Putrescine protects hulless barley from damage due to UV-B stress via H<Subscript>2</Subscript>S- and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-mediated signaling pathways

Key message

In hulless barley, H ₂ S mediated increases in H ₂ O ₂ induced by putrescine, and their interaction enhanced tolerance to UV-B by maintaining redox homeostasis and promoting the accumulation of UV-absorbing compounds.

Abstract

This study investigated the possible relationship between putrescence (Put), hydrogen sulfide (H₂S) and hydrogen peroxide (H₂O₂) as well as the underlying mechanism of their interaction in reducing UV-B induced damage. UV-B radiation increased electrolyte leakage (EL) and the levels of malondialdehyde (MDA) and UV-absorbing compounds but reduced antioxidant enzyme activities and glutathione (GSH) and ascorbic acid (AsA) contents. Exogenous application of Put, H₂S or H₂O₂ reduced some of the above-mentioned negative effects, but were enhanced by the addition of Put, H₂S and H₂O₂ inhibitors. Moreover, the protective effect of Put against UV-B radiation-induced damage to hulless barley was diminished by dl-propargylglycine (PAG, a H₂S biosynthesis inhibitor), hydroxylamine (HT, a H₂S scavenger), diphenylene iodonium (DPI, a PM-NADPH oxidase inhibitor) and dimethylthiourea (DMTU, a ROS scavenger), and the effect of Put on H₂O₂ accumulation was abolished by HT. Taken together, as the downstream component of the Put signaling pathway, H₂S mediated H₂O₂ accumulation, and H₂O₂ induced the accumulation of UV-absorbing compounds and maintained redox homeostasis under UV-B stress, thereby increasing the tolerance of hulless barley seedlings to UV-B stress.

     

External spermine prevents UVA-induced damage of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 via increased catalase activity and decreased H<Subscript>2</Subscript>O<Subscript>2</Subscript> and malonaldehyde levels

Common polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), are cationic compounds known as beneficial factors for many cellular processes including cell division, proliferation, differentiation, and stress response in all living organisms. Effects of exogenous Spm on the protective responses of Synechocystis sp. PCC 6803 exposed to UVA were investigated. The presence of 0.5 mM Spm in the culture medium significantly reduced cell growth after 60 min under white light condition but protected the cells after growing for 60 min under UVA. The stress-tolerant response of Synechocystis cells represented by the ratio of putrescine/spermidine (Put/Spd) showed about a 6-fold increase after 60 min UVA in the presence of Spm. In addition, those levels of chlorophyll a, carotenoids, and photosynthetic oxygen evolution were increased by Spm supplementation in UVA-treated cells. Exogenous Spm induced the activity of catalase but not superoxide dismutase in cells under UVA treatment. On the other hand, Spm treatment enabled cells to apparently decrease the intracellular free radical H₂O₂ and malonaldehyde (MDA) levels. Overall results suggested that Spm supplementation could protect Synechocystis sp. PCC 6803 cells via the increase of Put/Spd ratio and the reduction of both H₂O₂ and MDA levels in conjunction with the induction of catalase activity. Interestingly, UVA-treated cells as compared to non-treated cells with exogenous Spm showed a decrease of Spm with an increase of Put and no change in Spd. This suggested the back conversion of Spm to Spd and finally to Put as cellular mechanism in response to UVA.     

Halophilic rhizobacteria from <Emphasis Type="Italic">Distichlis spicata</Emphasis> promote growth and improve salt tolerance in heterologous plant hosts

Rhizobacteria are central components of the plant microbiome and influence root development and function. Desciphering how rhizobacteria contribute to plant performance under adverse environments is a major research challenge. The aims of the present study were to isolate and characterize rhizobacteria from the halophilic grass Distichlis spicata and to test their possible growth promoting and salt protective properties in Arabidopsis thaliana, Cucumis sativus, and Citrullus lanatus. To determine their possible plant growth promoting properties, 38 rhizobacterial isolates were co-cultivated with Arabidopsis seedlings in vitro. Out of these, two halophilic bacteria, LBEndo1 and KBEcto4, were selected following their strong shoot and root biostimulation. 16S rRNA sequencing identified LBEndo1 as Bacillus sp. and KBEcto4 as Pseudomonas lini. Both strains improved growth under standard and saline conditions, which correlated with IAA and siderophore production, as well as phosphate solubilization. Additionally, the KBEcto4 strain expresses the ACC deaminase enzyme (acdS gene), and slightly increases auxin redistribution within Arabidopsis roots expressing an auxin-inducible gene construct. These data reveal the potential of saltgrass (Distichlis spicata) rhizobacteria to promote growth and confer salt tolerance to Arabidopsis and crop plants.     

