Influence of a phyA Mutation on Polyamine Metabolism in Arabidopsis Depends on Light Spectral Conditions.

The aim of the study was to reveal the influence of phyA mutations on polyamine metabolism in Arabidopsis under different spectral compositions. Polyamine metabolism was also provoked with exogenous spermine. The polyamine metabolism-related gene expression of the wild type and phyA plants responded similarly under white and far-red light conditions but not at blue light. Blue light influences rather the synthesis side, while far red had more pronounced effects on the catabolism and back-conversion of the polyamines. The observed changes under elevated far-red light were less dependent on PhyA than the blue light responses. The polyamine contents were similar under all light conditions in the two genotypes without spermine application, suggesting that a stable polyamine pool is important for normal plant growth conditions even under different spectral conditions. However, after spermine treatment, the blue regime had more similar effects on synthesis/catabolism and back-conversion to the white light than the far-red light conditions. The additive effects of differences observed on the synthesis, back-conversion and catabolism side of metabolism may be responsible for the similar putrescine content pattern under all light conditions, even in the presence of an excess of spermine. Our results demonstrated that both light spectrum and phyA mutation influence polyamine metabolism.

Light Spectral Composition Modifies Polyamine Metabolism in Young Wheat Plants.

Although light-emitting diode (LED) technology has extended the research on targeted photomorphogenic, physiological, and biochemical responses in plants, there is not enough direct information about how light affects polyamine metabolism. In this study, the effect of three spectral compositions (referred to by their most typical characteristic: blue, red, and the combination of blue and red [pink] lights) on polyamine metabolism was compared to those obtained under white light conditions at the same light intensity. Although light quality induced pronounced differences in plant morphology, pigment contents, and the expression of polyamine metabolism-related genes, endogenous polyamine levels did not differ substantially. When exogenous polyamines were applied, their roborative effect were detected under all light conditions, but these beneficial changes were correlated with an increase in polyamine content and polyamine metabolism-related gene expression only under blue light. The effect of the polyamines on leaf gene expression under red light was the opposite, with a decreasing tendency. Results suggest that light quality may optimize plant growth through the adjustment of polyamine metabolism at the gene expression level. Polyamine treatments induced different strategies in fine-tuning of polyamine metabolism, which were induced for optimal plant growth and development under different spectral compositions.

Mutations in Rht-B1 Locus May Negatively Affect Frost Tolerance in Bread Wheat.

The wheat semi-dwarfing genes Rht (Reduced height) are widely distributed among the contemporary wheat varieties. These genes also exert pleiotropic effects on plant tolerance towards various abiotic stressors. In this work, frost tolerance was studied in three near-isogenic lines of the facultative variety 'April Bearded' (AB), carrying the wild type allele Rht-B1a (tall phenotype), and the mutant alleles Rht-B1b (semi-dwarf) and Rht-B1c (dwarf), and was further compared with the tolerance of a typical winter type variety, 'Mv Beres'. The level of freezing tolerance was decreasing in the order 'Mv Beres' > AB Rht-B1a > AB Rht-B1b > AB Rht-B1c. To explain the observed differences, cold acclimation-related processes were studied: the expression of six cold-related genes, the phenylpropanoid pathway, carbohydrates, amino acids, polyamines and compounds in the tricarboxylic acid cycle. To achieve this, a comprehensive approach was applied, involving targeted analyses and untargeted metabolomics screening with the help of gas chromatography/liquid chromatography­mass spectrometry setups. Several cold-related processes exhibited similar changes in these genotypes; indeed, the accumulation of eight putrescine and agmatine derivatives, 17 flavones and numerous oligosaccharides (max. degree of polymerization 18) was associated with the level of freezing tolerance in the 'April Bearded' lines. In summary, the mutant Rht alleles may further decrease the generally low frost tolerance of the Rht-B1a, and, based on the metabolomics study, the mechanisms of frost tolerance may differ for a typical winter variety and a facultative variety. Present results point to the complex nature of frost resistance.

Polyamine Metabolism under Different Light Regimes in Wheat.

