Effect of plant WEE1 on the cell cycle and development in Arabidopsis thaliana and Nicotiana tabacum

In eukaryotes the regulatory cell cycle gene, WEE1, encodes a protein kinase. In late G2, it inactivates cyclin-dependent kinase (CDKs) in the CDK-cyclinA/B complexes, by phosphorylating the CDK on tyrosine 15. This can result in a delay in mitosis. Expression of Arabidopsis thaliana homologue of WEE1 (AtWEEl) in fission yeast resulted in an elongated cell length phenotype in the same way as over expression of fission yeast weel. I have tested whether At WEE1 could also induce this effect in tobacco cells and Arabidopsis plant roots. The tobacco BY-2 cells have been transformed with AtWEEl, both under constitutive and inducible promoters. Phenotypic characteristics observed compared with the control are premature entry into mitosis and a reduced cell size through a shortening of the G2 phase with a compensatory increase in the duration of Gl phase. Hence, the phenotype and cell cycle response is the exact opposite of the known effect of expression of this gene in fission yeast. NtWEEl expression data revealed that the endogenous WEE1 expression is delayed in transgenic lines, this results in a non-inhibition of CDKA and CDKB1 which are already active in early S-phase. AtWEEl was also employed to transform Arabidopsis thaliana plants, both under constitutive and inducible promoters. The effect of AtWEEl over expression was investigated on primary root growth and lateral root development. In particular, AtWEEl over expression lead to less primary root growth and a reduction in the frequency of lateral root primordia initiated when compared with wild type. Arabidopsis transgenic plants initiated fewer primordia both per unit time and per cm of primary root.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Effect of plant WEE1 on the cell cycle and development in Arabidopsis thaliana and Nicotiana tabacum

In eukaryotes the regulatory cell cycle gene, WEE1, encodes a protein kinase. In late G2, it inactivates cyclin-dependent kinase (CDKs) in the CDK-cyclinA/B complexes, by phosphorylating the CDK on tyrosine 15. This can result in a delay in mitosis. Expression of Arabidopsis thaliana homologue of WEE1 (AtWEEl) in fission yeast resulted in an elongated cell length phenotype in the same way as over expression of fission yeast weel. I have tested whether At WEE1 could also induce this effect in tobacco cells and Arabidopsis plant roots. The tobacco BY-2 cells have been transformed with AtWEEl, both under constitutive and inducible promoters. Phenotypic characteristics observed compared with the control are premature entry into mitosis and a reduced cell size through a shortening of the G2 phase with a compensatory increase in the duration of Gl phase. Hence, the phenotype and cell cycle response is the exact opposite of the known effect of expression of this gene in fission yeast. NtWEEl expression data revealed that the endogenous WEE1 expression is delayed in transgenic lines, this results in a non-inhibition of CDKA and CDKB1 which are already active in early S-phase. AtWEEl was also employed to transform Arabidopsis thaliana plants, both under constitutive and inducible promoters. The effect of AtWEEl over expression was investigated on primary root growth and lateral root development. In particular, AtWEEl over expression lead to less primary root growth and a reduction in the frequency of lateral root primordia initiated when compared with wild type. Arabidopsis transgenic plants initiated fewer primordia both per unit time and per cm of primary roo

Expression of Arabidopsis WEE1 in Tobacco Induces Unexpected Morphological and Developmental Changes

WEE1 regulates the cell cycle by inactivating cyclin dependent protein kinases (CDKs) via phosphorylation. In yeast and animal cells, CDC25 phosphatase dephosphorylates the CDK releasing cells into mitosis, but in plants, its role is less clear. Expression of fission yeast CDC25 (Spcdc25) in tobacco results in small cell size, premature flowering and increased shoot morphogenetic capacity in culture. When Arath;WEE1 is over-expressed in Arabidopsis, root apical meristem cell size increases, and morphogenetic capacity of cultured hypocotyls is reduced. However expression of Arath;WEE1 in tobacco plants resulted in precocious flowering and increased shoot morphogenesis of stem explants, and in BY2 cultures cell size was reduced. This phenotype is similar to expression of Spcdc25 and is consistent with a dominant negative effect on WEE1 action. Consistent with this putative mechanism, WEE1 protein levels fell and CDKB levels rose prematurely, coinciding with early mitosis. The phenotype is not due to sense-mediated silencing of WEE1, as overall levels of WEE1 transcript were not reduced in BY2 lines expressing Arath;WEE1. However the pattern of native WEE1 transcript accumulation through the cell cycle was altered by Arath;WEE1 expression, suggesting feedback inhibition of native WEE1 transcription

Effect of plant WEE1 on the cell cycle and development in Arabidopsis thaliana and Nicotiana tabacum.

In eukaryotes the regulatory cell cycle gene, WEE1, encodes a protein kinase. In late G2, it inactivates cyclin-dependent kinase (CDKs) in the CDK-cyclinA/B complexes, by phosphorylating the CDK on tyrosine 15. This can result in a delay in mitosis. Expression of Arabidopsis thaliana homologue of WEE1 (AtWEE1) in fission yeast resulted in an elongated cell length phenotype in the same way as over expression of fission yeast wee1. I have tested whether AtWEE1 could also induce this effect in tobacco cells and Arabidopsis plant roots. The tobacco BY-2 cells have been transformed with AtWEE1, both under constitutive and inducible promoters. Phenotypic characteristics observed compared with the control are premature entry into mitosis and a reduced cell size through a shortening of the G2 phase with a compensatory increase in the duration of G1 phase. Hence, the phenotype and cell cycle response is the exact opposite of the known effect of expression of this gene in fission yeast. NtWEE1 expression data revealed that the endogenous WEE1 expression is delayed in transgenic lines, this results in a non-inhibition of CDKA and CDKB1 which are already active in early S-phase. AtWEE1 was also employed to transform Arabidopsis thaliana plants, both under constitutive and inducible promoters. The effect of AtWEE1 over expression was investigated on primary root growth and lateral root development. In particular, AtWEE1 over expression lead to less primary root growth and a reduction in the frequency of lateral root primordia initiated when compared with wild type. Arabidopsis transgenic plants initiated fewer primordia both per unit time and per cm of primary root

Effect of Elevated Atmospheric CO2 and Temperature on the Disease Severity of Rocket Plants Caused by Fusarium Wilt under Phytotron Conditions.

