Cytokinesis in flowering plants: cellular process and developmental integration (2023)

Phragmoplastin, a dynamin-like GTPase, is associated with cell plate formation in plants. We expressed a phragmoplastin-GFP construct (Phr^G) under the control of a 35S promoter in transgenic tobacco plants. High levels of expression of this chimeric protein in transgenic plants were confirmed by Western blot analysis. T1 seedlings formed morphologically normal cotyledons, but most were unable to develop beyond the first pair of true leaves. Only 5–10% of the seedlings could develop into small plants whose progenies continued to exhibit severe growth defects. The primary root growth was arrested after 7 days and no lateral roots were formed. The mature primary root meristem consisted of disorganized isodiametrically enlarged cells and no recognizable quiescent center or collumela. Adventitious root primordia were initiated in the hypocotyl, but completely arrested at an early stage and contained differentiated treachery elements. The orientation of cell plates was abnormal, suggesting a possible defect in its positioning. However, unlike other cytokinesis mutants, no binucleate or polyploid cells were found. Fluorescence image cytometry studies indicated that Phr^G root meristem cells had 2C levels of DNA and appear to be arrested in G1 stage of the cell cycle. Heavy callose deposition was observed at the nascent cell plate and callose accumulation persisted in new cell walls. Electron microscopy revealed unusual electron dense substances in the maturing cell plate and accumulation of multivesicular bodies around the cell plate. Since phragmoplastin interacts with callose synthase complex, its overexpression may affect accumulation of callose arresting plant growth due to perturbation in cell division progression.

The tobacco mitogen-activated protein kinase kinase kinase NPK1 regulates lateral expansion of the cell plate at cytokinesis. Here, we show that the kinesin-like proteins NACK1 and NACK2 act as activators of NPK1. Biochemical analysis suggests that direct binding of NACK1 to NPK1 stimulates kinase activity. NACK1 is accumulated specifically in M phase and colocalized with NPK1 at the phragmoplast equator. Overexpression of a truncated NACK1 protein that lacks the motor domain disrupts NPK1 concentration at the phragmoplast equator and cell plate formation. Incomplete cytokinesis is also observed when expression of NACK1 and NACK2 is repressed by virus-induced gene silencing and in embryonic cells from Arabidopsis mutants in which a NACK1 ortholog is disrupted. Thus, we conclude that expansion of the cell plate requires NACK1/2 to regulate the activity and localization of NPK1.

Plant cytokinesis starts in the center of the division plane, with vesicle fusion generating a new membrane compartment, the cell plate, that subsequently expands laterally by continuous fusion of newly arriving vesicles to its margin 1, 2, 3, 4. Targeted delivery of vesicles is assisted by the dynamic reorganization of a plant-specific cytoskeletal array, the phragmoplast, from a solid cylinder into an expanding ring-shaped structure [5]. This lateral translocation is brought about by depolymerization of microtubules in the center, giving way to the expanding cell plate, and polymerization of microtubules along the edge 6, 7. Whereas several components are known to mediate cytokinetic vesicle fusion 8, 9, 10, no gene function involved in phragmoplast dynamics has been identified by mutation. Mutations in the Arabidopsis HINKEL gene cause cytokinesis defects, such as enlarged cells with incomplete cell walls and multiple nuclei. Proper targeting of the cytokinesis-specific syntaxin KNOLLE [8] and lateral expansion of the phragmoplast are not affected. However, the phragmoplast microtubules appear to persist in the center, where vesicle fusion should result in cell plate formation. Molecular analysis reveals that the HINKEL gene encodes a plant-specific kinesin-related protein with a putative N-terminal motor domain and is expressed in a cell cycle-dependent manner similar to the KNOLLE gene. Our results suggest that HINKEL plays a role in the reorganization of phragmoplast microtubules during cell plate formation.

Industrial Crops and Products, Volume 81, 2016, pp. 20-29

Consumption of medicinal and aromatic plants is widespread and increasing worldwide. Yet, harvesting from the wild, the main source of raw material in developing countries, is causing loss of genetic diversity and habitat destruction. This situation makes imperative the development and application of breeding programs. Autopolyploidy has brought advantages for the improvement of agronomic traits of economically important plants. In this sense, obtaining polyploid individuals is an interesting strategy to achieve this objective. In the present study successful induction of polyploidy in Lippia integrifolia (“incayuyo”) was achieved by applying colchicine in the multiplication medium MS+2.2μM BAP. Induced autotetraploids showed significant differences from the field mother plant (size of the leaves, inflorescences, trichomes, stomatas, and pollen grains). In addition, essential oil yields were enhanced in tetraploids, and surprisingly, quantitative differences were detected in the composition of all recovered individuals from in vitro culture (tetraploids and diploids) with respect to the mother field plant. Due to the phenotypic differences, and enhanced essential oil yields and composition, tetraploid individuals became a new variety of incayuyo.

