Cytokinin Research Articles

Cytokinin-mediated shoot proliferation and its correlation with phytoremediation effects in Cd-hyperaccumulator ecotype of Sedum alfredii

The phytohormones cytokinins (CKs) are known to regulate apical/auxiliary meristems, control shoot growth and are associated with nutrient uptake and high biomass production. In this study, different cytokinins were tested on Sedum alfredii (S.alfredii) for shoot proliferation and growth performance as well as their correlation with phytoextraction efficiency. Among the tested cytokinins, Zeatin (ZTN) treatments produced the highest number of shoots (5–6 per explant) with 5 and 10 μM ZTN concentrations which are shown as zeatin (ZTN) > kinetin (KTN) > benzylaminopurine (BA) > thidiazuron (TDZ). Maximum biomass production was produced on these media. The maximum biomass (0.14 g) was found in 10 μM ZTN concentration with a 1-fold difference (mean value: 0.02 g) from CK (0.12 g). However, the lowest biomass (0.11 g) was found with 4 μM TDZ, with a 1-fold difference (mean value: 0.02 g) from CK (0.13 g) which suppressed shoot growth. The leaf area and leaf chlorophyll index were significantly increased in all cytokinins except TDZ, and the relation was ZTN > KTN > BA>CK > TDZ. Cadmium accumulation was significantly higher in treatments containing cytokinins as compared to cytokinin-free media. Zeatin at 10 μM concentration was the most effective for high biomass production and correlated with higher cadmium uptake efficiency. The results suggest that cytokinins particularly ZTN, play a crucial role in enhancing both biomass production and cadmium, uptake efficiency in S. alfredii. Therefore, in large-scale phytoremediation initiatives conducted in field conditions, cytokinins can be utilized as growth regulators to enhance biomass production and cadmium extraction efficiency in S.alfredii.

Profiling of adenine-derived signaling molecules, cytokinins, in myotubes reveals fluctuations in response to lipopolysaccharide-induced cell stress.

Cytokinins (CTKs) are a diverse collection of evolutionarily conserved adenine-derived signaling molecules classically studied as phytohormones; however, their roles and production have been less studied in mammalian systems. Skeletal muscles are sensitive to cellular cues such as inflammation and in response, alter their secretome to regulate the muscle stem cell and myofiber niche. Using cultured C2C12 muscle cells, we profiled CTK levels to understand (1) whether CTKs are part of the muscle secretome and (2) whether CTKs are responsive to cellular stress. To induce cellular stress, C2C12 myotubes were treated with lipopolysaccharides (LPS) for 24 h and then media and cell fractions were collected for ultra high-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-(ESI+)-HRMS/MS) for metabolomics and CTK profiling. Across LPS-treated and control cells, 11 CTKs were detected in the extracellular space while 6 were detected intracellularly. We found that muscle cells are enriched in isopentenyladenine (iP) species (from free base, riboside to nucleotide forms), and that extracellular levels are increased after LPS treatment. Our study establishes that muscle cells express various forms of CTKs, and that CTK levels are responsive to LPS-induced cell stress, suggesting a role for CTKs in intra- and extracellular signaling of mammalian cells.

Nitric Oxide (NO) Improves Wheat Growth under Dehydration Conditions by Regulating Phytohormone Levels and Induction of the Expression of the TADHN Dehydrin Gene