Ectomycorrhizae of <Emphasis Type="Italic">Tuber huidongense</Emphasis> and <Emphasis Type="Italic">T. liyuanum</Emphasis> with <Emphasis Type="Italic">Castanea mollissima</Emphasis> and <Emphasis Type="Italic">Pinus armandii</Emphasis>

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees—Castanea mollissima and Pinus armandii—under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.     

Molecular diversity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing PGPR from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) rhizosphere

Aims

The present study was planned to investigate the diversity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing bacteria from the rhizosphere of wheat plants and subsequent evaluation of selected PGPR on growth enhancement of wheat seedlings under drought and saline conditions.

Methods

ACC deaminase producing plant growth promoting rhizobacteria (PGPR) were isolated from the rhizosphere of wheat and identified using 16S rRNA gene sequence analysis. Isolates were evaluated for various direct and indirect plant growth promoting (PGP) traits. Plant inoculation experiment was conducted using isolates IG 19 and IG 22 in wheat to assess their plant growth promotion potential under salinity and drought stress.

Results

Thirty-eight ACC deaminase producing PGPR were isolated which belonged to 12 distinct genera and falling into four phyla γ-proteobacteria, β-proteobacteria, Flavobacteria and Firmicutes. Klebsiella sp. was the most abundant genera and followed by Enterobacter sp. The isolates exhibited ACC deaminase activities ranging from 0.106–0.980 μM α- ketobutyrate μg protein^?1 h^?1. The isolates showed multiple PGP traits such as IAA production, phosphate, zinc, potassium solubilization and siderophore production. Enterobacter cloacae (IG 19) and Citrobacter sp. (IG 22) inoculated wheat seedlings showed notable increases in fresh and dry biomass under non-stress as well as under stressed condition.

Conclusion

To the best of our knowledge this is the first report of presence of ACC deaminase activity and other PGP traits from the genus Citrobacter and Empedobacter. Our finding revealed that the γ-proteobacteria group dominated the wheat rhizosphere. Plant inoculation with PGPR could be a sustainable approach to alleviate abiotic stresses in wheat plants. These native PGPR isolates could be used as potential biofertilizers for sustainable agriculture.

     

Molecular characterization and homology modeling of spermidine synthase from <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7942

Spermidine synthase (Spds) catalyzes the formation of spermidine by transferring the aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The Synechococcus spds gene encoding Spds was expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 33 kDa and showed optimal activity at pH 7.5, 37?°C. The enzyme had higher affinity for dcSAM (K _m, 20 µM) than for putrescine (K _m, 111 µM) and was highly specific towards the diamine putrescine with no activity observed towards longer chain diamines. The three-dimensional structural model for Synechococcus Spds revealed that most of the ligand binding residues in Spds from Synechococcus sp. PCC 7942 are identical to those of human and parasite Spds. Based on the model, the highly conserved acidic residues, Asp89, Asp159 and Asp162, are involved in the binding of substrates putrescine and dcSAM and Pro166 seems to confer substrate specificity towards putrescine.     

The root growth of wheat plants,the water conservation and fertility status of sandy soils influenced by plant growth promoting rhizobacteria

Plant growth promoting rhizobacteria were isolated and characterized from sandy soils in Pakistan. The role of the rhizobacteria, in association with plant growth regulators, was studied on the roots of wheat grown under water stressed conditions. The plant growth promoting rhizobacteria were characterized on the basis of colony morphology, biochemical traits and identified on the basis of 16S-rRNA gene sequencing which identified the selected isolates Planomicrobium chinense, Bacillus cereus and Pseudomonas fluorescens. Antibacterial and antifungal activities were determined. The fresh cultures (24 h old) of isolates were used to soak the seeds for 2–3 h prior to sowing. The growth regulators salicylic acid and putrescine were applied to the plant as foliar spray at three leaf stage. The plant growth promoting rhizobacteria produced exopolysaccharides that formed soil aggregation around roots of the plants and significantly enhanced water holding capacity of sandy soil. The relative water content (80%) of leaves and root fresh (80%) and dry weight (68%) were higher in plant growth promoting rhizobacteria inoculated plants. The nutrient content of rhizosphere soil of treated plants was also enhanced (Ca 35%, K 34%, Mg 52% and Na 42%) over stressed controls. Integrative use of effective plant growth promoting rhizobacteria in combination with salicylic acid appears to be an effective eco-friendly approach to increase drought tolerance in wheat plants to combat desertification.     