Gene, Volume 529, Issue 1, 2013, pp. 45-49

Tyrosinemia type II, also designated as oculocutaneous tyrosinemia or Richner–Hanhart syndrome (RHS), is a very rare autosomal recessive disorder. In the present study, we report clinical features and molecular genetic investigation of the tyrosine aminotransferase (TAT) gene in two young patients, both born to consanguineous unions between first-degree cousins. These two unrelated families originated from Northern and Southern Tunisia. The clinical diagnosis was based on the observation of several complications related to Richner–Hanhart syndrome: recurrent eye redness, tearing and burning pain, photophobia, bilateral pseudodendritic keratitis, an erythematous and painful focal palmo-plantar hyperkeratosis and a mild delay of mental development. The diagnosis was confirmed by biochemical analysis. Sequencing of the TAT gene revealed the presence of a previously reported missense mutation (c.452G>A, p.Cys151Tyr) in a Tunisian family, and a novel G duplication (c.869dupG, p.Trp291Leufs*6). Early diagnosis of RHS and protein-restricted diet are crucial to reduce the risk and the severity of long-term complications of hypertyrosinemia such as intellectual disability.

Plant Physiology and Biochemistry, Volume 97, 2015, pp. 255-263

In plant evolution, because of its key role in sexual polyploidization or whole genome duplication events, diploid gamete formation is considered as an important component in diversification and speciation. Environmental stress often triggers unreduced gamete production. However, the molecular, cellular mechanisms and adverse temperature regulating diplogamete production in carnation remain poorly understood. Here, we investigate the cytological basis for 2n male gamete formation and describe the isolation and characterization of the first gene, DcPS1 (Dianthus Caryophyllus Parallel Spindle 1). In addition, we analyze influence of temperature stress on diploid gamete formation and transcript levels of DcPS1. Cytological evidence indicated that 2n male gamete formation is attributable to abnormal spindle orientation at male meiosis II. DcPS1 protein is conserved throughout the plant kingdom and carries domains suggestive of a regulatory function. DcPS1 expression analysis show DcPS1 gene probably have a role in 2n pollen formation. Unreduced pollen formation in various cultivation was sensitive to high or low temperature which was probably regulated by the level of DcPS1 transcripts. In a broader perspective, these findings can have potential applications in fundamental polyploidization research and plant breeding programs.

Toxicology and Applied Pharmacology, Volume 379, 2019, Article 114638

Our previous study has demonstrated that 4-amino-2-trifluoromethyl-phenyl Retinate (ATPR) can induce human leukemia NB4 cells differentiation and G0/G1 phase arrest, but the underlying mechanism is still unclear. In this study, we used proteomics to screen differentially expressed protein profiles in NB4 cells before and after ATPR treatment in vitro. We analyzed the peptides digested from total cellular proteins by reverse phase LC-MS/MS and then performed label-free quantitative analysis. We found 27 significantly up-regulated proteins in the ATPR group compared to the control group. NCF1 was the most significantly changed protein. Immunoprecipitation and double immunofluorescent staining showed that EBP50 bind to NCF1. We further explored the potential molecular mechanism of EBP50/NCF1 complex in ATPR-induced differentiation and G0/G1 phase arrest. The results showed that ATPR remarkably reduced the expression of EBP50 in vivo and in vitro. Interestingly, the reduction of EBP50 contributed to ROS release by modulating the subcellular localization of NCF1. The reduction of EBP50 also contributed to G0/G1 phase arrest by inhibiting CyclinD1, CyclinA2 and CDK4, as well as promoting the differentiation of NB4 cells by increasing the expression of CD11b. Furthermore, we found that the overexpression of EBP50 restrained the effects of ATPR on differentiation and G0/G1 phase arrest in NB4 cells. These results suggest that ATPR-induced differentiation and G0/G1 phase arrest in acute promyelocytic leukemia (APL) by repressing EBP50/NCF1 complex to promote the production of ROS, and the results from in vivo experiments were consistent with those from in vitro studies. Therefore, our finding results suggest that EBP50 may be a new target for ATPR in the treatment of APL.

Mitochondrion, Volume 22, 2015, pp. 9-16

The biogenesis of mitochondrial respiratory chain components is complex. Mammalian complex I (NADH:ubiquinone oxidoreductase) contains 44 different subunits, an FMN and seven iron–sulfur centers. Its assembly involves at least twelve additional proteins, called assembly factors. One of these is FOXRED1, a 486-amino acid FAD-dependent oxidoreductase. FOXRED1 is a member of the d-amino acid oxidase (DAO) family. A structural model of FOXRED1 reveals a large substrate-binding cavity and a putative oxygen-binding site. These features strongly suggest that FOXRED1 is catalytically active as an oxidoreductase. A metabolic role for FOXRED1 in the biogenesis of complex I should be considered.

Biochemical and Biophysical Research Communications, Volume 495, Issue 1, 2018, pp. 1041-1047

Protein disulfide isomerases (PDIs) can catalyze disulfide bond formation in nascent secretory proteins and membrane proteins and can introduce correct disulfide bonds into substrate proteins containing mispaired disulfides. The functions of mammalian PDIs have been extensively studied; however, relative to mammalian PDIs, the systematic characterization of PDIs for their oxidoreductase activity in plants is still lacking. Arabidopsis protein disulfide isomerases-11 (AtPDI11), with the structure of a-a'-D, has no ortholog in animals or yeast. In this study, we demonstrated that AtPDI11 has oxidoreductase activity invitro using a GSSG/GSH-mediated oxidative protein folding system. Moreover, the active site in the a' domain of AtPDI11 is critical for its oxidative folding activity. AtPDI11 is present in four redox forms invivo, which are determined by the active site cysteines (Cys52 and Cys55 in the a domain, and Cys171 and Cys174 in the a' domain). Genetic evidence suggests that AtPDI11 is required for plant growth under reducing conditions. Our work provides an example for studying the oxidoreductase function of other plant PDIs.