Nitric oxide (NO) is a universal signaling molecule with important regulatory functions in the plant’s life cycle and adaptation to a wide spectrum of environmental stresses including drought. The effect of pre-sowing seed treatment with the donor of NO sodium nitroprusside (SNP, 200 μM) on wheat Triticum aestivum L. plants subjected to dehydration (PEG-8000, 12%) was investigated. SNP pretreatment stimulated germination and seedling growth in normal conditions and protected them under dehydration. These effects were confirmed by percentage of seed germination, changes in fresh and dry weight of 5–6-day-old seedlings, as well as by seedlings’ linear dimensions, visual appearance, and mitotic index of the root apical meristem. Assessment of the transpiration intensity (TI) and relative water content (RWC) showed that SNP pretreatment helped to maintain the water status of seedlings subjected to dehydration stress. The data obtained by enzyme-linked immunosorbent assay (ELISA) suggested that the positive effects of SNP may be due to its influence on the phytohormonal system. SNP pretreatment induced an increase in the level of indolylacetic acid (IAA) and especially cytokinins (CK), while essential changes in ABA content were not detected. Water deficiency caused a substantial increase in ABA content and a decrease in the levels of CK and IAA. Pre-sowing SNP treatment decreased stress-induced fluctuations in the content of all studied phytohormones. Using reverse-transcription PCR (RT-PCR), we obtained data on the increase in expression of the TADHN dehydrin gene in SNP-pretreated seedlings under normal and, especially, under dehydration conditions. These findings may indicate the participation of dehydrins in NO-induced defense reactions in wheat plants under water stress. Furthermore, exogenous NO had a stabilizing effect on membrane cellular structures, as evidenced by the reduction of electrolyte leakage (EL) levels and malondialdehyde (MDA) content in dehydrated wheat seedlings under the influence of pre-sowing SNP treatment.

Knockout of the sugar transporter OsSTP15 enhances grain yield by improving tiller number due to increased sugar content in the shoot base of rice (Oryza sativa L.).

Sugar transporter proteins (STPs) play critical roles in regulating plant stress tolerance, growth, and development. However, the role of STPs in regulating crop yield is poorly understood. This study elucidates the mechanism by which knockout of the sugar transporter OsSTP15 enhances grain yield via increasing the tiller number in rice. We found that OsSTP15 is specifically expressed in the shoot base and vascular bundle sheath of seedlings and encodes a plasma membrane-localized high-affinity glucose efflux transporter. OsSTP15 knockout enhanced sucrose and trehalose-6-phosphate (Tre6P) synthesis in leaves and improved sucrose transport to the shoot base by inducing the expression of sucrose transporters. Higher glucose, sucrose, and Tre6P contents were observed at the shoot base of stp15 plants. Transcriptome and metabolome analyses of the shoot base demonstrated that OsSTP15 knockout upregulated the expression of cytokinin (CK) synthesis- and signaling pathway-related genes and increased CK levels. These findings suggest that OsSTP15 knockout represses glucose export from the cytoplasm and simultaneously enhances sugar transport from source leaves to the shoot base by promoting the synthesis of sucrose and Tre6P in leaves. Subsequent accumulation of glucose, sucrose, and Tre6P in the shoot base promotes tillering by stimulating the CK signaling pathway.

Rapid profiling of cytokinins using supercritical fluid chromatography coupled with tandem mass spectrometry

BackgroundThe determination of plant hormones is still a very challenging analytical discipline, mainly due to their low concentration in complex plant matrices. Therefore, the involvement of very sensitive high-throughput techniques is required. Cytokinins (CKs) are semi-polar basic plant hormones regulating plant growth and development. Modern methods for CK determination are currently based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), which enables the separation of CK isomeric forms occurring endogenously in plants. Here, ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry (UHPSFC-MS/MS) was used for the simultaneous determination of 37 CK metabolites. ResultsThe chromatographic conditions were tested on three different columns with various retention mechanisms. Hybrid silica modified with 2-picolylamine was selected as the stationary phase. Several parameters such as column temperature, back pressure regulation, mobile phase composition and make-up solvent were investigated to achieve efficient separation of CK isomers and reasonable sensitivity. Compared to UHPLC-MS/MS, a 9-min chromatographic analysis using a mobile phase of supercritical CO2 and 5 mM ammonia in methanol represents a three-fold acceleration of total run time. The quantification limit of UHPSFC-MS/MS method was in the range of 0.03–0.19 fmol per injection and the method validation showed high accuracy and precision (below 15 % for most analytes). The method was finally applied to the complex plant matrix of the model plant Arabidopsis thaliana and the obtained profiles of CK metabolites were compared with the results from the conventional UHPLC-MS/MS method. SignificanceThe presented work offers a novel approach for quantification of endogenous CKs in plants. Compared to the conventional UHPLC-MS/MS, the total run time is shorter and the matrix effect lower for the key CK metabolites. This approach opens the opportunity to utilize UHPSFC-MS/MS instrumentation for targeted plant hormonomics including other plant hormone families.

