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

A fitokróm fotoreceptorok indukálta jelátviteli lánc molekuláris vizsgálata Arabidopsis thalianában = Molecular analysis of phytochrome induced signal transduction in Arabidopsis thaliana

Bebizonyítottuk, hogy a fitokróm-A sejtmagba történő transzlokációjában és sejtmagi akkumulációjában, tehát a fitokróm-A szabályozta jelátviteli lánc aktiválásában az FHY1 fehérje meghatározó szerepet játszik. Kimutattuk, hogy a fitokróm-A autofoszforilációja negatívan szabályozza a jelátviteli lánc aktivitását és azonosítottunk egy olyan foszfatázt, ami specifikusan a fitokróm-A fotoreceptor defoszforilációjáért felelős. Bebizonyítottuk, hogy a PIF3 fehérje nem pozitív, hanem negatív regulátora a fotomorfogenezisnek, a PIF3 fehérje nem szükséges a cirkadián óra működéséhez, továbbá, hogy fény hatására a PIF3 fehérje gyorsan degradálódik és hogy a fitokróm- A-B és D fotoreceptorok együttesen szabályozzák a PIF3 transzkripciós faktor fény indukálta degradációját. | We have provided compelling evidence that nuclear translocation and accumulation of phytochrome-A requires the FHY1 protein and that FHY1 interacts with the phyA molecule in a conformation dependent fashion and this interaction is mediated by the very N-terminal domain of the photoreceptor. We demonstrated that PIF3 transcription factor negatively regulates phytochrome controlled photomorphogenesis, its function is not required for light dependent entrainment of the plant circadian clock and that phytochrome-A in harmony with phytochrome-B and D regulates light induced degradation of PIF3. Moreover, we showed that interaction of phytochrome-A with various components of the phytochrome-A dependent signalling cascade is negatively regulated by the autophosphorylation of the photoreceptor and that dephosphorylation of the photoreceptor is mediated by a specific phosphatase

Timely removal of exogenous cytokinin and the prevention of auxin transport from the shoot to the root affect the regeneration potential of Arabidopsis roots

In vitro regeneration of Arabidopsis from roots is generally achieved via indirect organogenesis. First, transdifferentiation of lateral root primordia to calli is achieved by a balanced auxin-to-cytokinin ratio that is followed by the induction of shoot meristem formation using a high cytokinin level. Here we demonstrate that if the root explants were transferred onto a hormone-free medium after a transient (4-days) cytokinin treatment, embryogenic marker genes (LEC1, LEC2, FUS3) started to be expressed. App. 50% of the regeneration foci developed into plantlets with trichome-less cotyledon-like leaves. Moreover, the somatic embryogenesis defective lec1 mutant could regenerate only shoots with trichome-bearing leaves under this condition. Based on these observations, the mixed accomplishment of shoot organogenesis and somatic embryogenesis is hypothesized in the Arabidopsis root explants cultured under hormone-free conditions following cytokinin induction. Using whole seedlings instead of root explants in the same experimental set up, no regenerates were formed on the roots. Applying the auxin transport inhibitor TIBA to the root-to-shoot junction of the seedlings, the regeneration ability of the root could be restored. The observations indicate that shoot-derived endogenous auxin blocks the cytokinin-induced regeneration process in the roots of whole seedlings. The expression of the wound-induced transcription factor WIND1 could be detected in the roots of unwounded seedlings if the shoot-to-root auxin transport was inhibited. Manipulating the exogenous cytokinin level together with the endogenous shoot-to-root auxin transport therefore could mimic the effect of wounding (removal of shoot) on plant regeneration from roots. Key message Transferring root explants from high cytokinin to hormone-free conditions resulted in the expression of embryogenic markers. Inhibiting the shoot-to-root auxin transport had similar effect on regeneration as wounding

CRK5 Protein Kinase Contributes to the Progression of Embryogenesis of Arabidopsis thaliana

The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins

Timely removal of exogenous cytokinin and the prevention of auxin transport from the shoot to the root affect the regeneration potential of Arabidopsis roots

In vitro regeneration of Arabidopsis from roots is generally achieved via indirect organogenesis. First, transdifferentiation of lateral root primordia to calli is achieved by a balanced auxin-to-cytokinin ratio that is followed by the induction of shoot meristem formation using a high cytokinin level. Here we demonstrate that if the root explants were transferred onto a hormone-free medium after a transient (4-days) cytokinin treatment, embryogenic marker genes (LEC1, LEC2, FUS3) started to be expressed. App. 50% of the regeneration foci developed into plantlets with trichome-less cotyledon-like leaves. Moreover, the somatic embryogenesis defective lec1 mutant could regenerate only shoots with trichome-bearing leaves under this condition. Based on these observations, the mixed accomplishment of shoot organogenesis and somatic embryogenesis is hypothesized in the Arabidopsis root explants cultured under hormone-free conditions following cytokinin induction. Using whole seedlings instead of root explants in the same experimental set up, no regenerates were formed on the roots. Applying the auxin transport inhibitor TIBA to the root-to-shoot junction of the seedlings, the regeneration ability of the root could be restored. The observations indicate that shoot-derived endogenous auxin blocks the cytokinin-induced regeneration process in the roots of whole seedlings. The expression of the wound-induced transcription factor WIND1 could be detected in the roots of unwounded seedlings if the shoot-to-root auxin transport was inhibited. Manipulating the exogenous cytokinin level together with the endogenous shoot-to-root auxin transport therefore could mimic the effect of wounding (removal of shoot) on plant regeneration from roots. Key message Transferring root explants from high cytokinin to hormone-free conditions resulted in the expression of embryogenic markers. Inhibiting the shoot-to-root auxin transport had similar effect on regeneration as wounding

