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Rice has a large number of nitrate or peptide transporter family (NPF) genes, but the effects of most members on rice growth and development are unknown. We report that OsNPF5.16, a nitrate transporter gene with natural variation in its promoter sequence, is essential for rice growth and yield. The promoter sequence showed various differences between indica and japonica cultivars, and higher expression of OsNPF5.16 was found in indica cultivars with higher plant weight and more tillers than japonica cultivars. OsNPF5.16 was highly expressed in roots, tiller basal parts, and leaf sheaths, and its protein was localized on the plasma membrane. In cRNA-injected Xenopus laevis oocytes, OsNPF5.16 transport of nitrate at high nitrate concentration depended on pH. Overexpression of OsNPF5.16 increased nitrate content and total nitrogen content in leaf sheath as well as biomass and tiller bud length in rice. Elevated expression of OsNPF5.16 increased rice tiller number and grain yield by regulating cytokinin levels. Inhibition of OsNPF5.16 expression showed the opposite effects. Regulating OsNPF5.16 expression has potential for improving rice grain yield.

Ovule initiation is a key step that strongly influences ovule number and seed yield. Notably, mutants with enhanced brassinosteroid (BR) and cytokinin (CK) signaling produce more ovules and have a higher seed number per silique (SNS) than wild-type plants. Here, we crossed BR- and CK-related mutants to test whether these phytohormones function together in ovule initiation. We determined that simultaneously enhancing BR and CK contents led to higher ovule and seed numbers than enhancing BR or CK separately, and BR and CK enhanced each other. Further, the BR-response transcription factor BZR1 directly interacted with the CK-response transcription factor ARABIDOPSIS RESPONSE REGULATOR1 (ARR1). Treatments with BR or BR plus CK strengthened this interaction and subsequent ARR1 targeting and induction of downstream genes to promote ovule initiation. Enhanced CK signaling partially rescued the reduced SNS phenotype of BR-deficient/insensitive mutants whereas enhanced BR signaling failed to rescue the low SNS of CK-deficient mutants, suggesting that BR regulates ovule initiation and SNS through CK-mediated and CK-independent pathways. Our study thus reveals that interaction between BR and CK promotes ovule initiation and increases seed number, providing important clues for increasing the seed yield of dicot crops. This article is protected by copyright. All rights reserved.

In plant tissue culture, callus forms from detached explants in response to a high-auxin-to-low-cytokinin ratio on callus-inducing medium. Callus is a group of pluripotent cells because it can regenerate either roots or shoots in response to a low level of auxin on root-inducing medium or a high-cytokinin-to-low-auxin ratio on shoot-inducing medium, respectively1. However, our knowledge of the mechanism of pluripotency acquisition during callus formation is limited. On the basis of analyses at the single-cell level, we show that the tissue structure of Arabidopsis thaliana callus on callus-inducing medium is similar to that of the root primordium or root apical meristem, and the middle cell layer with quiescent centre-like transcriptional identity exhibits the ability to regenerate organs. In the middle cell layer, WUSCHEL-RELATED HOMEOBOX5 (WOX5) directly interacts with PLETHORA1 and 2 to promote TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 expression for endogenous auxin production. WOX5 also interacts with the B-type ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and represses A-type ARRs to break the negative feedback loop in cytokinin signalling. Overall, the promotion of auxin production and the enhancement of cytokinin sensitivity are both required for pluripotency acquisition in the middle cell layer of callus for organ regeneration.

Plant hormone cytokinins are important regulators of plant development, response to environmental stresses and interplay with other plant hormones. Cytokinin dehydrogenases (CKXs) are proteins responsible for the irreversible break-down of cytokinins to the adenine and aldehyde. Even though plant CKXs have been extensively studied, homologous proteins from other taxa remain mainly uncharacterised. Here we present our study on the molecular evolution and divergence of the CKX from bacteria, fungi, amoeba and viridiplantae. Although CKXs are present in eukaryotes and prokaryotes, they are missing in algae and metazoan taxa. The prevalent domain architecture consists of the FAD-binding and cytokinin binding domains, whereas some bacteria appear to have only cytokinin binding domain proteins. The CKXs play important role in the various aspects of plant life including control of plant development, response to biotic and abiotic stress, influence nutrition. Results of our study suggested that CKX originates from the FAD-linked C-terminal oxidase and has a defence-oriented function. The obtained results significantly extend the current understanding of the cytokinin dehydrogenases structure–function from the relationship to homologues from other taxa and provide a starting point baseline for their future functional characterization.

As an essential element, copper (Cu) is involved in various metabolic processes in plants. However, this metal becomes a potential stress factor when the concentration reaches the threshold level. A comparative analysis was conducted to investigate the potential role of two cytokinins [kinetin (KIN) and 6-benzylaminopurine (BAP)] in Cu stress alleviation in Ricinus communis seedlings by assessing the metal bioaccumulation, antioxidation mechanisms, anatomical changes as well as analysis of various essential elements and bioactive compounds in the cotyledonary leaves. Application of KIN and BAP regulated the Cu toxicity in castor seedlings via modulation of metal uptake and antioxidation mechanisms. The increase in antioxidant enzyme activities during Cu stress was further increased upon exposure to cytokinin treatments, and the enhancement reached up to 25 folds for CAT, 16 folds for POD, and 8 folds for SOD over the control, which helps the plant to alleviate the toxic effects of Cu stress. The FTIR analysis of the cotyledonary leaves revealed that the functional groups associated with cell wall materials contributed to the Cu sequestration in the cell wall, supported by the xylem wall thickening as observed in the SEM analysis. The GCMS analysis revealed that most of the secondary metabolites identified were phenolic and flavonoid compounds with antioxidant properties, which help the castor seedlings withstand the Cu stress, and the presence of these compounds was more prominent in cytokinin-treated seedlings. Findings revealed that BAP was more effective than KIN to withstand the Cu stress effects in castor seedlings.

Legumes balance nitrogen acquisition from soil nitrate with symbiotic nitrogen fixation. Nitrogen fixation requires establishment of a new organ, which is a cytokinin dependent developmental process in the root. We found cytokinin biosynthesis is a central integrator, balancing nitrate signalling with symbiotic acquired nitrogen. Low nitrate conditions provide a permissive state for induction of cytokinin by symbiotic signalling and thus nodule development. In contrast, high nitrate is inhibitory to cytokinin accumulation and nodule establishment in the root zone susceptible to nodule formation. This reduction of symbiotic cytokinin accumulation was further exacerbated in cytokinin biosynthesis mutants, which display hypersensitivity to nitrate inhibition of nodule development, maturation and nitrogen fixation. Consistent with this, cytokinin application rescues nodulation and nitrogen fixation of biosynthesis mutants in a concentration dependent manner. These inhibitory impacts of nitrate on symbiosis occur in a Nlp1 and Nlp4 dependent manner and contrast with the positive influence of nitrate on cytokinin biosynthesis that occurs in species that do not form symbiotic root nodules. Altogether this shows that legumes, as exemplified by Lotus japonicus, have evolved a different cytokinin response to nitrate compared to non-legumes., Nodule development in legumes is a cytokinin dependent process. Here the authors show that high nitrate supply, which limits nodulation, suppresses cytokinin biosynthesis in Lotus japonicus which contrasts with the positive effect of nitrate on cytokinin biosynthesis in non-legumes

Chickpea shoot exogenously treated with cytokinin showed stunted phenotype of root, shoot and significantly reduced nodule numbers. Genome-wide identification of LRR-RLKs in chickpea and Medicago resulted in 200 and 371 genes respectively. Gene duplication analysis revealed that LRR-RLKs family expanded through segmental duplications in chickpea and tandem duplications in Medicago. Expression profiling of LRR-RLKs revealed their involvement in cytokinin signaling and plant organ development. Overexpression of KLAVIER ortholog of chickpea, Ca_LRR-RLK147, in roots revealed its localization in the membrane but showed no effect on root nodulation despite increased cle peptide levels. Two findings (i) drastic effect on nodule number by exogenous cytokinin treatment to only shoot and restoration to normal nodulation by treatment to both root and shoot tissue and (ii) no effect on nodule number by overexpression of Ca_LRR-RLK147 establishes the fact that despite presence of cle peptides in root, the function of Ca_LRR-RLK147 was shoot mediated during AON.