Hormonal Content and Gene Expression during Olive Fruit Growth and Ripening.

The cultivated olive (Olea europaea L. subsp. europaea var. europaea) is one of the most valuable fruit trees worldwide. However, the hormonal mechanisms underlying the fruit growth and ripening in olives remain largely uncharacterized. In this study, we investigated the physiological and hormonal changes, by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS), as well as the expression patterns of hormone-related genes, using quantitative real-time PCR (qRT-PCR) analysis, during fruit growth and ripening in two olive cultivars, 'Arbequina' and 'Picual', with contrasting fruit size and shape as well as fruit ripening duration. Hormonal profiling revealed that olive fruit growth involves a lowering of auxin (IAA), cytokinin (CKs), and jasmonic acid (JA) levels as well as a rise in salicylic acid (SA) levels from the endocarp lignification to the onset of fruit ripening in both cultivars. During olive fruit ripening, both abscisic acid (ABA) and anthocyanin levels rose, while JA levels fell, and SA levels showed no significant changes in either cultivar. By contrast, differential accumulation patterns of gibberellins (GAs) were found between the two cultivars during olive fruit growth and ripening. GA1 was not detected at either stage of fruit development in 'Arbequina', revealing a specific association between the GA1 and 'Picual', the cultivar with large sized, elongated, and fast-ripening fruit. Moreover, ABA may play a central role in regulating olive fruit ripening through transcriptional regulation of key ABA metabolism genes, whereas the IAA, CK, and GA levels and/or responsiveness differ between olive cultivars during olive fruit ripening. Taken together, the results indicate that the relative absence or presence of endogenous GA1 is associated with differences in fruit morphology and size as well as in the ripening duration in olives. Such detailed knowledge may be of help to design new strategies for effective manipulation of olive fruit size as well as ripening duration.

Cytokinin signaling promotes root secondary growth and bud formation in Panax ginseng

BackgroundPanax ginseng, one of the valuable perennial medicinal plants, stores numerous pharmacological substrates in its storage roots. Given its perennial growth habit, organ regeneration occurs each year, and cambium stem cell activity is necessary for secondary growth and storage root formation. Cytokinin (CK) is a phytohormone involved in the maintenance of meristematic cells for the development of storage organs; however, its physiological role in storage-root secondary growth remains unknown. MethodsExogenous CK was repeatedly applied to P. ginseng, and morphological and histological changes were observed. RNA-seq analysis was used to elucidate the transcriptional network of CK that regulates P. ginseng growth and development. The HISTIDINE KINASE 3 (PgHK3) and RESPONSE REGULATOR 2 (PgRR2) genes were cloned in P. ginseng and functionally analyzed in Arabidopsis as a two-component system involved in CK signaling. ResultsPhenotypic and histological analyses showed that CK increased cambium activity and dormant axillary bud formation in P. ginseng, thus promoting storage-root secondary growth and bud formation. The evolutionarily conserved two-component signaling pathways in P. ginseng were sufficient to restore CK signaling in the Arabidopsis ahk2/3 double mutant and rescue its growth defects. Finally, RNA-seq analysis of CK-treated P. ginseng roots revealed that plant-type cell wall biogenesis-related genes are tightly connected with mitotic cell division, cytokinesis, and auxin signaling to regulate CK-mediated P. ginseng development. ConclusionOverall, we identified the CK signaling-related two-component systems and their physiological role in P. ginseng. This scientific information has the potential to significantly improve the field-cultivation and biotechnology-based breeding of ginseng.

