Evaluation of type-B RR dimerization in poplar: A mechanism to preserve signaling specificity?

Plants possess specific signaling pathways, such as the MultiStep Phosphorelay (MSP), which is involved in cytokinin and ethylene sensing, and light, drought or osmotic stress sensing. These MSP comprise histidine-aspartate kinases (HKs) as receptors, histidine phosphotransfer (HPts) proteins acting as phosphorelay proteins, and response regulators (RRs), some of which act as transcription factors (type-B RRs). In previous studies, we identified partners of the poplar osmosensing signaling pathway, composed of two HKs, three main HPts, and six type-B RRs. To date, it is unresolved as to how cytokinin or osmotic stress signal specificity is achieved in the MSP in order to generate specific responses. Here, we present a large-scale interaction study of poplar type-B RR dimerization. Using the two-hybrid assay, we were able to show the homodimerization of type-B RRs, the heterodimerization of duplicated type-B RRs, and surprisingly, a lack of interaction between some type-B RRs belonging to different duplicates. The lack of interaction of the duplicates RR12-14 and RR18-19, which are involved in the osmosensing pathway has been confirmed by BiFC experiments. This study reveals, for the first time, an overview of type-B RR dimerization in poplar and makes way for the hypothesis that signal specificity for cytokinin or osmotic stress could be in part due to the fact that it is impossible for specific type-B RRs to heterodimerize.

Highlighting type A RRs as potential regulators of the dkHK1 multi-step phosphorelay pathway in Populus.

In previous studies, we highlighted a multistep phosphorelay (MSP) system in poplars composed of two hybrid-type Histidine aspartate Kinases, dkHK1a and dkHK1b, which interact with three Histidine Phosphotransfer proteins, dkHPt2, 7, and 9, which in turn interact with six type B Response Regulators. These interactions correspond to the dkHK1a-b/dkHPts/dkRRBs MSP. This MSP is putatively involved in an osmosensing pathway, as dkHK1a-b are orthologous to the Arabidopsis osmosensor AHK1, and able to complement a mutant yeast deleted for its osmosensors. Since type A RRs have been characterized as negative regulators in cytokinin MSP signaling due to their interaction with HPt proteins, we decided in this study to characterize poplar type A RRs and their implication in the MSP. For a global view of this MSP, we isolated 10 poplar type A RR cDNAs, and determined their subcellular localization to check the in silico prediction experimentally. For most of them, the in planta subcellular localization was as predicted, except for three RRAs, for which this experimental approach gave a more precise localization. Interaction studies using yeast two-hybrid and in planta BiFC assays, together with transcript expression analysis in poplar organs led to eight dkRRAs being singled out as partners which could interfere the dkHK1a-b/dkHPts/dkRRBs MSP identified in previous studies. Consequently, the results obtained in this study now provide an exhaustive view of dkHK1a-b partners belonging to a poplar MSP.

Pinoresinol-lariciresinol reductase gene expression and secoisolariciresinol diglucoside accumulation in developing flax (Linum usitatissimum) seeds.

The transcription activity of the pinoresinol-lariciresinol reductase (PLR) gene of Linum usitatissimum (so-called LuPLR), a key gene in lignan synthesis, was studied by RT-PCR and promoter-reporter transgenesis. The promoter was found to drive transcription of a GUSint reporter gene in the seed coats during the flax seed development. This fitted well with the tissue localization monitored by semi-quantitative RT-PCR of LuPLR expression. Accumulation of the main flax lignan secoisolariciresinol diglucoside was coherent with LuPLR expression during seed development. This three-way approach demonstrated that the LuPLR gene is expressed in the seed coat of flax seeds, and that the synthesis of PLR enzyme occurs where flax main lignan is found stored in mature seeds, confirming its involvement in SDG synthesis.

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures.

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4'-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8'-linked lignan or 8-5'-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens.

Isolation of a flax pectin methylesterase promoter and its expression in transgenic tobacco.

Pectin methylesterases (PME) catalyze the de-esterification of methoxylated pectins in plant cell walls. We have isolated a 1.9 kb regulatory region upstream from the Lupme3 coding sequence of Linum usitatissimum L. (flax) using a 'Polymerase Chain Reaction (PCR) walking' strategy. Two 5' truncated deletion fragments (1.5 and 0.44 kb) of this potential promoter sequence were inserted upstream of the gus reporter gene in order to study their expression in transgenic plants. These constructs were transferred into Nicotiana tabacum, a heterologous system using Agrobacterium tumefaciens. Expression of the reporter gene was analyzed in regenerated transgenic plants and calli to study the promoter activities of these sequences. This expression was observed in calli with both constructs. In contrast, expression in organs was only detected in tobacco plants transformed with the largest (1.5 kb) construct. This long fragment triggered expression in roots and immature or vitrified leaves. Expression in both organs was localized in the vasculature, but also detected in the root meristem. These results are the first evidence, to our knowledge, of the spatial and temporal regulation of a specific pme promoter of flax. Localization of Lupme3 promoter activity in vascular tissues of immature organs provides an insight into the role of this PME isoform in cell elongation and differentiation.

