Inverse correlation between TP53 gene status and PD-L1 protein levels in a melanoma cell model depends on an IRF1/SOX10 regulatory axis.

BACKGROUND: PD-L1 expression on cancer cells is an important mechanism of tumor immune escape, and immunotherapy targeting the PD-L1/PD1 interaction is a common treatment option for patients with melanoma. However, many patients do not respond to treatment and novel predictors of response are emerging. One suggested modifier of PD-L1 is the p53 pathway, although the relationship of p53 pathway function and activation is poorly understood. METHODS: The study was performed on human melanoma cell lines with various p53 status. We investigated PD-L1 and proteins involved in IFNγ signaling by immunoblotting and mRNA expression, as well as membrane expression of PD-L1 by flow cytometry. We evaluated differences in the ability of NK cells to recognize and kill target tumor cells on the basis of p53 status. We also investigated the influence of proteasomal degradation and protein half-life, IFNγ signaling and p53 activation on biological outcomes, and performed bioinformatic analysis using available data for melanoma cell lines and melanoma patients. RESULTS: We demonstrate that p53 status changes the level of membrane and total PD-L1 protein through IRF1 regulation and show that p53 loss influences the recently discovered SOX10/IRF1 regulatory axis. Bioinformatic analysis identified a dependency of SOX10 on p53 status in melanoma, and a co-regulation of immune signaling by both transcription factors. However, IRF1/PD-L1 regulation by p53 activation revealed complicated regulatory mechanisms that alter IRF1 mRNA but not protein levels. IFNγ activation revealed no dramatic differences based on TP53 status, although dual p53 activation and IFNγ treatment confirmed a complex regulatory loop between p53 and the IRF1/PD-L1 axis. CONCLUSIONS: We show that p53 loss influences the level of PD-L1 through IRF1 and SOX10 in an isogenic melanoma cell model, and that p53 loss affects NK-cell cytotoxicity toward tumor cells. Moreover, activation of p53 by MDM2 inhibition has a complex effect on IRF1/PD-L1 activation. These findings indicate that evaluation of p53 status in patients with melanoma will be important for predicting the response to PD-L1 monotherapy and/or dual treatments where p53 pathways participate in the overall response.

Comparative characterization of two monoclonal antibodies targeting canine PD-1.

Monoclonal antibodies targeting immune checkpoints have revolutionized oncology. Yet, the effectiveness of these treatments varies significantly among patients, and they are associated with unexpected adverse events, including hyperprogression. The murine research model used in drug development fails to recapitulate both the functional human immune system and the population heterogeneity. Hence, a novel model is urgently needed to study the consequences of immune checkpoint blockade. Dogs appear to be uniquely suited for this role. Approximately 1 in 4 companion dogs dies from cancer, yet no antibodies are commercially available for use in veterinary oncology. Here we characterize two novel antibodies that bind canine PD-1 with sub-nanomolar affinity as measured by SPR. Both antibodies block the clinically crucial PD-1/PD-L1 interaction in a competitive ELISA assay. Additionally, the antibodies were tested with a broad range of assays including Western Blot, ELISA, flow cytometry, immunofluorescence and immunohistochemistry. The antibodies appear to bind two distinct epitopes as predicted by molecular modeling and peptide phage display. Our study provides new tools for canine oncology research and a potential veterinary therapeutic.

Transcriptional changes during crown-root development and emergence in barley (Hordeum vulgare L.).