The Variations of C/N/P Stoichiometry, Endogenous Hormones, and Non-Structural Carbohydrate Contents in Micheliamaudiae ‘Rubicunda’ Flower at Five Development Stages

Michelia maudiae ‘Rubicunda’ (M. maudiae ‘Rubicunda’) is one of the most popular ornamental plants. However, relatively little is known regarding its floral development. Here, the variations of the mineral, endogenous hormone, and non-structural carbohydrate (NSC) contents in the petals and gynoecium and androecium (GA) at five developmental stages during M. maudiae ‘Rubicunda’ flower development were analyzed. The results suggested that the carbon (C), nitrogen (N), and phosphorus (P) endogenous hormones, NSC contents, and C/N/P stoichiometric ratios exhibit large variations during flower development. There were significant differences in N and P contents in the GA and petals among the five growth stages, while C contents did not change significantly. In the five flower development stages, the average N and P contents in the GA were higher than those in the petals. The maximum C/N and N/P ratios in the GA and petals were foundat the senescent flower stage (stage5) and green bud stage (stage1), respectively. The C/P ratio in petals reached its maximum value at the mature bud stage (stage 2), and the C/P ratio in the GA reached its maximum value at the senescent flower stage (stage 5). The C/N/P stoichiometric ratios in the petals were more sensitive to development stages than those in the GA. There were highly significant correlations between the NSC and C/N/P stoichiometric ratios in the GAand petals. Moreover, acetic acid (IAA), abscisic acid (ABA), gibberellic acid 3 (GA3), and cytokinin (CTK) contents in the petals exhibited significant changes in response to development stages. Principal component analysis (PCA) revealed significant correlations and clear differences in the test indexes between the development stage and organs, and the variation was explained by PC-1 (55.6%), PC-2 (23.3%), and the cumulative contribution percentage of the total biplot (78.9%). These studies can lay the foundation for elucidating the requirements and dynamic balance among C, N, P, NSC, and hormone contents during the flower development of M. maudiae‘ Rubicunda’.

Far-red light inhibits lateral bud growth mainly through enhancing apical dominance independently of strigolactone synthesis in tomato.

The ratio of red light to far-red light (R:FR) is perceived by light receptors and consequently regulates plant architecture. Regulation of shoot branching by R:FR ratio involves plant hormones. However, the roles of strigolactone (SL), the key shoot branching hormoneand the interplay of different hormones in the light regulation of shoot branching in tomato (Solanum lycopersicum) are elusive. Here, we found that defects in SL synthesis genes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CCD8 in tomato resulted in more lateral bud growth but failed to reverse the FR inhibition of lateral bud growth, which was associated with increased auxin synthesis and decreased synthesis of cytokinin (CK) and brassinosteroid (BR). Treatment of auxin also inhibited shoot branching in ccd mutants. However, CK released the FR inhibition of lateral bud growth in ccd mutants, concomitant with the upregulation of BR synthesis genes. Furthermore, plants that overexpressed BR synthesis gene showed more lateral bud growth and the shoot branching was less sensitive to the low R:FR ratio. The results indicate that SL synthesis is dispensable for light regulation of shoot branching in tomato. Auxin mediates the response to R:FR ratio to regulate shoot branching by suppressing CK and BR synthesis.

Leaf ontogeny modulates epinasty through shifts in hormone dynamics during waterlogging in tomato.

Waterlogging leads to hypoxic conditions in the root zone that subsequently cause systemic adaptive responses in the shoot, including leaf epinasty. Waterlogging-induced epinasty in tomato has long been ascribed to the coordinated action of ethylene and auxins. However, other hormonal signals have largely been neglected, despite evidence of their importance in leaf posture control. To cover a large group of growth regulators, we performed a tissue-specific and time-dependent hormonomics analysis. This revealed that multiple hormones are differentially affected throughout a 48 h waterlogging treatment, and that leaf age determines hormone homeostasis and modulates their changes during waterlogging. In addition, we distinguished early hormonal signals that contribute to fast responses to oxygen deprivation from those that potentially sustain the waterlogging response. We found that abscisic acid (ABA) levels peak in petioles within the first 12 h of the treatment, while its precursors only increase much later, suggesting that ABA transport is altered. At the same time, cytokinins (CKs) and their derivatives drastically decline during waterlogging in leaves of all ages. This drop in CKs possibly releases the inhibition of ethylene- and auxin-mediated cell elongation to establish epinastic bending. Auxins themselves rise substantially in the petiole of mature leaves, but mostly after 48 h of root hypoxia. Based on our hormone profiling, we propose that ethylene and ABA might act synergistically as an early signal to induce epinasty, while the balance of indole-3-acetic acid and CKs in the petiole ultimately regulates differential growth.

