Richard G. Anthony

A protein kinase target of a PDK1 signalling pathway is involved in root hair growth in Arabidopsis

Here we report on a lipid‐signalling pathway in plants that is downstream of phosphatidic acid and involves the Arabidopsis protein kinase, AGC2‐1, regulated by the 3′‐phosphoinositide‐dependent kinase‐1 (AtPDK1). AGC2‐1 specifically interacts with AtPDK1 through a conserved C‐terminal hydrophobic motif that leads to its phosphorylation and activation, whereas inhibition of AtPDK1 expression by RNA interference abolishes AGC2‐1 activity. Phosphatidic acid specifically binds to AtPDK1 and stimulates AGC2‐1 in an AtPDK1‐dependent manner. AtPDK1 is ubiquitously expressed in all plant tissues, whereas expression of AGC2‐1 is abundant in fast‐growing organs and dividing cells, and activated during re‐entry of cells into the cell cycle after sugar starvation‐induced G1‐phase arrest. Plant hormones, auxin and cytokinin, synergistically activate the AtPDK1‐regulated AGC2‐1 kinase, indicative of a role in growth and cell division. Cellular localisation of GFP‐AGC2‐1 fusion protein is highly dynamic in root hairs and at some stages confined to root hair tips and to nuclei. The agc2‐1 knockout mutation results in a reduction of root hair length, suggesting a role for AGC2‐1 in root hair growth and development.

Growth signalling pathways in Arabidopsis and the AGC protein kinases

Lipid-derived signals are central to regulating a multitude of cellular processes but, in plants, little is known of the downstream signalling pathways. The Arabidopsis 3-phosphoinositide-dependent protein kinase (PDK1) could couple lipid signals to the activation of several protein kinases of the so-called AGC kinase family. The Arabidopsis AGC kinases contain sequence motives required for the docking of PDK1 and phosphorylation of their activation loop in the kinase catalytic domain. It is becoming evident that specific members of the AGC kinases are implicated in key growth signalling pathways. For example, Arabidopsis p70(S6K) might be a nodal point able to integrate hormonal and developmental signals with nutritional inputs, together with the Arabidopsis Target of Rapamycin (TOR) protein.

The Actin-Interacting Protein AIP1 Is Essential for Actin Organization and Plant Development

Cell division, growth, and cytoplasmic organization require a dynamic actin cytoskeleton. The filamentous actin (F-actin) network is regulated by actin binding proteins that modulate actin dynamics. These actin binding proteins often have cooperative interactions. In particular, actin interacting protein 1 (AIP1) is capable of capping F-actin and enhancing the activity of the small actin modulating protein, actin depolymerising factor (ADF) in vitro. Here, we analyze the effect of the inducible expression of AIP1 RNAi in Arabidopsis plants to assess AIP1s role in vivo. In intercalary growing cells, the normal actin organization is disrupted, and thick bundles of actin appear in the cytoplasm. Moreover, in root hairs, there is the unusual appearance of actin cables ramifying the root hair tip. We suggest that the reduction in AIP1 results in a decrease in F-actin turnover and the promotion of actin bundling. This distortion of the actin cytoskeleton causes severe plant developmental abnormalities. After induction of the Arabidopis RNAi lines, the cells in the leaves, roots, and shoots fail to expand normally, and in the severest phenotypes, the plants die. Our data suggest that AIP1 is essential for the normal functioning of the actin cytoskeleton in plant development.

Herbicide resistance caused by spontaneous mutation of the cytoskeletal protein tubulin

The dinitroaniline herbicides (such as trifluralin and oryzalin) have been developed for the selective control of weeds in arable crops. However, prolonged use of these chemicals has resulted in the selection of resistant biotypes of goosegrass, a major weed. These herbicides bind to the plant tubulin protein but not to mammalian tubulin. Here we show that the major α-tubulin gene of the resistant biotype has three base changes within the coding sequence. These base changes swap cytosine and thymine, most likely as the result of the spontaneous deamination of methylated cytosine. One of these base changes causes an amino-acid change in the protein: normal threonine at position 239 is changed to isoleucine. This position is close to the site of interaction between tubulin dimers in the microtubule protofilament. We show that the mutated gene is the cause of the herbicide resistance by using it to transform maize and confer resistance to dinitroaniline herbicides. Our results provide a molecular explanation for the resistance of goosegrass to dinitroanaline herbicides, a phenomenon that has arisen, and been selected for, as a result of repeated exposure to this class of herbicide.

Green Fluorescent Protein-mTalin Causes Defects in Actin Organization and Cell Expansion in Arabidopsis and Inhibits Actin Depolymerizing Factor's Actin Depolymerizing Activity in Vitro

Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that GFP-mTalin colocalizes with the actin cytoskeleton, its effect on actin dynamics and cell expansion has not been studied in detail. We created Arabidopsis (Arabidopsis thaliana) plants harboring alcohol inducible GFP-mTalin constructs to assess the effect of GFP-mTalin expression in vivo. We focused on the growing root hair as this is a model cell for studying cell expansion and root hair tip growth that requires a highly dynamic and polar actin cytoskeleton. We show that alcohol inducible expression of GFP-mTalin in root hairs causes severe defects in actin organization, resulting in either the termination of growth, cell death, and/or changes in cell shape. Fluorescence recovery after photobleaching experiments demonstrate that the interaction of GFP-mTalin and actin filaments is highly dynamic. To assess how GFP-mTalin affects actin dynamics we performed cosedimentation assays of GFP-mTalin with actin on its own or in the presence of the actin modulating protein, actin depolymerizing factor. We show that that GFP-mTalin does not affect actin polymerization but that it does inhibit the actin depolymerizing activity of actin depolymerizing factor. These observations demonstrate that GFP-mTalin can affect cell expansion, actin organization, and the interaction of actin binding proteins with actin.

