Tissue‐specific inactivation by cytosine deaminase/uracil phosphoribosyl transferase as a tool to study plant biology

Recent advances in the study of plant developmental and physiological responses have benefited from tissue‐specific approaches, revealing the role of some cell types in these processes. Such approaches have relied on the inactivation of target cells using either toxic compounds or deleterious genes; however, both tissue‐specific and truly inducible tools are lacking in order to precisely target a developmental window or specific growth response. We engineered the yeast fluorocytosine deaminase (FCY1) gene by creating a fusion with the bacterial uracil phosphoribosyl transferase (UPP) gene. The recombinant protein converts the precursor 5‐fluorocytosine (5‐FC) into 5‐fluorouracyl, a drug used in the treatment of a range of cancers, which triggers DNA and RNA damage. We expressed the FCY‐UPP gene construct in specific cell types using enhancer trap lines and promoters, demonstrating that this marker acts in a cell‐autonomous manner. We also showed that it can inactivate slow developmental processes like lateral root formation by targeting pericycle cells. It also revealed a role for the lateral root cap and the epidermis in controlling root growth, a faster response. The 5‐FC precursor acts systemically, as demonstrated by its ability to inhibit stomatal movements when supplied to the roots in combination with a guard cell‐specific promoter. Finally, we demonstrate that the tissular inactivation is reversible, and can therefore be used to synchronize plant responses or to determine cell type‐specific functions during different developmental stages. This tool will greatly enhance our capacity to understand the respective role of each cell type in plant physiology and development.     

NADPH and oxidized thioredoxin mediate redox interconversion of calf-liver and Escherichia coli thioredoxin reductase

The activity of pure calf-liver and Escherichia coli thioredoxin reductases decreased drastically in the presence of NADPH or NADH, while NADP⁺, NAD⁺ and oxidized E. coli thioredoxin activated both enzymes significantly, particularly the bacterial one. The loss of activity under reducing conditions was time-dependent, thus suggesting an inactivation process: in the presence of 0.24 mM NADPH the half-lives for the E. coli and calf-liver enzymes were 13.5 and 2 min, respectively. Oxidized E. coli thioredoxin fully protected both enzymes from inactivation, and also promoted their complete reactivation after only 30 min incubation at 30° C. Lower but significant protection and reactivation was also observed with NADP⁺ and NAD⁺. EDTA protected thioredoxin reductase from NADPH inactivation to a great degree, thus indicating the participation of metals in the process; EGTA did not protect the enzyme from redox inactivation. Thioredoxin reductase was extensively inactivated by NADPH under aerobic and anaerobic conditions, thus excluding the participation of O₂ or oxygen active species in redox inactivation. The loss of thioredoxin reductase activity promoted by NADPH was much faster and complete in the presence of NAD⁺ glycohydrolase, thus suggesting that inactivation was related to full reduction of the redox-active disulfide. Those results indicate that thioredoxin reductase activity can be modulated in bacteria and mammals by the redox status of NADP(H) and thioredoxin pools, in a similar way to glutathione reductase. This would considerably expand the regulatory potential of the thioredoxin-thioredoxin reductase system with the enzyme being self-regulated by its own substrate, a regulatory protein.Abbreviations DTNB 5,5-dithiobis(2-nitrobenzoate) - EGTA Ethylenglycoltetraacetic Acid - TNB 5-thio-2-nitrobenzoate - Trx Thioredoxin - Trx(SH)2 Reduced Thioredoxin - Trx-S2 Oxidized Thioredoxin     

Mechanism of Fe uptake by the leaf symplast: Is Fe inactivation in leaf a cause of Fe deficiency chlorosis?