TOR coordinates cytokinin and gibberellin signals mediating development and defense.

Plants constantly perceive and process environmental signals and balance between the energetic demands of growth and defense. Growth arrest upon pathogen attack was previously suggested to result from a redirection of the plants' metabolic resources towards the activation of plant defense. The energy sensor Target of Rapamycin (TOR) kinase is a conserved master coordinator of growth and development in all eukaryotes. Although TOR is positioned at the interface between development and defense, little is known about the mechanisms by which TOR may potentially regulate the relationship between these two modalities. The plant hormones cytokinin (CK) and gibberellin (GA) execute various aspects of plant development and defense. The ratio between CK and GA was reported to determine the outcome of developmental programmes. Here, investigating the interplay between TOR-mediated development and TOR-mediated defense in tomato, we found that TOR silencing resulted in rescue of several different aberrant developmental phenotypes, demonstrating that TOR is required for the execution of developmental cues. In parallel, TOR inhibition enhanced immunity in genotypes with a low CK/GA ratio but not in genotypes with a high CK/GA ratio. TOR-inhibition mediated disease resistance was found to depend on developmental status, and was abolished in strongly morphogenetic leaves, while being strongest in mature, differentiated leaves. CK repressed TOR activity, suggesting that CK-mediated immunity may rely on TOR downregulation. At the same time, TOR activity was promoted by GA, and TOR silencing reduced GA sensitivity, indicating that GA signalling requires normal TOR activity. Our results demonstrate that TOR likely acts in concert with CK and GA signalling, executing signalling cues in both defense and development. Thus, differential regulation of TOR or TOR-mediated processes could regulate the required outcome of development-defense prioritisation.

The Cytokinins BAP and 2-iP Modulate Different Molecular Mechanisms on Shoot Proliferation and Root Development in Lemongrass (Cymbopogon citratus).

The known activities of cytokinins (CKs) are promoting shoot multiplication, root growth inhibition, and delaying senescence. 6-Benzylaminopurine (BAP) has been the most effective CK to induce shoot proliferation in cereal and grasses. Previously, we reported that in lemongrass (Cymbopogon citratus) micropropagation, BAP 10 µM induces high shoot proliferation, while the natural CK 6-(γ,γ-Dimethylallylamino)purine (2-iP) 10 µM shows less pronounced effects and developed rooting. To understand the molecular mechanisms involved, we perform a protein-protein interaction (PPI) network based on the genes of Brachypodium distachyon involved in shoot proliferation/repression, cell cycle, stem cell maintenance, auxin response factors, and CK signaling to analyze the molecular mechanisms in BAP versus 2-iP plants. A different pattern of gene expression was observed between BAP- versus 2-iP-treated plants. In shoots derived from BAP, we found upregulated genes that have already been demonstrated to be involved in de novo shoot proliferation development in several plant species; CK receptors (AHK3, ARR1), stem cell maintenance (STM, REV and CLV3), cell cycle regulation (CDKA-CYCD3 complex), as well as the auxin response factor (ARF5) and CK metabolism (CKX1). In contrast, in the 2-iP culture medium, there was an upregulation of genes involved in shoot repression (BRC1, MAX3), ARR4, a type A-response regulator (RR), and auxin metabolism (SHY2).

Phytohormonal modulation of the drought stress in soybean: outlook, research progress, and cross-talk.