The severity of F. oxysporum f.sp. conglutinans on rocket plants grown under simulated climate change conditions has been studied. The rocket plants were cultivated on an infested substrate (4 log CFU g-1) and a non-infested substrate over three cycles. Pots were placed in six phytotrons in order to simulate different environmental conditions: 1) 400-450 ppm CO2, 18-22°C; 2) 800-850 ppm CO2, 18-22°C; 3) 400-450 ppm CO2, 22-26°C, 4) 800-850 ppm CO2, 22-26°C, 5) 400-450 ppm CO2, 26-30°C; 6) 800-850 ppm CO2, 26-30°C. Substrates from the infested and control samples were collected from each phytotron at 0, 60 and 120 days after transplanting. The disease index, microbial abundance, leaf physiological performances, root exudates and variability in the fungal profiles were monitored. The disease index was found to be significantly influenced by higher levels of temperature and CO2. Plate counts showed that fungal and bacterial development was not affected by the different CO2 and temperature levels, but a significant decreasing trend was observed from 0 up to 120 days. Conversely, the F. oxysporum f.sp. conglutinans plate counts did not show any significantly decrease from 0 up to 120 days. The fungal profiles, evaluated by means of polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE), showed a relationship to temperature and CO2 on fungal diversity profiles. Different exudation patterns were observed when the controls and infested plants were compared, and it was found that both CO2 and temperature can influence the release of compounds from the roots of rocket plants. In short, the results show that global climate changes could influence disease incidence, probably through plant-mediated effects, caused by soilborne pathogens

Photosynthetic efficiency measurements.

<p>Effect of different CO₂ and temperature combinations on the photosynthetic efficiency of the leaves (PI) of rocket plants grown in a substrate artificially infested with <i>F</i>. <i>oxysporum</i> f.sp. <i>conglutinans</i> and the control. Tukey's HSD test (P < 0.05).</p

PLS-DA models based on DGGE similarity distance matrix.

<p>Plot A, PLS-DA models based on the DGGE similarity matrix as a function of CO₂: 400–450 ppm (blue), 800–850 ppm (yellow); Plot B, PLS-DA models based on the DGGE similarity matrix as a function of the temperature: 26°C (blue), 22°C (yellow), and 30°C (red): Plot C, PLS-DA models based on the DGGE similarity matrix as a function of the time: time 0 (red), time 60 (yellow) and time120 (blue); Plot D; PLS-DA models based on the DGGE similarity matrix as a function of the phytotrons: 1 (red), 2 (yellow), 3 (blue), 4 (black), 5 (green), 6 (pink).</p

Main root exudate components analyzed on rocket plants cultivated in infested and control substrates collected at the end of the cycle.

<p>^a The root exudate parameters were expressed as the mean values of three plants for three cultivation cycles. Values with different superscripts in the same row differ significantly according to Tukey's HSD test (P<0.05)</p><p>Main root exudate components analyzed on rocket plants cultivated in infested and control substrates collected at the end of the cycle.</p

Total bacterial counts (TBC) of mesophilic bacteria from the infested and control substrate samples following incubation in phytotrons for 120 days.

<p>^<b>a</b>The plate counts were calculated as the mean Log counts of the three replicates. Values with different superscripts differ significantly according to Tukey's HSD test (P<0.05)</p><p>Total bacterial counts (TBC) of mesophilic bacteria from the infested and control substrate samples following incubation in phytotrons for 120 days.</p

Tailed Forisomes of Canavalia gladiata: A New Model to Study Ca2+-driven Protein Contractility

Background and Aims Forisomes are Ca2+-dependent contractile protein bodies that form reversible plugs in sieve tubes of faboid legumes. Previous work employed Vicia faba forisomes, a not entirely unproblematic experimental system. The aim of this study was to seek to establish a superior model to study these intriguing actuators. Methods Existing isolation procedures were modified to study the exceptionally large, tailed forisomes of Canavalia gladiata by differential interference contrast microscopy in vitro. To analyse contraction/expansion kinetics quantitatively, a geometric model was devised which enabled the computation of time-courses of derived parameters such as forisome volume from simple parameters readily determined on micrographs. Key Results Advantages of C. gladiata over previously utilized species include the enormous size of its forisomes (up to 55 µm long), the presence of tails which facilitate micromanipulation of individual forisomes, and the possibility of collecting material repeatedly from these fast-growing vines without sacrificing the plants. The main bodies of isolated Canavalia forisomes were box-shaped with square cross-sections and basically retained this shape in all stages of contraction. Ca2+-induced a 6-fold volume increase within about 10-15 s; the reverse reaction following Ca2+-depletion proceeded in a fraction of that time. Conclusions The sword bean C. gladiata provides a superior experimental system which will prove indispensable in physiological, biophysical, ultrastructural and molecular studies on the unique ATP-independent contractility of forisomes