Indomethacin-induced G1/S phase arrest of the plant cell cycle

AbstractIn animal systems, indomethacin inhibits cAMP production via a prostaglandin-adenylyl cyclase pathway. To examine the possibility that a similar mechanism occurs in plants, the effect of indomethacin on the cell cycle of a tobacco bright yellow 2 (TBY-2) cell suspension was studied. Application of indomethacin during mitosis did not interfere with the M/G1 progression in synchronized BY-2 cells but it inhibited cAMP production at the beginning of the G1 phase and arrested the cell cycle progression at G1/S. These observations are discussed in relation to the putative involvement of cAMP biosynthesis in the cell cycle progression in TBY-2 cells

Metabolic Fate of Jasmonates in Tobacco Bright Yellow-2 Cells

Jasmonic acid and methyl jasmonate play an essential role in plant defense responses and pollen development. Their levels are temporarily and spatially controlled in plant tissue. However, whereas jasmonate biosynthesis is well studied, metabolic pathways downstream of jasmonic acid are less understood. We studied the uptake and metabolism of jasmonic acid and methyl jasmonate in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture. We found that upon uptake, jasmonic acid was metabolized to its Glc and gentiobiose esters, and hydroxylation at C-11 or C-12 occurred. Free hydroxylated jasmonates were the preferential fraction of the culture medium. Upon hydrolysis of methyl jasmonate to jasmonic acid, a similar set of conversions occurs. In contrast to jasmonic acid, none of its derivatives interfere with the G2/M transition in synchronized tobacco Bright Yellow-2 cells

Levels of endogenous cytokinins, indole-3-acetic acid and abscisic acid during the cell cycle of synchronized tobacco BY-2 cells

Correlation between cell cycle progression and endogenous levels of plant hormones was studied in synchronized tobacco BY-2 cell suspension cultures. Sixteen different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) were extracted using solid-phase anion exchange chromatography in combination with immunoaffinity purification, and quantified by mass spectrometry. No significant correlation could be identified for IAA and ABA, In contrast, there were sharp peaks in the levels of specific cytokinins (zeatin- and dihydrozeatin-type) at the end of the S phase and during mitosis. The levels of other cytokinins analyzed, including zeatins N- and O-glucosides, remained low, suggesting that the increased amounts of their corresponding non-glucosylated form resulted from de novo synthesis. These findings suggest that zeatin- and dihydrozeatin-type cytokinins might play a specific regulatory role in the progression of the plant cell cycle. One hypothesis to explain cytokinin action is based on a specific interaction with kinases that regulate cell cycle progression, as has been recently shown for the cytokinin analogue olomoucine

Differential Effect of Jasmonic Acid and Abscisic Acid on Cell Cycle Progression in Tobacco BY-2 Cells

Environmental stress affects plant growth and development. Several plant hormones, such as salicylic acid, abscisic acid (ABA), jasmonic acid (JA), and ethylene play a crucial role in altering plant morphology in response to stress. Developmental regulation often has the cell cycle machinery among its targets. We analyzed the effect of JA and ABA on cell cycle progression in synchronized tobacco (Nicotiana tabacum) BY-2 cells. Both compounds were found to prevent DNA replication, keeping the cells in the G1 stage, when applied just before the G1/S transition. However, ABA did not have any effect on subsequent phases of the cell cycle when applied at a later stage, whereas JA effectively prevented mitosis on application during DNA synthesis. This demonstrates that JA treatment can freeze synchronized BY-2 cells in both the G1 and G2 stages of the cell cycle. Jasmonate administered after the S-phase was less effective in decreasing the mitotic index, suggesting that cell sensitivity toward JA is dependent on the cell cycle phase. In cultures detained in the G2-phase, we observed a reduced histone H1 kinase activity of kinases associated with the p13(sucl) protein

Jasmonic acid prevents the accumulation of cyclin B1;1 and CDK-B in synchronized tobacco BY-2 cells

AbstractJasmonic acid (JA) plays a crucial role in plant fertility and defense responses. It exerts an inhibitory effect on plant growth when applied exogenously. This effect seems to be somehow related to a negative regulation of cell cycle progression in the meristematic tissues. In this report, we focus on the molecular events that occur during JA-induced G2 arrest. We demonstrate that JA prevents the accumulation of B-type cyclin-dependent kinases and the expression of cyclin B1;1, which are both essential for the initiation of mitosis. This feature suggests the existence of an early G2 checkpoint that is affected by JA