Phytohormones play vital roles in stress modulation and enhancing the growth of plants. They interact with one another to produce programmed signaling responses by regulating gene expression. Environmental stress, including drought stress, hampers food and energy security. Drought is abiotic stress that negatively affects the productivity of the crops. Abscisic acid (ABA) acts as a prime controller during an acute transient response that leads to stomatal closure. Under long-term stress conditions, ABA interacts with other hormones, such as jasmonic acid (JA), gibberellins (GAs), salicylic acid (SA), and brassinosteroids (BRs), to promote stomatal closure by regulating genetic expression. Regarding antagonistic approaches, cytokinins (CK) and auxins (IAA) regulate stomatal opening. Exogenous application of phytohormone enhances drought stress tolerance in soybean. Thus, phytohormone-producing microbes have received considerable attention from researchers owing to their ability to enhance drought-stress tolerance and regulate biological processes in plants. The present study was conducted to summarize the role of phytohormones (exogenous and endogenous) and their corresponding microbes in drought stress tolerance in model plant soybean. A total of n=137 relevant studies were collected and reviewed using different research databases.

Effects of AMF inoculation on the growth, photosynthesis and root physiological morphology of root-pruned Robinia pseudoacacia seedlings.

Root pruning hinders the absorption and utilization of nutrients and water by seedlings in the short term. Arbuscular mycorrhizal fungi (AMF) are an important source of nutrient and water for seedlings except for the root system. However, the mechanism by which AMF affect the physiological growth of seedlings after root pruning has rarely been studied. In this study, a pot experiment was conducted through a three-compartment partition system to clarify the effects of Funneliformis mosseae (F. mosseae) strain BGC XJ07A on the physiological growth of root-pruned Robinia pseudoacacia seedlings. Five root pruning treatments (zero, one-fifth, one-fourth, one-third and one-half of the taproot length were removed) were applied to noninoculated seedlings and those inoculated with F. mosseae. The results showed that the presence of F. mosseae significantly increased the shoot and root biomasses, leaf photosynthetic rate, stomatal conductance and transpiration rate. The root projected area, root surface area, average root diameter, root density, root volume and number of root tips of the inoculated seedlings were higher than those without inoculation in all root pruning treatments. The root cytokinin, gibberellins and indole-3-acetic acid concentrations, but root abscisic acid concentration, were higher than those measured in the absence of inoculation in all root pruning treatments. Moreover, the changes in the root endogenous hormone concentrations of the seedlings were closely related to the root morphological development and seedling biomass. The AMF increased the soil available nitrogen, soil available phosphorus, soil available potassium and soil organic matter concentrations compared with the noninoculated treatment. These results indicate that AMF can alleviate the adverse effects of root pruning on the physiological growth of R. pseudoacacia and soil properties, and can provide a basis for AMF application to forest cultivation and the sustainable development of forest ecosystems.

Virulence Factors of the Fungal Pathogen Stagonospora nodorum Manipulate Hormonal Signaling Pathways in Triticum aestivum L. by Regulating Host Plant MicroRNA Expressions.

Currently, the role of microRNAs in plant immune responses is being actively studied. Thus, our aim was to research the effect of Stagonospora nodorum (Berk.) NEs SnToxA and SnTox3 on the expression of miRNAs involved in the wheat-S. nodorum interaction and to determine the role of phytohormones in this process. The expressions of nine conserved microRNAs were studied by quantitative real-time polymerase chain reaction in three different wheat genotypes of bread spring wheat (Triticum aestivum L.) infected with S. nodorum. Phytohormone treatments (trans-zeatin, 2-chloroethylphosphonic acid (etefone is the chemical precursor of ethylene), and salicylic acid) were applied. The results were compared with disease symptoms, the redox status of plants, and the expression of fungal necrotrophic effector (NE) genes of SnToxA and SnTox3 and genes of SnPf2, SnStuA, alongside SnCon7 transcription factors (TFs). Salicylic acid (SA) and cytokinins (CK) are involved in the development of defense reactions in wheat plants against S. nodorum, by regulating the expression of fungal NEs and TFs genes, inducing an oxidative burst in all three wheat genotypes. Moreover, ethylene enhanced the virulence of the pathogen by increasing the expression of fungal NE and TF genes, thereby resulting in a decrease in the generation of reactive oxygen species in all three cultivars. The nine miRNAs played a role in the development of wheat resistance against S. nodorum. NE SnTox3 mainly suppressed the expression of three miRNAs: miR159, miR393, and miR408, while NE SnToxA suppressed miR166 expression. Conversely, treatment with CK and SA increased the expression of miR159 and miR408; treatment with CK increased the expression of miR393 and miR166. Ethylene inhibited the expression of miR159, miR408, miR393, and miR166. Suppression of miP159 expression by NE SnTox3 was most likely associated with the activation of the ethylene signaling pathway. NEs SnToxA and SnTox3 suppressed the expression of miR408, whose role most likely consisted of inhibiting the catalase activity, via SA and CK regulation. In addition, NE SnToxA hijacked the SA signaling pathway and manipulated it for fungal growth and development. Fungal TFs SnPf2 and SnStuA could be involved in the regulation of these processes indirectly through the regulation of the expression of NE genes. The results of this work show, for the first time, the role of microRNAs in the development of wheat resistance against S. nodorum and the effect of S. nodorum NEs SnToxA and SnTox3 on the activity of plant microRNAs.

