of WD-40 repeats. Among the WD-40 repeat proteins, BGB1 showed high similarity (63% identity) to a rat intracellular receptor for protein kinase C (RACK1) that functions in the translocation of activated protein kinase C (PKC) from the cytosolic fraction to the membrane fraction. BGB1 also had two sequence motifs involved in binding of RACK1 to PKC. The cDNA clone, when carried in a Xenopus oocyte expression vector and injected into Xenopus laevis oocytes, inhibited insulin-induced maturation of the oocytes, a PKC-mediated pathway, and this inhibition was accompanied by reduction of PKC in the membrane fraction, as in the case of mammalian RACKs. The data show that BGB1 shares some common functional characteristics with the mammalian RACK1 along with the structural similarity, suggesting that a mammalian RACK1-related cellular process might be operating in plants. Southern blot analyses of the genome of B. napus and Arabidopsis thaliana (L.) Heynh. revealed that BGB1-related genes constitute a small multigene family in both species. An approximately 1.4-kb transcript was constitutively expressed in all organs examined.

The promoter activity of sen 1, a senescence-associated gene of Arabidopsis, is repressed by sugars

Summary A senescence-associated gene of Arabidopsis thaliana, sen 1 , was previously shown to be distinctively regulated by several senescence-inducing factors including dark treatment. In this study, the promoter region of the sen 1 gene was fused to the β-glucuronidase (GUS) reporter gene and introduced into tobacco plants to examine the regulation mechanism of this gene by dark treatment. The sen 1 gene expression was highly induced by dark and abscisic acid treatment in transgenic tobacco as in Arabidopsis . The promoter activity was rapidly induced by dark treatment but the values of senescence parameters (chlorophyll content, photochemical efficiency, soluble protein content) changed only slightly during dark incubation for 4 days, showing that dark activation of the senl promoter was not closely associated with major symptoms of leaf senescence. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) treatment of the excised transgenic tobacco leaves in light caused activation of the sen 1 promoter and this was concomitant with a decrease in sugar levels, indicating that sugar content may be related to the regulation of the senl promoter. Darkinduced activation of the sen 1 promoter was highly suppressed by addition of exogenous sucrose at physiological concentrations. Glucose and fructose were also effective in suppressing the activity of the sen 1 promoter. However, mannitol or 3-O-methyl-D-glucose, a non-metabolizable analog of glucose did not suppress the promoter activity. We further confirmed that the dark activation of the sen 1 promoter was associated with a decrease in sugar content. Our results clearly demonstrate that the promoter of the sen 1 gene is negatively regulated by sugar and the dark-induced activation of the sen 1 promoter is due to release of the suppression of the sen 1 promoter activity by sugar. These data imply that the senescence-associated gene, sen 1 , is activated upon sugar starvation, recognizing sugar starvation as one of senescence signals.