BACKGROUND: Roots play an important role during plant growth and development, ensuring water and nutrient uptake. Understanding the mechanisms regulating their initiation and development opens doors towards root system architecture engineering. RESULTS: Here, we investigated by RNA-seq analysis the changes in gene expression in the barley stem base of 1 day-after-germination (DAG) and 10DAG seedlings when crown roots are formed. We identified 2,333 genes whose expression was lower in the stem base of 10DAG seedlings compared to 1DAG seedlings. Those genes were mostly related to basal cellular activity such as cell cycle organization, protein biosynthesis, chromatin organization, cytoskeleton organization or nucleotide metabolism. In opposite, 2,932 genes showed up-regulation in the stem base of 10DAG seedlings compared to 1DAG seedlings, and their function was related to phytohormone action, solute transport, redox homeostasis, protein modification, secondary metabolism. Our results highlighted genes that are likely involved in the different steps of crown root formation from initiation to primordia differentiation and emergence, and revealed the activation of different hormonal pathways during this process. CONCLUSIONS: This whole transcriptomic study is the first study aiming at understanding the molecular mechanisms controlling crown root development in barley. The results shed light on crown root emergence that is likely associated with a strong cell wall modification, death of the cells covering the crown root primordium, and the production of defense molecules that might prevent pathogen infection at the site of root emergence.

Biochemical evidence for conformational variants in the anti-viral and pro-metastatic protein IFITM1.

Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.

N-glycan profiling of tissue samples to aid breast cancer subtyping.

Breast cancer is a highly heterogeneous disease. Its intrinsic subtype classification for diagnosis and choice of therapy traditionally relies on the presence of characteristic receptors. Unfortunately, this classification is often not sufficient for precise prediction of disease prognosis and treatment efficacy. The N-glycan profiles of 145 tumors and 10 healthy breast tissues were determined using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry. The tumor samples were classified into Mucinous, Lobular, No-Special-Type, Human Epidermal Growth Factor 2 + , and Triple-Negative Breast Cancer subtypes. Statistical analysis was conducted using the reproducibility-optimized test statistic software package in R, and the Wilcoxon rank sum test with continuity correction. In total, 92 N-glycans were detected and quantified, with 59 consistently observed in over half of the samples. Significant variations in N-glycan signals were found among subtypes. Mucinous tumor samples exhibited the most distinct changes, with 28 significantly altered N-glycan signals. Increased levels of tri- and tetra-antennary N-glycans were notably present in this subtype. Triple-Negative Breast Cancer showed more N-glycans with additional mannose units, a factor associated with cancer progression. Individual N-glycans differentiated Human Epidermal Growth Factor 2 + , No-Special-Type, and Lobular cancers, whereas lower fucosylation and branching levels were found in N-glycans significantly increased in Luminal subtypes (Lobular and No-Special-Type tumors). Clinically normal breast tissues featured a higher abundance of signals corresponding to N-glycans with bisecting moiety. This research confirms that histologically distinct breast cancer subtypes have a quantitatively unique set of N-glycans linked to clinical parameters like tumor size, proliferative rate, lymphovascular invasion, and metastases to lymph nodes. The presented results provide novel information that N-glycan profiling could accurately classify human breast cancer samples, offer stratification of patients, and ongoing disease monitoring.

IFITM protein regulation and functions: Far beyond the fight against viruses.

Interferons (IFNs) are important cytokines that regulate immune responses through the activation of hundreds of genes, including interferon-induced transmembrane proteins (IFITMs). This evolutionarily conserved protein family includes five functionally active homologs in humans. Despite the high sequence homology, IFITMs vary in expression, subcellular localization and function. The initially described adhesive and antiproliferative or pro-oncogenic functions of IFITM proteins were diluted by the discovery of their antiviral properties. The large set of viruses that is inhibited by these proteins is constantly expanding, as are the possible mechanisms of action. In addition to their beneficial antiviral effects, IFITM proteins are often upregulated in a broad spectrum of cancers. IFITM proteins have been linked to most hallmarks of cancer, including tumor cell proliferation, therapeutic resistance, angiogenesis, invasion, and metastasis. Recent studies have described the involvement of IFITM proteins in antitumor immunity. This review summarizes various levels of IFITM protein regulation and the physiological and pathological functions of these proteins, with an emphasis on tumorigenesis and antitumor immunity.

Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition.

