CgIPT1 is required for synthesis of cis-zeatin cytokinins and contributes to stress tolerance and virulence in Colletotrichum graminicola.

We have previously shown that the maize pathogen Colletotrichum graminicola is able to synthesise cytokinins (CKs). However, it remained unsettled whether fungal CK production is essential for virulence in this hemibiotrophic fungus. Here, we identified a candidate gene, CgIPT1, that is homologous to MOD5 of Saccharomyces cerevisiae and genes from other fungi and plants, which encode tRNA-isopentenyltransferases (IPTs). We show that the wild type strain mainly synthesises cis-zeatin-type (cisZ) CKs whereas ΔCgipt1 mutants are severely impeded to do so. The spectrum of CKs produced confirms bioinformatical analyses predicting that CgIpt1 is a tRNA-IPT. The virulence of the ΔCgipt1 mutants is moderately reduced. Furthermore, the mutants exhibit increased sensitivities to osmotic stress imposed by sugar alcohols and salts, as well as cell wall stress imposed by Congo red. Amendment of media with CKs did not reverse this phenotype suggesting that fungal-derived CKs do not explain the role of CgIpt1 in mediating abiotic stress tolerance. Moreover, the mutants still cause green islands on senescing maize leaves indicating that the cisZ-type CKs produced by the fungus do not cause this phenotype.

Cytokinin-induced parthenocarpy of San Pedro type fig (Ficus carica L.) main crop: explained by phytohormone assay and transcriptomic network comparison.

KEY MESSAGE: CPPU-induced San Pedro type fig main crop parthenocarpy exhibited constantly increasing IAA content and more significantly enriched KEGG pathways in the receptacle than in female flowers. N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) was applied to San Pedro fig (Ficus carica L.) main crop to induce parthenocarpy; the optimal effect was obtained with 25 mg L-1 application to syconia when female flowers were at anthesis. To elucidate the key expression changes in parthenocarpy conversion, significant changes in phytohormone level and transcriptome of fig female flowers and receptacles were monitored. HPLC-MS revealed increased IAA content in female flowers and receptacle 2, 4 and 10 days after treatment (DAT), decreased zeatin level in the receptacle 2, 4 and 10 DAT, decreased GA3 content 2 and 4 DAT, and increased GA3 content 10 DAT. ABA level increased 2 and 4 DAT, and decreased 10 DAT. CPPU-treated syconia released more ethylene than the control except 2 DAT. RNA-Seq and bioinformatics analysis revealed notably more differentially expressed KEGG pathways in the receptacle than in female flowers. In the phytohormone gene network, GA-biosynthesis genes GA20ox and GA3ox were upregulated, along with GA signal-transduction genes GID1 and GID2, and IAA-signaling genes AUX/IAA and GH3. ABA-biosynthesis gene NCED and signaling genes PP2C and ABF were downregulated 10 DAT. One ACO gene showed consistent upregulation in both female flowers and receptacle after CPPU treatment, and more than a dozen of ERFs demonstrated opposing changes in expression. Our results revealed early-stage spatiotemporal phytohormone and transcriptomic responses in CPPU-induced San Pedro fig main crop parthenocarpy, which could be valuable for further understanding the nature of the parthenocarpy of different fig types.

Analysis of transcripts and splice isoforms in Medicago sativa L. by single-molecule long-read sequencing.

KEY MESSAGE: The full-length transcriptome of alfalfa was analyzed with PacBio single-molecule long-read sequencing technology. The transcriptome data provided full-length sequences and gene isoforms of transcripts in alfalfa, which will improve genome annotation and enhance our understanding of the gene structure of alfalfa. As an important forage, alfalfa (Medicago sativa L.) is world-wide planted. For its complexity of genome and unfinished whole genome sequencing, the sequences and complete structure of mRNA transcripts remain unclear in alfalfa. In this study, single-molecule long-read sequencing was applied to investigate the alfalfa transcriptome using the Pacific Biosciences platform, and a total of 113,321 transcripts were obtained from young, mature and senescent leaves. We identified 72,606 open reading frames including 46,616 full-length ORFs, 1670 transcription factors from 54 TF families and 44,040 simple sequence repeats from 30,797 sequences. A total of 7568 alternative splicing events was identified and the majority of alternative splicing events in alfalfa was intron retention. In addition, we identified 17,740 long non-coding RNAs. Our results show the feasibility of deep sequencing full-length RNA from alfalfa transcriptome on a single-molecule level.

Oligo-carrageenan kappa-induced reducing redox status and activation of TRR/TRX system increase the level of indole-3-acetic acid, gibberellin A3 and trans-zeatin in Eucalyptus globulus trees.