The Arabidopsis Protein Kinase PTI1-2 Is Activated by Convergent Phosphatidic Acid and Oxidative Stress Signaling Pathways Downstream of PDK1 and OXI1

Arabidopsis PDK1 activity is regulated by binding to the lipid phosphatidic acid (PA) resulting in activation of the oxidative stress-response protein kinase OXI1/AGC2-1. Thus there is an inferred link between lipid signaling and oxidative stress signaling modules. Among a panel of hormones and stresses tested, we found that, in addition to PA, the fungal elicitor xylanase activated PDK1, suggesting that PDK1 has a role in plant pathogen defense mechanisms. The downstream OXI1 was activated by additional stress factors, including PA, H2O2, and partially by xylanase. We have isolated an interacting partner of OXI1, a Ser/Thr kinase (PTI1-2), which is downstream of OXI1. Its sequence closely resembles the tomato Pti kinase, which has been implicated in the hypersensitive response, a localized programmed cell death that occurs at the site of pathogen infection. PTI1-2 is activated by the same stresses/elicitors as OXI1 and additionally flagellin. We have used RNA interference to knock out the expression of PDK1 and OXI1 and to study the effects on PTI1-2 activity. We show that specific lipid signaling pathways converge on PTI1-2 via the PDK1-OXI1 axis, whereas H2O2 and flagellin signals to OXI1-PTI1-2 via a PDK1-independent pathway. PTI1-2 represents a new downstream component that integrates diverse lipid and reactive oxygen stress signals and functions closely with OXI1.

Dinitroaniline herbicide resistance and the microtubule cytoskeleton

Dinitroaniline herbicides have been used for pre-emergence weed control for the past 25 years in cotton, soybean, wheat and oilseed crops. Considering their long persistence and extensive use, resistance to dinitroanilines is fairly rare. However, the most widespread dinitroaniline-resistant weeds, the highly resistant (R) and the intermediate (I) biotypes of the invasive goosegrass Eleusine indica, are now infesting more than 1000 cotton fields in the southern states of the USA. The molecular basis of this resistance has been identified, and found to be a point mutation in a major microtubule cytoskeletal protein, alpha-tubulin. These studies have served both to explain the establishment of resistance and to reveal fundamental properties of tubulin gene expression and microtubule structure.

Dinitroaniline herbicide-resistant transgenic tobacco plants generated by co-overexpression of a mutant |[alpha]|-tubulin and a |[beta]|-tubulin

Dinitroaniline herbicides are used for the selective control of weeds in arable crops. Dinitroaniline herbicide resistance in the invasive weed goosegrass was previously shown to stem from a spontaneous mutation in an α-tubulin gene. We transformed and regenerated tobacco plants with an α/β-tubulin double gene construct containing the mutant α-tubulin gene and showed that expression of this construct confers a stably inherited dinitroaniline-resistant phenotype in tobacco. In all transformed lines, the transgene α- and β-tubulins increased the cytoplasmic pool of tubulin approximately 1.5-fold while repressing endogenous α- and β-tubulin synthesis by up to 45% in some tissues. Transgene α- and β-tubulin were overexpressed in every plant tissue analyzed and comprised approximately 66% of the total tubulin in these tissues. Immunolocalization studies revealed that transgene α- and β-tubulins were incorporated into all four microtubule arrays, indicating that they are functional. The majority of the α/β- tubulin pools are encoded by the transgenes, which implies that the mutant α-tubulin and the β-tubulin can perform the majority, if not all, of the roles of microtubules in both juvenile and adult tobacco plants.

A Topoisomerase II-Dependent Checkpoint in G2-Phase Plant Cells Can Be Bypassed by Ectopic Expression of Mitotic Cyclin B2

DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclin-dependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin. Key Words: Plant cyclin B2, Topoisomerase II, ICRF-193, G2 checkpoint, Microtubules

Dinitroaniline Herbicide Resistant Transgenic Plants

There is a strict aerobic bacterium, Vitreoscilla stercoraria, living in stagnant ponds and decaying vegetable matter that produces a hemoglobin (VHb) to sustain these oxygen-limiting conditions. The gene coding for this protein has been cloned and expressed in a variety of prokaryotic organisms, but also transferred in a few eukaryotic hosts leading to improved growth, increased production of recombinant proteins and enhanced yields of secondary metabolites. To further investigate these properties a randomly mutated expression library in E. coli based on the Vitreoscillahemoglobin gene, vhb, was created by error-prone PCR. Several mutants with altered phenotype were obtained. Four mutants were able to reach 22-155% higher final cell densities in microaerobic fed-batch cultivations than an E. coli clone expressing native VHb. The expression levels of mutated VHb proteins were similar to the amounts produced by the controls expressing native VHb. The clones also synthesized functional VHb, as their CO-binding absorbance spectra indicated absorption characteristics analogous to native VHb having an absorption maximum at 419 nm and a minimum at 437 nm. This indicates that the amino acid mutations within the vhb gene are able to slightly change the structure of VHb. However, the change of structure of mutant VHbs was able to improve the growth of E. coli under microaerobic conditions. By-product formation of these mutants was also decreased, for acetate to approximately 18-40% and for ethanol up to 60% of final concentration in cultures expressing native VHb. These findings indicate that there is a correlation between a decreased by-product formation and an improved respiratory efficiency in the E. coli cell caused by mutations within the vhb gene. Furthermore, the production of a recombinant marker protein β-lactamase was increased up to five times for mutants compared to the strain producing native VHb. These results show that an E. coli cell expressing mutant VHb is able to direct more carbon towards cell growth and recombinant protein production relative to a control cell. Our intention is now to study the effects of the most interesting mutant VHbs in higher eukaryotes. Poster #2