Although the relationship between polyamines and photosynthesis has been investigated at several levels, the main aim of this experiment was to test light-intensity-dependent influence of polyamine metabolism with or without exogenous polyamines. First, the effect of the duration of the daily illumination, then the effects of different light intensities (50, 250, and 500 µmol m-2 s-1) on the polyamine metabolism at metabolite and gene expression levels were investigated. In the second experiment, polyamine treatments, namely putrescine, spermidine and spermine, were also applied. The different light quantities induced different changes in the polyamine metabolism. In the leaves, light distinctly induced the putrescine level and reduced the 1,3-diaminopropane content. Leaves and roots responded differently to the polyamine treatments. Polyamines improved photosynthesis under lower light conditions. Exogenous polyamine treatments influenced the polyamine metabolism differently under individual light regimes. The fine-tuning of the synthesis, back-conversion and terminal catabolism could be responsible for the observed different polyamine metabolism-modulating strategies, leading to successful adaptation to different light conditions.

Validation of Reference Genes for Studying Different Abiotic Stresses in Oat (Avena sativa L.) by RT-qPCR.

Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

The effects of putrescine pre-treatment on osmotic stress responses in drought-tolerant and drought-sensitive wheat seedlings.

Recent studies have demonstrated that exogenous polyamines have protective effects under various stress condition. A broader understanding of the role of the polyamine pool fine regulation and the alterations of polyamine-related physiological processes could be obtained by comparing the stress effects in different genotypes. In this study, the impact of pre-treatment with putrescine in response to osmotic stress was investigated in the drought-tolerant Katya and drought-sensitive Zora wheat (Triticum aestivum) cultivars. Photosynthetic performance, in vivo thermoluminescence emission from leaves, leaf temperature, polyamine and salicylic acid levels, contents of osmoprotectants, and activities of antioxidant enzymes in the leaves were investigated not only to reveal differences in the physiological processes associated to drought tolerance, but to highlight the modulating strategies of polyamine metabolism between a drought-tolerant and a drought-sensitive wheat genotype. Results showed that the tolerance of Katya under osmotic stress conditions was characterized by higher photosynthetic ability, stable charge separation across the thylakoid membrane in photosystem II, higher proline accumulation and antioxidant activity. Thermoluminescence also revealed differences between the two varieties - a downshift of the B band and an increase of the afterglow band under osmotic stress in Zora, providing original complementary information to leaf photosynthesis. Katya variety exhibited higher constitutive levels of the signaling molecules putrescine and salicylic acid compared to the sensitive Zora. However, responses to exogenous putrescine were more advantageous for the sensitive variety under PEG treatment, which may be in relation with the decreased catabolism of polyamines, suggesting the increased need for polyamine under stress conditions.

Acclimation of photosynthetic processes and metabolic responses to elevated temperatures in cereals.

The aim of the present work was to better understand the molecular mechanisms of heat acclimation processes in cereals. A large number of winter and spring wheat, barley and oat varieties were grown under either control conditions (22/20°C) or under a mild heat stress (30°C) that induce the acclimation processes. The temperature dependence of chlorophyll a fluorescence induction and gas exchange parameters showed that heat acclimation increased the thermotolerance of the photosynthetic apparatus, but these changes did not differ sharply in the winter-spring type cereals. Similarly, to wheat, elevated temperature also led to increasing transpiration rate and reduced water use efficiency in barley and oat plants. A non-targeted metabolomic analysis focusing on polar metabolites in two selected barley (winter type Mv Initium and spring type Conchita) and in two oat varieties (winter type Mv Hópehely and spring type Mv Pehely) revealed substantial differences between both the two species and between the acclimated and non-acclimated plants. Several compounds, including sugars, organic acids, amino acids and alcohols could be separated and detected. The expression level of the CYP707, HSP90, galactinol synthase, raffinose synthase and α-galactosidase genes showed genotype-dependent changes after 1 day; however, the CYP707 was the only one, which was still upregulated in at least some of the genotypes. Results suggest that heat acclimation itself does not require general induction of primary metabolites. However, induction of specific routes, e.g. the induction of the raffinose family oligosaccharides, especially the synthesis of galactinol, may also contribute the improved heat tolerance in cereals.

