The involvement of metallothioneins and stress markers in response to osmotic stress in Avena sativa L.

Abstract

Osmotic stress frequently caused by drought is the most threatening environmental stress that remarkably reduces crop yield. Oat (Avena sativa L.) is sensitive to water deficiency during growth. Plant metallothioneins (pMTs) show tremendous promise in enhancing general stress tolerance in plants. This study aimed to verify whether pMTs and elements of the antioxidant defence system protect oat against osmotic stress.Coding and genomic regions of A. sativa L. MTs belonging to three different types were cloned. To evaluate the role of MTs in osmotic stress, the expression of genes encoding AsMT1-3 was checked by qRT-PCR in the roots and shoots of oat plants growing in a hydroponic culture in the presence of polyethylene glycol (PEG 6000) and mannitol. The expression of AsMT1-3 changed in response to osmotic stress; however, the changes depended on the type of MT and the treatment. The amount of AsMT3 transcript was about five-fold and nine-fold higher in shoots and roots, respectively, in the presence of mannitol. To further analyse the response of oat to osmotic stress, the level of phenolic compounds, soluble sugars, abscisic acid, and the activity of antioxidant enzymes were tested using spectrophotometric and chromatographic methods. During osmotic stress, the content of phenols, soluble sugars, abscisic acid, and the activity of catalase and peroxidase in shoots increased. In roots treated with PEG 6000, the amount of phenolic compounds was higher than that in roots treated with mannitol. The activity of superoxide dismutase was about 5-fold higher in roots than in shoots. MTs are involved in plant response to osmotic stress. In the future, insight provided in this study will lead to application in agriculture either by using MTs as molecular markers for stress-resistant crop cultivars or by a generation of genetically modified crops overexpressing MTs.

Changes in physio-biochemical parameters and expression of metallothioneins in Avena sativa L. in response to drought

“…8C). In our previous study 66 , in oat seedlings subjected to osmotic stress, CAT and PX had the highest activity in the treated plants and SOD www.nature.com/scientificreports/ had the lowest. A similar observation was reported by Chakraborty and Pradhan 109 in Triticum aestivum L., where SOD activity showed a general decline in activity and the activity of PX increased greatly during water deficit.…”

“…[57][58][59][60] , Nicotiana tabacum L. 61 , Ipomoea nil (L.) Roth 22 , Brassica napus L. 24,38,62 , Cucumis sativus L. 63 , Oryza sativa L. 64 , and Zea mays L. 65 . However, only one report on A. sativa metallothioneins has been published so far 66 . This may be because oat is an allohexaploid species (2n = 6× = 42, AACCDD) with a large genome (12.5 Gb), which makes it difficult to work with on the gene level 67 .…”

“…In silico analyses of promoters of A. sativa L. MT genes. We have previously cloned three oat metallothionein partial cDNA sequences 66 . Since then, the genome assembly of A. sativa has been published in the PanOat database (https:// wheat.…”

“…Reverse transcription reaction was performed using a RevertAid Reverse Transcriptase (Thermo Fisher Scientific, US) according to the manufacturer's protocol using 250 ng oligo (dT) 20 primer and 200 ng random hexamers. RT-qPCR was performed in a total volume of 10 μL using a Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, US) 66 . The reaction mixture contained 4 μL of 10 × diluted cDNA and 0.3 μM genespecific primers (Table 2).…”

“…The qPCR reaction was conducted in a LightCycler 480 Instrument (Roche, Germany). The thermal cycling conditions were as follows: 95 °C for 10 min for initial denaturation, 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s 66,119 . Differences in the target gene expression were evaluated by a relative quantification method normalising the data to the reference genes for eukaryotic initiation factor 4A-3 (EIF4A) and heterogeneous nuclear ribonucleoprotein 27C (HNR) 120 .…”