Together with auxins, cytokinins are the main plant hormones involved in many different physiological processes. Given this knowledge, cytokinin levels can be manipulated by genetic modification in order to improve agronomic parameters of cereals in relation to, for example, morphology, yield, and tolerance to various stresses. The barley (Hordeum vulgare) cultivar Golden Promise was transformed using the cytokinin dehydrogenase 1 gene from Arabidopsis thaliana (AtCKX1) under the control of mild root-specific β-glucosidase promoter from maize. Increased cytokinin degradation activity was observed positively to affect the number and length of lateral roots. The impact on morphology depended upon the recombinant protein's subcellular compartmentation. While assumed cytosolic and vacuolar targeting of AtCKX1 had negligible effect on shoot growth, secretion of AtCKX1 protein to the apoplast had a negative effect on development of the aerial part and yield. Upon the application of severe drought stress, all transgenic genotypes maintained higher water content and showed better growth and yield parameters during revitalization. Higher tolerance to drought stress was most caused by altered root morphology resulting in better dehydration avoidance.
- MeSH
- aklimatizace genetika fyziologie MeSH
- biotechnologie MeSH
- cytokininy genetika metabolismus MeSH
- fenotyp MeSH
- fyziologický stres MeSH
- geneticky modifikované rostliny MeSH
- ječmen (rod) genetika růst a vývoj fyziologie MeSH
- kořeny rostlin fyziologie MeSH
- metabolické sítě a dráhy MeSH
- období sucha MeSH
- oxidoreduktasy genetika MeSH
- proteiny huseníčku genetika fyziologie MeSH
- regulátory růstu rostlin genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- rostlinné geny MeSH
- rostlinné proteiny genetika MeSH
- stanovení celkové genové exprese MeSH
- upregulace MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cytokinin oxidase MeSH Prohlížeč
- cytokininy MeSH
- oxidoreduktasy MeSH
- proteiny huseníčku MeSH
- regulátory růstu rostlin MeSH
- rekombinantní proteiny MeSH
- rostlinné proteiny MeSH
Barley (Hordeum vulgare L.) is one of the founder crops of agriculture, and today it is the fourth most important cereal grain worldwide. Barley is used as malt in brewing and distilling industry, as an additive for animal feed, and as a component of various food and bread for human consumption. Progress in stable genetic transformation of barley ensures a potential for improvement of its agronomic performance or use of barley in various biotechnological and industrial applications. Recently, barley grain has been successfully used in molecular farming as a promising bioreactor adapted for production of human therapeutic proteins or animal vaccines. In addition to development of reliable transformation technologies, an extensive amount of various barley genetic resources and tools such as sequence data, microarrays, genetic maps, and databases has been generated. Current status on barley transformation technologies including gene transfer techniques, targets, and progeny stabilization, recent trials for improvement of agricultural traits and performance of barley, especially in relation to increased biotic and abiotic stress tolerance, and potential use of barley grain as a protein production platform have been reviewed in this study. Overall, barley represents a promising tool for both agricultural and biotechnological transgenic approaches, and is considered an ancient but rediscovered crop as a model industrial platform for molecular farming.
- Klíčová slova
- Barley, Molecular pharming, Pathogen resistance, Stress tolerance, Transgenesis, Yield improvement,
- MeSH
- biotechnologie * MeSH
- fyziologická adaptace MeSH
- geneticky modifikované rostliny * MeSH
- ječmen (rod) * MeSH
- odolnost vůči nemocem MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The cytokinin dehydrogenases (CKX; EC 1.5.99.12) are a protein family that maintains the endogenous levels of cytokinins in plants by catalyzing their oxidative degradation. The CKX family in maize (Zea mays L.) has thirteen members, only two of which--ZmCKX1 and ZmCKX10--have previously been characterized in detail. In this study, nine further maize CKX isoforms were heterologously expressed in Escherichia coli, purified by affinity and ion-exchange chromatography and biochemically characterized. ZmCKX6 and ZmCKX9 could only be expressed successfully after the removal of putative sequence-specific vacuolar sorting signals (LLPT and LPTS, respectively), suggesting that these proteins are localized to the vacuole. Substrate specificity analyses revealed that the CKX isoforms can be grouped into two subfamilies: members of the first strongly prefer cytokinin free bases while members of the second degrade a broad range of substrates. The most active isoform was found to be ZmCKX1. One of the studied isoforms, ZmCKX6, seemed to encode a nonfunctional enzyme due to a mutation in a conserved HFG protein domain at the C-terminus. Site-directed mutagenesis experiments revealed that this domain is essential for CKX activity. The roles of the maize CKX enzymes in the development of maize seedlings during the two weeks immediately after radicle emergence were also investigated. It appears that ZmCKX1 is a key regulator of active cytokinin levels in developing maize roots. However, the expression of individual CKX isoforms in the shoots varied and none of them seemed to have strong effects on the cytokinin pool.