Plant somatic cells can be reprogrammed into a pluripotent cell mass, called callus, which can be subsequently used for de novo shoot regeneration through a two-step in vitro tissue culture method. MET1-dependent CG methylation has been implicated in plant regeneration in Arabidopsis, because the met1-3 mutant exhibits increased shoot regeneration compared with the wild-type. To understand the role of MET1 in de novo shoot regeneration, we compared the genome-wide DNA methylomes and transcriptomes of wild-type and met1-3 callus and leaf. The CG methylation patterns were largely unchanged during leaf-to-callus transition, suggesting that the altered regeneration phenotype of met1-3 was caused by the constitutively hypomethylated genes, independent of the tissue type. In particular, MET1-dependent CG methylation was observed at the blue light receptor genes, CRYPTOCHROME 1 (CRY1) and CRY2, which reduced their expression. Coexpression network analysis revealed that the CRY1 gene was closely linked to cytokinin signaling genes. Consistently, functional enrichment analysis of differentially expressed genes in met1-3 showed that gene ontology terms related to light and hormone signaling were overrepresented. Overall, our findings indicate that MET1-dependent repression of light and cytokinin signaling influences plant regeneration capacity and shoot identity establishment.

The development of secondary vascular tissue enhances the transport capacity and mechanical strength of plant bodies, while contributing a huge proportion of the world’s biomass in the form of wood. Cell divisions in the cambium, which constitutes the vascular meristem, provide progenitors from which conductive xylem and phloem are derived. The cambium is a somewhat unusual stem cell population in two respects, making it an interesting subject for developmental research. Firstly, it arises post-germination, and thus represents a model for understanding stem cell initiation beyond embryogenesis. Secondly, xylem and phloem differentiate on opposing sides of cambial stem cells, making them bifacial in nature. Recent discoveries in Arabidopsis thaliana have provided insight into the molecular mechanisms that regulate the initiation, patterning, and maintenance of the cambium. In this review, the roles of intercellular signalling via mobile transcription factors, peptide–receptor modules, and phytohormones are described. Crosstalk between these regulatory pathways is becoming increasingly apparent, yet the underlying mechanisms are not fully understood. Future study of the interaction between multiple independently identified regulators, as well as the functions of their orthologues in trees, will deepen our understanding of radial growth in plants., We review the literature describing the molecular mechanisms by which the vascular cambium is initiated and maintained in Arabidopsis.

Phytohormones play important roles in controlling leaf size and in the modulation of various stress responses, including drought. In this study, hormone profiling analyses by ultra high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-MS/MS) was performed in leaves collected at three stages of active leaf growth to unravel which phytohormones modulate leaf size in sesame (Sesamum indicum L.) plants, an important oil-rich crop. Furthermore, endogenous contents of phytohormones were measured in parallel to various stress markers in sesame plants exposed to mild water deficit conditions by withholding water in potted plants for one week. Results revealed a major role of cytokinins and auxin in the modulation of leaf growth in sesame plants (which increased by 21.5 and 2.1-fold, respectively, with leaf growth), as well as a putative antagonistic response between jasmonic acid and salicylic acid during leaf development. Furthermore, growth arrest during water deficit stress appeared to be modulated by cytokinins, the endogenous contents of which decreased (by 48%) in parallel with ABA increases (by 59%). Reductions in the contents of the active cytokinin trans-zeatin occurred in parallel with increases in isopentenyladenine contents under drought, which suggests a partial metabolic limitation in cytokinin biosynthesis in leaves upon water deficit stress. These results provide useful information for the hormonal modulation of leaf size and the improvement of leaf growth and production in sesame plants through manipulation of the levels of key regulatory phytohormones.

In this study, an efficient method for in vitro regeneration of Plumbago europaea was developed using direct and indirect organogenesis. Accordingly, micropropagation and regeneration were obtained on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators. The effects of explant type and plant growth regulators on shoot organogenesis of P. europaea were evaluated. For the nodal explants, MS medium containing 0.5 mg/l TDZ (11.62 shoots per node) was the best medium for high frequency of micropropagation. In comparison, the highest percentage of direct organogenesis (70%) and number of shoots per explants (14.6) were acquired for the internode explants using 0.5 mg/l TDZ and 0.1 mg/l IAA. The obtained data revealed that TDZ is the most effective cytokinin for the direct shoot organogenesis. The highest indirect organogenesis rate was observed using 2 mg/l BA and 0.1 mg/l NAA for the internode explant. The maximum number of roots was distinguished on ½ MS medium containing 0.5 mg/l IBA (6.42). The rooted plantlets were gradually hardened and acclimatized under ex vitro conditions. As an important outcome, the active compound plumbagin was found mainly in the root tissues of the micro-propagated and regenerated plantlets. Taken all together, this study achieved a successful protocol for in vitro regeneration of P. europrea and could be considered for large-scale multiplication of this important medicinal plant.

Plant micropropagation comprises biotechnological tools used for the conservation and mass propagation of orchid species. However, there are few reports of its use for orchids of the subtribe Pleurothallidinae. The present study evaluated the effects of 6-benzylaminopurine (BAP) on shoots formation and development of Dryadella zebrina (Porsch) Luer. For this, seeds were in vitro germinated, and the plantlets were submitted to different concentrations of BAP (0, 3, 6, 9, 12, and 15 µM). The plantlets derived from the treatments 0, 6, and 15 µM of BAP were collected after 60 days in culture and subjected to light microscopy analysis. Our results indicated that BAP increased the formation of the new shoots of D. zebrina, especially in treatment with 6 µM, and its use is indicated for the in vitro multiplication phase. The anatomical analyses of the roots showed a deleterious effect of 15 µM BAP on the meristematic region, with the presence of more vacuolated cells in this zone. Our results represent the first successful report of in vitro propagation for the genus Dryadella and may serve as a basis for further studies of in vitro propagation of phylogenetically related species.

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula., Precise spatial and temporal characterization of cytokinin (CK) responses reveals the function of the CK biosynthesis gene ISOPENTENYLTRANSFERASE 3 during nodule development in Medicago truncatula.

Summary The critical role of cytokinin in early nodulation in legumes is well known. In our study, exogenous cytokinin application to roots of the important crop legume, chickpea (Cicer arietinum L.), led to the formation of pseudo‐nodules even in the absence of rhizobia. Hence, a genome‐wide analysis of the cytokinin signalling, two‐component system (TCS) genes, was conducted in chickpea, Medicago and Cajanus cajan. The integrated phylogenetic, evolutionary and expression analysis of the TCS genes was carried out, which revealed that histidine kinases (HKs) were highly conserved, whereas there was diversification leading to neofunctionalization at the level of response regulators (RRs) especially the TypeB RRs. Further, the functional role of the CaHKs in nodulation was established by complementation of the sln1Δ mutant of yeast and cre1 mutants of (Medicago) which led to restoration of the nodule‐deficient phenotype. Additionally, the highest expressing TypeB RR of chickpea, CaRR13, was functionally characterized. Its localization in the nucleus and its Y1H assay‐based interaction with the promoter of the early nodulation gene CaNSP2 indicated its role as a transcription factor regulating early nodulation. Overexpression, RNAi lines and complementation of cre1 mutants with CaRR13 revealed its critical involvement as an important signalling molecule regulating early events of nodule organogenesis in chickpea.