The mechanism of iron (Fe) uptake from the leaf apoplast into leaf mesophyll cells was studied to evaluate the putative Fe inactivation as a possible cause of Fe deficiency chlorosis. For this purpose, sunflower (Helianthus annuus L.) and faba bean plants (Vicia faba L.) were precultured with varied Fe and bicarbonate (HCO ₃ ^- ) supply in nutrient solution. After 2–3 weeks preculture, Fe^III reduction and ⁵⁹Fe uptake by leaf discs were measured in solutions with Fe supplied as citrate or synthetic chelates in darkness. The data clearly indicate that Fe^III reduction is a prerequisite for Fe uptake into leaf cells and that the Fe nutritional status of plants does not affect either process. In addition, varied supply of Fe and HCO ₃ ^- to the root medium during preculture had no effect on pH of the xylem sap and leaf apoplastic fluid. A varied pH of the incubation solution had no significant effect on Fe^III reduction and Fe uptake by leaf discs in the physiologically relevant pH range of 5.0–6.0 as measured in the apoplastic leaf fluid. It is concluded that Fe inactivation in the leaf apoplast is not a primary cause of Fe deficiency chlorosis induced by bicarbonate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inactivation of epoxide hydrolase by catalysis-induced formation of isoaspartate

Epoxide hydrolases catalyze hydrolytic epoxide ring-opening, most often via formation of a covalent hydroxyalkyl-enzyme intermediate. A mutant of Agrobacterium radiobacter epoxide hydrolase, in which the phenylalanine residue that flanks the invariant catalytic aspartate nucleophile is replaced by a threonine, exhibited inactivation during conversion when the (R)-enantiomer of para-nitrostyrene epoxide was used as substrate. HPLC analysis of tryptic fragments of the epoxide hydrolase, followed by MALDI-TOF and TOF/TOF analysis, indicated that inactivation was due to conversion of the nucleophilic aspartate into isoaspartate, which represents a novel mechanism of catalysis-induced autoinactivation. Inactivation occurred at a lower rate with the (S)-enantiomer of para-nitrostyrene epoxide, indicating that it is related to the structure of the covalent hydroxyalkyl-enzyme intermediate.     

溶血磷脂酸对β-淀粉样蛋白片段AβP31-35所致小鼠大脑皮层离体神经元凋亡的神经保护作用

已报道低浓度溶血磷脂酸(lysophosphatidic acid,LPA)对去血清培养所致的神经元凋亡有神经保护作用.为了进一步观察LPA是否对β-amyloid peptide fragment 31-35(AβP31-35)所致的神经元凋亡也起类似的作用,本研究应用DNA电泳分析、HO33342和TUNEL染色法等技术,对培养的小鼠大脑皮层神经元进行了观察.结果显示,只有使用较低浓度的LPA(1～10μmol/L)、并且将此剂量的LPA比AβP31-35提前12～24 h加入培养液时,才可看到LPA明显削弱了AβP31-35所致的神经元凋亡.以上结果表明,适当浓度的LPA在长时间预作用的条件下,可对AβP31-35所致的皮层神经元凋亡起保护因子或抗凋亡因子的作用,但其作用途径可能较在去血清培养所致的凋亡时更为复杂,因为在去血清的同时加入LPA就能制止去血清所致的凋亡.     

Effects of chronic iTBS‐rTMS and enriched environment on visual cortex early critical period and visual pattern discrimination in dark‐reared rats

Early cortical critical period resembles a state of enhanced neuronal plasticity enabling the establishment of specific neuronal connections during first sensory experience. Visual performance with regard to pattern discrimination is impaired if the cortex is deprived from visual input during the critical period. We wondered how unspecific activation of the visual cortex before closure of the critical period using repetitive transcranial magnetic stimulation (rTMS) could affect the critical period and the visual performance of the experimental animals. Would it cause premature closure of the plastic state and thus worsen experience‐dependent visual performance, or would it be able to preserve plasticity? Effects of intermittent theta‐burst stimulation (iTBS) were compared with those of an enriched environment (EE) during dark‐rearing (DR) from birth. Rats dark‐reared in a standard cage showed poor improvement in a visual pattern discrimination task, while rats housed in EE or treated with iTBS showed a performance indistinguishable from rats reared in normal light/dark cycle. The behavioral effects were accompanied by correlated changes in the expression of brain‐derived neurotrophic factor (BDNF) and atypical PKC (PKCζ/PKMζ), two factors controlling stabilization of synaptic potentiation. It appears that not only nonvisual sensory activity and exercise but also cortical activation induced by rTMS has the potential to alleviate the effects of DR on cortical development, most likely due to stimulation of BDNF synthesis and release. As we showed previously, iTBS reduced the expression of parvalbumin in inhibitory cortical interneurons, indicating that modulation of the activity of fast‐spiking interneurons contributes to the observed effects of iTBS. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 19–33, 2016     