- Klíčová slova
- 2,6-dichlorophenolindophenol, AtCKX, CKX, Cytokinin, Cytokinin dehydrogenase, DCPIP, DHZ, DMAPP, EDTA, Escherichia coli IMPACT expression system, IPT, N(6)-(Δ(2)-isopentenyl)adenine, N(6)-(Δ(2)-isopentenyl)adenine-N9-glucoside, N(6)-(Δ(2)-isopentenyl)adenosine, N(6)-(Δ(2)-isopentenyl)adenosine-5′-monophosphate, Substrate preference, Zea mays L., ZmCKX, cZ, cZ9G, cZRMP, cis-zeatin, cis-zeatin riboside-5′-monophosphate, cis-zeatin-N9-glucoside, cytokinin dehydrogenase, cytokinin dehydrogenase from Arabidopsis thaliana, cytokinin dehydrogenase from Zea mays, dihydrozeatin, dimethylallyl diphosphate, ethylenediaminetetraacetic acid, iP, iP9G, iPR, iPRMP, isopentenyl transferase, tZ, tZ9G, tZR, tZRMP, trans-zeatin, trans-zeatin riboside, trans-zeatin riboside-5′-monophosphate, trans-zeatin-N9-glucoside,
- MeSH
- chromatografie afinitní MeSH
- chromatografie iontoměničová MeSH
- cytokininy metabolismus MeSH
- DNA primery MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fylogeneze MeSH
- kukuřice setá enzymologie růst a vývoj metabolismus MeSH
- molekulární sekvence - údaje MeSH
- mutageneze cílená MeSH
- oxidoreduktasy chemie genetika metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- regulace genové exprese enzymů * MeSH
- regulace genové exprese u rostlin * MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cytokinin oxidase MeSH Prohlížeč
- cytokininy MeSH
- DNA primery MeSH
- oxidoreduktasy MeSH
Cytokinins (CKs) are ubiquitous phytohormones that participate in development, morphogenesis and many physiological processes throughout plant kingdom. In higher plants, mutants and transgenic cells and tissues with altered activity of CK metabolic enzymes or perception machinery, have highlighted their crucial involvement in different agriculturally important traits, such as productivity, increased tolerance to various stresses and overall plant morphology. Furthermore, recent precise metabolomic analyses have elucidated the specific occurrence and distinct functions of different CK types in various plant species. Thus, smooth manipulation of active CK levels in a spatial and temporal way could be a very potent tool for plant biotechnology in the future. This review summarises recent advances in cytokinin research ranging from transgenic alteration of CK biosynthetic, degradation and glucosylation activities and CK perception to detailed elucidation of molecular processes, in which CKs work as a trigger in model plants. The first attempts to improve the quality of crop plants, focused on cereals are discussed, together with proposed mechanism of action of the responses involved.