The purpose of the study is to determine the effect of concentration and frequency of application of banana weevil solution kepok against red chili plants is roofed with translucent ultraviolet plastic 70% which acts as a natural growth regulator. Research on the provision of banana weevil solution covered in 70% translucent ultraviolet plastic in open land has not been found before. The growing regulators from the gibberellin and cytokinin groups were able to boost the growth and yield of chili peppers. The method of using a random design of factorial groups, as the first factor is age consists of 9 levels, namely T1: age (14) HSPT, T2: age (14.21) HSPT, T3: age (14,21,28) HSPT, T4: age (14,21,28, 35) HSPT, T5 : age (14,21,28,35, 42) HSPT, T6: age (14,21,28,35,42,49) HSPT, T7: age (14,21,28,35,42,49,56) HSPT, T8: age (14,21,28,35,42,49,56,63) HSPT, and control (T0). The second factor is concentration consists of 3 levels, namely K1 (30%), K2 (45%), and K0 (0%). The results of the study obtained concentration of K1 (30%) with the amount of T6 administration is the best result in vegetative growth of the plant height change, the number of branches, and the width of the header, while the concentration of K1 (30%) give the best results on the length of the fruit, the dry weight of the fruit, and the diameter of the fruit from other treatments. Conclusion of the application of concentration and frequency of the sea of banana weevil kepok containing regulatory substances growing on red chili plants in the field covered with ultraviolet plastic roof significantly increases the growth and yield of red chili peppers.

CKS (cyclin-dependent kinase subunit) is one of cell cycle genes, and cell cycle in apple (Malus × domestic Borkh.) fruit controlled cell number and fruit size. In order to explore CKS expression during the development of apple fruit size, the cultivar ‘Fuji’ and ‘Ralls’ whose fruit size had obvious difference were used for MdCKS1 gene cloning and expressing. In ‘Fuji’ and ‘Ralls’, the greatest growth rates of fruit size and weight occurred during 7–21 days after full bloom (DAFB), but in ‘Fuji’ during 21–35 DAFB the growth rates of fruit size and weight was still faster, and ‘Fuji’ remained much rapider growth rate of fruit size and weight than ‘Ralls’ within 7–35 DAFB, so the majority of cell production in ‘Ralls’ occurred between 7–21 DAFB and in ‘Fuji’ between 7–35 DAFB, and the biggest difference of fruit size and weight between ‘Fuji’ and ‘Ralls’ occurred at 35 DAFB. MdCKS1 sequence was identical with MdCKS1;1 (CO868566) and apple CKS1 (EB141951). MdCKS1 expression peak in ‘Fuji’ was two-fold higher than in ‘Ralls’; MdCKS1 expression peak in ‘Ralls’ occurred at 21 DAFB and in ‘Fuji’ at 35 DAFB which coincided respectively with rapid growth rate of early fruit. During 140–161 DAFB, ‘Fuji’ fruit had MdCKS1 expression peak again, but ‘Ralls’ not; ‘Fuji’ fruit size and weight had a larger extent of increase than ‘Ralls’, and the second significant difference of fruit size and weight between ‘Fuji’ and ‘Ralls’ occurred at 161 DAFB. In callus, MdCKS1 expression was much higher in ‘Fuji’ than in ‘Ralls’, up-regulated by auxin (NAA) or cytokinin (6-BA) or co-existence of them, and depressed by lower culture temperature. Altogether, ‘Fuji’ fruit had much rapider growth rate than ‘Ralls’ which resulted in significant difference of fruit size and weight between ‘Fuji’ and ‘Ralls’ respectively at 35 DAFB and 161 DAFB, and contributed to their fruit size difference; MdCKS1 expression in ‘Fuji’ fruit was much higher than in ‘Ralls’ both at early stage for fruit cell division and later stage for fruit cell expansion; MdCKS1 expression in callus was up-regulated by auxin or/and cytokinin. Hence, difference of fruit size between ‘Fuji’ and ‘Ralls’ was correlated with growth rate during fruit development, MdCKS1 expression correlated positively with growth rate and cell production of early fruit, and involved in fruit size which maybe was regulated by endogenous auxin or/and cytokine level of genotype.

Numerous advances have been made to mitigate the repercussion of toxic metals on photosynthetic prokaryotes, i.e. cyanobacteria. One strategy is exogenous supplementation of plant growth regulators (PGRs). This perhaps intensifies the resistance ability of cyanobacteria to encounter the environmental stresses by augmenting their physiological functioning and decontaminant efficiency. Thus, the aim of the present study was to determine if the PGRs auxin (indole-3 acetic acid, IAA; 300 nM) and cytokinin (kinetin, KN; 10 nM) are able to mitigate the toxic environmentally relevant doses of chromium (CrVI; 100 and 150 µM) in cyanobacteria (Nostoc muscorum ATCC 27,683 and Anabaena sp. PCC 7120). The response was assessed by estimating oxidative stress [in vitro and in vivo (superoxide radical; O2·−, hydrogen peroxide H2O2 and malondialdehyde equivalents contents; MDA)], feasible alteration in whole cell oxygen evolution, ascorbate–glutathione cycle and their metabolites. Intracellular Cr accumulation raised the production of ROS by down-regulating the photosynthetic rate due to decreased pigment contents (Chl a; chlorophyll a, Car; carotenoids and PC; phycocyanin) and reduced growth despite the accelerated activity of the ascorbate–glutathione (AsA-GSH) cycle enzymes (viz. APX; ascorbate peroxidase, GR; glutathione reductase and DHAR; dehydroascorbate reductase). Concurrently, exogenous PGRs (auxin and cytokinin) moderated the negative effects of Cr on the growth, by lowering the cellular Cr accumulation and ROS level and by modulating the pigment system, photosynthesis and a further rise in the efficiency of AsA-GSH cycle, thereby acting as positive regulators of the metal detoxification machinery. Thus, exogenous application of PGRs (auxin and cytokinin) could improve the growth in cyanobacteria in Cr-polluted environments, hence maintaining productivity in paddy cultivation as well as having potential to be used as bio-remediator in metal-contaminated aquatic systems.

DWARF53 (D53) in rice (Oryza sativa) and its homologs in Arabidopsis (Arabidopsis thaliana), SUPPRESSOR OF MAX2-LIKE 6 (SMXL6), SMXL7 and SMXL8, are well established negative regulators of strigolactone signalling involved in shoot branching. Little is known of pea (Pisum sativum) homologs and whether D53 and related SMXLs are specific to strigolactone signalling pathways. Here, we identify two allelic pea mutants, dormant3 (dor3), and demonstrate through gene mapping and sequencing that DOR3 corresponds to a homolog of D53 and SMXL6/SMXL7, designated PsSMXL7. Phenotype analysis, gene expression, protein and hormone quantification assays were performed to determine the role of PsSMXL7 in bud outgrowth regulation and the role of PsSMXL7 and D53 in integrating strigolactone and cytokinin responses. Like D53 and related SMXLs, we show that PsSMXL7 is strigolactone degradable, and induces feedback upregulation of PsSMXL7 transcript. Here we reveal a system conserved in pea and rice, whereby CK also upregulates PsSMXL7/D53 transcripts, providing a clear mechanism for strigolactone and cytokinin cross-talk in the regulation of branching. Further deepening our understanding of the branching network in pea, we provide evidence that strigolactone acts via PsSMXL7 to modulate auxin content via PsAFB5, which itself regulates expression of strigolactone biosynthesis genes. We therefore show that PsSMXL7 is key to a triple hormone network involving an auxin-SL feedback mechanism and SL-CK crosstalk.

Trichoderma is an important genus of symbiotic fungi, commonly used around the world as biocontrol agents and as biofertilizer. Although their beneficial effects are well known and are successfully exploited in sustainable agriculture practices, the biochemical mechanisms of plant growth-promoting actions of Trichoderma and their anti-pathogen characteristics are not well understood. This study biochemically surveyed 22 strains of Trichoderma and shows that Trichoderma produces cytokinins (CKs), which has not been reported to date. The phytohormone profiles ranged from 5.34 to 379.99 pmol CKs released to 10 mL of the growth medium and comprised riboside and nucleotide derivatives of cis-zeatin (cZ) and isopentenyladenine (iP), suggesting that fungal CKs originate from a tRNA degradation pathway. We reveal a connection between the levels of free base cZ produced by Trichoderma and the inhibition rate against the pathogen Fusarium graminearum among the tested strains. Furthermore, we analyzed CK profiles of Arabidopsis plants cultured in vitro in the presence of Trichoderma strains. The inoculated plants showed increased levels of cZ-type (cZR, cZROG) and iP-type (iP, iPR) CKs—the forms which dominated CK profiles of all the fungal in vitro cultures tested in this study. The increase in the levels of cZ derivatives was accompanied by a significant reduction in plant trans-zeatin (tZ)-type CKs (tZR, tZNT, tZOG, tZ7G, tZ9G) in Arabidopsis when co-cultured with the fungus. Our work suggests that CKs produced by plant symbiotic Trichoderma strains can be used for plant growth stimulation, may impact the colonization strategy of symbiotic fungi, and include alterations to the host plant phytohormones for enhanced plant resistance against pathogens.

