Structure and function of cytokinin oxidase/dehydrogenase genes of maize,rice,<Emphasis Type="Italic"> Arabidopsis</Emphasis> and other species

Cytokinin oxidases/dehydrogenases (CKX) catalyze the irreversible degradation of the cytokinins isopentenyladenine, zeatin, and their ribosides in a single enzymatic step by oxidative side chain cleavage. To date the sequences of 17 fully annotated CKX genes are known, including two prokaryotic genes. The CKX gene families of Arabidopsis thaliana and rice comprise seven and at least ten members, respectively. The main features of CKX genes and proteins are summarized in this review. Individual proteins differ in their catalytic properties, their subcellular localization and their expression domains. The evolutionary development of cytokinin-catabolizing gene families and the individual properties of their members indicate an important role for the fine-tuned control of catabolism to assure proper regulation of cytokinin functions. The use of CKX genes as a tool in studies of cytokinin biology and biotechnological applications is discussed.     

Elevated auxin and reduced cytokinin contents in rootstocks improve their performance and grafting success

Plant grafting is an important technique for horticultural and silvicultural production. However, many rootstock plants suffer from undesirable lateral bud outgrowth, low grafting success rates or poor rooting. Here, we used a root‐predominant gene promoter (SbUGT) to drive the expression of a tryptophan‐2‐monooxygenase gene (iaaM) from Agrobacterium tumefaciens to increase auxin levels in tobacco. The transgenic plants, when used as a rootstock, displayed inhibited lateral bud outgrowth, enhanced grafting success rate and improved root initiation. However, root elongation and biomass of SbUGT::iaaM transgenic plants were reduced compared to those of wild‐type plants. In contrast, when we used this same promoter to drive CKX (a cytokinin degradation gene) expression, the transgenic tobacco plants displayed enhanced root elongation and biomass. We then made crosses between the SbUGT::CKX and SbUGT::iaaM transgenic plants. We observed that overexpression of the CKX gene neutralized the negative effects of auxin overproduction on root elongation. Also, the simultaneous expression of both the iaaM and CKX genes in rootstock did not disrupt normal growth and developmental patterns in wild‐type scions. Our results demonstrate that expression of both the iaaM and CKX genes predominantly in roots of rootstock inhibits lateral bud release from rootstock, improves grafting success rates and enhances root initiation and biomass.     

Response of <Emphasis Type="Italic">Pisum Sativum</Emphasis> Cytokinin Oxidase/Dehydrogenase Expression and Specific Activity to Drought Stress and Herbicide Treatments

The expression of cytokinin oxidase/dehydrogenase (CKX EC: 1.5.99.12) is subject to fine regulation and it provides a rapid turnover of cytokinins, which serves as a signal for triggering developmental events during plant growth. The activity of this enzyme is believed to be responsible for the changes in cytokinin pool under adverse environmental conditions. CKX gene-specific assay to measure the expression in response to different stress treatments in the tissues of Pisum sativum plants was developed. Pea CKX genes were amplified and sequenced using primers designed from the sequences of Medicago truncatula CKX genes. Expression of two P. sativum CKX genes was assessed using relative-quantification in real time two-step RT-PCR, in leaves and roots of drought-, glufosinate- and atrazine-treated cv. Manuela pea plants. Varied CKX responses support the existence of complicated regulating mechanism of cytokinin oxidase/ dehydrogenase gene expression.     

Enhanced cytokinin degradation in leaf primordia of transgenic Arabidopsis plants reduces leaf size and shoot organ primordia formation

