Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells

The yeast SKP1 gene and its human homolog p19 ^skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle. In orchids we identified a cDNA (O108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1. Based on the orchid O108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh. cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1. The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1. The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia. In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium. Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity. Received: 6 June 1997 / Accepted: 2 July 1997     

Phytophthora capsici homologue of the cell cycle regulator SDA1 is required for sporangial morphology,mycelial growth and plant infection

SDA1 encodes a highly conserved protein that is widely distributed in eukaryotic organisms. SDA1 is essential for cell cycle progression and organization of the actin cytoskeleton in yeasts and humans. In this study, we identified a Phytophthora capsici orthologue of yeast SDA1, named PcSDA1. In P. capsici, PcSDA1 is strongly expressed in three asexual developmental states (mycelium, sporangia and germinating cysts), as well as late in infection. Silencing or overexpression of PcSDA1 in P. capsici transformants affected the growth of hyphae and sporangiophores, sporangial development, cyst germination and zoospore release. Phalloidin staining confirmed that PcSDA1 is required for organization of the actin cytoskeleton. Moreover, 4′,6‐diamidino‐2‐phenylindole (DAPI) staining and PcSDA1‐green fluorescent protein (GFP) fusions revealed that PcSDA1 is involved in the regulation of nuclear distribution in hyphae and sporangia. Both silenced and overexpression transformants showed severely diminished virulence. Thus, our results suggest that PcSDA1 plays a similar role in the regulation of the actin cytoskeleton and nuclear division in this filamentous organism as in non‐filamentous yeasts and human cells.     

The Arabidopsis SMO2, a homologue of yeast TRM112, modulates progression of cell division during organ growth

Cell proliferation is integrated into developmental progression in multicellular organisms, including plants, and the regulation of cell division is of pivotal importance for plant growth and development. Here, we report the identification of an Arabidopsis SMALL ORGAN 2 (SMO2) gene that functions in regulation of the progression of cell division during organ growth. The smo2 knockout mutant displays reduced size of aerial organs and shortened roots, due to the decreased number of cells in these organs. Further analyses reveal that disruption of SMO2 does not alter the developmental timing but reduces the rate of cell production during leaf and root growth. Moreover, smo2 plants exhibit a constitutive activation of cell cycle‐related genes and over‐accumulation of cells expressing CYCB1;1:β‐glucuronidase (CYCB1;1:GUS) during organogenesis, suggesting that smo2 has a defect in G₂–M phase progression in the cell cycle. SMO2 encodes a functional homologue of yeast TRM112, a plurifunctional component involved in a few cellular events, including tRNA and protein methylation. In addition, the mutation of SMO2 does not appear to affect endoreduplication in Arabidopsis leaf cells. Taken together we postulate that Arabidopsis SMO2 is a conserved yeast TRM112 homologue and SMO2‐mediated cellular events are required for proper progression of cell division in plant growth and development.     

Functional characterization of the Arabidopsis eukaryotic translation initiation factor 5A-2 that plays a crucial role in plant growth and development by regulating cell division, cell growth, and cell death

The eukaryotic translation initiation factor 5A (eIF-5A) is a highly conserved protein found in all eukaryotic organisms. Although originally identified as a translation initiation factor, recent studies in mammalian and yeast (Saccharomyces cerevisiae) cells suggest that eIF-5A is mainly involved in RNA metabolism and trafficking, thereby regulating cell proliferation, cell growth, and programmed cell death. In higher plants, the physiological function of eIF-5A remains largely unknown. Here, we report the identification and characterization of an Arabidopsis (Arabidopsis thaliana) mutant fumonisin B(1)-resistant12 (fbr12). The fbr12 mutant shows an antiapoptotic phenotype and has reduced dark-induced leaf senescence. Moreover, fbr12 displays severe defects in plant growth and development. The fbr12 mutant plant is extreme dwarf with substantially reduced size and number of all adult organs. During reproductive development, fbr12 causes abnormal development of floral organs and defective sporogenesis, leading to the abortion of both female and male germline cells. Microscopic studies revealed that these developmental defects are associated with abnormal cell division and cell growth. Genetic and molecular analyses indicated that FBR12 encodes a putative eIF-5A-2 protein. When expressed in a yeast mutant strain carrying a mutation in the eIF-5A gene, FBR12 cDNA is able to rescue the lethal phenotype of the yeast mutant, indicating that FBR12 is a functional eIF-5A. We propose that FBR12/eIF-5A-2 is fundamental for plant growth and development by regulating cell division, cell growth, and cell death.     