The Arabidopsis RLCK VI_A2 Kinase Controls Seedling and Plant Growth in Parallel with Gibberellin

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation

PlantSize Offers an Affordable, Non-destructive Method to Measure Plant Size and Color in Vitro

Plant size, shape and color are important parameters of plants, which have traditionally been measured by destructive and time-consuming methods. Non-destructive image analysis is an increasingly popular technology to characterize plant development in time. High throughput automatic phenotyping platforms can simultaneously analyze multiple morphological and physiological parameters of hundreds or thousands of plants. Such platforms are, however, expensive and are not affordable for many laboratories. Moreover, determination of basic parameters is sufficient for most studies. Here we describe a non-invasive method, which simultaneously measures basic morphological and physiological parameters of in vitro cultured plants. Changes of plant size, shape and color is monitored by repeated photography with a commercial digital camera using neutral white background. Images are analyzed with the MatLab-based computer application PlantSize, which simultaneously calculates several parameters including rosette size, convex area, convex ratio, chlorophyll and anthocyanin contents of all plants identified on the image. Numerical data are exported in MS Excel-compatible format. Subsequent data processing provides information on growth rates, chlorophyll and anthocyanin contents. Proof-of-concept validation of the imaging technology was demonstrated by revealing small but significant differences between wild type and transgenic Arabidopsis plants overexpressing the HSFA4A transcription factor or the hsfa4a knockout mutant, subjected to different stress conditions. While HSFA4A overexpression was associated with better growth, higher chlorophyll and lower anthocyanin content in saline conditions, the knockout hsfa4a mutant showed hypersensitivity to various stresses. Morphological differences were revealed by comparing rosette size, shape and color of wild type plants with phytochrome B (phyB-9) mutant. While the technology was developed with Arabidopsis plants, it is suitable to characterize plants of other species including crops, in a simple, affordable and fast way. PlantSize is publicly available (http://www.brc.hu/pub/psize/index.html)

Image_1.JPEG

<p>Plant size, shape and color are important parameters of plants, which have traditionally been measured by destructive and time-consuming methods. Non-destructive image analysis is an increasingly popular technology to characterize plant development in time. High throughput automatic phenotyping platforms can simultaneously analyze multiple morphological and physiological parameters of hundreds or thousands of plants. Such platforms are, however, expensive and are not affordable for many laboratories. Moreover, determination of basic parameters is sufficient for most studies. Here we describe a non-invasive method, which simultaneously measures basic morphological and physiological parameters of in vitro cultured plants. Changes of plant size, shape and color is monitored by repeated photography with a commercial digital camera using neutral white background. Images are analyzed with the MatLab-based computer application PlantSize, which simultaneously calculates several parameters including rosette size, convex area, convex ratio, chlorophyll and anthocyanin contents of all plants identified on the image. Numerical data are exported in MS Excel-compatible format. Subsequent data processing provides information on growth rates, chlorophyll and anthocyanin contents. Proof-of-concept validation of the imaging technology was demonstrated by revealing small but significant differences between wild type and transgenic Arabidopsis plants overexpressing the HSFA4A transcription factor or the hsfa4a knockout mutant, subjected to different stress conditions. While HSFA4A overexpression was associated with better growth, higher chlorophyll and lower anthocyanin content in saline conditions, the knockout hsfa4a mutant showed hypersensitivity to various stresses. Morphological differences were revealed by comparing rosette size, shape and color of wild type plants with phytochrome B (phyB-9) mutant. While the technology was developed with Arabidopsis plants, it is suitable to characterize plants of other species including crops, in a simple, affordable and fast way. PlantSize is publicly available (http://www.brc.hu/pub/psize/index.html).</p

Presentation_1.PDF

<p>Plant size, shape and color are important parameters of plants, which have traditionally been measured by destructive and time-consuming methods. Non-destructive image analysis is an increasingly popular technology to characterize plant development in time. High throughput automatic phenotyping platforms can simultaneously analyze multiple morphological and physiological parameters of hundreds or thousands of plants. Such platforms are, however, expensive and are not affordable for many laboratories. Moreover, determination of basic parameters is sufficient for most studies. Here we describe a non-invasive method, which simultaneously measures basic morphological and physiological parameters of in vitro cultured plants. Changes of plant size, shape and color is monitored by repeated photography with a commercial digital camera using neutral white background. Images are analyzed with the MatLab-based computer application PlantSize, which simultaneously calculates several parameters including rosette size, convex area, convex ratio, chlorophyll and anthocyanin contents of all plants identified on the image. Numerical data are exported in MS Excel-compatible format. Subsequent data processing provides information on growth rates, chlorophyll and anthocyanin contents. Proof-of-concept validation of the imaging technology was demonstrated by revealing small but significant differences between wild type and transgenic Arabidopsis plants overexpressing the HSFA4A transcription factor or the hsfa4a knockout mutant, subjected to different stress conditions. While HSFA4A overexpression was associated with better growth, higher chlorophyll and lower anthocyanin content in saline conditions, the knockout hsfa4a mutant showed hypersensitivity to various stresses. Morphological differences were revealed by comparing rosette size, shape and color of wild type plants with phytochrome B (phyB-9) mutant. While the technology was developed with Arabidopsis plants, it is suitable to characterize plants of other species including crops, in a simple, affordable and fast way. PlantSize is publicly available (http://www.brc.hu/pub/psize/index.html).</p