The TGF-ß signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-ß signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-ß effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated AGR2 knockout with and without TGF-ß treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of COL4A1, COL4A2, FLNA, VAV3, VEGFA, and VINC mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-ß treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-ß have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both AGR2 knockout and TGF-ß treatment and were validated by RT-qPCR of GPX2, PTGS2, and PLA2G4A. Since PGE2 is a product of arachidonic acid metabolism, its lowered concentration in media from AGR2-knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-ß have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway.

DNA and RNA Binding Proteins: From Motifs to Roles in Cancer.

DNA and RNA binding proteins (DRBPs) are a broad class of molecules that regulate numerous cellular processes across all living organisms, creating intricate dynamic multilevel networks to control nucleotide metabolism and gene expression. These interactions are highly regulated, and dysregulation contributes to the development of a variety of diseases, including cancer. An increasing number of proteins with DNA and/or RNA binding activities have been identified in recent years, and it is important to understand how their activities are related to the molecular mechanisms of cancer. In addition, many of these proteins have overlapping functions, and it is therefore essential to analyze not only the loss of function of individual factors, but also to group abnormalities into specific types of activities in regard to particular cancer types. In this review, we summarize the classes of DNA-binding, RNA-binding, and DRBPs, drawing particular attention to the similarities and differences between these protein classes. We also perform a cross-search analysis of relevant protein databases, together with our own pipeline, to identify DRBPs involved in cancer. We discuss the most common DRBPs and how they are related to specific cancers, reviewing their biochemical, molecular biological, and cellular properties to highlight their functions and potential as targets for treatment.

Transcriptomic Profiling Revealed Lnc-GOLGA6A-1 as a Novel Prognostic Biomarker of Meningioma Recurrence.

BACKGROUND: Meningioma is the most common primary central nervous system neoplasm, accounting for about a third of all brain tumors. Because their growth rates and prognosis cannot be accurately estimated, biomarkers that enable prediction of their biological behavior would be clinically beneficial. OBJECTIVE: To identify coding and noncoding RNAs crucial in meningioma prognostication and pathogenesis. METHODS: Total RNA was purified from formalin-fixed and paraffin-embedded tumor samples of 64 patients with meningioma with distinct clinical characteristics (16 recurrent, 30 nonrecurrent with follow-up of >5 years, and 18 with follow-up of <5 years without recurrence). Transcriptomic sequencing was performed using the HiSeq 2500 platform (Illumina), and biological and functional differences between meningiomas of different types were evaluated by analyzing differentially expression of messenger RNA (mRNA) and long noncoding RNA (IncRNA). The prognostic value of 11 differentially expressed RNAs was then validated in an independent cohort of 90 patients using reverse transcription quantitative (real-time) polymerase chain reaction. RESULTS: In total, 69 mRNAs and 108 lncRNAs exhibited significant differential expression between recurrent and nonrecurrent meningiomas. Differential expression was also observed with respect to sex (12 mRNAs and 59 lncRNAs), World Health Organization grade (58 mRNAs and 98 lncRNAs), and tumor histogenesis (79 mRNAs and 76 lncRNAs). Lnc-GOLGA6A-1, ISLR2, and AMH showed high prognostic power for predicting meningioma recurrence, while lnc-GOLGA6A-1 was the most significant factor for recurrence risk estimation (1/hazard ratio = 1.31; P = .002). CONCLUSION: Transcriptomic sequencing revealed specific gene expression signatures of various clinical subtypes of meningioma. Expression of the lnc-GOLGA61-1 transcript was found to be the most reliable predictor of meningioma recurrence.

The effects of p53 gene inactivation on mutant proteome expression in a human melanoma cell model.