Usage of strain-producers of adhesin RapA1 from <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> for the creation of binary biofertilizers

Binary biofertilizers consisting of a rhizobial strain specific to the plant Rhizobium leguminosarum and a strain-producer of the RapA1 adhesin protein, which enhances the adhesion of rhizobacteria to the plant roots, have been designed. A system consisting of highly productive bacteria strains and additional strains that enhance the action of the main component have been obtained.     

Metabolic evolution and a comparative omics analysis of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for putrescine production

Putrescine is widely used in the industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Because the highest titer of putrescine is much lower than that of its precursor l-ornithine reported in microorganisms to date, further work is needed to increase putrescine production in Corynebacterium glutamicum. We first compared 7 ornithine decarboxylase genes and found that the Enterobacter cloacae ornithine decarboxylase gene speC1 was most suitable for putrescine production in C. glutamicum. Increasing NADPH availability and blocking putrescine oxidation and acetylation were chosen as targets for metabolic engineering. The putrescine producer C. glutamicum PUT4 was first constructed by deleting puo, butA and snaA genes, and replacing the fabG gene with E. cloacae speC1. After adaptive evolution with C. glutamicum PUT4, the evolved strain C. glutamicum PUT-ALE, which produced an 96% higher amount of putrescine compared to the parent strain, was obtained. The whole genome resequencing indicates that the SNPs located in the odhA coding region may be associated with putrescine production. The comparative proteomic analysis reveals that the pentose phosphate and anaplerotic pathway, the glyoxylate cycle, and the ornithine biosynthetic pathway were upregulated in the evolved strain C. glutamicum PUT-ALE. The aspartate family, aromatic, and branched chain amino acid and fatty acid biosynthetic pathways were also observed to be downregulated in C. glutamicum PUT-ALE. Reducing OdhA activity by replacing the odhA native start codon GTG with TTG and overexpression of cgmA or pyc458 further improved putrescine production. Repressing the carB, ilvH, ilvB and aroE expression via CRISPRi also increased putrescine production by 5, 9, 16 and 19%, respectively.     

Response of the predatory mite <Emphasis Type="Italic">Cydnoseius negevi</Emphasis> (Acari: Phytoseiidae) to webbing of the date palm mite, <Emphasis Type="Italic">Oligonychus afrasiaticus</Emphasis> (Acari: Tetranychidae), on date palm fruits and leaves

The behavioral response of a generalist phytoseiid predator, Cydnoseius negevi (Swirskii & Amitai) (Acari: Phytoseiidae) to the complicated webbing of the date palm mite (DPM) Oligonychus afrasiaticus (McGregor) (Acari: Tetranychidae) was evaluated for the first time on date palm leaves and fruits, both in the laboratory and on field-collected samples. Cydnoseius negevi used its first pair of legs in a swimming-like to-and-fro movement to penetrate the complicated webbing of DPM and demonstrated three attack behaviors, i.e., patrolling (PG), web invasion (WI), and web penetration (WP), against the webbing of DPM on date fruits and leaves. The time spent by the predator on attack behaviors was significantly longer in the laboratory for treatments where either more prey females or immature stages along with females were present. The time spent by C. negevi on response behaviors such as searching, grooming, and resting increased with increased number of DPM females and with the addition of immature stages along with DPM females. Cydnoseius negevi, even though being a generalist phytoseiid predator, showed its potential towards penetrating the complex dense webs of DPM on date palm fruits and leaves. If released in suitable numbers prior to establishment of DPM colonies, C. negevi may prove to be an effective biological control agent.     

