Different reactions of wheat, maize, and rice plants to putrescine treatment.

Polyamines play an important role in growth and differentiation by regulating numerous physiological and biochemical processes at the cellular level. In addition to their roborative effect, their essential role in plant stress responses has been also reported. However, the positive effect may depend on the fine-tuning of polyamine metabolism, which influences the production of free radicals and/or signalling molecules. In the present study, 0.3 mM hydroponic putrescine treatment was tested in wheat, maize, and rice in order to reveal differences in their answers and highlight the relation of these with polyamine metabolism. In the case of wheat, the chlorophyll content and the actual quantum yield increased after putrescine treatment, and no remarkable changes were detected in the stress markers, polyamine contents, or polyamine metabolism-related gene expression. Although, in maize, the actual quantum yield decreased, and the root hydrogen peroxide content increased, no other negative effect was observed after putrescine treatment due to activation of polyamine oxidases at enzyme and gene expression levels. The results also demonstrated that after putrescine treatment, rice with a higher initial polyamine content, the balance of polyamine metabolism was disrupted and a significant amount of putrescine was accumulated, accompanied by a detrimental decrease in the level of higher polyamines. These initial differences and the putrescine-induced shift in polyamine metabolism together with the terminal catabolism or back-conversion-induced release of a substantial quantity of hydrogen peroxide could contribute to oxidative stress observed in rice.

Changes in polyamine contents during Fusarium graminearum and Fusarium verticillioides inoculation in maize seedlings with or without seed-priming.

Maize (Zea mays L.) is the most produced field crop all over the world. One of its most critical diseases that results in economic loss is ear rot caused by various Fusarium species. Previous researches have shown that polyamines, found in all living cells, play crucial role in biotic stress responses. At the same time, biosynthesis of polyamines is of paramount importance not only for plants but also for their pathogens to promote stress tolerance and pathogenicity. In our study, we investigated the polyamine content changes induced in the seedlings of two maize genotypes of different susceptibility by isolates of Fusarium verticillioides and Fusarium graminearum, two Fusarium species of different lifestyles. Apart from that, it was examined how infection efficiency and changes in polyamine contents were modified by salicylic acid or putrescine seed soaking pre-treatments. Our observations confirmed that initial and stress-induced changes in the polyamine contents are not directly related to tolerance in either coleoptile or radicle. However, the two pathogens with different lifestyles induced remarkably distinct changes in the polyamine contents. The effect of the seed soaking pre-treatments depended on the pathogens and plant resistance as well: both salicylic acid and putrescine seed soaking had positive results against F. verticillioides, while in the case of infection with F. graminearum, seed soaking with distilled water alone affected biomass parameters positively in the tolerant genotype.

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.

The role of methyl salicylate in plant growth under stress conditions.

Methyl salicylate is a volatile compound, the synthesis of which takes place via the salicylic acid pathway in plants. Both compounds can be involved in the development of systemic acquired resistance and they play their role partly independently. Salicylic acid transport has an important role in long-distance signalling, but methyl salicylate has also been suggested as a phloem-based mobile signal, which can be demethylated to form salicylic acid, inducing the de-novo synthesis of salicylic acid in distal tissue. Despite the fact that salicylic acid has a protective role in abiotic stress responses and tolerance, very few investigations have been reported on the similar effects of methyl salicylate. In addition, as salicylic acid and methyl salicylate are often treated simply as the volatile and non-volatile forms of the same compound, and in several cases they also act in the same way, it is hard to highlight the differences in their mode of action. The main aim of the present review is to reveal the individual role and action mechanism of methyl salicylate in systemic acquired resistance, plant-plant communication and various stress conditions in fruits and plants.

Improvement of Drought Tolerance by Exogenous Spermidine in Germinating Wheat (Triticum aestivum L.) Plants Is Accompanied with Changes in Metabolite Composition.

Drought is one of the most important environmental factors reducing the yield and production of crops, including wheat. Polyamines are closely associated with plant stress tolerance. The present study investigated the mechanisms through seed germination with spermidine protecting wheat varieties from drought stress. In the first experiment, the effects of spermidine on the germination of wheat varieties, namely Rakhshan, Mihan, Sirvan and Pishgam, were investigated in three drought levels, namely 0, -2, and -4 MPa induced by polyethylene glycol 6000. Analysis of variance indicated that spermidine, drought stress and interaction between varieties and drought stress were significant for all traits, and with severity of stress, all traits significantly decreased. In the second experiment, detailed gene expression and non-targeted metabolomics analyses were carried out using the Rakhshan and Mihan varieties after germination, with or without spermidine treatment and/or drought stress. According to the biomass parameters, the Mihan variety showed relatively better growth compared to the other variety, but the Rakhshan one showed more pronounced responses at gene expression level to exogenous spermidine than the Mihan variety. Overall, these results showed that spermidine increased the drought tolerance of wheat at the germination stage, due to specific role of polyamine metabolism in the development of effective responses under drought stress.

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.

Methylglyoxal induces stress signaling and promotes the germination of maize at low temperature.