Molecular background of cadmium tolerance in Rht dwarf wheat mutant is related to a metabolic shift from proline and polyamine to phytochelatin synthesis.

Plant height is among the most important agronomic traits influencing crop yield. Wheat lines carrying Rht genes are important in plant breeding due to their both higher yield capacity and better tolerance to certain environmental stresses. However, the effects of dwarf-inducing genes on stress acclimation mechanisms are still poorly understood. Under the present conditions, cadmium stress induced different stress responses and defence mechanisms in the wild-type and dwarf mutant, and the mutant with the Rht-B1c allele exhibited higher tolerance. In the wild type after cadmium treatment, the abscisic acid synthesis increased in the leaves, which in turn might have induced the polyamine and proline metabolisms in the roots. However, in the mutant line, the slight increment in the leaf abscisic acid content accompanied by relatively high salicylic acid accumulation was not sufficient to induce such a great accumulation of proline and putrescine. Although changes in proline and polyamines, especially putrescine, showed similar patterns, the accumulation of these compounds was antagonistically related to the phytochelatin synthesis in the roots of the wild type after cadmium stress. In the dwarf genotype, a favourable metabolic shift from the synthesis of polyamine and proline to that of phytochelatin was responsible for the higher cadmium tolerance observed.

Exogenous methylglyoxal enhances the reactive aldehyde detoxification capability and frost-hardiness of wheat.

Cold-acclimation is essential for the development of adequate frost-hardiness in cereals and therefore sudden freezes can cause considerable damage to the canopy. However, timely adding of an appropriate signal in the absence of cold acclimation may also harden wheat for the upcoming freeze. The feasibility of the promising signal molecule methylglyoxal was tested here for such applications and the signal mechanism was studied in bread wheat (Triticum aestivum L.) and durum wheat (Triticum turgidum L. ssp. durum). Spraying with 10 mM methylglyoxal did not decrease the fresh weight and photosynthetic parameters in most wheat varieties at growth temperature (21 °C). Photosynthetic parameters even improved and chlorophyll content increased in some cases. Increased transcript level of glutathione-S-transferases and omega-3 fatty acid desaturases was detected by qPCR 6 h after the last methylglyoxal spray. Aldo-keto reductase and glyoxalase enzyme activities, as well as sorbitol content of wheat plants increased 24 h after the last 10 mM methylglyoxal spray in most of the cultivars. These mechanisms may explain the increased freezing survival of methylglyoxal pretreated wheat plants from less than 10% to over 30%. Our results demonstrate that exogenous methylglyoxal treatment can be safely added to wheat plants as preparatory treatment without detrimental effects but inducing some of the stress-protective mechanisms, which contribute to frost-hardiness.

Polyamine-Induced Hormonal Changes in eds5 and sid2 Mutant Arabidopsis Plants.

Polyamines are multifaceted compounds which play a role in regulating plant growth and stress tolerance in interactions with plant hormones. The aim of the present study was to reveal how exogenous polyamines influence the synthesis of salicylic acid, with a special emphasis on the effect of salicylic acid deficiency on the polyamine metabolism and polyamine-induced changes in other plant hormone contents. Our hypothesis was that the individual polyamines induced different changes in the polyamine and salicylic acid metabolism of the wild type and salicylic acid-deficient Arabidopsis mutants, which in turn influenced other hormones. To our knowledge, such a side-by-side comparison of the influence of eds5-1 and sid2-2 mutations on polyamines has not been reported yet. To achieve our goals, wild and mutant genotypes were tested after putrescine, spermidine or spermine treatments. Polyamine and plant hormone metabolism was investigated at metabolite and gene expression levels. Individual polyamines induced different changes in the Arabidopsis plants, and the responses were also genotype-dependent. Polyamines upregulated the polyamine synthesis and catabolism, and remarkable changes in hormone synthesis were found especially after spermidine or spermine treatments. The sid2-2 mutant showed pronounced differences compared to Col-0. Interactions between plant hormones may also be responsible for the observed differences.

