In plants, nitrogen is the most important nutritional factor limiting the yield of cultivated crops. Since nitrogen is essential for synthesis of nucleotides, amino acids and proteins, studies on gene expression in plants cultivated under different nitrogen availability require particularly careful selection of suitable reference genes which are not affected by nitrogen limitation. Therefore, the objective of this study was to select the most reliable reference genes for qPCR analysis of target cucumber genes under varying nitrogen source and availability. Among twelve candidate cucumber genes used in this study, five are highly homologous to the commonly used internal controls, whereas seven novel candidates were previously identified through the query of the cucumber genome. The expression of putative reference genes and the target CsNRT1.1 gene was analyzed in roots, stems and leaves of cucumbers grown under nitrogen deprivation, varying nitrate availability or different sources of nitrogen (glutamate, glutamine or NH3). The stability of candidate genes expression significantly varied depending on the tissue type and nitrogen supply. However, in most of the outputs genes encoding CACS, TIP41, F-box protein and EFα proved to be the most suitable for normalization of CsNRT1.1 expression. In addition, our results suggest the inclusion of 3 or 4 references to obtain highly reliable results of target genes expression in all cucumber organs under nitrogen-related stress.

Background and Aims NRT1 proteins are H + -coupling nitrate transporters which belong to the family of peptide transporters (PTRs) and facilitate low and high affinity nitrate transport systems in a model plant Arabidopsis thaliana. In this study, we present the first inventory of the Cucumis sativus NRT1 family together with the transcriptional profile of CsNRT1 genes suggesting the physiological function of the family members in cucumber. Methods Semiquantitative RT-PCR was used to analyze the level and organ-distribution of expression of CsNRT1 genes. The response of those CsNRT1s, whose transcripts were clearly detectable in vegetative tissues to different level of nitrate supply was examined through real-time PCR assays. ResultsThe newly identified cucumber NRT1s were given the designation according to their homology to A. thaliana AtNRT1s. The comparison of the Arabidopsis and cucumber NRT gene families, similarly to the previous comparison of NRT1s in Arabidopsis, poplar and grasses, reveals some striking differences in genes' structure and quantity. Conclusions The putative function of particular CsNRT1 proteins is discussed, considering the results obtained here as well as the already published studies on A. thaliana NRT1 transporters.

There are 15 and 23 members of the pleiotropic drug resistance (PDR) proteins, a subfamily of ATP-binding cassette (ABC) transporters, in Arabidopsis thaliana and Oryza sativa, respectively. Until recently, only a few members of Arabidopsis thaliana PDRs (AtPDRs) had been characterized in detail but growing reports indicate that proteins of this family may participate in growth regulator-mediated signaling and phytohormone transport. The profile of Oryza sativa PDRs (OsPDRs) expression in response to different stimuli also suggests that some rice PDRs are associated with a phytohormone-mediated response to environmental changes. Due to the lack of full genome resources, only individual members of PDRs in other plants (Nicotiana, Oryza, Glycine or Spirodela) have been studied. We have previously cloned two full cDNA sequences encoding for cucumber homologs of Arabidopsis PDR8 and PDR12. Here, we present the genomic organization of 16 cucumber PDRs and the phylogeny of the predicted Cucumis sativus PDR (CsPDR) proteins. Further analysis of CsPDR8 and CsPDR12 reveal that these cucumber genes are constitutively expressed mainly in roots of young seedlings and flowering plants (CsPDR8 and CsPDR12), and in flowers (CsPDR12). In roots, the transcript of CsPDR8 decreased in response to ABA and markedly raised in the presence of ACC, salicylic acid or jasmonic acid. In comparison, the expression of CsPDR12 was not affected by ACC and significantly increased upon the addition of jasmonic acid, salicylic acid, 2,4-D, kinetin or ABA to the growth media. These data suggest that CsPDR8 and CsPDR12 may be involved in a phytohormone-mediated response of plants to different stimuli by sharing different signaling pathways.

Studies in the last few years have shed light on the process of nitrate accumulation within plant cells, achieving molecular identification and partial characterization of the genes and proteins involved in this process. However, contrary to the plasma membrane-localized nitrate transport activities, the kinetics of active nitrate influx into the vacuole and its adaptation to external nitrate availability remain poorly understood. In this work, we have investigated the activity and regulation of the tonoplast-localized H+/NO3 − antiport in cucumber roots in response to N starvation and NO3 − induction. The time course of nitrate availability strongly influenced H+/NO3 − antiport activity at the tonoplast of root cells. However, under N starvation active nitrate accumulation within the vacuole still occurred. Hence, either a constitutive H+-coupled transport system specific for nitrate operates at the tonoplast, or nitrate uses another transport protein of broader specificity to different anions to enter the vacuole via a proton-dependent process. H+/NO3 − antiport in cucumber was significantly stimulated in NO3 −-induced plants that were supplied with nitrate for 24 hours following 6-day-long N starvation. The cytosolic fraction isolated from the roots of NO3 −-induced plants significantly stimulated H+/NO3 − antiport in tonoplast membranes isolated from cucumbers growing on nitrate. The stimulatory effect of the cytosolic fraction was completely abolished by EGTA and the protein kinase inhibitor staurosporine and slightly enhanced by the phosphatase inhibitors okadaic acid and cantharidin. Hence, we conclude that stimulation of H+/NO3 − antiport at the tonoplast of cucumber roots in response to nitrate provision may occur through the phosphorylation of a membrane antiporter involving Ca-dependent, staurosporine-sensitive protein kinase.

Full-size members of the ABCG (ATP-binding cassette, subfamily G) subfamily of ABC transporters have been found only in plants and fungi. The plant genes encoding full-size ABCGs identified so far appeared to be differentially regulated under various environmental constraints, plant growth regulators, and microbial elicitors, indicating a broad functional role of these proteins in plant responses to abiotic and biotic stress. Nevertheless, the structure and physiological function of full-size ABCGs in many plant species are still unknown. We have recently identified 16 genes encoding full-size ABCG proteins in cucumber and found that the transcripts of two of them, CsABCG36 (CsPDR8) and CsABCG40 (CsPDR12), are most abundant in roots and are significantly affected by phytohormones and auxin herbicide. In this study, we analyzed the structure and phylogeny of all the full-size cucumber ABCG transporters and studied the organ expression profiles of the remaining 14 CsABCG genes. In addition, we investigated the effect of different plant growth regulators and the diterpene sclareolide on CsABCG expression in cucumber roots. Until now, the full-size plant ABCG transporters have been grouped into five different clusters. The new phylogenetic analysis of full-size ABCGs from model plants and cucumber clustered these proteins into six different subgroups. Interestingly, the expression profiles of cucumber ABCG genes assigned to the same clusters were not correlated, suggesting functional diversification or different regulatory mechanisms of the full-size cucumber ABCG proteins.Electronic supplementary materialThe online version of this article (doi:10.1007/s11105-015-0956-9) contains supplementary material, which is available to authorized users.

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