Stressztoleranciát biztosító gének azonosítása a halofita Lepidium crassifolium-ból

Extreme environmental conditions limit plant growth and impose abiotic stress to plants. Land degradation, including desertification, drought and salinity affects around one third of the global land surface (Jarraud 2005). Adaptation of plants to suboptimal conditions requires extensive physiological and molecular reprogramming, leading to major changes in metabolic, proteomic and transcript profiles. Research on model organisms such as Arabidopsis thaliana and application of system biology approaches has identified a number of genes and regulatory hubs which control the networks linking stress perception and metabolic or developmental responses (Ahuja et al. 2010). However, study of a stress sensitive model has limitations in understanding tolerance to harsh environments. Extremophile plants, such as xerophytes and halophytes can grow in arid regions or on saline soils, which are otherwise lethal to nonadapted species. Halophytes represent 1% of all plant species; can optimally thrive in the presence of 50–250mM NaCl, whilst some withstand salt concentrations up to 600mM NaCl (Flowers and Colmer 2008). While the physiology of halophytes has been extensively studied, molecular regulation of the extremophile character still remains to be understood. Eutrema salsugineum (previously called Thellungiella salsuginea) is a salt tolerant relative of Arabidopsis, which has been used in a number of comparative studies to reveal the genetic and molecular basis of halophytism (Amtmann 2009). Natural genetic variability of extremophiles is an attractive genetic resource to improve tolerance of crops to adverse environments (Nevo and Chen 2010). Transfer of tolerance traits to other species is however usually hampered by incompatibility. Transformation of genomic or cDNA libraries can facilitate random gene transfer between different species. Examples include a cDNA library of E. salsugineum, expressed in Arabidopsis, leading to the identification of several Eutrema genes which improved salt tolerance (Du et al. 2008). A binary bacterial artificial chromosome library was used to transfer large genomic fragments of E. salsugineum to Arabidopsis and screen for salt tolerance (Wang et al. 2010). Here, we describe the novel version of the Conditional cDNA Overexpressing System (COS), which was developed to randomly transfer and express cDNA clones in Arabidopsis under the control of a chemically inducible promoter system (Papdi et al. 2008; Rigó et al. 2012). The cDNA library was derived from the less-known halophyte of the Brassicaceae family Lepidium crassifolium, which naturally grows on salty-sodic soils in Central Europe and Asia. Random transfer and overexpression of L. crassifolium cDNA in Arabidopsis could facilitate the identification of novel tolerance genes. Here, we demonstrate that regulated expression of several L. crassifolium cDNA could enhance salt, osmotic or oxidative stress tolerance of Arabidopsis. The COS system is therefore suitable for interspecific gene transfer and can be employed to identify valuable genes from less-known wild species

A brasszinoszteroidok szerepének vizsgálata a növényi szervek morfogenezisének szabályozásában = The regulatory role of brassinosteroids in the morphogenic determination of plant organs

A brasszinoszteroidok (BR-ok) növényi szteroid hormonok, amelyek fontos szerepet játszanak az egyedfejlődési és reproduktív funkciók szabályozásában. Munkánk célja olyan tényezők azonosítása volt, amelyek a sejtek szenzitizálása, ill. a hormon felhalmozása révén befolyásolhatják a BR válaszreakciókat. Kimutattuk, hogy a feltételezett egyenletes kifejeződéssel szemben a BR receptort kódoló BRI1 gén differenciált szerv- és fejlődés-specifikus expressziót mutat. BRI1 promóter-riporter fúziókat hordozó transzgenikus Arabidopsis vonalainkban a fokozott hormonérzékenység szoros korrelációt mutatott a receptor erős kifejeződésével, jelezve a receptor sűrűség és a válaszreakció kialakulása közti kapcsolatot. Másrészt meghatároztuk az aktív BR formák korábban ismeretlen szintjét az Arabidopsis egyes szerveiben, igazolva, hogy a hormon felhalmozódás mértékét jórészt a bioszintetikus gének transzkripciós szintű szabályozása határozza meg. A sebesség-meghatározó CPD/CYP90A1 enzimről episztázis analízissel kimutattuk, hogy az a szintézis hatékonyságát közvetlenül a BR szintézis első reakcióját követően kontrollálja. Az anyagcsereút utolsó, még tisztázatlan enzim funkcióját karakterizálva meghatároztuk, hogy a CPD/CYP90A1 a szteroid váz C-3 pozíciójának oxidációjáért felelős. Ezzel egyúttal kísérletes bizonyítékot szolgáltattunk egy új, a korábban ismertnél hatékonyabb BR szintézisút in vivo jelentőségére. | Brassinosteroids (BRs) are steroidal plant hormones controlling morphogenesis and reproductive development. The aim of our project was to elucidate basic mechanisms that influence BR responses by modulating cellular susceptibility or accumulation of the biologically active hormone. We demonstrated that, in contrast to an earlier concept of ubiquitous expression, the BRI1 gene encoding the BR receptor shows differential organ-specific and developmental activity. Our BRI1 promoter-reporter constructs revealed that in Arabidopsis elevated BRI1 expression coincides with increased BR responsiveness, suggesting an important role for receptor abundance in the initiation of BR signaling. In a different approach we determined the hitherto unknown distribution of active BRs in the various organs of Arabidopsis, and provided evidence that the transcriptional control of BR biosynthetic genes efficiently regulates hormone accumulation. We have shown that CPD/CYP90A1 is a key enzyme of BR biosynthesis and, using epistasis analysis, we demonstrated that it controls intermediate flow immediately downstream of the first committed step of the BR pathway. Characterizing the last unknown enzyme function in the BR pathway, we clarified by in vitro enzyme assays that CPD/CYP90A catalyzes the C-3 oxidation of early BR intermediates. Based on this result we proposed an enzymologically well supported novel BR biosynthetic pathway