Pogostemon cablin (Blanco) Benth. or “Patchouli” is a prized medicinal herb used in various ethno-medicinal preparations and perfume industry. However due to over-collection and nematode infections in the roots of P. cablin, the wild populations are facing a steady decline and are at risk. In the present research, we report a fast clonally stable regeneration protocol for P. cablin with the utilization of meta-topolin (mT), a prized aromatic cytokinin (CK) for the first time. The highest rate of shoot proliferation i.e. 45 shoots/explant was observed after 4 weeks from leaf segments on MS media fortified with 1.0 mg/l mT. The highest rate of rooting was observed when the regenerated micro shoots were cultured in MS medium supplemented with 1.5 mg/l IBA. Direct root regeneration was tested upon with the applications of three conventional auxins i.e., Naphthalene acetic acid (NAA), Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA) using the same explants. The genetic homogeneity of the regenerated clones was ascertained, using two gene targeted molecular marker systems namely Start Codon Targeted Polymorphism (SCoT) and Inter-Retrotransposon Amplified Polymorphism (IRAP) ascertaining genetic stability amongst the regenerates and the donor plant. In precise, the present research provides vital insights into the role of aromatic CKs like mT in the high frequency regeneration of medicinally important plants like P. cablin which might serve as important baseline information for the pharma industry where supply of Quality Planting Material (QPM) is a real challenge.

Expansins are cell wall proteins that, due to changes in pH, causing the expansion of the cell walls. In this study, a previously gene construct designed based on a root-specific gene, AtEXPA18, was utilized to assess its potential roles on different morphological, physiological, and cellular levels of generated transgenic tobacco plants in response to moderate and severe drought stress. AtEXPA18 gene was successfully transferred to the tobacco plants through an agrobacterium-mediate transformation system. Upon obtaining the second generation, tobacco transgenic plants were confirmed by conventional polymerase chain reaction (PCR) technique alongside reverse transcription PCR (RT-PCR) using specific primers. Under drought stress, the transgenic lines showed remarkable growth and significantly improved based on morphological traits such as height and stem diameter, leaf area, leaf number, root dry weight, and Abscisic acid (ABA) levels of leaves compared control plants. As a result, the Cytokinin content of transgenic plants has increased under severe stress levels. Notably, the area's expansion for abaxial epidermal cells under the microscope confirmed in transgene cells compared with the -transgene cells. These results, altogether, could support the AtEXPA18 gene implication in cell expansion and improving tolerance capacity of transgenic crops under drought stress.

Early-spring plants are a special type of plant that complete their life cycle promptly in cold, early spring. Very little effort has been made into researching the flowering mechanism of this type. Vernalization, as an indispensable process for flowering induction at low temperature, is likely to be closely related to life cycle transition in early-spring plants. To explore the flowering strategy at low temperature, this study selected two local early-spring flowering plant species (Hylomecon japonica (Thunb.) Prantl, Smilacina japonica A. Gray) and two early-spring unflowered plants species (Cimicifuga foetida L., Veratrum nigrum L.) as subjects. Firstly, the ratio of abscisic acid (ABA), gibberellic acid (GA3), indole-3-acetic acid (IAA), and cytokinin (CTK) hormones was analyzed through high-performance liquid chromatography–mass spectrometry, results of which indicated that early-spring flowering plants have completed vernalization and thus entered the flowering stage. IAA content in early-spring flowering plants was significantly lower than that of unflowered plants, while GA3 and ABA contents were higher. Secondly, gas chromatography coupled with mass spectrometry (GC–MS) was adopted to profile metabolite richness across the two types of plants. Integrated key metabolic pathway analysis suggested that the contents of D-ribose, L-valine, L-isoleucine, alanine as well as various other compounds participating in the key pathways of early-spring flowering plants significantly reduced, demonstrating that early-spring flowering plants consume a significant amount of energy to regulate flowering, that is sourced from mass carbohydrate consumption. These results provide a greater understanding of early-spring flowering plants and their metabolic mechanism of vernalization, laying the foundation for further exploration on early-spring plants as well as the processing mechanism of the plant life cycle.

Cytokinins regulate diverse aspects of plant growth and development, primarily through modulation of gene expression. The cytokinin-responsive transcriptome has been thoroughly described in dicots, especially Arabidopsis, but much less so in monocots. Here, we present a meta-analysis of five different transcriptomic analyses of rice (Oryza sativa) roots treated with cytokinin, including three previously unpublished experiments. We developed a treatment method in which hormone is added to the media of rice seedlings grown in sterile hydroponic culture under a continuous airflow, which resulted in minimal perturbation of the seedlings, thus greatly reducing changes in gene expression in the absence of exogenous hormone. We defined a core set of 205 upregulated and 86 downregulated genes that were differentially expressed in at least three of the transcriptomic datasets. This core set includes genes encoding the type-A response regulators (RRs) and cytokinin oxidases/dehydrogenases, which have been shown to be primary cytokinin response genes. GO analysis revealed that the upregulated genes were enriched for terms related to cytokinin/hormone signaling and metabolism, while the downregulated genes were significantly enriched for genes encoding transporters. Variations of type-B RR binding motifs were significantly enriched in the promoters of the upregulated genes, as were binding sites for other potential partner transcription factors. The promoters of the downregulated genes were generally enriched for distinct cis-acting motifs and did not include the type-B RR binding motif. This analysis provides insight into the molecular mechanisms underlying cytokinin action in a monocot and provides a useful foundation for future studies of this hormone in rice and other cereals.

Key message Root-specific expression of a cytokinin-degrading CKX gene in maize roots causes formation of a larger root system leading to higher element content in shoot organs. Abstract The size and architecture of the root system is functionally relevant for the access to water and soil nutrients. A great number of mostly unknown genes are involved in regulating root architecture complicating targeted breeding of plants with a larger root system. Here, we have explored whether root-specific degradation of the hormone cytokinin, which is a negative regulator of root growth, can be used to genetically engineer maize (Zea mays L.) plants with a larger root system. Root-specific expression of a CYTOKININ OXIDASE/DEHYDROGENASE (CKX) gene of Arabidopsis caused the formation of up to 46% more root dry weight while shoot growth of these transgenic lines was similar as in non-transgenic control plants. The concentration of several elements, in particular of those with low soil mobility (K, P, Mo, Zn), was increased in leaves of transgenic lines. In kernels, the changes in concentration of most elements were less pronounced, but the concentrations of Cu, Mn and Zn were significantly increased in at least one of the three independent lines. Our data illustrate the potential of an increased root system as part of efforts towards achieving biofortification. Taken together, this work has shown that root-specific expression of a CKX gene can be used to engineer the root system of maize and alter shoot element composition.

SummaryThe hemiparasitic plant Phtheirospermum japonicum is a nutritional specialist that supplements its nutrient requirements by parasitizing other plants through haustoria. During parasitism, the Phtheirospermum haustorium transfers hypertrophy-inducing cytokinins (CKs) to the infected host root. The CK biosynthesis genes required for haustorium-derived CKs and the induction of hypertrophy are still unknown. We searched for haustorium-expressed isopentenyltransferases (IPTs) that catalyse the first step of CK biosynthesis, confirmed the specific expression by in vivo imaging of a promoter-reporter, and further analysed the subcellular localization, the enzymatic function, and contribution to inducing hypertrophy by studying CRISPR-Cas9 induced Phtheirospermum mutants. PjIPT1a was expressed in intrusive cells of the haustorium close to the host vasculature. PjIPT1a and its closest homolog PjIPT1b located to the cytosol and showed isopentenyltransferases activity in vitro with differences in substrate specificity. Mutating PjIPT1a abolished parasite-induced CK responses in the host. A homolog of PjIPT1a with shared characteristics was also identified in the related weed Striga hermonthica. We propose that PjIPT1a exemplifies how parasitism-related functions evolve through gene duplications and neofunctionalization.