Maize is sensitive to cold injury, especially during germination. Since cold causes oxidative stress, compounds that promote the accumulation of free radical forms, such as the reactive aldehyde (RA) methylglyoxal (MG), may be suitable to trigger a systemic defense response. In this study, maize seeds were soaked in MG solution for one night at room temperature, before germination test at 13°C. The exogenous MG enhanced the germination and photosynthetic performance of maize at low temperature. Transcriptome analysis, hormonal, and flavonoid profiling indicated MG-induced changes in photosystem antenna proteins, pigments, late embryogenesis abundant proteins, abscisic acid (ABA) derivatives, chaperons, and certain dihydroflavonols, members of the phenylpropanoid pathway. MG-response of the two maize cultivars (A654 and Cm174) were somewhat different, but we recorded higher endogenous hydrogen peroxide (H2 O2 ) and lower nitric oxide (NO) level in at least one of the treated genotypes. These secondary signal molecules may provoke some of the changes in the hormonal, metabolic and gene expression profile. Decreased auxin transport, but increased ABA degradation and cytokinin and jasmonic acid (JA) synthesis, as well as an altered carbohydrate metabolism and transport (amylases, invertases, and SWEET transporters) could have promoted germination of MG-pretreated seeds. While LEA accumulation could have protected against osmotic stress and catalase expression and production of many antioxidants, like para-hydroxybenzoic acid (p-HBA) and anthocyanins may have balanced the oxidative environment for maize germination. Our results showed that MG-pretreatment could be an effective way to promote cold germination and its effect was more pronounced in the originally cold-sensitive maize genotype.

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.

Unfinished story of polyamines: Role of conjugation, transport and light-related regulation in the polyamine metabolism in plants.

Polyamines play a fundamental role in the functioning of all cells. Their regulatory role in plant development, their function under stress conditions, and their metabolism have been well documented as regards both synthesis and catabolism in an increasing number of plant species. However, the majority of these studies concentrate on the levels of the most abundant polyamines, sometimes providing data on the enzyme activity or gene expression levels during polyamine synthesis, but generally making no mention of the fact that changes in the polyamine pool are very dynamic, and that other processes are also involved in the regulation of actual polyamine levels. Differences in the distribution of individual polyamines and their conjugation with other compounds were described some time ago, but these have been given little attention. In addition, the role of polyamine transporters in plants is only now being recognised. The present review highlights the importance of conjugated polyamines and also points out that investigations should not only deal with the polyamine metabolism itself, but should also cover other important questions, such as the relationship between light perception and the polyamine metabolism, or the involvement of polyamines in the circadian rhythm.

Abiotic Stress Response of Near-Isogenic Spring Durum Wheat Lines under Different Sowing Densities.

A detailed study was made of changes in the plant development, morphology, physiology and yield biology of near-isogenic lines of spring durum wheat sown in the field with different plant densities in two consecutive years (2013-2014). An analysis was made of the drought tolerance of isogenic lines selected for yield QTLs (QYld.idw-2B and QYld.idw-3B), and the presence of QTL effects was examined in spring sowings. Comparisons were made of the traits of the isogenic pairs QYld.idw-3B++ and QYld.idw-3B-- both within and between the pairs. Changes in the polyamine content, antioxidant enzyme activity, chlorophyll content of the flag leaf and the normalized difference vegetation index (NDVI) of the plot were monitored in response to drought stress, and the relationship between these components and the yield was analyzed. In the case of moderate stress, differences between the NIL++ and NIL-- pairs appeared in the early dough stage, indicating that the QYld.idw-3B++ QTL region was able to maintain photosynthetic activity for a longer period, resulting in greater grain number and grain weight at the end of the growing period. The chlorophyll content of the flag leaf in phenophases Z77 and Z83 was significantly correlated with the grain number and grain weight of the main spike. The grain yield was greatly influenced by the treatment, while the genotype had a significant effect on the thousand-kernel weight and on the grain number and grain weight of the main spike. When the lines were compared in the non-irrigated treatment, significantly more grains and significantly higher grain weight were observed in the main spike in NIL++ lines, confirming the theory that the higher yields of the QYld.idw-3B++ lines when sown in spring and exposed to drought stress could be attributed to the positive effect of the "Kofa" QTL on chromosome 3B.

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.

Salicylic Acid Signalling in Plants.

Ten articles published in the "Special Issue: Salicylic Acid Signalling in Plants" are summarized, in order to get a global picture about the mode of action of salicylic acid in plants, and about its interaction with other stress-signalling routes. Its ecological aspects and possible practical use are also discussed.

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.

Involvement of Salicylic Acid and Other Phenolic Compounds in Light-Dependent Cold Acclimation in Maize.