Trypanosome TOR as a major regulator of cell growth and autophagy

Trypanosomatid protozoa parasites are responsible for tropical diseases, and undergo complex life cycles involving developmental forms adapted to insect vectors and vertebrate hosts. During their life cycle these parasites proceed through different forms in response to dramatic environmental changes and/or developmentally regulated programs. Successful progression of the parasite through its life cycle is highly dependent on the capacity of adaptation to distinct stresses involving processes such as autophagy. In eukaryotes, Target Of Rapamycin (TOR) protein kinases act as a sensor, which integrates the nutritional and energetic status, adjusting cell metabolism and growth. Compromising cell viability in yeast and mammals leads to a reduction of TOR function, triggering processes aimed to overcome unfavourable conditions. This is partly achieved by TOR-mediated regulation of protein synthesis and recycling of cellular components by autophagy. In the last few years, autophagy has been described during developmental differentiation processes in Trypanosomatids. However, no link between TOR signalling, autophagy and differentiation has been described so far. This addendum is a commentary to the work published by our group,¹ where we discuss the possible role of TOR kinases, as a controller of cell growth and autophagy, in the regulation of differentiation processes during Trypanosomatids life cycles.     

The TIP GROWTH DEFECTIVE1 S-acyl transferase regulates plant cell growth in Arabidopsis

TIP GROWTH DEFECTIVE1 (TIP1) of Arabidopsis thaliana affects cell growth throughout the plant and has a particularly strong effect on root hair growth. We have identified TIP1 by map-based cloning and complementation of the mutant phenotype. TIP1 encodes an ankyrin repeat protein with a DHHC Cys-rich domain that is expressed in roots, leaves, inflorescence stems, and floral tissue. Two homologues of TIP1 in yeast (Saccharomyces cerevisiae) and human (Homo sapiens) have been shown to have S-acyl transferase (also known as palmitoyl transferase) activity. S-acylation is a reversible hydrophobic protein modification that offers swift, flexible control of protein hydrophobicity and affects protein association with membranes, signal transduction, and vesicle trafficking within cells. We show that TIP1 binds the acyl group palmitate, that it can rescue the morphological, temperature sensitivity, and yeast casein kinase2 localization defects of the yeast S-acyl transferase mutant akr1Delta, and that inhibition of acylation in wild-type Arabidopsis roots reproduces the Tip1- mutant phenotype. Our results demonstrate that S-acylation is essential for normal plant cell growth and identify a plant S-acyl transferase, an essential research tool if we are to understand how this important, reversible lipid modification operates in plant cells.     

The Arabidopsis cyclin-dependent kinase-activating kinase CDKF;1 is a major regulator of cell proliferation and cell expansion but is dispensable for CDKA activation

Cyclin-dependent kinases (CDKs) play an essential role in cell cycle regulation during the embryonic and post-embryonic development of various organisms. Full activation of CDKs requires not only binding to cyclins but also phosphorylation of the T-loop domain. This phosphorylation is catalysed by CDK-activating kinases (CAKs). Plants have two distinct types of CAKs, namely CDKD and CDKF; in Arabidopsis, CDKF;1 exhibits the highest CDK kinase activity in vitro . We have previously shown that CDKF;1 also functions in the activation of CDKD;2 and CDKD;3 by T-loop phosphorylation. Here, we isolated the knockout mutants of CDKF;1 and showed that they had severe defects in cell division, cell elongation and endoreduplication. No defect was observed during embryogenesis, suggesting that CDKF;1 function is primarily required for post-embryonic development. In the cdkf;1 mutants, T-loop phosphorylation of CDKA;1, an orthologue of yeast Cdc2/Cdc28p, was comparable to that in wild-type plants, and its kinase activity did not decrease. In contrast, the protein level and kinase activity of CDKD;2 were significantly reduced in the mutants. Substitution of threonine-168 with a non-phosphorylatable alanine residue made CDKD;2 unstable in Arabidopsis tissues. These results indicate that CDKF;1 is dispensable for CDKA;1 activation but is essential for maintaining a steady-state level of CDKD;2, thereby suggesting the quantitative regulation of a vertebrate-type CAK in a plant-specific manner.     