The plant hormone cytokinin is a key morphogenic factor controlling cell division and differentiation, and thus the formation and growth rate of organs during a plant's life cycle. In order to explore the relevance of cytokinin during the initial phase of leaf primordia formation and its impact on subsequent leaf development, we increased cytokinin degradation in young shoot organ primordia of Arabidopsis thaliana by expressing a cytokinin oxidase/dehydrogenase (CKX) gene under control of the AINTEGUMENTA (ANT) promoter. The final leaf size in ANT:CKX3 plants was reduced to ∼27% of the wild-type size and the number of epidermal cells was reduced to ∼12% of the wild type. Kinematic analysis revealed that cell proliferation ceased earlier and cell expansion was accelerated in ANT:CKX3 leaves, demonstrating that cytokinin controls the duration of the proliferation phase by delaying the onset of cell differentiation. The reduction of the cell number was partially compensated by an increased cell expansion. Interestingly, ANT:CKX3 leaf cells became about 60% larger than those of 35S:CKX3 leaves, indicating that cytokinin has an important function during cell expansion as well. Furthermore, ANT:CKX3 expression significantly reduced the capacity of both the vegetative as well as the generative shoot apical meristem to initiate the formation of new leaves and flowers, respectively. We therefore hypothesize that the cytokinin content in organ primordia is important for regulating the activity of the shoot meristem in a non-autonomous fashion.     

RcRR1, a Rosa canina Type-A Response Regulator Gene,Is Involved in Cytokinin-Modulated Rhizoid Organogenesis

In vitro, a new protocol of plant regeneration in rose was achieved via protocorm-like bodies (PLBs) induced from the root-like organs named rhizoids that developed from leaf explants. The development of rhizoids is a critical stage for efficient regeneration, which is triggered by exogenous auxin. However, the role of cytokinin in the control of organogenesis in rose is as yet uncharacterized. The aim of this study was to elucidate the molecular mechanism of cytokinin-modulated rhizoid formation in Rosa canina. Here, we found that cytokinin is a key regulator in the formation of rhizoids. Treatment with cytokinin reduced callus activity and significantly inhibited rhizoid formation in Rosa canina. We further isolated the full-length cDNA of a type-A response regulator gene of cytokinin signaling, RcRR1, from which the deduced amino acid sequence contained the conserved DDK motif. Gene expression analysis revealed that RcRR1 was differentially expressed during rhizoid formation and its expression level was rapidly up-regulated by cytokinin. In addition, the functionality of RcRR1 was tested in Arabidopsis. RcRR1 was found to be localized to the nucleus in GFP-RcRR1 transgenic plants and overexpression of RcRR1 resulted in increased primary root length and lateral root density. More importantly, RcRR1 overexpression transgenic plants also showed reduced sensitivity to cytokinin during root growth; auxin distribution and the expression of auxin efflux carriers PIN genes were altered in RcRR1 overexpression plants. Taken together, these results demonstrate that RcRR1 is a functional type-A response regulator which is involved in cytokinin-regulated rhizoid formation in Rosa canina.     

Regulated Expression of a Cytokinin Biosynthesis Gene IPT Delays Leaf Senescence and Improves Yield under Rainfed and Irrigated Conditions in Canola (Brassica napus L.)

Delay of leaf senescence through genetic modification can potentially improve crop yield, through maintenance of photosynthetically active leaves for a longer period. Plant growth hormones such as cytokinin regulate and delay leaf senescence. Here, the structural gene (IPT) encoding the cytokinin biosynthetic enzyme isopentenyltransferase was fused to a functionally active fragment of the AtMYB32 promoter and was transformed into canola plants. Expression of the AtMYB32xs::IPT gene cassette delayed the leaf senescence in transgenic plants grown under controlled environment conditions and field experiments conducted for a single season at two geographic locations. The transgenic canola plants retained higher chlorophyll levels for an extended period and produced significantly higher seed yield with similar growth and phenology compared to wild type and null control plants under rainfed and irrigated treatments. The yield increase in transgenic plants was in the range of 16% to 23% and 7% to 16% under rainfed and irrigated conditions, respectively, compared to control plants. Most of the seed quality parameters in transgenic plants were similar, and with elevated oleic acid content in all transgenic lines and higher oil content and lower glucosinolate content in one specific transgenic line as compared to control plants. The results suggest that by delaying leaf senescence using the AtMYB32xs::IPT technology, productivity in crop plants can be improved under water stress and well-watered conditions.     

Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.     

