Wu-Sheng Liang

Systemic Induction and Role of Mitochondrial Alternative Oxidase and Nitric Oxide in a Compatible Tomato–Tobacco mosaic virus Interaction

The role of mitochondrial alternative oxidase (AOX) and the relationship between AOX and nitric oxide (NO) in virus-induced systemic defense to Tobacco mosaic virus (TMV) were investigated in susceptible tomato (Solanum lycopersicum) plants. TMV inoculation to the lower leaves induced a rapid NO synthesis and AOX activation in upper uninoculated leaves as early as 0.5 day postinoculation. Application of exogenous potassium cyanide (KCN, a cytochrome pathway inhibitor) at nonlethal concentrations and NO donor diethylamine NONOate (DEA/NO) to the upper uninoculated leaves greatly induced accumulation of AOX transcript, reduced TMV viral RNA accumulation, and increased the leaf photochemical quantum yield at photosystem II. Pretreatment with NO scavenger almost completely blocked TMV-induced AOX induction and substantially increased TMV susceptibility. Salicylhydroxamic acid (SHAM, an AOX inhibitor) pretreatment reduced the DEA/NO-induced cyanide-resistant respiration and partially compromised induced resistance to TMV. Conversely, KCN and SHAM pretreatment had very little effect on generation of NO, and pretreatment with NO scavenger did not affect KCN-induced AOX induction and TMV resistance. These results suggest that TMV-induced NO generation acts upstream and mediates AOX induction which, in turn, induces mitochondrial alternative electron transport and triggers systemic basal defense against the viral pathogen.

The reduction of reactive oxygen species formation by mitochondrial alternative respiration in tomato basal defense against TMV infection

The role of mitochondrial alternative oxidase (AOX) and the relationship between systemic AOX induction, ROS formation, and systemic plant basal defense to Tobacco mosaic virus (TMV) were investigated in tomato plants. The results showed that TMV inoculation significantly increased the level of AOX gene transcripts, ubiquinone reduction levels, pyruvate content, and cyanide-resistant respiration (CN-resistant R) in upper, un-inoculated leaves. Pretreatment with potassium cyanide (KCN, a cytochrome pathway inhibitor) greatly increased CN-resistant R and reduced reactive oxygen species (ROS) formation, while application of salicylhydroxamic acid (SHAM, an AOX inhibitor) blocked the AOX activity and enhanced the production of ROS in the plants. Furthermore, TMV systemic infection was enhanced by SHAM and reduced by KCN pretreatment, as compared with the un-pretreated TMV counterpart. In addition, KCN application significantly diminished TMV-induced increase in antioxidant enzyme activities and dehydroascorbate/total ascorbate pool, while an opposite change was observed with SHAM-pretreated plants. These results suggest that the systemic induction of the mitochondrial AOX pathway plays a critical role in the reduction of ROS to enhance basal defenses. Additional antioxidant systems were also coordinately regulated in the maintenance of the cellular redox homeostasis.

Drought stress increases both cyanogenesis and β-cyanoalanine synthase activity in tobacco

Abstract Results of relative water content (RWC) detection and morphology observation indicated that tobacco ( Nicotiana tabaccum L. cv. Samsun N/N) plants could resist drought stress for 2 days. Ethylene production rates of the tobacco leaves were increased by 9–12-folds during the 2 days of drought stress. Ethylene production rate of the tobacco roots was also increased by about 10-folds during the first day of drought stress. In the meantime, after 1 day of drought stress the cyanide levels in the tobacco leaves and roots were increased by 6.3 and 3.5-folds, respectively. However, during the second day of drought stress the cyanide levels declined to similar levels as those in the unstressed tobacco. Upon drought stress activity of β-cyanoalanine synthase (CAS, EC 4.4.1.9), the main enzyme for cyanide detoxification in plants, sharply increased in the tobacco leaves and roots, but declined once the tobacco plants were re-watered. Activity detection on gel following non-denaturing polyacrylamide gel electrophoresis (PAGE) showed that activities of the two CAS isozymes in tobacco were both induced by drought stress. Spraying 10 μmol l −1 KCN solution onto the leaves of normally watered tobacco plants could obviously induce the CAS activity in the leaves, showing a close relationship between cyanide detoxification necessity and CAS activity therein. The above results indicate that cyanogenesis may be induced by drought stress in plants and CAS may play an important role during the defense of plants against drought stress by cyanide detoxification.