The exposure of plants to non-lethal low temperatures may increase their tolerance to a subsequent severe chilling stress. To some extent, this is also true for cold-sensitive species, including maize. In the present work, based on our previous microarray experiment, the differentially expressed genes with phenylpropanoid pathways in the focus were further investigated in relation to changes in certain phenolic compounds and other plant growth regulators. Phenylalanine ammonia lyase (PAL) was mainly activated under limited light conditions. However, light-induced anthocyanin accumulation occurred both in the leaves and roots. Chilling stress induced the accumulation of salicylic acid (SA), but this accumulation was moderated in the cold-acclimated plants. Acclimation also reduced the accumulation of jasmonic acid (JA) in the leaves, which was rather induced in the roots. The level of abscisic acid (ABA) is mainly related to the level of the stress, and less indicated the level of the acclimation. The highest glutathione (GSH) amount was observed during the recovery period in the leaves of plants that were cold acclimated at growth light, while their precursors started to accumulate GSH even during the chilling. In conclusion, different light conditions during the cold acclimation period differentially affected certain stress-related mechanisms in young maize plants and changes were also light-dependent in the root, not only in the leaves.

Interaction between elevated temperature and different types of Na-salicylate treatment in Brachypodium dystachion.

Salicylic acid (SA) plays a role in several physiological processes in plants. Exogenously applied SA is a promising tool to reduce stress sensitivity. However, the mode of action may depend on how the treatment was performed and environmental conditions may alter the effects of SA. In the present study the physiological and biochemical effects of different modes of application (soaking seeds prior sowing; spraying leaves with 0.5 mM NaSA) were compared at normal and moderately elevated temperatures (4 h; 35°C) in Brachypodium distachyon (L.) P. Beauv. plants. While soaking the seeds stimulated plant growth, spraying caused mild stress, as indicated by the chlorophyll-a fluorescence induction parameters and changes in certain protective compounds, such as glutathione, flavonoids or antioxidant enzymes. Elevated temperature also caused an increase in the glutathione-S-transferase activity, and this increase was more pronounced in plants pre-treated with NaSA. Both seed soaking or spraying with NaSA and exposure to heat treatment at 35°C reduced the abscisic acid levels in the leaves. In contrast to abscisic acid, the jasmonic acid level in the leaves were increased by both spraying and heat treatment. The present results suggest that different modes of application may induce different physiological processes, after which plants respond differently to heat treatment. Since these results were obtained with a model plants, further experiments are required to clarify how these changes occur in crop plants, especially in cereals.

Comparative study of salicylic acid contents in young wheat and rice plants and their anticancer activities in HepG2 and Caco-2 cells.

Some recent results reported that aspirin (acetylsalicylic acid) had a positive effect on the treatment of certain types of cancer. However, the results cannot be generalized and it is not always clear whether it is a direct anticancer effect or a general health effect. Since plants produce different amounts of salicylic acid, we have sought a relationship between the salicylic acid content of some plant extracts and their anticancer activity. Growing of wheat and rice plants were carried out under controlled conditions. The salicylic acid content was determined by high-performance liquid chromatography. The viability and cell cycle assays were performed on HepG2 and Caco-2 cell lines. Despite the high content of salicylic acid, the extracts from rice plants did not show significant anticancer activity. In spite of the low salicylic acid content, the positive effect of wheat germ was confirmed in both tests. There is no direct relationship between the salicylic acid content of the plant extracts and their anticancer activity. However, it has been proven that young wheat germ is more effective than mature leaf.

Metabolic responses of wheat seedlings to osmotic stress induced by various osmolytes under iso-osmotic conditions.

Many environmental stresses cause osmotic stress which induces several metabolic changes in plants. These changes often vary depending on the genotype, type and intensity of stress or the environmental conditions. In the current experiments, metabolic responses of wheat to osmotic stress induced by different kinds of osmolytes were studied under iso-osmotic stress conditions. A single wheat genotypes was treated with PEG-6000, mannitol, sorbitol or NaCl at such concentrations which reduce the osmotic potential of the culture media to the same level (-0.8MPa). The metabolic changes, including the accumulation of proline, glycine betaine (GB) and sugar metabolites (glucose, fructose, galactose, maltose and sucrose) were studied both in the leaves and roots together with monitoring the plant growth, changes in the photosynthetic activity and chlorophyll content of the leaves. In addition, the polyamine metabolism was also investigated. Although all osmolytes inhibited growth similarly, they induced different physiological and metabolic responses: the CO2 assimilation capacity, RWC content and the osmotic potential (ψπ) of the leaves decreased intensively, especially after mannitol and sorbitol treatments, followed by NaCl treatment, while PEG caused only a slight modification in these parameters. In the roots, the most pronounced decrease of ψπ was found after salt-treatments, followed by PEG treatment. Osmotic stress induced the accumulation of proline, glycine betaine and soluble sugars, such as fructose, glucose, sucrose and galactose in both the root and leaf sap. Specific metabolic response of roots and leaves under PEG included accumulation of glucose, fructose and GB (in the roots); sucrose, galactose and proline synthesis were dominant under NaCl stress while exposure to mannitol and sorbitol triggered polyamine metabolism and overproduction of maltose. The amount of those metabolites was time-dependent in the manner that longer exposure to iso-osmotic stress conditions stimulated the sugar metabolic routes. Our results showed that the various osmolytes activated different metabolic processes even under iso-osmotic stress conditions and these changes also differed in the leaves and roots.