Transgenic Tobacco plants with T-DNA Phytohormone synthesis genes

Agrobacterium tumefaciens binary vectors carrying kanamycin resistance gene and either C58 T-DNA gene 4 for cytokinin synthesis or genes 1 and 2 for auxin synthesis were constructed and used for transformation of a short-day tobacco Maryland Mammoth. Kanamycin resistant plants were regenerated from a small fraction of transformed tissue and the presence of T-DNA in their genome was verified by Southern blotting. The level of endogenous cytokinin in plants transgenic for gene 4 and the level of endogenous IAA in those transgenic for genes 1 and 2 increased by more than 100 %. A number of morphological characteristics distinguish them from untransformed controls.     

Identification and Characterization of ATP/ADP Isopentenyltransferases (ATP/ADP PpIPTs) Genes in Peach

ATP/ADP isopentenyltransferase (IPTs) genes encode key enzymes involved in cytokinin synthesis. In this study, the functions of ATP/ADP PpIPTs in peach were investigated. According to the genome sequence, we have found and verified that there are four members of this gene family in peach, namely, PpIPT1, PpIPT3, PpIPT5, and PpIPT7. Overexpression of each of these genes in Arabidopsis resulted in increased levels of cytokinins in the transgenic plants, confirming their roles in cytokinin synthesis. Numerous altered phenotypes were observed in the transgenic plants, including vigorous growth and enhanced salt resistance. ATP/ADP PpIPTs were expressed in tissues throughout the plant, but the expression patterns differed between the genes. Only PpIPT3 was upregulated within 2 h after the application of nitrate to N-deprived peach seedlings, and the increase was resistant to pre-treatment of a specific nitrate metabolism inhibitor. Results showed that ATP/ADP PpIPT expression levels decreased significantly in pulp within 2 weeks after flowering and remained low. However, pulp cytokinin levels were quite high during this time. Only PpIPT5 in seed increased significantly within 2 weeks after flowering, which was consistent with cytokinin levels during early fruit development, suggesting that PpIPT5 in seed is the key gene for cytokinin biosynthesis during early fruit development. ATP/ADP PpIPT expression also increased significantly during later fruit development in seed.

     

Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.     

The stay-green phenotype of wheat mutant <Emphasis Type="Italic">tasg1</Emphasis> is associated with altered cytokinin metabolism

Key message

By measuring the cytokinin content directly and testing the sensitivity to the cytokinin inhibitor lovastatin, we demonstrated that tasg1 cytokinin metabolism is different from wild-type.

Abstract

Our previous studies have indicated that compared with wild-type (WT) plants, a wheat stay-green mutant tasg1 exhibited delayed senescence. In this study, we found that the root development of tasg1 occurred later than that of WT. The number of lateral roots was fewer, but the lateral root length was longer in tasg1 than in WT, which resulted in a lower root to shoot ratio in tasg1 than WT. The levels of cytokinin (CK), CK activity, and expression of CK metabolic genes were measured. We found that the total CK content in the root tips and leaf of tasg1 was greater than in WT. The accumulation of mRNA of the CK synthetic gene (TaIPT) in tasg1 was higher than in WT at 9 and 11 days during seedling growth, but the expression of CK oxidase gene (TaCKX) was significantly lower in tasg1. Furthermore, the CK inhibitor lovastatin was used to inhibit CK activity. When treated with lovastatin, both the chlorophyll content and thylakoid membrane protein stability were significantly lower in tasg1 than WT, consistent with the inhibited expression of senescence-associated genes (TaSAGs) in tasg1. Lovastatin treatment also inhibited the antioxidative capability of wheat seedlings, and tasg1 was more sensitive to lovastatin than WT, as indicated by the MDA content, protein carbonylation, and antioxidant enzyme activity. The decreased antioxidative capability after lovastatin treatment may be related to the down-regulation of some antioxidase genes. These results suggest that the CK metabolism was altered in tasg1, which may play an important role in its ability to delay senescence.

     

Expression and functional analysis of genes encoding cytokinin receptor-like histidine kinase in maize (Zea mays L.)