BcXTH1, a Brassica campestris homologue of Arabidopsis XTH9, is associated with cell expansion

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a group of the enzymes that are responsible for reorganization of the cellulose–xyloglucan framework by catalyzing cleavage and religation of the xyloglucan chains in the plant cell wall. In this study, we report the isolation and characterization of a XTH gene from a pistil cDNA library of Brassica campestris. Sequence analysis of the gene, designated BcXTH1, revealed that it is homologous to the XTH9 gene of Arabidopsis. The highly conserved domain (DEIDFEFLG) found among all XTHs was also present in BcXTH1 but with the two amino acid substitutions (NEFDFEFLG) also found in Arabidopsis XTH9. These results suggest that BcXTH1 is the B. campestris homologue of XTH9. Expression analysis of BcXTH1 revealed that it was expressed in most of the plant organs. In situ hybridization showed that the gene is highly expressed in the floral primodia, especially in the epidermal cell layer. Southern blot analysis indicated that the BcXTH1 gene exists as a multi-copy gene in the B. campestris genome. The function of the BcXTH1 gene was deduced from using an overexpression strategy in Arabidopsis. Interestingly, the transgenic plants showed a pronounced cell expansion phenotype. Immunoelectron microscopy shows that BcXTH1 is localized almost exclusively to the cell wall, supporting our conclusion that it participates in the regulation of cell expansion in B. campestris.     

Latent membrane protein 2A, a viral B cell receptor homologue, induces CD5+ B-1 cell development

The latent membrane protein 2A (LMP2A) of EBV plays a key role in regulating viral latency and EBV pathogenesis by functionally mimicking a constitutively active B cell Ag receptor. When expressed as a B cell-specific transgene in mice, LMP2A drives B cell development, resulting in the bypass of normal developmental checkpoints. In this study, we have demonstrated that expression of LMP2A in transgenic mice results in B cell development that exclusively favors B-1 cells. This switch to B-1 cell development occurs at the pre-B-cell stage of normal B cell development in the bone marrow, a B cell stage much earlier than appreciated for B-1 commitment. This finding indicates that all pre-B cells have the capacity to assume a B-1 cell phenotype if they encounter the appropriate signal during normal development. Furthermore, these studies offer insight into EBV latency and pathogenesis in the human host.     

In vitro genotoxicity assessment of the synthetic plant growth regulator, 1-naphthaleneacetamide

1-Naphthaleneacetamide (NAAm) is a synthetic plant growth regulator in the auxin family that is widely used in agriculture to promote the growth of numerous fruits, for root cuttings and as a fruit thinning agent. The potential genotoxic effects of NAAm were investigated in vitro by the chromosome aberrations (CAs), and cytokinesis-block micronucleus assays in human peripheral blood lymphocytes (PBLs) for the first time. The human PBLs were treated with 20, 40, 80, and 160 µg/mL of NAAm for 24 and 48 h. The results of this study showed that NAAm significantly induced the formation of structural CA and MN for all concentrations (20, 40, 80 and 160 µg/mL) and treatment periods (24 and 48 h) when compared with the negative and the solvent control. In addition, the higher concentrations of NAAm (80 and 160 µg/mL) caused a statistically significant increase in nuclear bud (NBUD) formation for both 24 and 48 h treatment times. With regard to the cell cycle kinetics, at all the tested concentrations, NAAm caused a statistically significant reduction in the mitotic index (MI) only for 48 h treatment period and also in the nuclear division index (NDI) for both 24 and 48 h treatment periods as compared to the control groups. The reductions in the MI and NDI occured in a concentration-dependent manner for both treatment times. In conclusion, the present results indicate that in the tested experimental conditions, NAAm was genotoxic and cytotoxic on human PBLs in vitro.     