Inactivation of beef heart mitochondrial F1-ATPase by the 2',3'-dialdehyde derivatives of adenine nucleotides

Beef heart mitochondrial F1-ATPase was inactivated by the 2',3'-dialdehyde derivatives of ATP, ADP and AMP (oATP, oADP, oAMP). In the absence of Mg2+, inactivation resulted from the binding of 1 mol nucleotide analog per active unit of F1. The most efficient analog was oADP, followed by oAMP and oATP. Complete inactivation was correlated with the binding of about 11 mol [14C]oADP/mol F1. After correction for non-specific labeling, the number of specifically bound [14C]oADP was 2-3 mol per mol F1. By SDS-polyacrylamide gel electrophoresis, [14C]oADP was found to bind covalently mainly to the alpha and beta subunits. In the presence of Mg2+, oATP behaved as a substrate and was slowly hydrolyzed.     

Cellulose microfibril alignment recovers from DCB-induced disruption despite microtubule disorganization

Cellulose microfibril deposition patterns define the direction of plant cell expansion. To better understand how microfibril alignment is controlled, we examined microfibril orientation during cortical microtubule disruption using the temperature-sensitive mutant of Arabidopsis thaliana, mor1-1. In a previous study, it was shown that at restrictive temperature for mor1-1, cortical microtubules lose transverse orientation and cells lose growth anisotropy without any change in the parallel arrangement of cellulose microfibrils. In this study, we investigated whether a pre-existing template of well-ordered microfibrils or the presence of well-organized cortical microtubules was essential for the cell to resume deposition of parallel microfibrils. We first transiently disrupted the parallel order of microfibrils in mor1-1 using a brief treatment with the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB). We then analysed the alignment of recently deposited cellulose microfibrils (by field emission scanning electron microscopy) as cellulose synthesis recovered and microtubules remained disrupted at the mor1-1 mutant's non-permissive culture temperature. Despite the disordered cortical microtubules and an initially randomized wall texture, new cellulose microfibrils were deposited with parallel, transverse orientation. These results show that transverse cellulose microfibril deposition requires neither accurately transverse cortical microtubules nor a pre-existing template of well-ordered microfibrils. We also demonstrated that DCB treatments reduced the ability of cortical microtubules to form transverse arrays, supporting a role for cellulose microfibrils in influencing cortical microtubule organization.     

Methanol metabolism in yeasts: Regulation of the synthesis of catabolic enzymes

The regulation of the synthesis of four dissimilatory enzymes involved in methanol metabolism, namely alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase and catalase was investigated in the yeasts Hansenula polymorpha and Kloeckera sp. 2201. Enzyme profiles in cell-free extracts of the two organisms grown under glucose limitation at various dilution rates, suggested that the synthesis of these enzymes is controlled by derepression — represion rather than by induction — repression. Except for alcohol oxidase, the extent to which catabolite repression of the catabolic enzymes was relieved at low dilution rates was similar in both organisms. In Hansenula polymorpha the level of alcohol oxidase in the cells gradually increased with decreasing dilution rate, whilst in Kloeckera sp. 2201 derepression of alcohol oxidase synthesis was only observed at dilution rates below 0.10 h^–1 and occurred to a much smaller extent than in Hansenula polymorpha.Derepression of alcohol oxidase and catalase in cells of Hansenula polymorpha was accompanied by synthesis of peroxisomes. Moreover, peroxisomes were degraded with a concurrent loss of alcohol oxidase and catalase activities when excess glucose was introduced into the culture. This process of catabolite inactivation of peroxisomal enzymes did not affect cytoplasmic formaldehyde dehydrogenase.