Toxicity and Detoxification Mechanism of Black Pepper and Its Major Constituent in Controlling <Emphasis Type="Italic">Rhynchophorus ferrugineus</Emphasis> Olivier (Curculionidae: Coleoptera)

The survival, feeding response, and detoxification mechanism of Rhynchophorus ferrugineus Olivier, a key pest responsible for destruction of date palm, was examined with different extracts of Piper nigrum and its major constituent (piperine) identified by GC-MS. In the present study, toxicity of different extracts of black pepper was evaluated by incorporating diffferent doses of extracts into the artificial diet of red palm weevil larvae. All extracts showed dose-dependent insecticidal activity to the tested eighth-instar red palm weevil larvae. Among all the extracts, maximum larvicidal activity was exhibited by chloroform (LD₅₀ = 342.62 mg/l), followed by dichloromethane (LD₅₀ = 357.78 mg/l), acetone (LD₅₀ = 372.57 mg/l), and ethanol (LD₅₀ = 408.88 mg/l). However, piperine, a major constituent of all black pepper extracts identified by GC-MS in the present work, was found to be the most potent treatment exhibiting the least LD₅₀ (219.88 mg/l). In addition, nutritional indices evaluated by calculating the efficiency of the conversion of ingested food (ECI) and digested food (ECD) at the same dose (219.88 mg/l) showed that there was maximum reduction in the ECI (49.90%) and ECD (62.21%) index of larvae fed diets incorporated with piperine. Larvae that were fed diets incorporated with different black pepper extracts experienced increases in the expression of detoxification genes (glutathione S-transferase and cytochrome P450), and this upregulation in detoxification genes (glutathione S-transferase, cytochrome P450 and esterase) was tremendously high in larvae fed diets incorporated with piperine. Results suggest that piperine is a promising bio-pesticide agent for the control of R. ferrugineus Olivier.     

Ameliorative effects of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> on monocot crops under hydroponic saline environment

Ameliorative effects of Trichoderma harzianum (Th-6) on monocot crops under saline environment using hydroponic system were examined. Both rice and maize seeds were coated with T. harzianum (Th-6) and used for the saline and non-saline treatment. Germination and seedling growth performance were studied. T. harzianum (Th-6)-treated seeds showed constantly faster and more uniform germination as compared to untreated seeds. Moreover, seeds treated with Trichoderma improved plants’ growth and physiological performance under hydroponic saline environment compared to control. The treatments showed higher relative water content (RWC), dark-adapted quantum yield (F _v/F _m ratio), performance index (PI_ABS), photochemical quenching (q _P), stomatal conductance (g _s), pigments concentrations and antioxidant enzymes as compared to untreated saline environment. Application of endophyte inhibited the Na⁺ and Cl^? ion uptake in leaves when plants were exposed to saline environment. However, H₂O₂ contents of both treated crops declined under hydroponic salt stress environment. Physiological mechanism of T. harzianum (Th-6) application in mitigating the salt-related consequences of both monocot crops was discussed.     

<Emphasis Type="Italic">Rhizobium</Emphasis> as plant probiotic for strawberry production under microcosm conditions

There is increasing interest in the use of plant growth-promoting rhizobacteria (PGPR) as environmental-friendly and healthy biofertilizers. Strawberries (Fragraria x ananassa) are mainly consumed fresh and hence any PGPRs used for biofertilization must be safe for humans, which is the case for members of the genus Rhizobium. In this study, the effects of inoculation of strawberry plants with Rhizobium sp. strain PEPV16, which belongs to the phylogenetic group of R. leguminosarum, and whose plant growth promotion ability has been reported previously for lettuce (Lactuca sativa) and carrots (Daucus carota), was examined. The results demonstrated that PEPV16 promotes strawberry growth through significant increases in the number of stolons, flowers and fruits as compared with uninoculated controls. Compared to uninoculated controls, the fruits of the inoculated plants had higher concentrations of Fe, Zn, Mn and Mo, and they also had higher concentrations of organic acids, such as citric and malic acid, and lower amounts of ascorbic acid than fruits. Although decreases in ascorbic acid have previously been described after the inoculation of strawberry with strains from different PGPR genera, this is the first study to report increases in organic acids after PGPR inoculation.     