Comparative transcriptome analysis of high- and low-embryogenic Hevea brasiliensis genotypes reveals involvement of phytohormones in somatic embryogenesis

BackgroundRubber plant (Hevea brasiliensis) is one of the major sources of latex. Somatic embryogenesis (SE) is a promising alterative to its propagation by grafting and seed. Phytohormones have been shown to influence SE in different plant species. However, limited knowledge is available on the role of phytohormones in SE in Hevea. The anther cultures of two Hevea genotypes (Yunyan 73477-YT and Reken 628-RT) with contrasting SE rate were established and four stages i.e., anthers (h), anther induced callus (y), callus differentiation state (f), and somatic embryos (p) were studied. UPLC-ESI-MS/MS and transcriptome analyses were used to study phytohormone accumulation and related expression changes in biosynthesis and signaling genes.ResultsYT showed higher callus induction rate than RT. Of the two genotypes, only YT exhibited successful SE. Auxins, cytokinins (CKs), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), gibberellins (GAs), and ethylene (ETH) were detected in the two genotypes. Indole-3-acetic acid (IAA), CKs, ABA, and ETH had notable differences in the studied stages of the two genotypes. The differentially expressed genes identified in treatment comparisons were majorly enriched in MAPK and phytohormone signaling, biosynthesis of secondary metabolites, and metabolic pathways. The expression changes in IAA, CK, ABA, and ETH biosynthesis and signaling genes confirmed the differential accumulation of respective phytohormones in the two genotypes.ConclusionThese results suggest potential roles of phytohormones in SE in Hevea.

MdAIL5 overexpression promotes apple adventitious shoot regeneration by regulating hormone signaling and activating the expression of shoot development-related genes.

Adventitious shoot (AS) regeneration is a significant factor in the genetic transformation of horticultural plants. It is also a noteworthy approach to their vegetative propagation. AS regeneration remains highly dependent on the genotype or maturity of explants. We here found that the AS regeneration abilities of apple leaves were positively correlated with MdAIL5 expression. MdAIL5 overexpression dramatically increased AS regeneration efficiency. Notably, MdAIL5 overexpression could restore the AS formation ability of explants to a certain extent, which was lost with an increase in maturity. Endogenous hormone detection revealed that MdAIL5 overexpression changed the contents of auxin, cytokinin (CK), and other hormones in apple leaves. Transcriptome analysis revealed that many genes related to auxin, CK, and brassinolide signaling pathways were significantly and differentially expressed between MdAIL5-overexpressing transgenic apple and wild-type apple plants. Yeast one-hybrid assays, the electrophoretic mobility shift assay, and the dual-luciferase reporter assay revealed that MdAIL5 directly binds to MdARF9 and MdHB14 promoters and positively affects their expression. We here established a model of MdAIL5 regulating AS formation, which acts as a theoretical basis for facilitating genotype- or explant maturity-independent AS regeneration in the future.

The cytokinin receptor OHK4/OsHK4 regulates inflorescence architecture in rice via an IDEAL PLANT ARCHITECTURE1/WEALTHY FARMER'S PANICLE-mediated positive feedback circuit.