- MeSH
- alkyltransferasy a aryltransferasy metabolismus MeSH
- cytokininy genetika metabolismus MeSH
- fyziologický stres MeSH
- genetické inženýrství metody MeSH
- geneticky modifikované rostliny genetika MeSH
- glykosylace MeSH
- histidinkinasa MeSH
- oxidoreduktasy metabolismus MeSH
- proteinkinasy metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- signální transdukce MeSH
- zemědělské plodiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- adenylate isopentenyltransferase MeSH Prohlížeč
- alkyltransferasy a aryltransferasy MeSH
- cytokinin oxidase MeSH Prohlížeč
- cytokininy MeSH
- histidinkinasa MeSH
- oxidoreduktasy MeSH
- proteinkinasy MeSH
- rostlinné proteiny MeSH
Barley is one of the most important cereal crops grown worldwide. It has numerous applications, but its utility could potentially be extended by genetically manipulating its hormonal balances. To explore some of this potential we identified gene families of cytokinin dehydrogenases (CKX) and isopentenyl transferases, enzymes that respectively irreversibly degrade and synthesize cytokinin (CK) plant hormones, in the raw sequenced barley genome. We then examined their spatial and temporal expression patterns by immunostaining and qPCR. Two CKX-specific antibodies, anti-HvCKX1 and anti-HvCKX9, predominantly detect proteins in the aleurone layer of maturing grains and leaf vasculature, respectively. In addition, two selected CKX genes were used for stable, Agrobacterium tumefaciens-mediated transformation of the barley cultivar Golden Promise. The results show that constitutive overexpression of CKX causes morphological changes in barley plants and prevents their transition to flowering. In all independent transgenic lines roots proliferated more rapidly and root-to-shoot ratios were higher than in wild-type plants. Only one transgenic line, overexpressing CKX under the control of a promoter from a phosphate transporter gene, which is expressed more strongly in root tissue than in aerial parts, yielded progeny. Analysis of several T1-generation plants indicates that plants tend to compensate for effects of the transgene and restore CK homeostasis later during development. Depleted CK levels during early phases of development are restored by down-regulation of endogenous CKX genes and reinforced de novo biosynthesis of CKs.
- MeSH
- Agrobacterium tumefaciens MeSH
- cytokininy biosyntéza genetika MeSH
- exprese genu * MeSH
- fertilita genetika MeSH
- geneticky modifikované rostliny enzymologie genetika MeSH
- ječmen (rod) enzymologie genetika MeSH
- kořeny rostlin embryologie genetika MeSH
- oxidoreduktasy biosyntéza genetika MeSH
- rostlinné proteiny biosyntéza genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cytokinin oxidase MeSH Prohlížeč
- cytokininy MeSH
- oxidoreduktasy MeSH
- rostlinné proteiny MeSH
Nutritional quality of human and animal foodstuffs is determined by the content of essential amino acids. Barley is the fourth most important cereal of the world and the second most important cereal grown in the Czech Republic. Cereal grains such as barley contain insufficient levels of some essential amino acids, especially lysine. Dihydrodipicolinate synthase is the key enzyme involved in the regulatory step for lysine biosynthesis. Two constructs pBract214::sTPdapA and pBract214::mdapA containing the dapA gene from Escherichia coli coding for the bacterial dihydrodipicolinate synthase were used for transformation of barley. An Agrobacterium-mediated technique was used for transformation of immature embryos of spring barley cv. Golden Promise. Transgenic barley plants of the T0 and T1 generations were evaluated by PCR, real-time PCR, gel electrophoresis, and Western blot. Amino acid content was analyzed by HPLC after HCl hydrolysis. The lysine content in leaves of the T1 generation plant no. 5/5 was 50% higher than in wild-type plants; the lysine content in seeds of T2 generation plant no. 5/16 was 30% higher than in wild-type seeds of spring barley cv. Golden Promise.
- MeSH
- aminokyseliny analýza metabolismus MeSH
- chromatografie iontoměničová MeSH
- dehydratasy biosyntéza genetika metabolismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- geneticky modifikované rostliny chemie enzymologie genetika MeSH
- hydrolýza MeSH
- ječmen (rod) chemie enzymologie genetika MeSH
- listy rostlin chemie MeSH
- lysin analýza metabolismus MeSH
- polymerázová řetězová reakce MeSH
- proteiny z Escherichia coli genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- western blotting MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 4-hydroxy-tetrahydrodipicolinate synthase MeSH Prohlížeč
- aminokyseliny MeSH
- dehydratasy MeSH
- lysin MeSH
- proteiny z Escherichia coli MeSH
- rekombinantní proteiny MeSH
