Fiziološki i morfološki efekti ekspresije AtCKX gena u transformisanim biljkama krompira (Solanum tuberosum L.) gajenim in vitro

Abstract

Krompir (Solanum tuberosum L.) je po značaju četvrta poljoprivredna kultura na svetu gajena za potrebe ljudske ishrane. Ekonomski značaj krompira potiče od krtola, jestivih podzemnih modifikacija bočnih izdanaka koji omogućavaju nakupljanje rezervi skroba. Brojna istraživanja potvrdila su da biljni hormoni citokinini igraju značajnu ulogu u kontroli tuberizacije. Citokinin oksidaza/dehidrogenaza (CKX) je ključni enzim katabolizma citokinina kod biljaka, a transgene biljke koje eksprimiraju gene za ovaj enzim predstavljaju dragocen model sistem za izučavanje citokininske homeostaze. Krompir (sorta "Désirée") transformisan je genima AtCKX1 i AtCKX2 poreklom iz Arabidopsis thaliana u cilju utvrđivanja efekata promenjene citokininske homeostaze na morfološke i fiziološke parametre transformisanih biljaka krompira gajenih in vitro, a naročito na proces tuberizacije. Dobijene su dve AtCKX1 i tri AtCKX2 linije sa visokim nivoom ekspresije transgena, odnosno aktivosti CKX u proteinskim ekstraktima izdanaka i korenova. Ekspresija gena AtCKX1 i AtCKX2 dovela je do značajnih promena u hormonskoj homeostazi transformisanih biljaka krompira gajenih in vitro, ali ne i do snižavanja nivoa ukupnih endogenih citokinina. Umesto toga, kod većine transformisanih linija dolazi do značajnog sniženja nivoa bioaktivnih citokinina – slobodnih baza i ribozida, kao i citokininskih nukleotida. Pored toga, značajno je promenjen i nivo endogene indol-sirćetne kiseline (IAA). Izmenjena hormonska homeostaza odražava se na morfologiju biljaka, kao i na procese vezane za tuberizaciju, pa kod pojedinih transformisanih linija dolazi do izmena u regulaciji tuberizacije in vitro, kako u uslovima dugog dana, tako i u kontinuiranom mraku. Kod tri od pet transformisanih linija došlo je do prevazilaženja inhibitornog uticaja svetlosti na indukciju krtola, pa su se krtole pojavile već u toku prvih 30 dana pri uslovima dugog dana, što se nikada ne događa kod kontrolnih biljaka ove sorte krompira. Transformisane AtCKX biljke formirale su u proseku manji broj krtola po izdanku nego kontrolne biljke, što ukazuje na smanjenu inicijaciju krtola kod biljaka sa sniženim nivoima bioaktivnih citokinina, a može biti posledica povećanog odnosa između endogene IAA i bioaktivnih citokinina. Efekat na inicijaciju krtola je mnogo izraženiji u uslovima kontinuiranog mraka, nego dugog dana. Prečnik i masa krtola kod većine transformisanih linija se nisu značajno razlikovali od vrednosti kod kontrolnih biljaka. Samo jedna od pet transformisanih linija, AtCKX1-36a imala je u uslovima dugog dana krtole manjeg prečnika i mase. Citokinini različito utiču na pojedine faze u procesu tuberizacije kod krompira. Sniženje nivoa bioaktivnih citokinina dovodi do ranije indukcije krtola kod AtCKX biljaka, ali ovaj efekat nije ujedno praćen stimulatornim efektima na inicijaciju i uvećavanje krtola. Potrebna su dalja istraživanja transgenih biljaka koje bi eksprimirale kako kataboličke (AtCKX) tako i gene biosinteze citokinina (ipt), kako bismo identifikovali regulatorne mehanizme pomoću kojih citokinini utiču na različite faze procesa tuberizacije kako u indukujućim, tako i u neindukujućim uslovima.

Potato (Solanum tuberosum L.) is the world's fourth most important food crop. Its economical importance arises from its potential to develop tubers, edible storage organs derived from modified underground lateral shoots, which accumulate starch. Extensive research has provided evidence that plant hormones cytokinins have an important role in control of tuberization. Cytokinin oxidase/dehydrogenase (CKX) is the key enzyme of cytokinin catabolism in plants, thus transgenic plants expressing CKX genes have provided a valuable model system for the research of cytokinin homeostasis. Potato (cv. "Désirée") plants have been transformed with AtCKX1 and AtCKX2 genes from Arabidopsis thaliana, in order to determine the effects of altered cytokinin homeostasis on the morphological and physiological features of the transformed potato plants grown in vitro, with particular emphasis on the tuberization process. A high level of transgene expression and/or CKX activity in shoots and roots have been confirmed in two AtCKX1 and three AtCKX2 lines. The expression of AtCKX genes has reflected on significant changes in the hormonal homeostasis of the transformed potato plants grown in vitro, but did not lead to the decrease of total endogenous cytokinin levels. However in the majority of the AtCKX lines, the levels of bioactive cytokinins (nucleobases and ribosides), as well as cytokinin nucleotides, were significantly decreased. In addition, the level of endogenous indole-3-acetic acid (IAA) was also significantly changed. The altered hormonal homeostasis reflected on the morphological level, including the tuberization process. Tuberization was altered in AtCKX potato lines both in long-day conditions, and in continuous darkness. In three of the five AtCKX lines, the overcoming of the inhibitory effect of light on tuber induction has been observed. In these lines, tubers appeared already during the first 30 days in long-day conditions, which has never been observed in control plants of this potato cultivar. The AtCKX plants developed less tubers per shoot in comparison to control plants, indicating decreased tuber initiation in plants with lower endogenous levels of bioactive cytokinins. It is possible that this was a consequence of the increased ratio of IAA vs. bioactive cytokinins. The effect on tuber initiation was more pronounced in continuous darkness, than in long-day conditions. Tuber diameter and mass did not significantly differ from control values in the majority of transformed AtCKX lines. Only one of the five transformed lines, AtCKX1-36a developed tubers of smaller diameter and mass. Cytokinins have different effects on particular steps in the tuberization process in potato. Lower bioactive cytokinin levels lead to early tuber induction in AtCKX plants, but this effect is not followed by enhanced tuber initiation and growth. Further research on potato plants that would express either cytokinin catabolism (AtCKX) or biosynthesis (ipt) transgenes, is required to identify the regulation mechanisms by which cytokinins affect particular steps of the tuberization process in both tuber-inducing and noninducing conditions.