Genome-Wide Identification of QTL for Seed Yield and Yield-Related Traits and Construction of a High-Density Consensus Map for QTL Comparison in Brassica napus

Seed yield (SY) is the most important trait in rapeseed, is determined by multiple seed yield-related traits (SYRTs) and is also easily subject to environmental influence. Many quantitative trait loci (QTLs) for SY and SYRTs have been reported in Brassica napus; however, no studies have focused on seven agronomic traits simultaneously affecting SY. Genome-wide QTL analysis for SY and seven SYRTs in eight environments was conducted in a doubled haploid population containing 348 lines. Totally, 18 and 208 QTLs for SY and SYRTs were observed, respectively, and then these QTLs were integrated into 144 consensus QTLs using a meta-analysis. Three major QTLs for SY were observed, including cqSY-C6-2 and cqSY-C6-3 that were expressed stably in winter cultivation area for 3 years and cqSY-A2-2 only expressed in spring rapeseed area. Trait-by-trait meta-analysis revealed that the 144 consensus QTLs were integrated into 72 pleiotropic unique QTLs. Among them, all the unique QTLs affected SY, except for uq.A6-1, including uq.A2-3, uq.C1-2, uq.C1-3, uq.C6-1, uq.C6-5, and uq.C6-6 could also affect more than two SYRTs. According to the constructed high-density consensus map and QTL comparison from literatures, 36 QTLs from five populations were co-localized with QTLs identified in this study. In addition, 13 orthologous genes were observed, including five each gene for SY and thousand seed weight, and one gene each for biomass yield, branch height, and plant height. The genomic information of these QTLs will be valuable in hybrid cultivar breeding and in analyzing QTL expression in different environments.

Stimulation of ethylene production in aged potato tuber slices by salicylic acid

The influence of salicylic acid (SA) on ethylene formation in aged potato (Solanum tuberosum) tuber slices was investigated. SA treatments significantly stimulated ethylene production of the slices during 24 hr of ageing. Up to 90 μM SA (the highest concentration tested), the stimulation was positively correlated with concentrations. SA showed stimulation effects on ethylene production at pH 5.4, 6.4 and 7.4, with the greatest stimulation at pH 6.4. These results show that SA enhances endogenous ethylene formation in aged potato tuber slices. This stimulation effect of SA is different from the general conception that SA ultimately inhibits ethylene biosynthesis in plants.

Effects of modifying alternative respiration on nitric oxide-induced virus resistance and PR1 protein accumulation.

Nitric oxide (NO) is an important defensive signal in plants but its effects on virus infection are not well understood. Administration of NO-releasing compounds immediately before inoculation of tobacco leaves with potato virus X and tobacco mosaic virus decreased the accumulation of virus, indicating that NO can induce resistance rapidly. Resistance induction was inhibited by co-administration with an NO-scavenging compound or when experiments were done in transgenic tobacco plants expressing increased alternative respiratory pathway capacity due to constitutive expression of the plant mitochondrial enzyme, alternative oxidase (AOX). These results indicate that NO, which inhibits electron transport chain activity, is triggering defensive signalling by inducing changes in mitochondrial reactive oxygen species levels that are in turn regulated by AOX. Experiments using nahG-transgenic plants, which cannot accumulate the defensive plant hormone salicylic acid (SA) showed that NO rapidly induces resistance to virus infection independently of SA. However, this initial state of resistance may be transient. Subsequently, by 5 days post-treatment, NO had caused an increase in pathogenesis-related protein 1 (PR1) expression (a proxy for increased SA biosynthesis), which correlated with a longer-term state of resistance to virus infection. The induction by NO of PR1 accumulation was modified in AOX-transgenic plants. This indicates that the influence of NO on defensive gene expression is in part mediated through its effects on mitochondria.