To overcome nitrogen deficiency, legume roots establish symbiotic interactions with nitrogen-fixing rhizobia that are fostered in specialized organs (nodules). Similar to other organs, nodule formation is determined by a local maximum of the phytohormone auxin at the primordium site. However, how auxin regulates nodule development remains poorly understood. Here, we found that in soybean, (Glycine max), dynamic auxin transport driven by PIN-FORMED (PIN) transporter GmPIN1 is involved in nodule primordium formation. GmPIN1 was specifically expressed in nodule primordium cells and GmPIN1 was polarly localized in these cells. Two nodulation regulators, (iso)flavonoids trigger expanded distribution of GmPIN1b to root cortical cells, and cytokinin rearranges GmPIN1b polarity. Gmpin1abc triple mutants generated with CRISPR-Cas9 showed the impaired establishment of auxin maxima in nodule meristems and aberrant divisions in the nodule primordium cells. Moreover, overexpression of GmPIN1 suppressed nodule primordium initiation. GmPIN9d, an ortholog of Arabidopsis thaliana PIN2, acts together with GmPIN1 later in nodule development to acropetally transport auxin in vascular bundles, fine-tuning the auxin supply for nodule enlargement. Our findings reveal how PIN-dependent auxin transport modulates different aspects of soybean nodule development and suggest that the establishment of auxin gradient is a prerequisite for the proper interaction between legumes and rhizobia., In soybean, nodule primordium formation involves GmPIN1-mediated polar auxin transport within primordium cells, and nodule enlargement involves the collaboration of GmPIN9d and GmPIN1-dependent auxin transport within nodule vasculature.

Moso bamboo (Phyllostachys edulis) is a fast-growing species with uneven growth and lignification from lower to upper segments within one internode. MicroRNAs (miRNAs) play a vital role in post-transcriptional regulation in plants. However, how miRNAs regulate fast growth in bamboo internodes is poorly understood. In this study, one moso bamboo internode was divided during early rapid growth into four segments called F4 (bottom) to F1 (upper) and these were then analysed for transcriptomes, miRNAs and degradomes. The F4 segment had a higher number of actively dividing cells as well as a higher content of auxin (IAA), cytokinin (CK) and gibberellin (GA) compared with the F1 segment. RNA-seq analysis showed DNA replication and cell division-associated genes highly expressed in F4 rather than in F1. In total, 63 miRNAs (DEMs) were identified as differentially expressed between F4 and F1. The degradome and the transcriptome indicated that many downstream transcription factors and hormonal responses genes were modulated by DEMs. Several miR-target interactions were further validated by tobacco co-infiltration. Our findings give new insights into miRNA-mediated regulatory pathways in bamboo, and will contribute to a comprehensive understanding of the molecular mechanisms governing rapid growth.

Cytokinin is a plant hormone and an important regulator of root growth. Reduced cytokinin levels increase root systems, which can be beneficial for crops grown on nutrient poor and/or dry soils. Our study analyzed the effects of increased cytokinin oxidase 5 (OsCKX5) expression in rice roots on root morphology, architecture, and nutrient uptake. We fused the OsCKX5 gene to the strong, root-specific RCc3 rice promoter and overexpressed it in rice plants. The root-specific expression of OsCKX5 in the transgenic plants resulted in positive effects on root development without compromising shoot growth. The root system of the transgenic plants exhibited greater volume, length, projection area, a higher number of tips, and enhanced surface area. OsCKX5 overexpression also resulted in an increased uptake ability of certain macro- and micronutrients, a greater root biomass, deeper root system, and a higher root:shoot ratio, when cultivated on low-fertility soils. We conclude that the enhanced root growth and development by root-specific expression of OsCKX5 could be advantageous for crops grown on oxidic soils in the tropical regions, with a low availability of certain nutrients, especially P and Zn.

Chichi is a unique biological phenomenon observed in Ginkgo biloba L.. In this study, multi-omics analysis was used to compare basal chichi (C) with roots (R) and stems (S) to explore the regulatory mechanisms of basal chichi ontogenesis. The results showed that compared with roots and stems, the tZ, SA and ABA contents in basal chichi were the highest, and the ratio of IAA/tZ was the lowest. Nucleotides and their derivatives in basal chichi were upregulated, and phenylpropane metabolites were downregulated. Some differentially expressed genes (DEGs) strongly correlated to plant hormones were screened. We speculate that auxin and cytokinin are involved in the morphogenesis of basal chichi and that cytokinin plays a major role. The ontogenesis of basal chichi is closely related to environmental stress, and it may be a coping strategy of G. biloba in the face of environmental stress.

The density and morphology of glandular trichomes in rose geranium (Pelargonium graveolens L’Her.) are often correlated to the essential oil biosynthesis. However, whether the different organs in the same rose geranium plant are affected by hail damage, or if they recover similarly following hail damage is still unknown concerning the essential oil biosynthesis. The objective of this study was to evaluate the effects of split-applying cytokinin (CK), gibberellic acid (GA) and defoliation on rose geranium trichome morphology and density, and essential oil biosynthesis. The experiment was carried out in a hail-net covered tunnel structure during 2017-2018 growing season. The experimental design was a complete randomized blocks with a 3x3 factorial arrangement of treatments: three defoliation levels (0, 50, and 100%), two levels of split-application of CK and GA (0.32 mg L-1 CK + 150 mg L-1 GA; and 0.64 mg L-1 CK + 300 mg L-1 GA), and a control. The attenuate, peltate, and capitate trichome groups were identified in the leaf. Morphologically, the attenuate trichome group was less dense on both recovered and new plant leaves, despite split-applied CK and GA treatment. With the peltate group, brevicollate trichome density declined in all plant leaves treated with 0.64 mg L-1 CK + 300 mg L-1 GA on both leaf surfaces. Application of 0.64 mg L-1 CK + 300 mg L-1 GA led to high density of the elongated-capitate type on leaves recovered from 50 and 100% defoliation. The essential oil yield was not affected by density and morphological changes of the trichomes. However, the application of 0.32 mg L-1 CK + 150 mg L-1 GA increased the citronellol content where plants endured 100% defoliation. Results demonstrated that the effects of hail damage stress and subsequent split-applied CK and GA could transform the morphology of trichomes, subsequently increasing the density.

As one of the most popular bulbous plant with high ornamental value, Lilium spp. has been used as cut flower or green plants in urban landscapes and home gardening. We noticed a cultivar ‘Elodie’ show weak double-flowered phenotype, by which whole 3 can be classified into three different types including stamen, partially petaloid stamen (PPetSta) and totally petaloid stamen (TPetSta). To understand the molecular basis of this homeotically conversion variation, quantitative RNA-Seq analysis was conducted to screen the important unigenes underlying this phenomenon. Meanwhile, endogenous hormone contents including auxin, cytokinin, GA and ABA were detected at the same time. In total, we assembled and annotated 53,182 unigenes. 8453, 21,281 and 7646 DEGs (differentially expressed genes) were identified from PPetSta vs Stamen, TPetSta vs Stamen, and TPetSta vs PPetSta comparisons, in which 2241 unigenes were shared DEGs. 20 transcription factors in MADS-box and MYB families, 43 unigenes in signaling and synthesis pathways of auxin, cytokinin, GA, ABA and ethylene were differentially expressed. Meanwhile, endogenous hormone contents including auxin, cytokinin, GA and ABA were declined in partially petaloidy flower and totally petaloidy flower when compared with the normal stamen. The expression of GA20ox3 and CYP735A2 encode the biosynthesis genes of GA and cytokinin, respectively were downregulated, which was consistent with the hormone determination results. The expression of SAUR50, SLR5, PYL9, ERF4/27 involved in the signaling pathways of IAA, GA, ABA, ethylene and transcription factors MYB44/108, AG2, AGL104, MGS1 had the same expression profiles corresponding with the endogenous hormone content, indicating that they may play a positive role during the process of stamen petalization. This work will help us to unravel several specific genes and provide a theoretical basis for double flower breeding of Lilium species.