BACKGROUND: The identification of mutated proteins in human cancer cells-termed proteogenomics, requires several technologically independent research methodologies including DNA variant identification, RNA sequencing, and mass spectrometry. Any one of these methodologies are not optimized for identifying potential mutated proteins and any one output fails to cover completely a specific landscape. METHODS: An isogenic melanoma cell with a p53-null genotype was created by CRISPR/CAS9 system to determine how p53 gene inactivation affects mutant proteome expression. A mutant peptide reference database was developed by comparing two distinct DNA and RNA variant detection platforms using these isogenic cells. Chemically fractionated tryptic peptides from lysates were processed using a TripleTOF 5600+ mass spectrometer and their spectra were identified against this mutant reference database. RESULTS: Approximately 190 mutated peptides were enriched in wt-p53 cells, 187 mutant peptides were enriched in p53-null cells, with an overlap of 147 mutated peptides. STRING analysis highlighted that the wt-p53 cell line was enriched for mutant protein pathways such as CDC5L and POLR1B, whilst the p53-null cell line was enriched for mutated proteins comprising EGF/YES, Ubiquitination, and RPL26/5 nodes. CONCLUSION: Our study produces a well annotated p53-dependent and p53-independent mutant proteome of a common melanoma cell line model. Coupled to the application of an integrated DNA and RNA variant detection platform (CLCbio) and software for identification of proteins (ProteinPilot), this pipeline can be used to detect high confident mutant proteins in cells. GENERAL SIGNIFICANCE: This pipeline forms a blueprint for identifying mutated proteins in diseased cell systems.

Aromatic Cytokinin Arabinosides Promote PAMP-like Responses and Positively Regulate Leaf Longevity.

Cytokinins are plant hormones with biological functions ranging from coordination of plant growth to the regulation of biotic and abiotic stress-related responses and senescence. The components of the plant immune system can learn from past elicitations by microbial pathogens and herbivores and adapt to new threats. It is known that plants can enter the primed state of enhanced defense induced by either natural or synthetic compounds. While the involvement of cytokinins in defense priming has been documented, no comprehensive model of their action has been provided to date. Here, we report the functional characterization of two aromatic cytokinin derivatives, 6-benzylaminopurine-9-arabinosides (BAPAs), 3-methoxy-BAPA and 3-hydroxy-BAPA, that proved to be effective in delaying senescence in detached leaves while having low interactions with the cytokinin pathway. An RNA-seq profiling study on Arabidopsis leaves treated with 3-methoxy-BAPA revealed that short and extended treatments with this compound shifted the transcriptional response markedly toward defense. Both treatments revealed upregulation of genes involved in processes associated with plant innate immunity such as cell wall remodeling and upregulation of specific MAP kinases, most importantly MPK11, which is a MAPK module involved in stress-related signaling during the pathogen-associated molecular patterns (PAMPs) response. In addition, elevated levels of JA and its metabolites, jasmonate/ethylene-driven upregulation of PLANT DEFENSIN 1.2 (PDF1.2) and other defensins, and also temporarily elevated levels of reactive oxygen species marked the plant response to 3-methoxy-BAPA treatment. Synergistic interactions were observed when plants were cotreated with 3-hydroxy-BAPA and the flagellin-derived bacterial PAMP peptide (flg22), leading to the enhanced expression of the PAMP-triggered immunity (PTI) marker gene FRK1. Our data collectively show that some BAPAs can sensitively prime the PTI responses in a low micromolar range of concentrations while having no observable negative effects on the overall fitness of the plant.

SATrans: New Free Available Software for Annotation of Transcriptome and Functional Analysis of Differentially Expressed Genes.

Recent technological advances have made next-generation sequencing (NGS) a popular and financially accessible technique allowing a broad range of analyses to be done simultaneously. A huge amount of newly generated NGS data, however, require advanced software support to help both in analyzing the data and biologically interpreting the results. In this article, we describe SATrans (Software for Annotation of Transcriptome), a software package providing fast and robust functional annotation of novel sequences obtained from transcriptome sequencing. Moreover, it performs advanced gene ontology analysis of differentially expressed genes, thereby helping to interpret biologically-and in a user-friendly form-the quantitative changes in gene expression. The software is freely available and provides the possibility to work with thousands of sequences using a standard personal computer or notebook running on the Linux operating system.

