Uticaj dehidratacije i rehidratacije na fiziološki odgovor i ekspresiju gena za akvaporine i metabolizam apscisinske kiseline kod Impatiens walleriana

Abstract

Drought is one of the abiotic stress facotors and one of the most common and biggest problems today. Drought has a negative impact on the growth and development of numerous plant species, and thus directly or indirectly affects their abundance and distribution, leaving negative consequences on the biodiversity of the entire flora and fauna. This study was carried out to examine the dehydration caused by drought, and rehydration effect on development, physiological, biochemical and molecular parameters in Impatiens walleriana, as well as the effects of the methyl jasmonate (MeJA) elicitation in order to overcome the stress caused by dehydration. I. walleriana (Balsaminaceae) is one of the three Impatiens species (beside I. hawkeri and I. balsamina) which are commercially produced in Serbia for many years. Due to its decorative traits and long flowering period, I. walleriana belongs to the most popular horticultural species all over the world. Plants have high requirements for the presence of water in the substrate, which deficiency leads to a rapid drop in cell turgor and tissue dehydration which is the main problem in commercial production and market placement of these plants. In the first part of the experiment design there were three treatments: control plant groups grown under optimal watering with 35–37% of soil moisture content, stressed plant groups non-irrigated to reach 15 and 5% of soil moisture content and recovered plant groups rehydrated for four days to reach optimal soil moisture content. Dehydration reduced fresh weight, total leaf area, as well as dry weight of I. walleriana shoots. Dehydration up-regulated expression of abscisic acid (ABA) biosynthesis genes 9-cis-epoxycarotenoid dioxygenase 4 (IwNCED4) and abscisic aldehyde oxidase 2 (IwAAO2) and catabolic gene ABA 8′-hydroxylase 3 (IwABA8ox3) which was followed by increased ABA content in the leaves. Decrement in water potential of shoots during the dehydration was not accompanied with increased amino acid proline content. Increament in total chlorophyll, carotenoid, polyphenols and flavonols content, as well as malondialdehyde (MDA), hydrogen peroxide (H2O2) and DPPH (1,1′-diphenyl-2-picrylhydrazyl) activity, was observed in plants during dehydration. Increased antioxidant enzyme activities (superoxide-dismutase – SOD, peroxidase – POX and catalase – CAT) throughout dehydration were also determined. Rehydration was significant for overcoming dehydration effect on growth parameters, shoot water potential, endogenous proline content and genes expression. Also, analysis of molecular structure and gene expression of aquaporins (AQP) in I. walleriana leaves were estimated. Aquaporins comprise a large group of transmembrane proteins responsible for water transport, which is crucial for plant survival under stress conditions. Despite the vital role of AQPs, nothing is known about this protein family in I. walleriana. In the present doctoral dissertation, attention is given to the molecular characterization of AQPs in I. walleriana and their expression during drought stress and recovery. We identified four I. walleriana AQPs: IwPIP1;4, IwPIP2;2, IwPIP2;7 and IwTIP4;1. All of them had conserved NPA motifs (Asparagine-Proline-Alanine), transmembrane helices (TMh), pore characteristics, stereochemical properties and tetrameric structure of holoprotein. Dehydration and rehydration treatment affected the AQPs expression in I. walleriana leaves, which was up- or downregulated depending on stress intensity. Expression of IwPIP2;7 was the most affected of all analyzed I. walleriana AQPs. At 15 and 5% soil moisture and recovery from 15 and 5% soil moisture, IwPIP2;7 expression significantly decreased and increased, respectively. Aquaporins IwPIP1;4 and IwTIP4;1 had lower expression in comparison to IwPIP2;7, with moderate expression changes in response to dehydration and rehydration, while IwPIP2;2 expression was significantly increased only in recovered plants. Insight into the molecular structure of I. walleriana AQPs expanded knowledge about plant AQPs, while its expression during dehydration and rehydration contributed to I. walleriana dehydration tolerance mechanisms and re-acclimation. In the second part of the experiment, the effect of MeJA elicitation on overcome dehydration effects on the growth and development of I. walleriana was evaluated. The effect of MeJA elicitation in concentrations of 5 and 50 μM was manifested in both control and plants exposed to dehydration (at 15 and 5% soil moisture content) and rehydration. A positive effect of MeJA was observed on the growth parameters (fresh weight of shoots, total leaf area and dry weight of shoots) in control and plants exposed to different dehydration intensities. Methyl jasmonate induced increament in the expression of genes involved in ABA de novo synthesis during dehydration, as well as the change in the expression of gene involved in ABA catabolism. Also, reducing effect of MeJA elicitation on the stomatal conductivity was noted in control and dehydrated plants. Elicitation increased chlorophyll a/b ratio during lower and higher dehydration intensity, as well as the total chlorophyll and carotenoid content during intensive dehydration. In plant groups treated with MeJA, a decrease in the concentration of MDA and H2O2, as well as the lower content of total polyphenols and flavonols and changed activity of antioxidant enzymes SOD, POX and CAT were observed during dehydration. Elicitation with MeJA differently affected the expression of the AQPs genes IwPIP;4, IwPIP2;2, IwPIP2;7 and IwTIP4;1 during dehydration and rehydration, indicating the importance of the water channels involved in adapting water transport processes according to physiological state of the organism.