The HD-Zip IV gene GaHOX1 from cotton is a functional homologue of the Arabidopsis GLABRA2

Most of the plant homeodomain-containing proteins play important roles in organ patterning and development, and Arabidopsis GLABRA2 (GL2), a member of the class IV homeodomain-leucine zipper (HD-ZIP) proteins, is a trichome and non-root hair cell regulator. Here we report the analysis of two cotton homeodomain-containing proteins, GaHOX1 and GaHOX2, isolated from the diploid cotton Gossypium arboreum . Both GaHOX1 and GaHOX2 belong to the class IV HD-ZIP family. When expressed under the control of the GL2 promoter, GaHOX1 rescued trichome development of an Arabidopsis glabrous mutant of gl2-2 (SALK_130213), whereas GaHOX2 did not. On the other hand, expression of GaHOX1 with a Cauliflower mosaic virus (CaMV) 35S promoter in the wild-type Arabidopsis plants suppressed the trichome development just as the GL2 ectopic expression. Expression analysis by Northern, RT-PCR and in situ hybridization indicated that GaHOX1 is predominantly expressed in cotton fiber cells at early developmental stages, consistent with its putative role in regulating cotton fiber development, while GaHOX2 is expressed in both fiber and other ovular tissues, including outer and inner integuments. Our results suggest that GaHOX1 is a functional homolog of GL2 in plant trichome development.     

The major leaf ferredoxin Fd2 regulates plant innate immunity in Arabidopsis

Ferredoxins, the major distributors for electrons to various acceptor systems in plastids, contribute to redox regulation and antioxidant defence in plants. However, their function in plant immunity is not fully understood. In this study, we show that the expression of the major leaf ferredoxin gene Fd2 is suppressed by Pseudomonas syringae pv. tomato (Pst) DC3000 infection, and that knockout of Fd2 (Fd2‐KO) in Arabidopsis increases the plant's susceptibility to both Pst DC3000 and Golovinomyces cichoracearum. On Pst DC3000 infection, the Fd2‐KO mutant accumulates increased levels of jasmonic acid and displays compromised salicylic acid‐related immune responses. Fd2‐KO also shows defects in the accumulation of reactive oxygen species induced by pathogen‐associated molecular pattern‐triggered immunity. However, Fd2‐KO shows enhanced R‐protein‐mediated resistance to Pst DC3000/AvrRpt2 infection, suggesting that Fd2 plays a negative role in effector‐triggered immunity. Furthermore, Fd2 interacts with FIBRILLIN4 (FIB4), a harpin‐binding protein localized in chloroplasts. Interestingly, Fd2, but not FIB4, localizes to stromules that extend from chloroplasts. Taken together, our results demonstrate that Fd2 plays an important role in plant immunity.     

The green algal pigment caulerpin as a plant growth regulator

Caulerpin, a green algal pigment possessing a unique bis-indole structure, has been shown to be a plant growth regulator. Root growth assays were conducted with caulerpin and its hydrolysis product, and the results were compared with data obtained with indole-3-acetic acid (IAA), indole-3-pyruvic acid (IPA) and indole-3-acrylic acid (IAcA). This study has indicated that caulerpin, in essence a dimer of indole-3-acrylic acid, behaves much like the indole auxins.     

The carboxyl terminal extension of the Drosophila insulin receptor homologue binds IRS-1 and influences cell survival.