Genome sequencing of Rhinorhipus Lawrence exposes an early branch of the Coleoptera.

BACKGROUND: Rhinorhipidae Lawrence, 1988 is an enigmatic beetle family represented by a single species, Rhinorhipus tamborinensis Lawrence, 1988, from Australia, with poorly established affinities near the superfamily Elateroidea (click beetles, soldier beetles and fireflies) or the more inclusive series (infraorder) Elateriformia. Its evolutionary position may inform the basal relationships of the suborder Polyphaga, the largest clade of Coleoptera. RESULTS: We analyzed four densely sampled DNA datasets of major coleopteran lineages for mitogenomes, rRNA genes and single copy nuclear genes. Additionally, genome sequencing was used for incorporation of R. tamborinensis into a set of 4220 orthologs for 24 terminals representing 12 polyphagan superfamilies. Topologies differed to various degrees, but all consistently refute the proposed placement of Rhinorhipidae in Elateroidea and instead indicate either sister relationships with other Elateriformia, frequently together with Nosodendridae, another divergent small family hitherto placed in Derodontoidea, or in an isolated position among the deepest lineages of Polyphaga. The phylogenomic analyses recovered Rhinorhipus in a sister position to all other Elateriformia composed of five superfamilies. Therefore, we erect the new superfamily Rhinorhipoidea Lawrence, 1988, stat. Nov., with the type-family Rhinorhipidae. The origins of the Rhinorhipidae were dated to the Upper Triassic/Lower Jurassic at the very early phase of polyphagan diversification. CONCLUSIONS: Thus, Rhinorhipidae adds another example to several recently recognized ancient relict lineages which are interspersed within contemporaneous hugely species-rich lineages of Coleoptera.

Light influences cytokinin biosynthesis and sensing in Nostoc (cyanobacteria).

Cytokinins are an important group of plant hormones that are also found in other organisms, including cyanobacteria. While various aspects of cytokinin function and metabolism are well understood in plants, the information is limited for cyanobacteria. In this study, we first experimentally confirmed a prenylation of tRNA by recombinant isopentenyl transferase NoIPT2 from Nostoc sp. PCC 7120, whose encoding gene we previously identified in Nostoc genome along with the gene for adenylate isopentenyl transferase NoIPT1. In contrast to NoIPT2, the transcription of NoIPT1 was strongly activated during the dark period and was followed by an increase in the cytokinin content several hours later in the light period. Dominant cytokinin metabolites detected at all time points were free bases and monophosphates of isopentenyladenine and cis-zeatin, while N-glucosides were not detected at all. Whole transcriptome differential expression analysis of cultures of the above Nostoc strain treated by cytokinin compared to untreated controls indicated that cytokinin together with light trigger expression of several genes related to signal transduction, including two-component sensor histidine kinases and two-component hybrid sensors and regulators. One of the affected histidine kinases with a cyclase/histidine kinase-associated sensory extracellular domain similar to the cytokinin-binding domain in plant cytokinin receptors was able to modestly bind isopentenyladenine. The data show that the genetic disposition allows Nostoc not only to produce free cytokinins and prenylate tRNA but also modulate the cytokinin biosynthesis in response to light, triggering complex changes in sensing and regulation.

Dataset for transcriptional response of barley (Hordeum vulgare) exposed to drought and subsequent re-watering.