Background Raising nitrogen use efficiency of crops by improving root system architecture is highly essential not only to reduce costs of agricultural production but also to mitigate climate change. The physiological mechanisms of how biochar affects nitrogen assimilation by crop seedlings have not been well elucidated. Results Here, we report changes in root system architecture, activities of the key enzymes involved in nitrogen assimilation, and cytokinin (CTK) at the seedling stage of cotton with reduced urea usage and biochar application at different soil layers (0–10 cm and 10–20 cm). Active root absorption area, fresh weight, and nitrogen agronomic efficiency increased significantly when urea usage was reduced by 25% and biochar was applied in the surface soil layer. Glutamine oxoglutarate amino transferase (GOGAT) activity was closely related to the application depth of urea/biochar, and it increased when urea/biochar was applied in the 0–10 cm layer. Glutamic-pyruvic transaminase activity (GPT) increased significantly as well. Nitrate reductase (NR) activity was stimulated by CTK in the very fine roots but inhibited in the fine roots. In addition, AMT1;1, gdh3, and gdh2 were significantly up-regulated in the very fine roots when urea usage was reduced by 25% and biochar was applied. Conclusion Nitrogen assimilation efficiency was significantly affected when urea usage was reduced by 25% and biochar was applied in the surface soil layer at the seedling stage of cotton. The co-expression of gdh3 and gdh2 in the fine roots increased nitrogen agronomic efficiency. The synergistic expression of the ammonium transporter gene and gdh3 suggests that biochar may be beneficial to amino acid metabolism.

Background Rhizobium–legume symbiosis is a specific, coordinated interaction that results in the formation of a root nodule, where biological nitrogen fixation occurs. NADPH oxidases, or Respiratory Burst Oxidase Homologs (RBOHs) in plants, are enzymes that generate superoxide (O2•−). Superoxide produces other reactive oxygen species (ROS); these ROS regulate different stages of mutualistic interactions. For example, changes in ROS levels are thought to induce ROS scavenging, cell wall remodeling, and changes in phytohormone homeostasis during symbiotic interactions. In common bean (Phaseolus vulgaris), PvRbohB plays a key role in the early stages of nodulation. Results In this study, to explore the role of PvRbohB in root nodule symbiosis, we analyzed transcriptomic data from the roots of common bean under control conditions (transgenic roots without construction) and roots with downregulated expression of PvRbohB (by RNA interference) non-inoculated and inoculated with R. tropici. Our results suggest that ROS produced by PvRBOHB play a central role in infection thread formation and nodule organogenesis through crosstalk with flavonoids, carbon metabolism, cell cycle regulation, and the plant hormones auxin and cytokinin during the early stages of this process. Conclusions Our findings provide important insight into the multiple roles of ROS in regulating rhizobia–legume symbiosis.

Steroid alkaloid solasodine is a nitrogen analogue of diosgenin and has great importance in the production of steroidal medicines. Solanum erianthum D. Don (Solanaceae) is a good source of solasodine. The aim of this study was to evaluate the effect of different cytokinins on the production of secondary metabolites, especially solasodine in the in vitro culture of S. erianthum. For solasodine estimation, field-grown plant parts and in vitro tissues were extracted thrice and subjected to high-performance liquid Chromatography. Quantitative analysis of different secondary metabolites showed that the amount was higher in the in vitro regenerated plantlets compared to callus and field-grown plants. The present study critically evaluates the effect of the type of cytokinin used in the culture medium on solasodine accumulation in regenerated plants. The highest solasodine content (46.78±3.23 mg g-1) was recorded in leaf extracts of the in vitro grown plantlets in the presence of 6-γ,γ-dimethylallylamino purine in the culture medium and the content was 3.8-fold higher compared to the mother plant.

In view of extending the relatively brief postharvest life of flowers by a range of technologies, the present study elucidates the implication of 6-benzylamino purine (BAP), kinetin (KN) and thidiazuron (TDZ) on postharvest performance and flower longevity of isolated flowers of Calendula officinalis. BAP and KN belong to adenine group cytokinins while as TDZ is a diphenyl urea compound having cytokinin like activity. The harvested flowers were supplemented with BAP, KN and TDZ at various concentrations viz., 25, 50, 75 and 100 µM at one day before anthesis (cup shaped) stage. The control was designated by a distinct set of flowers held in distilled water (DW). Our findings revealed substantial enhancement in flower longevity by application of various growth regulators as compared to the control. Vase solutions containing BAP and KN at 50 µM and TDZ at 75 µM (individually) were most effective in improving the longevity of cut Calendula flowers. Improvement in flower longevity was primarily associated with high membrane stability index (MSI), upregulated activities of various antioxidant enzymes viz., catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), besides an attenuated lipoxygenase (LOX) activity in the petals. As compared to control, the treated flowers exhibited higher values of soluble proteins, total phenols and total sugars, besides lower α-amino acid content in the petal tissues. However, BAP outplayed TDZ and KN in improving the flower longevity of Calendula officinalis by maintaining higher physiological and biochemical stability in petals. Resumo Visando a estender a vida pós-colheita relativamente breve de flores de corte por uma gama de tecnologias, o presente estudo elucida a implicação de 6-benzilamino purina (BAP), cinetina (KN) e tidiazuron (TDZ) no desempenho pós-colheita e longevidade de flores isoladas flores de Calendula officinalis. O BAP e o KN pertencem às citocininas do grupo adenina, enquanto o TDZ é um composto de difenilureia com atividade semelhante à da citocinina. As flores foram suplementadas com BAP, KN e TDZ em várias concentrações 25, 50, 75 e 100 µM um dia antes do estágio de antese (em forma de copo). O controle foi designado por um conjunto distinto de flores mantidas em água destilada (DW). Nossos resultados revelaram o aumento substancial na longevidade das flores pela aplicação de vários reguladores de crescimento em comparação com o controle. Soluções de vaso contendo 50 µM de BAP e KN e 75 µM de TDZ (individualmente) foram mais eficazes em melhorar a longevidade das flores de calêndula cortadas. A melhora na longevidade das flores foi principalmente associada com alto índice de estabilidade de membrana (MSI), o aumento das atividades de várias enzimas antioxidantes viz., Catalase (CAT), superóxido dismutase (SOD) e ascorbato peroxidase (APX), além de uma atividade atenuada de lipoxigenase (LOX) em as pétalas. Em relação ao controle, as flores exibiram maiores valores de proteínas solúveis, fenóis e açúcares totais, além de menor teor de α-aminoácidos. No entanto, o BAP superou o TDZ e o KN na melhoria da longevidade da flor de Calendula officinalis, mantendo maior estabilidade fisiológica e bioquímica nos tecidos das pétalas.

Phyto-hormones play a key role in regulating plant responses to stress. Cytokines are a type of phyto-hormones involved in the regulation of many important biological processes related to growth, development, and response to environmental variables. The exogenous application of cytokines increases the possibility of delaying senescence; however, this is a physiological process, and, under certain conditions, degradation processes may be triggered. The effect of 6-bencilaminopurine application and the endogenous hormonal changes involved in lilies floral stalks after their cutting were studied. In order to improve vase life and quality of Lilium longiflorum ‘Brindisi’ flower stalks, they were sprayed with 6-BAP, at a concentration of 300 ppm at pre-harvest, post-harvest, and pre- and post-harvest stages. After that, they were compared to non-sprayed control stalks. The application of 6-BAP caused endogenous hormonal changes in abscisic acid and cytokinin levels, and the most effective treatment was pre-harvest spraying. This treatment proved to be an appropriate method to improve the stalk tolerance to post-harvest stress as it delayed the appearance of senescence symptoms and reduced the speed of chlorophyll degradation with differences of up to 10% with respect to untreated stalks. In addition, the opening of flowers was delayed by up to 2 days, although there were no significant differences in total vase life. Resumo Os fitormônios desempenham um papel fundamental na regulação das respostas das plantas ao estresse. As citocinas são um tipo de fitormônios envolvidos na regulação de muitos processos biológicos importantes relacionados ao crescimento, desenvolvimento e resposta a variáveis ambientais. A aplicação exógena de citocinas aumenta a possibilidade de retardar a senescência; entretanto, este é um processo fisiológico e, sob certas condições, processos de degradação podem ser desencadeados. Foi estudado o efeito da aplicação de 6-benzilaminopurina e as alterações hormonais endógenas envolvidas, nas hastes florais de lírio japonês após seu corte. Para melhorar a vida de vaso e a qualidade das hastes florais de Lilium longiflorum ‘Brindisi’, estes foram pulverizados com 6-BAP na concentração de 300 ppm nas fases de pré-colheita, pós-colheita e pré e pós-colheita e foram comparados com hastes não pulverizadas. A aplicação de 6-BAP causou alterações hormonais endógenas nos níveis de ácido abscísico e citocinina, e o tratamento mais eficaz foi a pulverização pré-colheita. Este tratamento mostrou-se um método adequado para melhorar a tolerância da haste ao estresse pós-colheita, pois retardou o aparecimento dos sintomas de senescência e reduziu a velocidade de degradação da clorofila com diferenças de até 10% em relação às hastes não tratadas. Além disso, a abertura das flores foi atrasada em até 2 dias, embora não houvesse diferenças significativas na vida total de vaso.