Cytokinin signaling is vital for plant growth and development which function via the two-component system (TCS). As one of the key component of TCS, transmembrane histidine kinases (HK) are encoded by a small gene family in plants. In this study, we focused on expression and functional analysis of cytokinin receptor-like HK genes (ZmHK) in maize. Firstly, bioinformatics analysis revealed that seven cloned ZmHK genes have different expression patterns during maize development. Secondly, ectopic expression by CaMV35S promoter in Arabidopsis further revealed that functional differentiation exists among these seven members. Among them, the ZmHK1a2-OX transgenic line has the lowest germination rate in the dark, ZmHK1-OX and ZmHK2a2-OX can delay leaf senescence, and seed size of ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX was obviously reduced as compared to wild type. Additionally, ZmHK genes play opposite roles in shoot and root development; all ZmHK-OX transgenic lines display obvious shorter root length and reduced number of lateral roots, but enhanced shoot development compared with the wild type. Most notably, Arabidopsis response regulator ARR5 gene was up-regulated in ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX as compared to wild type. Although the causal link between ZmHK genes and cytokinin signaling pathway is still an area to be further elucidated, these findings reflected that the diversification of ZmHK genes expression patterns and functions occurred in the course of maize evolution, indicating that some ZmHK genes might play different roles during maize development.     

phiC31 Integrase-Mediated Site-Specific Recombination in Barley

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F₁, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.     

Cytokinin dehydrogenase: a genetic target for yield improvement in wheat

The plant hormone group, the cytokinins, is implicated in both qualitative and quantitative components of yield. Cytokinins have opposing actions in shoot and root growth—actions shown to involve cytokinin dehydrogenase (CKX), the enzyme that inactivates cytokinin. We revise and provide unambiguous names for the CKX gene family members in wheat, based on the most recently released wheat genome database, IWGSC RefSeq v1.0 & v2.0. We review expression data of CKX gene family members in wheat, revealing tissue‐specific gene family member expression as well as sub‐genome‐specific expression. Manipulation of CKX in cereals shows clear impacts on yield, root growth and orientation, and Zn nutrition, but this also emphasizes the necessity to unlink promotive effects on grain yield from negative effects of cytokinin on root growth and uptake of mineral nutrients, particularly Zn and Fe. Wheat is the most widely grown cereal crop globally, yet is under‐research compared with rice and maize. We highlight gaps in our knowledge of the involvement of CKX for wheat. We also highlight the necessity for accurate analysis of endogenous cytokinins, acknowledging why this is challenging, and provide examples where inadequate analyses of endogenous cytokinins have led to unjustified conclusions. We acknowledge that the allohexaploid nature of bread wheat poses challenges in terms of uncovering useful mutations. However, we predict TILLING followed by whole‐exome sequencing will uncover informative mutations and we indicate the potential for stacking mutations within the three genomes to modify yield components. We model a wheat ideotype based on CKX manipulation.     

Transformation of kiwifruit using the <Emphasis Type="Italic">ipt</Emphasis> gene alters tree architecture

To manipulate the architecture of woody plants by controlling endogenous cytokinin levels, the isopentenyl transferase gene (ipt) from Agrobacterim tumefaciens was introduced to kiwifruit using stable transformation. Consequently, eight transgenic lines were obtained. Transgenic shoots harboring the ipt gene were recalcitrant to rooting under tissue-culture conditions; thus, their in vitro-cultivated shoots were directly grafted onto potted wild-type kiwifruit seedlings to evaluate their morphological features, and three lines (tmr2-4, tmr2-G, tmr3-C) were successfully grafted. The grafted transgenic plants had dwarfing and branching phenotypes, both of which are typical features of cytokinin overproduction. In addition, the number of buds increased and internode length was shorter in the grafted transgenic plants. The content of a precursor, trans-zeatin riboside, and an active cytokinin, trans-zeatin, increased in one transgenic line, in which the level of ipt gene expression was high, indicating that morphological changes were related to expression levels of the ipt gene and cytokinin content. Possibilities for potential utilization of the ipt gene in manipulating tree shape are discussed.