Modified hormonal balance in rooting-recalcitrant rac mutant tobacco shoots

The rooting-recalcitrant rac tobacco mutant has been multiplied in vitro via outgrowth of axillary buds in parallel to the DS wild-type. The mutant shoots grew at a lower rate and did not root whatever the treatments, whereas the wild-type shoots rooted spontaneously during the culture cycle without auxin treatment. The mutant and wild-type shoots showed similar peroxidase variations along the culture cycle (21 days) but with higher levels of activity for the rac mutant: minimum peroxidase activity occurrey at day 14 in whole shoots of both tobacco genotypes, but already at day 7 in the basal parts of the stems (where roots appear) of the wild-type tobacco, while it was delayed in the mutant. Free and conjugated auxin and polyamine levels were also determined in whole shoots and basal parts of the stems. The rac mutant was characterised by higher auxin and polyamine contents. A peak of auxins and polyamines appeared at day 14 in the whole shoots whatever the tobacco genotype. This peak was delayed in the basal parts of the rac stems compared to the wild-type ones. The mutant shoots contained higher levels of benzyladenine and isopentenyladenosine at the end of the culture cycle, whereas zeatin riboside was more abundant in wild-type shoots. In response to increasing concentrations of indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), only the wild-type shoots responded by an increase in growth rate followed by inhibition at high concentrations. The rac shoot responses were very low or nonexistent. Peroxidase activity was also measured in E basal parts of tobacco stems grown in the presence of IBA. Results suggest growth inhibition related to auxin accumulation, possibly combined with elevated putrescine content. Second, rooting induction seems to take place in both tobacco genotypes; however, the process of root formation is blocked in the mutant. The lack of initiation and expression phases of rooting in relation to auxin content in the mutant is discussed

Zeatin is indispensable for the G₂-M transition in tobacco BY-2 cells

AbstractThe importance of N6-isoprenoid cytokinins in the G2-M transition of Nicotiana tabacum BY-2 cells was investigated. Both cytokinin biosynthesis and entry in mitosis were partially blocked by application at early or late G2 of lovastatin (10 μM), an inhibitor of mevalonic acid synthesis.LC-MS/MS quantification of endogenous cytokinins proved that lovastatin affects cytokinin biosynthesis by inhibiting HMG-CoA reductase. Out of eight different aminopurines and a synthetic auxin tested for their ability to override lovastatin inhibition of mitosis, only zeatin was active. Our data point to a key role for a well-defined cytokinin (here, zeatin) in the G2-M transition of tobacco BY-2 cells

A Comparative Molecular-Physiological Study of Submergence Response in Lowland and Deepwater Rice

Survival of rice (Oryza sativa) upon an extreme rise of the water level depends on rapid stem elongation, which is mediated by ethylene. A genomic clone (OS-ACS5) encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, which catalyzes a regulatory step in ethylene biosynthesis, has been isolated from cv IR36, a lowland rice variety. Expression was induced upon short- and long-term submergence in cv IR36 and in cv Plai Ngam, a Thai deepwater rice variety. Under hypoxic conditions, abscisic acid and gibberellin had a reciprocal opposite effect on the activity of OS-ACS5. Gibberellin up-regulated and abscisic acid down-regulated OS-ACS5 mRNA accumulation. Growth experiments indicated that lowland rice responded to submergence with a burst of growth early on, but lacked the ability to sustain elongation growth. Sustained growth, characteristic for deepwater rice, was correlated with a prolonged induction of OS-ACS5. In addition, a more pronounced capacity to convert ACC to ethylene, a limited ACC conjugation, and a high level of endogenous gibberellin(20) were characteristic for the deepwater variety. An elevated level of OS-ACS5 messenger was found in cv IR36 plants treated with exogenous ACC. This observation was concomitant with an increase in the capacity of converting ACC to ethylene and in elongation growth, and resulted in prolonged survival. In conclusion, OS-ACS5 is involved in the rapid elongation growth of deepwater rice by contributing to the initial and long-term increase in ethylene levels. Our data also suggest that ACC limits survival of submerged lowland rice seedlings