Eucalyptus globulus trees treated with oligo-carrageenan (OC) kappa showed an increase in NADPH, ascorbate and glutathione levels and activation of the thioredoxin reductase (TRR)/thioredoxin (TRX) system which enhance photosynthesis, basal metabolism and growth. In order to analyze whether the reducing redox status and the activation of thioredoxin reductase (TRR)/thioredoxin (TRX) increased the level of growth-promoting hormones, trees were treated with water (control), with OC kappa, or with inhibitors of ascorbate synthesis, lycorine, glutathione synthesis, buthionine sulfoximine (BSO), NADPH synthesis, CHS-828, and thioredoxin reductase activity, auranofine, and with OC kappa, and cultivated for four additional months. Eucalyptus trees treated with OC kappa showed an increase in the levels of the auxin indole 3-acetic acid (IAA), gibberellin A3 (GA3) and the cytokinin trans-zeatin (t-Z) as well as a decrease in the level of the brassinosteroid epi-brassinolide (EB). In addition, treatment with lycorine, BSO, CHS-828 and auranofine inhibited the increase in IAA, GA3 and t-Z as well as the decrease in EB levels. Thus, the reducing redox status and the activation of TRR/TRX system induced by OC kappa increased the levels of IAA, GA3 and t-Z levels determining, at least in part, the stimulation of growth in Eucalyptus trees.

Uniconazole, a cytochrome P450 inhibitor, inhibits trans-zeatin biosynthesis in Arabidopsis.

Cytokinin (CK) is a plant hormone that plays important regulatory roles in many aspects of plant growth and development. Although functions of CK and its biosynthesis pathway have been studied extensively, there is still no efficient biosynthesis inhibitor, which would be useful for studying CK from a chemical genetic approach. Here, CK biosynthesis inhibitor candidates were searched for using a systematic approach. In silico screening of candidates were carried out using genome-wide gene expression profiles and prediction of target sites using global CK accumulation profile analysis. As a result of these screenings, it was found that uniconazole, a well known inhibitor of cytochrome P450 monooxygenase, prevents the biosynthesis of trans-zeatin, and that its target is CYP735As in Arabidopsis.

The type IV secretion system component VirB5 binds to the trans-zeatin biosynthetic enzyme Tzs and enables its translocation to the cell surface of Agrobacterium tumefaciens.

VirB5 is a minor component of the extracellular T pilus determined by the Agrobacterium tumefaciens type IV secretion system. To identify proteins that interact with VirB5 during the pilus assembly process, we purified VirB5 as a recombinant fusion protein and, by using a gel overlay assay, we detected a 26-kDa interacting protein in Agrobacterium cell lysates. The VirB5-binding protein was purified from A. tumefaciens and identified as the cytokinin biosynthetic enzyme Tzs. The VirB5-Tzs interaction was confirmed using pulldown assays with purified proteins and the yeast two-hybrid system. An analysis of the subcellular localization in A. tumefaciens showed that Tzs was present in the soluble as well as the membrane fraction. Tzs was extracted from the membranes with the mild detergent dodecyl-beta-D-maltoside in complexes of different molecular masses, and this association was strongly reduced in the absence of VirB5. Using immunoelectron microscopy, we also detected Tzs on the Agrobacterium cell surface. A functional type IV secretion system was required for efficient translocation to the surface, but Tzs was not secreted into the cell supernatant. The fact that Tzs localizes on the cell surface suggests that it may contribute to the interaction of Agrobacterium with plants.

Phytohormone production by three strains of Bradyrhizobium japonicum and possible physiological and technological implications.

The aim of this work was to evaluate phytohormone biosynthesis, siderophores production, and phosphate solubilization in three strains (E109, USDA110, and SEMIA5080) of Bradyrhizobium japonicum, most commonly used for inoculation of soybean and nonlegumes in USA, Canada, and South America. Siderophore production and phosphate solubilization were evaluated in selective culture conditions, which had negative results. Indole-3-acetic acid (IAA), gibberellic acid (GA(3)), and abscisic acid (ABA) production were analyzed by gas chromatography-mass spectrometry (GC-MS). Ethylene and zeatin biosynthesis were determined by GS-flame ionization detection and high-performance liquid chromatography (HPLC-UV), respectively. IAA, zeatin, and GA(3) were found in all three strains; however, their levels were significantly higher (p < 0.01) in SEMIA5080 (3.8 microg ml(-1)), USDA110 (2.5 microg ml(-1)), and E109 (0.87 microg ml(-1)), respectively. ABA biosynthesis was detected only in USDA110 (0.019 microg ml(-1)). Ethylene was found in all three strains, with highest production rate (18.1 ng ml(-1) h(-1)) in E109 cultured in yeast extract mannitol medium plus L-methionine. This is the first report of IAA, GA(3), zeatin, ethylene, and ABA production by B. japonicum in pure cultures, using quantitative physicochemical methodology. The three strains have differential capability to produce the five major phytohormones and this fact may have an important technological implication for inoculant formulation.