Promotion of Peanut Growth by Co-inoculation with Selected Strains of <Emphasis Type="Italic">Bradyrhizobium</Emphasis> and <Emphasis Type="Italic">Azospirillum</Emphasis>

The ability of inoculated rhizobial strains to increase root nodulation of host legumes often depends on their competitiveness with existing native soil strains. Results of studies to date on rhizobial inoculation for improvement of peanut (Arachis hypogaea L.) production in Argentina have been inconsistent and controversial. In many cases, nodulation and yield of peanut crops have been increased by inoculation of specific rhizobial strains. Native peanut-nodulating strains are generally present in soils of agricultural areas, but their growth-promoting effect is often lower than that of inoculated strains. Many species of the genus Bradyrhizobium interact in a host-specific manner with legume species and form nitrogen-fixing root nodules. Other free-living rhizobacteria such as species of the genus Azospirillum are facultatively capable of interacting with legume roots and promoting plant growth. We evaluated and compared the effects of various single inoculation and co-inoculation treatments on peanut growth parameters in greenhouse and field experiments. In the greenhouse studies, co-inoculation with various Bradyrhizobium strains (native 15A and PC34, and recommended peanut inoculant C145), and Azospirillum brasilense strain Az39 generally resulted in increases in the measured parameters. The growth-promoting effect of 15A was similar to or higher than that of C145. In the field studies, 15A-Az39 co-inoculation had a greater promoting effect on measured growth parameters than did C145-Az39 co-inoculation. Our findings indicate that careful selection of native rhizobacterial strains adapted to peanut soils is useful in strategies for growth promotion, and that 15A in particular is a promising candidate for future inoculant formulation.     

Mycorrhizal synthesis between <Emphasis Type="Italic">Lactarius deliciosus</Emphasis> and <Emphasis Type="Italic">Arbutus unedo</Emphasis> L.

Arbutoid mycorrhizae were synthesized in vitro between Arbutus unedo L. and two isolates of Lactarius deliciosus. The fungal isolates were obtained from sporocarps collected under Pinus sylvestris and in a mixed forest stand of Quercus suber and Pinus pinea. Synthesis tubes filled with a mixture of sterilized peat, vermiculite, and perlite imbibed with nutrient solution were used. Two inoculation methods using solid and liquid media were tested. Shoots from an adult selected clone of A. unedo were used after in vitro rooting by auxin dipping. After 3 months of shoots transfer to the substrate, the root systems were examined for arbutoid mycorrhizae formation and later on ex vitro conditions, 9 months after acclimatization. The inoculum treatment with liquid medium improved the mycorrhizal development for both isolates, in vitro. Sterilized substrate for plant acclimatization increased the mycorrhizal development. The arbutoid mycorrhizae were observed in vitro as well as 9 months after acclimatization. Standard arbutoid mycorrhiza features were observed: pale yellow mantle, typical cruciform appearance, Hartig net (HN), and intracellular hyphal complexes, both confined to the epidermis. L. deliciosus mycorrhizae synthetized in vitro persisted 9 months after plant acclimatization. Morphological observations were confirmed by molecular techniques.     

Diversity and characterization of <Emphasis Type="Italic">Azotobacter</Emphasis> isolates obtained from rice rhizosphere soils in Taiwan

Azotobacter species, free-living nitrogen-fixing bacteria, have been used as biofertilizers to improve the productivity of non-leguminous crops, including rice, due to their various plant growth-promoting traits. The purposes of this study were to characterize Azotobacter species isolated from rice rhizospheres in Taiwan and to determine the relationship between the species diversity of Azotobacter and soil properties. A total of 98 Azotobacter isolates were isolated from 27 paddy fields, and 16S rRNA gene sequences were used to identify Azotobacter species. The characteristics of these Azotobacter strains were analyzed including carbon source utilization and plant growth-promoting traits such as nitrogen fixation activity, indole acetic acid production, phosphate-solubilizing ability, and siderophore secretion. Of the 98 strains isolated in this study, 12 were selected to evaluate their effects on rice growth. Four species of Azotobacter were identified within these 98 strains, including A. beijerinckii, A. chroococcum, A. tropicalis, and A. vinelandii. Of these four species, A. chroococcum was predominant (51.0%) but A. beijerinckii had the highest level of nucleotide diversity. Strains within individual Azotobacter species showed diverse profiles in carbon source utilization. In addition, the species diversity of Azotobacter was significantly related to soil pH, Mn, and Zn. Members of the same Azotobacter species showed diverse plant growth-promoting traits, suggesting that the 98 strains isolated in this study may not equally effective in promoting rice growth. Of the 12 strains evaluated, A. beijerinckii CHB 461, A. chroococcum CHB 846, and A. chroococcum CHB 869 may be used to develop biofertilizers for rice cultivation because they significantly promoted rice growth. This study contributes to the selection of suitable Azotobacter strains for developing biofertilizer formulations and soil management strategies of Azotobacter for paddy fields.     

