Endogenous Polyamines and Ethylene Biosynthesis in Relation to Germination of Osmoprimed Brassica napus Seeds under Salt Stress

Currently, seed priming is reported as an efficient and low-cost approach to increase crop yield, which could not only promote seed germination and improve plant growth state but also increase abiotic stress tolerance. Salinity represents one of the most significant abiotic stresses that alters multiple processes in plants. The accumulation of polyamines (PAs) in response to salt stress is one of the most remarkable plant metabolic responses. This paper examined the effect of osmopriming on endogenous polyamine metabolism at the germination and early seedling development of Brassica napus in relation to salinity tolerance. Free, conjugated and bound polyamines were analyzed, and changes in their accumulation were discussed with literature data. The most remarkable differences between the corresponding osmoprimed and unprimed seeds were visible in the free (spermine) and conjugated (putrescine, spermidine) fractions. The arginine decarboxylase pathway seems to be responsible for the accumulation of PAs in primed seeds. The obvious impact of seed priming on tyramine accumulation was also demonstrated. Moreover, the level of ethylene increased considerably in seedlings issued from primed seeds exposed to salt stress. It can be concluded that the polyamines are involved in creating the beneficial effect of osmopriming on germination and early growth of Brassica napus seedlings under saline conditions through moderate changes in their biosynthesis and accumulation.

Plant Growth-Promoting Endophytic Bacillus Aryabhattai Triggers Novel Genes to Induce Plant Growth

Background: In nature, plants interact with a wide range of microorganisms. Most of these microorganisms have the ability to promote plant growth through the induction of important molecular pathways. The current work evaluated whether the endophytic bacterium Bacillus aryabhattai encourages plant growth and how transcriptional changes might be implicated in this effect.Results: The endophytic bacterium showed a significant effect on plant growth. Our results revealed that B. aryabhattai promotes the growth of Arabidopsis and tobacco plants. Notably, transcriptional changes in Arabidopsis plants treated with the bacterium were identified. Genes such as cinnamyl alcohol dehydrogenase, apyrase, thioredoxin H8, benzaldehyde dehydrogenase, indoleacetaldoxime dehydratase, berberine bridge enzyme-like and gibberellin-regulated protein were highly expressed. Additionally, endophytic bacterial genes such as arginine decarboxylase, D-hydantoinase, ATP synthase gamma chain and 2-hydroxyhexa-2,4-dienoate hydratase were activated during the interaction with Arabidopsis.Conclusions: The results show that new plant growth-related genes are induced during the interaction endophytic bacterium B. aryabhattai, and these changes may promote plant growth in sustainable agriculture.

Effect of arginine, putrescine and spermidine on the polyamine, proline and chlorophyll content of tobacco (Nicotiana tabacum L.)

Polyamines, such as spermidine (Spd) spermine (Spm) and their direct precursor, the diamine putrescine (Put) are vital and essential aliphatic amines which are also present in plants. Although ethylene and polyamines are also involved in fruit ripening, the genes coding them must also take part in other biosynthetic pathways. In the ethylene and polyamines play an important role in development of salt stress tolerance, and in responses for biotic and abiotic stresses. Exogenous application of all three main polyamines (Put, Spd, Spm) increase salt tolerance of plants, but, accordingly to previous experiments, spermidine has the main effect on the enhancement of salt tolerance. Nicotiana tabacum L. plants were grown in vitro on MS medium, the treatments were as follows: arginine (150 mg l-1), putrescine (10 mg l-1), spermidine (10 mg l-1). Proline, chlorophyll a, b and polyamine contents were measured. The obtained results show that the arginine decarboxylase and the spermidine synthase genes involved in polyamine metabolism, cannot be enhanced by exogenous addition of their precursor molecules. On the contrary, the spermine synthase gene has a positive effect to the lower-class forms of polyamines.

Improvement of putrescine production through the arginine decarboxylase pathway in Escherichia coli K-12

In the bio-based polymer industry, putrescine is in the spotlight for use as a material. We constructed strains of Escherichia coli to assess its putrescine production capabilities through the arginine decarboxylase pathway in batch fermentation. N-Acetylglutamate (ArgA) synthase is subjected to feedback inhibition by arginine. Therefore, the 19th amino acid residue, Tyr, of argA was substituted with Cys to desensitize the feedback inhibition of arginine, resulting in improved putrescine production. The inefficient initiation codon GTG of argA was substituted with the effective ATG codon, but its replacement did not affect putrescine production. The essential genes for the putrescine production pathway, speA and speB, were cloned into the same plasmid with argAATG Y19C to form an operon. These genes were introduced under different promoters; lacIp, lacIqp, lacIq1p, and T5p. Among these, the T5 promoter demonstrated the best putrescine production. In addition, disruption of the puuA gene encoding enzyme of the first step of putrescine degradation pathway increased the putrescine production. Of note, putrescine production was not affected by the disruption of patA, which encodes putrescine aminotransferase, the initial enzyme of another putrescine utilization pathway. We also report that the strain KT160, which has a genomic mutation of YifEQ100TAG, had the greatest putrescine production. At 48 h of batch fermentation, strain KT160 grown in terrific broth with 0.01 mM IPTG produced 19.8 mM of putrescine.

Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (>89.0% yield based on 100 mM of Arg within 5 h).

Analysis of the expression of polyamine biosynthesis genes in nodules of the garden pea (Pisum sativum L.) and the effect of exogenous treatment with polyamines on their development

BACKGROUND: Polyamines are acting as signaling molecules during adaptation to stressful environment and as regulators of plant development. In plants, polyamines are represented mainly by putrescine, spermidine and spermine. The concentration of polyamines in symbiotic nodules of some legumes is 510 times higher than in the other organs, which indicates their important role in the formation and functioning of symbiotic nodules. MATERIALS AND METHODS: We analyzed the expression of genes encoding polyamine biosynthesis enzymes in symbiotic nodules, as well as the effect of exogenous polyamines on the nodule number and the average nodule weight in wild-type SGE plants and symbiotic pea mutants SGEFix-1 (sym40-1) and SGEFix-2 (sym33-3). RESULTS: The comparable expression level of arginine decarboxylase gene (PsADC) was observed in all analyzed nodules, whereas the expression level of ornithine decarboxylase gene (PsODC), was highly increased in nodules of SGEFix-2 (sym33-3) mutant. Treatment of the root system with a 0.1 mM solution of polyamines mixture led to an increase in the average weight of the nodule in wild-type plants and in the SGEFix-2 (sym33-3) mutant plants. CONCLUSIONS: It was shown that the main pathway of putrescine synthesis in wild-type pea symbiotic nodules is the arginine pathway, while the ornithine pathway is probably associated with activation of plant defense reactions. Polyamines acting, apparently, through ethylene, affect the functioning of the nodule meristem.

Citrus sinensis CBF1 Functions in Cold Tolerance by Modulating Putrescine Biosynthesis Through Regulation of ARGININE DECARBOXYLASE

C-repeat (CRT) binding factors (CBFs) are well known to act as crucial transcription factors that function in cold stress response. Arginine decarboxylase (ADC)-mediated putrescine biosynthesis has been reported to be activated in plants exposed to cold conditions, but it remains elusive whether CBFs can regulate ADC expression and putrescine accumulation. In this study, we show that cold up-regulated ADC gene (CsADC) and elevation of endogenous putrescine content in sweet orange (Citrus sinensis). Promoter of CsADC contains two CRT sequences that are canonical elements recognized by CBFs. Sweet orange genome contains four CBFs (CsCBF1-4), in which CsCBF1 was significantly induced by cold. CsCBF1, located in the nucleus, was demonstrated to bind directly and specifically to the promoter of CsADC and acted as a transcriptional activator. Overexpression of CsCBF1 led to notable elevation of CsADC and putrescine level in sweet orange transgenic plants, along with remarkably enhanced cold tolerance, relative to the wild type (WT). However, pretreatment with D-arginine, an ADC inhibitor, caused prominent reduction of endogenous putrescine level in the overexpressing lines, accompanied by greatly compromised cold tolerance. Taken together, these results demonstrate that CBF1 of sweet orange directly regulates ADC expression and modulates putrescine synthesis for orchestrating the cold tolerance. Our findings shed light into the transcriptional regulation of putrescine accumulation through targeting the ADC gene in the presence of cold stress. Meanwhile, this study illustrates a new mechanism underlying the CBF-mediated cold stress response.

Polyamine Metabolism in Scots Pine Embryogenic Cells under Potassium Deficiency

Polyamines (PA) have a protective role in maintaining growth and development in Scots pine during abiotic stresses. In the present study, a controlled liquid Scots pine embryogenic cell culture was used for studying the responses of PA metabolism related to potassium deficiency. The transcription level regulation of PA metabolism led to the accumulation of putrescine (Put). Arginine decarboxylase (ADC) had an increased expression trend under potassium deficiency, whereas spermidine synthase (SPDS) expression decreased. Generally, free spermidine (Spd) and spermine (Spm)/ thermospermine (t-Spm) contents were kept relatively stable, mostly by the downregulation of polyamine oxidase (PAO) expression. The low potassium contents in the culture medium decreased the potassium content of the cells, which inhibited cell mass growth, but did not affect cell viability. The reduced growth was probably caused by repressed metabolic activity and cell division, whereas there were no signs of H2O2-induced oxidative stress or increased cell death. The low intracellular content of K+ decreased the content of Na+. The decrease in the pH of the culture medium indicated that H+ ions were pumped out of the cells. Altogether, our findings emphasize the specific role(s) of Put under potassium deficiency and strict developmental regulation of PA metabolism in Scots pine.