The Drosophila insulin receptor (INR) homolog includes an extension of approximately 400 amino acids at the carboxyl-terminal end of its beta subunit containing several tyrosine-based motifs known to mediate interactions with signaling proteins. In order to explore the role of this extension in INR function, mammalian expression vectors encoding either the complete INR beta subunit (beta-Myc) or the INR beta subunit without the carboxyl-terminal extension (betaDelta) were constructed, and the membrane-bound beta subunits were expressed in 293 and Madin-Darby canine kidney cells in the absence of the ligand-binding alpha subunits. beta-Myc and betaDelta proteins were constitutively active tyrosine kinases of 180 and 102 kDa, respectively. INR beta-Myc co-immunoprecipitated a phosphoprotein of 170 kDa identified as insulin receptor substrate-1 (IRS-1), whereas INR betaDelta did not, suggesting that the site of interaction was within the carboxyl-terminal extension. IRS-1 was phosphorylated on tyrosine to a much greater extent in cells expressing INR beta-Myc than in parental or INR betaDelta cells. Despite this, a variety of PTB or SH2 domain-containing signaling proteins, including IRS-2, mSos-1, Shc, p85 subunit of phosphatidylinositol 3-kinase, SHP-2, Raf-1, and JAK2, were not associated with the INR beta-Myc.IRS-1 complex. Overexpression of INR beta-Myc and betaDelta kinases conferred an equivalent increase in cell proliferation in both 293 and Madin-Darby canine kidney cells, indicating that this growth response is independent of the carboxyl-terminal extension. However, INR beta-Myc-expressing cells exhibited enhanced survival relative to parental and betaDelta cells, suggesting that the carboxyl-terminal extension, through its interaction with IRS-1, plays a role in the regulation of cell death.     

The liver kinase B1 is a central regulator of T cell development, activation, and metabolism

T cell activation leads to engagement of cellular metabolic pathways necessary to support cell proliferation and function. However, our understanding of the signal transduction pathways that regulate metabolism and their impact on T cell function remains limited. The liver kinase B1 (LKB1) is a serine/threonine kinase that links cellular metabolism with cell growth and proliferation. In this study, we demonstrate that LKB1 is a critical regulator of T cell development, viability, activation, and metabolism. T cell-specific ablation of the gene that encodes LKB1 resulted in blocked thymocyte development and a reduction in peripheral T cells. LKB1-deficient T cells exhibited defects in cell proliferation and viability and altered glycolytic and lipid metabolism. Interestingly, loss of LKB1 promoted increased T cell activation and inflammatory cytokine production by both CD4(+) and CD8(+) T cells. Activation of the AMP-activated protein kinase (AMPK) was decreased in LKB1-deficient T cells. AMPK was found to mediate a subset of LKB1 functions in T lymphocytes, as mice lacking the α1 subunit of AMPK displayed similar defects in T cell activation, metabolism, and inflammatory cytokine production, but normal T cell development and peripheral T cell homeostasis. LKB1- and AMPKα1-deficient T cells each displayed elevated mammalian target of rapamycin complex 1 signaling and IFN-γ production that could be reversed by rapamycin treatment. Our data highlight a central role for LKB1 in T cell activation, viability, and metabolism and suggest that LKB1-AMPK signaling negatively regulates T cell effector function through regulation of mammalian target of rapamycin activity.     

The Arabidopsis TRM1-TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes

Land plant cells assemble microtubule arrays without a conspicuous microtubule organizing center like a centrosome. In Arabidopsis thaliana, the TONNEAU1 (TON1) proteins, which share similarity with FOP, a human centrosomal protein, are essential for microtubule organization at the cortex. We have identified a novel superfamily of 34 proteins conserved in land plants, the TON1 Recruiting Motif (TRM) proteins, which share six short conserved motifs, including a TON1-interacting motif present in all TRMs. An archetypal member of this family, TRM1, is a microtubule-associated protein that localizes to cortical microtubules and binds microtubules in vitro. Not all TRM proteins can bind microtubules, suggesting a diversity of functions for this family. In addition, we show that TRM1 interacts in vivo with TON1 and is able to target TON1 to cortical microtubules via its C-terminal TON1 interaction motif. Interestingly, three motifs of TRMs are found in CAP350, a human centrosomal protein interacting with FOP, and the C-terminal M2 motif of CAP350 is responsible for FOP recruitment at the centrosome. Moreover, we found that TON1 can interact with the human CAP350 M2 motif in yeast. Taken together, our results suggest conservation of eukaryotic centrosomal components in plant cells.