Barley (Hordeum vulgare) is an economically important species, which can be cultivated in environmentally adverse conditions due to its higher tolerance in contrast to other cereal crops. The draft of H. vulgare genome is available already for couple of years; however its functional annotation is still incomplete. All available databases were searched to expand current annotation. The improved annotation was used to describe processes and genes regulated in transgenic lines showing higher tolerance to drought in our associated article, doi:10.1016/j.nbt.2016.01.010 (Vojta et al., 2016) [1]. Here we present whole transcriptome response, using extended annotation, to severe drought stress and subsequent re-watering in wild-type barley plants in stem elongation phase of growth. Up- and down-regulated genes fall into distinct GO categories and these enriched by stress and revitalization are highlighted. Transcriptomic data were evaluated separately for root and aerial tissues.

Cytokinin metabolism in maize: Novel evidence of cytokinin abundance, interconversions and formation of a new trans-zeatin metabolic product with a weak anticytokinin activity.

Cytokinins (CKs) are an important group of phytohormones. Their tightly regulated and balanced levels are essential for proper cell division and plant organ development. Here we report precise quantification of CK metabolites and other phytohormones in maize reproductive organs in the course of pollination and kernel maturation. A novel enzymatic activity dependent on NADP(+) converting trans-zeatin (tZ) to 6-(3-methylpyrrol-1-yl)purine (MPP) was detected. MPP shows weak anticytokinin properties and inhibition of CK dehydrogenases due to their ability to bind to an active site in the opposite orientation than substrates. Although the physiological significance of tZ side-chain cyclization is not anticipated as the MPP occurrence in maize tissue is very low, properties of the novel CK metabolite indicate its potential for utilization in plant in vitro tissue culture. Furthermore, feeding experiments with different isoprenoid CKs revealed distinct preferences in glycosylation of tZ and cis-zeatin (cZ). While tZ is preferentially glucosylated at the N9 position, cZ forms mainly O-glucosides. Since O-glucosides, in contrast to N9-glucosides, are resistant to irreversible cleavage catalyzed by CK dehydrogenases, the observed preference of maize CK glycosyltransferases to O-glycosylate zeatin in the cis-position might be a reason why cZ derivatives are over-accumulated in different maize tissues and organs.

Whole transcriptome analysis of transgenic barley with altered cytokinin homeostasis and increased tolerance to drought stress.

Cytokinin plant hormones have been shown to play an important role in plant response to abiotic stresses. Herein, we expand upon the findings of Pospísilová et al. [30] regarding preparation of novel transgenic barley lines overexpressing cytokinin dehydrogenase 1 gene from Arabidopsis under the control of mild root-specific promotor of maize ß-glycosidase. These lines showed drought-tolerant phenotype mainly due to alteration of root architecture and stronger lignification of root tissue. A detailed transcriptomic analysis of roots of transgenic plants subjected to revitalization after drought stress revealed attenuated response through the HvHK3 cytokinin receptor and up-regulation of two transcription factors implicated in stress responses and abscisic acid sensitivity. Increased expression of several genes involved in the phenylpropanoid pathway as well as of genes encoding arogenate dehydratase/lyase participating in phenylalanine synthesis was found in roots during revitalization. Although more precursors of lignin synthesis were present in roots after drought stress, final lignin accumulation did not change compared to that in plants grown under optimal conditions. Changes in transcriptome indicated a higher auxin turnover in transgenic roots. The same analysis in leaves revealed that genes encoding putative enzymes responsible for production of jasmonates and other volatile compounds were up-regulated. Although transgenic barley leaves showed lower chlorophyll content and down-regulation of genes encoding proteins involved in photosynthesis than did wild-type plants when cultivated under optimal conditions, they did show a tendency to return to initial photochemical activities faster than did wild-type leaves when re-watered after severe drought stress. In contrast to optimal conditions, comparative transcriptomic analysis of revitalized leaves displayed up-regulation of genes encoding enzymes and proteins involved in photosynthesis, and especially those encoded by the chloroplast genome. Taken together, our results indicate that the partial cytokinin insensitivity induced in barley overexpressing cytokinin dehydrogenase contributes to tolerance to drought stress.

Transgenic barley overexpressing a cytokinin dehydrogenase gene shows greater tolerance to drought stress.

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 ß-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.