Exogenous spermidine alleviates fruit granulation in a <Emphasis Type="Italic">Citrus</Emphasis> cultivar (<Emphasis Type="Italic">Huangguogan</Emphasis>) through the antioxidant pathway

The role of exogenous spermidine (Spd) in alleviating fruit granulation in the grafted seedlings of a Citrus cultivar (Huangguogan) was investigated. Granulation resulted in increased electrical conductivity, cell membrane permeability, and total pectin, soluble pectin, cellulose, and lignin contents. However, it decreased the activities of superoxide dismutase, peroxidase, and catalase, as well as the (Spd + Spm):Put ratio. The application of exogenous Spd onto Huangguogan seedlings significantly increased proline and ascorbate contents, but decreased the H₂O₂ and O ₂ ^?· levels, which suggested that exogenous Spd provided some protection from oxidative damage. In addition, exogenous Spd decreased cell membrane permeability and MDA content, and increased the (Spd + Spm):Put ratio. The activities of antioxidant enzymes, such as catalase, peroxidase, and superoxide dismutase, were increased in Spd-treated seedlings affected by fruit granulation, resulting in a decrease in oxidative stress levels. The protective effects of Spd were reflected by a decrease in superoxide levels through osmoregulation, increased proline and ascorbate contents, and increased antioxidant activities. Our observations reveal the importance of exogenous Spd in alleviating citrus fruit granulation.     

Putrescine enhances chilling tolerance of tomato (Lycopersicon esculentum Mill.) through modulating antioxidant systems

Seedlings of two tomato (Lycopersicon esculentum Mill.) cultivars, cv. Mawa (chilling-resistant) and cv. Moneymaker (chilling-sensitive) were used to investigate the effects of exogenous putrescine (Put) on chilling tolerance as well as on changes of physiological features and the fluctuation of free and conjugated endogenous polyamines (PAs) contents in the leaves under chilling stress. During chilling stress, accumulation of hydrogen peroxide (H₂O₂) was obviously detected in the leaves of both cultivars, but it was fewer in cv. Mawa. Meanwhile, d-arginine (d-Arg), a Put biosynthesis inhibitor caused more H₂O₂ accumulation in both cultivars, especially in cv. Moneymaker. By adding back Put to leaves, accumulation of H₂O₂ obviously reduced in two cultivars. Put was also involved in the increase of Fv/Fm and the decrease of malondialdehyde (MDA) in two cultivars under chilling stress. Despite the two cultivars displaying differential behavior towards enzymic antioxidants, enzymes and components of the ascorbate–glutathione (AsA–GSH) cycle in responses to chilling stress, d-Arg treatment diminished the enzyme activities and antioxidant contents induced by chilling stress and its reversion was performed by adding Put in both cultivars. During chilling stress, free and conjugated endogenous PA contents increased in two cultivars. d-Arg treatment inhibited the increases, and exogenously applied Put enhanced the increases in two cultivars. These results suggested that Put played important roles in the tolerance of tomato against chilling stress, which was most likely achieved by modulating antioxidant system as well as increasing free and conjugated PAs.     

Putrescine promotes changes in the endogenous polyamine levels and proteomic profiles to regulate organogenesis in <Emphasis Type="Italic">Cedrela fissilis</Emphasis> Vellozo (Meliaceae)

Improvements to in vitro organogenesis are essential for optimizing shoot development and understanding basic physiological processes. The addition of polyamines (PAs) to the culture medium has been used to modulate organogenesis in plants, and this work evaluated the effects of exogenous PAs on direct organogenesis from apical and cotyledonary nodal Cedrela fissilis explants as well as the effects of putrescine (Put) on endogenous PA levels and variations in protein abundance. The effects of exogenous Put, spermidine, and spermine at 0, 0.5, 1, 2.5, or 5 mM on shoot development were tested. The comparison of the tested PAs to the control treatment revealed that 2.5 mM Put significantly increased the length of shoots from cotyledonary nodal explants, which are more sensitive than apical nodal explants, and treatment with 2.5 mM Put significantly increased the endogenous total free-PA and free-Put levels in shoots compared with the control (no Put). A comparative proteomic analysis of shoots indicated that 2.5 mM Put significantly changed the abundance of proteins, primarily metabolic and cellular proteins associated with stress and energy processes such as cell division. These results show that Put functions in endogenous PA metabolism and alters protein abundance, thereby contributing to shoot development in C. fissilis.