Role of polyamines in plant growth regulation of Rht wheat mutants.

Besides their protective role, polyamines also serve as signalling molecules. However, further studies are needed to elucidate the polyamine signalling pathways, especially to identify polyamine-regulated mechanisms and their connections with other regulatory molecules. Reduced height (Rht) genes in wheat are often used in breeding programs to increase harvest index. Some of these genes are encoding DELLA proteins playing role in gibberellic acid signalling. The aim of the present paper was to reveal how the mutations in Rht gene modify the polyamine-regulated processes in wheat. Wild type and two Rht mutant genotypes (Rht 1: semi-dwarf; Rht 3: dwarf mutants) were treated with polyamines. Polyamine treatments differently influenced the polyamine metabolism, the plant growth parameters and certain hormone levels (salicylic acid and abscisic acid) in these genotypes. The observed distinct metabolism of Rht 3 may more likely reflect more intensive polyamine exodus from putrescine to spermidine and spermine, and the catabolism of the higher polyamines. The lower root to shoot translocation of putrescine can contribute to the regulation of polyamine pool, which in turn may be responsible for the observed lack of growth inhibition in Rht 3 after spermidine and spermine treatments. Lower accumulation of salicylic acid and abscisic acid, plant hormones usually linked with growth inhibition, in leaves may also be responsible for the diminished negative effect of higher polyamines on the shoot growth parameters observed in Rht 3. These results provide an insight into the role of polyamines in plant growth regulation based on the investigation of gibberellin-insensitive Rht mutants.

Interaction of polyamines, abscisic acid and proline under osmotic stress in the leaves of wheat plants.

The exact relationship between polyamine, abscisic acid and proline metabolisms is still poorly understood. In the present study, the effects of putrescine and abscisic acid treatments alone or in combination with polyethylene glycol-induced osmotic stress were investigated in young wheat plants. It was observed that abscisic acid plays a role in the coordinated regulation of the proline and polyamine biosynthetic pathways, which compounds are related to each other through a common precursor. Abscisic acid pre-treatment induced similar alteration of polyamine contents as the osmotic stress, namely increased the putrescine, but decreased the spermidine contents in the leaves. These changes were mainly related to the polyamine cycle, as both the synthesis and peroxisomal oxidation of polyamines have been induced at gene expression level. Although abscisic acid and osmotic stress influenced the proline metabolism differently, the highest proline accumulation was observed in the case of abscisic acid treatments. The proline metabolism was partly regulated independently and not in an antagonistic manner from polyamine synthesis. Results suggest that the connection, which exists between polyamine metabolism and abscisic acid signalling leads to the controlled regulation and maintenance of polyamine and proline levels under osmotic stress conditions in wheat seedlings.

Comparative study on the effects of putrescine and spermidine pre-treatment on cadmium stress in wheat.

In several cases a correlation was found between polyamines and abiotic stress tolerance. However, the individual polyamines may have different effects, which also vary depending on the type of treatment. When applied as seed soaking or added hydroponically 0.5mM putrescine and spermidine, different changes were induced during 50µM cadmium stress in wheat plants. Seed-soaked plants were exposed to cadmium immediately after germination for 5 days, while plants pre-treated with polyamines hydroponically were stressed at age of 14 days for 7 days. Putrescine pre-treatment was beneficial both as seed soaking and applied hydroponically, while spermidine only had a protective effect in the case of seed soaking, enhancing the Cd-induced oxidative stress when were pre-treated hydroponically. The differences observed were related to the polyamine metabolism. The accumulation of endogenous putrescine beyond a certain amount may be in relation with the negative effect of hydroponic spermidine pre-treatment during Cd stress. The increased putrescine content was also correlated with the highest accumulation of Cd, salicylic acid and proline contents in plants treated with a combination of spermidine and Cd. However, the expression level of the gene encoding phytochelatin synthase was only influenced by hydroponically applied spermidine, which decreased it under cadmium stress. Changes in the activities of antioxidant enzymes, diamine and polyamine oxidases were also discussed.