Increase of HCN and β-cyanoalanine synthase activity during ageing of potato tuber slices

The evolution of HCN increased significantly during the ageing process of slices of potato (Solanum tuberosum L.) tubers in 24 h. Concomitantly, the activity of β-cyanoalanine synthase (CAS)(E.C. 4.4.1.9) was greatly enhanced in the same ageing period. Both the activity of CAS and the evolution of HCN were stimulated by 1-aminocyclopropane-1-carboxylic acid (ACC) and inhibited by salicylic acid (SA). It was also shown that the production of endogenous ethylene in aged slices exhibited a similar increase. Ethylene production was also affected by ACC and SA. A possible relationship between ethylene production, HCN evolution and the stimulation of CAS activity is discussed.

The two β-cyanoalanine synthase isozymes of tobacco showed different antioxidative abilities

Abstract β-Cyanoalanine synthase (CAS, EC 4.4.1.9) catalyzes the conversion of cyanide and cysteine to β-cyanoalanine and plays pivotal roles in cyanide detoxification of plants. Both of tobacco leaves and roots showed some CAS activities, although with a much higher value in leaves. Crude CAS extracted from tobacco leaves or roots using buffers containing such reductants as cysteine (Cys), dithiothreitol (DTT) and ascorbic acid (AsA), produced two CAS bands on gel when detected with lead acetate following non-denaturing polyacrylamide gel electrophoresis (PAGE). They were designated CAS 1 and CAS 2 , respectively, CAS 2 with a relatively higher migration rate than CAS 1 . CAS 1 had higher activities than CAS 2 in both tobacco leaves and roots. Activity detection on gel showed that both CAS isozymes existed in the isolated cytosol from tobacco leaves, CAS 2 having a higher activity than CAS 1 . But only CAS 1 was observed in the isolated mitochondria and no CAS was found in the isolated chloroplast. When measured with N , N -dimethyl- p -phenylenediamine (DMPDA) CAS activities were also found in the isolated cytosol and mitochondria, but not in the isolated chloroplast. Crude CAS extracted from tobacco leaves or roots using buffers without any of the above reductants produced only CAS 1 on gel when detected with lead acetate following non-denaturing PAGE. Reductants also showed significant protective effects on the activity of the CAS extracts when measured with DMPDA. In vitro treatment of the crude CAS samples extracted in the absence of any reductants with cysteine of 10–1000 mM could not produce CAS 2 on gel. These results indicate that CAS 2 isozyme of tobacco is very sensitive to oxidation, but CAS 1 isozyme possesses a distinctly higher antioxidative ability than CAS 2 . Reductants such as cysteine, dithiothreitol, and ascorbic acid have significant protective effects on CAS 2 during tissue disruption. The difference in antioxidative ability may be used to distinguish different CAS isozymes, and give new clues for their variant physiological significances in plants.

Comparison of the measurement of the in vivo activity of the alternative pathway in aged potato tuber slices

Summary Recently it was reported that the use of the hydroxarnate-inhibicing method leads to an underestimation of the in vivo alternative pathway (AP) activity and its contribution to total respiration (V t ) in plants. However, there is still a dispute about the extent of these possible underestimations. In this paper the oxygen-isotope-discrimination (OID) technique and the hydroxamate-inhibiting method have been compared for aged potato ( Solanum tuberosum L.) tuber slices. As a result, the in vivo contributions of AP activity to V t , were calculated to be 15.7% and 16.8% with the hydroxamate-inhibiting method, and 30.2% and 34.9% with the OID method in slices aged for 12 and 24 h, respectively. The observed underestimations of about one-fold confirm the suggestions with respect to the unsuitability of the hydroxamate-inhibiting method for the determination of the in vivo AP activity.