Adrenalectomy prevents the development of alcohol preference in male rats.

By forcing adrenalectomized (ADX) and sham-operated (SHAM) rats to chronically drink ethanol by mean of presentation of only one drinking bottle containing 10% ethanol, no differences occurred between both groups. ADX and SHAM rats were then exposed to chronic alcoholization using an inhalation procedure. After sejourning 3 weeks into the alcoholization chamber, rats were submitted to a free-choice paradigm [water vs. a 10% (v/v) ethanol solution]. The sham-operated rats presented an alcohol-induced behavioral preference towards alcohol whereas adrenalectomized animals never exhibited a preference to ethanol. In the adrenalectomized rats treatment with hydrocortisone (30 micrograms/ml) given orally during the pulmonary alcoholization failed to modify this preference whereas treatment with corticosterone (25 micrograms/ml) given orally abolished the difference with SHAM animals. These data showed that adrenalectomy prevented the development of ethanol preference and the clear involvement of the hypothalamo-pituitary-axis in alcohol preference.

Adrenalectomy protects ethanol-withdrawn rats from harmine-induced tremor.

A growing number of studies have implicated the hypothalamic-pituitary-adrenal (HPA) axis in acute and chronic alcoholization and in ethanol withdrawal. In order to study the ethanol/HPA axis interaction during alcohol withdrawal, we performed experiments using adrenalectomized (ADX) male rats alcoholized by a chronic pulmonary alcoholization procedure. Eight hours after the 3 weeks of the alcoholization procedure, the rats were evaluated for a tremor activity. In order to reduce the great variability of the withdrawal tremors, we estimated the supersensitivity of the withdrawn rats to the tremorogenic compound harmine. We also studied the effect of a hydrocortisone treatment given in the drinking bottle during the alcoholization procedure on the harmine-induced tremors of ADX and sham rats. Alcohol withdrawal resulted in increased tremor response to 10 mg/kg harmine, and a protective effect of adrenalectomy on this effect was observed. Hydrocortisone administration to ADX or sham rats did not affect the tremor profile of the alcohol withdrawn rats.

N-tert-butyl-alpha-phenylnitrone administration fails to modify alcohol dependence and alcohol induced hypervascularization.

Chronic alcoholization by ethyl-alcohol inhalation was used to study the properties of a spin-trapping agent on different alcohol manifestations in rat. The spin-trapping agent, i.e. N-tert-butyl-alpha-phenylnitrone (PBN), was given at the dose of 32 mg/kg twice a day during the whole alcoholization procedure. The blood alcohol level, the hypermotility which accompanied the ethanol withdrawal, the behavioral dependence as estimated by a free-choice program and the hypervascularization which is developed after a chronic pulmonary alcoholization were quantified. The rats treated with PBN differ from the control rats only by their higher blood alcohol level at the end of the chronic alcoholization while the other quantified alcohol-induced manifestations remained unchanged.

Modulation of alcohol preference by NMDA antagonists in male rats.

Chronic alcoholization by alcohol inhalation was used to study the properties of magnesium, a non-competitive NMDA receptor antagonist, and CGP 39551, a competitive NMDA receptor antagonist, on behavioural dependence as estimated by the free-choice paradigm [alcohol 10% (v/v) vs. water], on the hypermotility after alcohol withdrawal, and finally on the cortical vascularization. The first experimental group received the drugs per os during the whole alcoholization period. Magnesium (20 mg/kg/day) decreased the alcohol dependence while CGP 39551 (5 and 10 mg/kg/day) increased, in a dose-dependent manner, the dependence to alcohol. A second group of animals received the same drugs at the same dosages, not simultaneously during chronic alcoholization, but immediately after alcoholization in one shot i.p. injection. In this case, rats receiving 5 mg/kg CGP 39551 never showed any dependence towards alcohol, while 10 mg/kg CGP 39551 or 20 mg/kg magnesium prolonged the number of days of alcohol dependence. These results thus indicate the close interaction between NMDA receptor function and dependence for alcohol. Magnesium had no effects on hypermotility, while CGP 39551-treated animals presented a decrease in the hypermotility observed after alcohol withdrawal. Neither drug affected the hypervascularization accompanying the chronic alcoholization.