The Australian Finger Lime (Citrus australasica) is a type of citrus from the Rutaceae family, endemic to the east coast of Australia. The finger lime, loaded with numerous vitamins and renders a unique taste, has also been backed by science to contain essential amounts of antioxidants that are beneficial for cell protection, immune response, cancer prevention, ageing, arthritis and prevention of kidney stones. Current propagation attempts still rely on conventional methods that are less efficient and resulted in the slow establishment of farms for finger lime especially for commercialization purposes. This study focuses on the induction of shoots from shoot tip explants using 6-Benzylaminopurine (BAP) and Kinetin. Aseptic explants were inoculated into Murashige and Skoog (MS) medium of full-strength and half-strength followed by full-strength MS media supplemented with different concentrations of BAP and Kinetin. Results obtained in this study showed no significant differences in terms of the number of axillary shoots produced between explants cultured in full and half-strength MS media. However, the highest number of shoots and increment in shoot length were obtained from MS media supplemented with 2.0 mg/L BAP with the values 1.80 ± 0.27 and 2.56 ± 0.36 cm, respectively. In conclusion, MS media supplemented with 2.0 mg/L BAP was found optimal in the induction of shoots and shoot elongation of C. australasica cv. Tasty Green.

Cytokinins play a central role in delaying senescence, reducing oxidative damage and maintaining plant growth during drought. This study showed that the ectopic expression of ProRE-deleted MdIPT5b, a key enzyme involved in cytokinin metabolism, increased the drought tolerance of transgenic Malus domestica (apple) callus and Solanum lycopersicum (tomato) seedlings by maintaining cytokinin homeostasis, and thus maintaining redox balance. Under restricted watering regimes, the yields of transgenic tomato plants were enhanced. Heterodimers of C/S1 bZIP are involved in the cytokinin-mediated drought response. The heterodimers bind the ProRE of MdIPT5b promoter, thus directly suppressing gene transcription. Single C/S1 bZIP members could not independently function as suppressors. However, specific paired members (heterodimers of MdbZIP80 with MdbZIP2 or with MdbZIP39) effectively suppressed transcription. The α-helical structure is essential for the heterodimerization of C/S1 bZIP members and for synergistic transcriptional suppression. As negative regulators of drought tolerance, suppressing either MdbZIP2 or MdbZIP39 alone does not improve the expression of MdIPT5b and did not increase the drought tolerance of transgenic apple callus. However, this could be achieved when they were co-suppressed. The suppression of MdbZIP80 alone could improve MdIPT5b expression and increase the drought tolerance of transgenic apple callus. However, these effects were reversed in response to the cosuppression of MdbZIP80 and MdIPT5b. Similar results were also observed during delayed dark-induced senescence in apple leaves. In conclusion, the apple C/S1 bZIP network (involving MdbZIP2, MdbZIP39 and MdbZIP80) directly suppressed the expression of MdIPT5b, thus negatively modulating drought tolerance and dark-induced senescence in a functionally redundant manner.

Moniliophthora perniciosa causes witches' broom disease of cacao and inflicts symptoms suggestive of hormonal imbalance. We investigated whether infection of the tomato (Solanum lycopersicum) model system Micro-Tom (MT) by the Solanaceae (S)-biotype of Moniliophthora perniciosa, which causes stem swelling and hypertrophic growth of axillary shoots, results from changes in host cytokinin metabolism. Inoculation of an MT-transgenic line that overexpresses the Arabidopsis CYTOKININ OXIDASE-2 gene (35S::AtCKX2) resulted in a reduction in disease incidence and stem diameter. RNA-sequencing analysis of infected MT and 35S::AtCKX2 revealed the activation of cytokinin-responsive marker genes when symptoms were conspicuous. The expression of an Moniliophthora perniciosa tRNA-ISOPENTENYL-TRANSFERASE suggests the production of isopentenyladenine (iP), detected in mycelia grown in vitro. Inoculated MT stems showed higher levels of dihydrozeatin and trans-zeatin but not iP. The application of benzyladenine induced symptoms similar to infection, whereas applying the cytokinin receptor inhibitors LGR-991 and PI55 decreased symptoms. Moniliophthora perniciosa produces iP that might contribute to cytokinin synthesis by the host, which results in vascular and cortex enlargement, axillary shoot outgrowth, reduction in root biomass and an increase in fruit locule number. This strategy may be associated with the manipulation of sink establishment to favour infection by the fungus.

Inula britannica L. is of great importance for Traditional Chinese and Kampo medicinal practices.Shoot cultures of Bulgarian accessions of the plant were developed and an experiment for elucidation of the combined effect of vitamins and auxin and cytokinin on their biomass formation, physiological status and secondary metabolite productivity was conducted. While Gamborg vitamins stimulated plant height, the Murashige and Skoog formulation increased leaf area and root length. Addition of benzyl adenine strongly stimulated axillary rosettes formation, however it suppressed aerial and root length and caused callusogenesis at explant base, this effect being slightly alleviated by 1-Naphthaleneacetic acid addition. Murashige and Skoog vitamins stimulated phenolic acids in the control and benzyl adenine treatments, while Gamborg—in the combined benzyl adenine and 1-Naphthaleneacetic acid ones. The capacity of production of britannin through gaillardin was stimulated by Gamborg vitamins in plant growth regulators free plants, but auxin and cytokinin supplementation reversed this effect. Plant growth regulators treatments generally lowered lipid peroxidation as compared with the controls at both vitamin supplementations. Elevation of hydrogen peroxide was related to preservation of lower malondyaldehyde levels, which was indicative of hydrogen peroxide role as a mediator to alleviate long-term lipid peroxidation in vitro. In all samples the chlorophyll a/b ratios remained at comparable levels indicating the physiological flexibility of the plant irrespectively of the different plant tissue manipulations. The results are indicative of the relations between growth, development and secondary metabolite productivity in I. britannica L. shoot cultures. The species showed a potential to target desired phytopharmaceuticals by modification of growth and development in vitro. The comparative study of vitamins and plant growth regulators treatments on Inula britannica L. shoot cultures showed their differential effect on biomass formation, physiological status and secondary metabolite formation.

Summary The cytokinins, which are N6‐substituted adenine derivatives, control key aspects of crop productivity. Cytokinin levels are controlled via biosynthesis by isopentenyl transferase (IPT), destruction by cytokinin oxidase/dehydrogenase (CKX), and inactivation via glucosylation by cytokinin glucosyl transferases (CGTs). While both yield components and tolerance to drought and related abiotic stressors have been positively addressed via manipulation of IPT and/or CKX expression, much less attention has been paid to the CGTs. As naming of the CGTs has been unclear, we suggest COGT, CNGT, CONGT and CNOGT to describe the O‐, N‐ and dual function CGTs. As specific CGT mutants of both rice and arabidopsis showed impacts on yield components, we interrogated the wheat genome database, IWGSC RefSeq v1.0 & v2.0, to investigate wheat CGTs. Besides providing unambiguous names for the 53 wheat CGTs, we show their expression patterns in 70 developmental tissues and their response characteristics to various stress conditions by reviewing more than 1000 RNA‐seq data sets. These revealed various patterns of responses and showed expression generally being more limited in reproductive tissues than in vegetative tissues. Multiple cis‐regulatory elements are present in the 3 kb upstream of the start codons of the 53 CGTs. Elements associated with abscisic acid, light and methyl jasmonate are particularly over‐represented, indicative of the responsiveness of CGTs to the environment. These data sets indicate that CGTs have potential value for wheat improvement and that these could be targeted in TILLING or gene editing wheat breeding programmes.