Inflorescence architecture is important for rice (Oryza sativa) grain yield. The phytohormone cytokinin (CK) has been shown to regulate rice inflorescence development; however, the underlying mechanism mediated by CK perception is still unclear. Employing a forward genetic approach, we isolated an inactive variant of the CK receptor OHK4/OsHK4 gene named panicle length1, which shows decreased panicle size due to reduced inflorescence meristem (IM) activity. A 2-amino acid deletion in the long α-helix stalk of the sensory module of OHK4 impairs the homodimerization and ligand-binding capacity of the receptor, even though the residues do not touch the ligand-binding domain or the dimerization interface. This deletion impairs CK signaling that occurs through the type-B response regulator OsRR21, which acts downstream of OHK4 in controlling inflorescence size. Meanwhile, we found that IDEAL PLANT ARCHITECTURE1(IPA1)/WEALTHY FARMER'S PANICLE (WFP), encoding a positive regulator of IM development, acts downstream of CK signaling and is directly activated by OsRR21. Additionally, we revealed that IPA1/WFP directly binds to the OHK4 promoter and upregulates its expression through interactions with 2 TCP transcription factors, forming a positive feedback circuit. Altogether, we identified the OHK4-OsRR21-IPA1 regulatory module, providing important insights into the role of CK signaling in regulating rice inflorescence architecture.

Increase in ENHANCER OF SHOOT REGENERATION2 expression by treatment with strigolactone-related inhibitors and kinetin during adventitious shoot formation in ipecac.

Increase of ENHANCER OF SHOOT REGENERATION 2 expression was consistent to treatment with kinetin, TIS108, and KK094 in adventitious shoot formation of ipecac. Unlike many plant species, ipecac (Carapichea ipecacuanha (Brot.) L. Andersson) can form adventitious shoots in tissue culture without cytokinin (CK) treatment. Strigolactone (SL) biosynthesis and signaling inhibitors stimulate adventitious shoot formation in ipecac, suggesting their potential use as novel growth regulators in plant tissue culture, but the molecular mechanism of their action is unclear. In this study, we compared the effects of SL-related inhibitors (TIS108 and KK094) and CKs (2iP, tZ, and kinetin) on adventitious shoot formation in ipecac. Exogenously applied SL-related inhibitors and CKs stimulated adventitious shoot formation. Combinations of SL-related inhibitors and kinetin also promoted adventitious shoot formation, but without additive effects. We also analyzed the expression of CK biosynthesis genes in ipecac. TIS108 increased the expression of the ipecac homolog of ISOPENTENYL TRANSFERASE 3 (CiIPT3) but decreased that of LONELY GUY 7 homolog (CiLOG7), presumably resulting in no change in 2iP-type CK levels. KK094 and kinetin increased CiLOG7 expression, elevating 2iP-type CK levels. Among pluripotency- and meristem-related genes, TIS108, KK094, and kinetin consistently increased the expression of ENHANCER OF SHOOT REGENERATION 2 homolog (CiESR2), which has a key role in shoot regeneration, in the internodal segment region that formed adventitious shoots. We propose that CiESR2 might be a key stimulator of adventitious shoot formation in ipecac.

Antioxidant system was triggered to alleviate salinity stress by cytokinin oxidase/dehydrogenase gene GhCKX6b-Dt in cotton

Cytokinin oxidase/dehydrogenase (CKX) is a key regulatory enzyme for the irreversible degradation of the plant hormone cytokinin (CK), which is important in growth and development and response to abiotic stresses in cotton. In this study, 27, 28, 14 and 14 CKXs were screened by FAD structural domain and cytokinin binding structural domain in Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii, respectively. Their phylogenetic relationships and expression patterns were analyzed, and most GhCKXs were found to be tissue-specific and responsive to various abiotic stresses such as cold, heat, salt and PEG. GhCKX6b-Dt was selected for gene silencing in evolutionary branch II for salt stress, because its expression increased after salt stress in cotton plants. An increase in PRO and MDA content and a decrease in SOD activity due to this gene were found after inducing salt stress, contributing to oxidative damage and decreased salt tolerance. In this study, CKXs were analyzed to reveal the possible role of GhCKXs against abiotic stresses in cotton, which provides a basis for further understanding of the biological functions of CK in plants such as growth and development and stress resistance.