Effect of dehydration and rehydration on physiological response and the expression of aquaporin genes and abscisic acid metabolism in Impatiens walleriana

Suša se ubraja u abiotičke stresogene faktore i kao takva predstavlja jedan od najzastupljenijih i najvećih problema današnjice. Suša ispoljava negativan uticaj na rastenje i razviće brojnih biljnih vrsta, pa tako direktno ili indirektno utiče na njihovu brojnost i rasprostranjenost, ostavljajući negativne posledice na biodiverzitet celokupne flore i faune. Istraživanja u okviru ove doktorske disertacije vršena su sa ciljem ispitivanja uticaja dehidratacije uzrokovane sušom i rehidratacije na rastenje, fiziološke, biohemijske i molekularne parametre kod vrste Impatiens walleriana, kao i uticaja elicitacije metil jasmonatom (MeJA) u cilju ublažavanja ili otklanjanja negativnih efekata dehidratacije. I. walleriana (Balsaminaceae) je jedna od tri vrste roda Impatiens (pored I. hawkeri i I. balsamina) koje se komercijlno proizvode u Srbiji dugi niz godina. S obzirom na ornamentalne karakteristike biljaka i dug period cvetanja, I. walleriana je jedna od najpopularnijih hortikulturnih vrsta širom sveta. Biljke imaju visoke zahteve za prisustvom vode u podlozi, čiji nedostatak uzrokuje brz pad turgora u ćelijama i dehidrataciju tkiva, što je glavni problem u komercijalnoj proizvodnji i tržišnom plasmanu biljaka. Prvi deo eksperimenta obuhvatao je sledeće tretmane: kontrolne grupe biljaka gajene pri optimalnoj vlažnosti supstrata od 35-37% tokom eksperimentalnog perioda; grupe biljaka izložene stresu isušivanjem supstrata do 15 i 5%; oporavljene grupe biljaka tj. rehidratisane biljke, zalivane četiri dana do postizanja optimalne vlažnosti supstrata. Dehidratacija je redukovala masu svežih izdanaka, ukupnu površinu listova kao i masu suvih izdanaka. Zabeležena je povećana ekspresija ABA (apscisinska kiselina) biosintetskih gena 9-cis-epoxycarotenoid dioxygenase 4 (IwNCED4) i abscisic aldehyde oxidase 2 (IwAAO2) i kataboličkog gena ABA 8′-hydroxylase 3 (IwABA8ox3), što je praćeno povećanom koncentracijom ABA u listovima I. walleriana pri dehidrataciji. Redukovanje vodnog potencijala izdanaka pri dehidrataciji nije bilo praćeno povećanom akumulacijom aminokiseline prolina. Detektovano je povećanje koncentracije ukupnih hlorofila, karotenoida, ukupnih polifenola, flavonola, kao i malondialdehida (MDA), vodonik peroksida (H2O2) i DPPH aktivnosti u biljkama tokom dehidratacije. Povećana aktivnost antioksidativnih enzima superoksid-dismutaze (SOD), peroksidaze (POX) i katalaze (CAT) pri dehidrataciji je takođe zabeležena. Rehidratacija biljaka je bila značajna u ublažavanju negativnih uticaja dehidratacije na parametre rastenja, vodni potencijal izdanaka, koncentraciju endogenog prolina i ekspresiju gena. Takođe, analizirane su molekularne strukture akvaporina (AQP) i ekspresija gena za AQP u listovima I. walleriana pri dehidrataciji i rehidrataciji. Akvaporini predstavljaju veliku grupu transmembranskih proteina odgovornih za transport vode, što je ključno za opstanak biljaka u stresnim uslovima. Uprkos značajnoj ulozi AQP, do danas nisu poznate informacije o ovoj proteinskoj grupi kod I. walleriana. U ovoj doktorskoj disertaciji pažnja je bila usmerena na molekularnu karakterizaciju AQP kod I. walleriana kao i na ekspresiju gena odgovornih za sintezu ovih proteina prilikom dehidratacije i rehidratacije. Identifikovana su četiri AQP u transkriptomu I. walleriana: IwPIP1;4, IwPIP2;2, IwPIP2;7 i IwTIP4;1. Svi AQP su imali NPA motive (Asparagin-Prolin-Alanin), transmembranske regione (TMh), odgovarajuće karakteristike kanala, stereohemijske osobine i tetramernu strukturu holoproteina. Dehidratacija i rehidratacija uticale su na ekspresiju gena za akvaporine u listovima I. walleriana, koja je bila povišena ili snižena u zavisnosti od intenziteta stresa. Ekspresija IwPIP2;7 gena bila je najviše promenjena od svih analiziranih AQP I. walleriana. Pri vlažnosti supstrata od 15 i 5% i rehidrataciji nakon isušivanja supstrata do 15 i 5%, ekspresija IwPIP2;7 gena se značajno smanjila, odnosno povećala...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..