BackgroundCold stress is the main abiotic stress in rice, which seriously affects the growth and yield of rice. Identification of cold tolerance genes is of great significance for rice to solve these problems. GATA-family transcription factors involve diverse biological functions, however, their role in cold tolerance in rice remains unclear.ResultsIn this study, a GATA-type zinc finger transcription factorOsGATA16, which can improve cold tolerance, was isolated and characterized from rice. OsGATA16 belongs to OsGATA subfamily-II and contains 11 putative phosphorylation sites, a nuclear localization signal (NLS), and other several conserved domains.OsGATA16was expressed in all plant tissues, with the strongest in panicles. It was induced by cold and ABA treatments, but was repressed by drought, cytokinin and JA, and acted as a transcriptional suppressor in the nucleus. Overexpression ofOsGATA16improves cold tolerance of rice at seedling stage. Under cold stress treatments, the transcription of four cold-related genesOsWRKY45–1,OsSRFP1,OsCYL4, andOsMYB30was repressed inOsGATA16-overexpressing (OE) rice compared with wild-type (WT). Interestingly, OsGATA16 bound to the promoter ofOsWRKY45–1and repressed its expression.In addition, haplotype analysis showed thatOsGATA16polarized between the two major rice subspeciesjaponicaandindica, and had a non-synonymous SNP8 (336G) associated with cold tolerance.ConclusionOsGATA16 is a GATA transcription factor, which improves cold tolerance at seedling stage in rice. It acts as a positive regulator of cold tolerance by repressing some cold-related genes such asOsWRKY45–1,OsSRFP1,OsCYL4andOsMYB30. Additionally, OsGATA16 has a non-synonymous SNP8 (336G) associated with cold tolerance on CDS region. This study provides a theoretical basis for elucidating the mechanism of cold tolerance in rice and new germplasm resources for rice breeding.

The cytokinin (CK) phytohormones have long been known to activate cell proliferation in plants. However, how CKs regulate cell division and cell expansion remains unclear. Here, we reveal that a basic helix–loop–helix transcription factor, CYTOKININ-RESPONSIVE GROWTH REGULATOR (CKG), mediates CK-dependent regulation of cell expansion and cell cycle progression in Arabidopsis thaliana. The overexpression of CKG increased cell size in a ploidy-independent manner and promoted entry into the S phase of the cell cycle, especially at the seedling stage. Furthermore, CKG enhanced organ growth in a pleiotropic fashion, from embryogenesis to reproductive stages, particularly of cotyledons. In contrast, ckg loss-of-function mutants exhibited smaller cotyledons. CKG mainly regulates the expression of genes involved in the regulation of the cell cycle including WEE1. We propose that CKG provides a regulatory module that connects cell cycle progression and organ growth to CK responses.

Root-synthesized cytokinins are transported to the shoot and regulate the growth, development, and stress responses of aerial tissues. Previous studies have demonstrated that Arabidopsis (Arabidopsis thaliana) ATP binding cassette (ABC) transporter G family member 14 (AtABCG14) participates in xylem loading of root-synthesized cytokinins. However, the mechanism by which these root-derived cytokinins are distributed in the shoot remains unclear. Here, we revealed that AtABCG14-mediated phloem unloading through the apoplastic pathway is required for the appropriate shoot distribution of root-synthesized cytokinins in Arabidopsis. Wild-type rootstocks grafted to atabcg14 scions successfully restored trans-zeatin xylem loading. However, only low levels of root-synthesized cytokinins and induced shoot signaling were rescued. Reciprocal grafting and tissue-specific genetic complementation demonstrated that AtABCG14 disruption in the shoot considerably increased the retention of root-synthesized cytokinins in the phloem and substantially impaired their distribution in the leaf apoplast. The translocation of root-synthesized cytokinins from the xylem to the phloem and the subsequent unloading from the phloem is required for the shoot distribution and long-distance shootward transport of root-synthesized cytokinins. This study revealed a mechanism by which the phloem regulates systemic signaling of xylem-mediated transport of root-synthesized cytokinins from the root to the shoot.

The addition of cytokinin drastically increases shoot proliferation in Melaleuca. Individual in vitro shoots previously treated with BA showed better rooting development than BA-free culture medium. Reduction of the osmotic potential in the culture medium decreased dehydration of micropropagated plantlets. Melaleuca alternifolia, known as tea tree, is an Australian medicinal plant widely used in the cosmetic and pharmaceutical industries due to its antibacterial and antifungal properties. Propagation of Melaleuca is limited due to low rates of seed germination and multiplication and the poor rooting of stem cuttings. Thus, micropropagation can be an alternative to the propagation of this woody medicinal species. In this study, different concentrations of 6-benzyladenine (BA) (0, 0.55, 1.11 and 2.22 µM) were tested during the in vitro multiplication phase. It was observed that even the lowest concentration of BA (0.55 µM) could drastically increase the multiplication rate in Melaleuca, compared to the BA-free treatment, due to multiple shoot proliferation. At the rooting stage, in the culture medium without phyto-regulators, individual shoots previously treated with BA had a higher rooting percentage (91–97%) and considerable height growth compared to those of the control treatment (without BA) (66%). However, none of the in vitro plantlets survived to acclimatization stage due to excessive and rapid dehydration of the plantlets under ex vitro conditions, making it the most challenging phase for the micropropagation of Melaleuca. Therefore, a second experimental setup was designed, which included treatments with sucrose, sucrose + sorbitol and sucrose + mannitol in the culture medium at − 0.2170, − 0.3255 and − 0.4340 MPa, respectively, to determine the effects of these osmotic agents on the development of Melaleuca in rooting and acclimatization stages. Sorbitol with sucrose at Ψπ = − 0.4340 decreased stomatal density in leaves and reduced dehydration of plantlets under ex vitro conditions, but was not enough to provide successful plantlets acclimatization.

Plants have evolved a complex network of components that sense and respond to diverse signals. In the present study, we have characterized OsRR6, a type-A response regulator, which is part of the two-component sensor-regulator machinery in rice. The expression of OsRR6 is induced by exogenous cytokinin and various abiotic stress treatments, including drought, cold and salinity stress. Organ-specific expression analysis revealed that its expression is high in anther and low in shoot apical meristem. The Arabidopsis plants constitutively expressing OsRR6 (OsRR6OX) exhibited reduced cytokinin sensitivity, adventitious root formation and enhanced anthocyanin accumulation in seeds. OsRR6OX plants were more tolerant to drought and salinity conditions when compared to wild-type. The hypocotyl growth in OsRR6OX seedlings was significantly inhibited under red, far-red and blue-light conditions and also a decline in transcript levels of OsRR6 was observed in rice under the above monochromatic as well as white light treatments. Transcriptome profiling revealed that the genes associated with defense responses and anthocyanin metabolism are up-regulated in OsRR6OX seedlings. Comparative transcriptome analysis showed that the genes associated with phenylpropanoid and triterpenoid biosynthesis are enriched among differentially expressed genes in OsRR6OX seedlings of Arabidopsis, which is in conformity with reanalysis of the transcriptome data performed in rice transgenics for OsRR6. Further, genes like DREB1A/CBF3, COR15A, KIN1, ERD10 and RD29A are significantly upregulated in OsRR6OX seedlings when subjected to ABA and abiotic stress treatments. Thus, a negative regulator of cytokinin signaling, OsRR6, plays a positive role in imparting abiotic stress tolerance.