[Effects of Cd2+ on seedling growth and phytohormone contents of Glycine max].

A laboratory incubation experiment was conducted to study the effects of different Cd2+ concentrations on seedling growth and phytohormone contents of Glycine max through determining some physiological and biochemical indexes. The results showed as follows: (1) Different Cd2+ concentrations inhibited the synthesis of indoleacetic acid (IAA) and gibberellins (GA3) in roots and stimulated the synthesis of zeatin (Z) and abscisic acid (ABA) not only in roots but also in aerial parts of Glycine Max. Cd2+ stimulated the synthesis of IAA and GA3 in aerial parts at lower concentrations, but inhibited the synthesis of IAA and GA3 at higher concentrations. (2) Cd2+ stress for 84h increased the root vitality and the contents of chlorophyll a and b, but no significant difference was found in carotinoid contents compared with the control. (3) Cd2+ stress decreased POD activities and MAD contents atlower concentrations and increased POD activities and MAD contents at higher concentrations. With increasing Cd2+ concentrations, this increase became significant gradually. (4) Cd2+ stress with lower concentrations stimulated the physiological activity of Glycine max seedling in a short time. When Cd2+ concentration was 0.50 mg/L, the root vitality, IAA and GA3 contents of Glycine max were the highest, and POD activity was the lowest.

Cytokinin production by Agrobacterium and Pseudomonas spp.

The production of cytokinins by plant-associated bacteria was examined by radioimmunoassay. Strains producing trans-zeatin were identified in the genera Agrobacterium and Pseudomonas. Agrobacterium tumefaciens strains containing nopaline tumor-inducing plasmids, A. tumefaciens Lippia isolates, and Agrobacterium rhizogenes strains produced trans-zeatin in culture at 0.5 to 44 micrograms/liter. Pseudomonas solanacearum and Pseudomonas syringae pv. savastanoi produced trans-zeatin at levels of up to 1 mg/liter. In vitro cytokinin biosynthetic activity was measured for representative strains and was found to correlate with trans-zeatin production. The genetic locus for trans-zeatin secretion (tzs) was cloned from four strains: A. tumefaciens T37, A. rhizogenes A4, P. solanacearum K60, and P. syringae pv. savastanoi 1006. Southern blot analysis showed substantial homology of the Agrobacterium tzs genes to each other but not to the two Pseudomonas genes.

Conversion of N6-isopentenyladenine to zeatin by Actinidia tissues.

Tissue cultures grown from stem explants of three Actinidia species and a hybrid species rapidly converted N6-isopentenyladenine (i6Ade) to zeatin (io6Ade), a potent hydroxylated cytokinin. Within 24 h on 50 uM i6Ade, callus tissues of A. chinensis x arguta accumulated 83 +/- 6 nmol/g io6Ade which was purified using HPLC and identified by its characteristic UV and mass spectra. Activity converting i6Ade to io6Ade was also demonstrated in stem segments from intact plants where it was low in the tip (3 cm), highest in the region corresponding to rapid leaf growth and very low in the mature stem. Root segments converted i6Ade to io6Ade almost as rapidly as the most active region of the stem while leaf petioles produced little io6Ade. Fruits of A. arguta and A. chinensis produced little or no io6Ade, respectively.

Relationship between adenylate cytokinin production and Ti plasmid of Agrobacterium tumefaciens.

To know whether the tumor-inducing plasmid of Agrobacterium tumefaciens carries genetic information of the biosynthesis of cytokinins, the levels of 6-(3-methyl-2-butenyl-amino)purine (iPAde) and its 4-hydroxy derivative trans-zeatin (trans-Z) and its p-beta-D-ribofuranoside (trans-ZR) produced in media by wild-type virulent strain, plasmid-cured avirulent strain and the deletion mutant were compared. The highest levels of iPAde and trans-Z were found in the culture filtrate of late-log phase growth of plasmid-containing virulent strain, then the levels of iPAde and trans-Z were reduced rapidly at stationary phase. The plasmid-cured avirulent strain and deletion mutant had low levels of iPAde and trans-Z throughout the growth. Results obtained here showed Ti plasmid plays an important role in cytokinin biosynthesis.