Accumulation pattern of endogenous cytokinins and phenolics in different organs of 1‐year‐old cytokinin pre‐incubated plants: implications for conservation

A better understanding of phytohormone physiology can provide an essential basis to coherently achieve a conservation drive/strategy for valuable plant species. We evaluated the distribution pattern of cytokinins (CKs) and phenolic compounds in different organs of 1‐year‐old greenhouse‐grown Tulbaghia simmleri pre‐treated (during micropropagation) with three aromatic CKs (benzyladenine = BA, meta‐topolin = mT, meta‐topolin riboside = mTR). The test species is highly valuable due to its medicinal and ornamental uses. Based on UHPLC‐MS/MS quantification, mT and mTR pre‐treated plants had the highest total CK, mostly resulting from the isoprenoid CK‐type, which occurred at highest concentrations in the roots. Although occurring in much lower concentrations when compared to isoprenoid CKs, aromatic CKs were several‐fold more abundant in the root of mT pre‐treated plants than with other treatments. Possibly related to the enhanced aromatic CKs, free bases and ribonucleotides, plants pre‐treated with mT generally displayed better morphology than the other treatments. A total of 12 bioactive phenolic compounds, including four hydroxybenzoic acids, five hydroxycinnamic acids and three flavonoids at varying concentrations, were quantified in T. simmleri. The occurrence, distribution and levels of these phenolic compounds were strongly influenced by the CK pre‐treatments, thereby confirming the importance of CKs in phenolic biosynthesis pathways.     

Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis

Cytokinins (CKs) regulate plant growth and development via a complex network of CK signaling. Here, we perform functional analyses with CK-deficient plants to provide direct evidence that CKs negatively regulate salt and drought stress signaling. All CK-deficient plants with reduced levels of various CKs exhibited a strong stress-tolerant phenotype that was associated with increased cell membrane integrity and abscisic acid (ABA) hypersensitivity rather than stomatal density and ABA-mediated stomatal closure. Expression of the Arabidopsis thaliana ISOPENTENYL-TRANSFERASE genes involved in the biosynthesis of bioactive CKs and the majority of the Arabidopsis CYTOKININ OXIDASES/DEHYDROGENASES genes was repressed by stress and ABA treatments, leading to a decrease in biologically active CK contents. These results demonstrate a novel mechanism for survival under abiotic stress conditions via the homeostatic regulation of steady state CK levels. Additionally, under normal conditions, although CK deficiency increased the sensitivity of plants to exogenous ABA, it caused a downregulation of key ABA biosynthetic genes, leading to a significant reduction in endogenous ABA levels in CK-deficient plants relative to the wild type. Taken together, this study provides direct evidence that mutual regulation mechanisms exist between the CK and ABA metabolism and signals underlying different processes regulating plant adaptation to stressors as well as plant growth and development.     

New insights into gibberellin signaling in regulating flowering in Arabidopsis

In angiosperms,floral transition is a key developmental transition from the vegetative to reproductive growth,and requires precise regulation to maximize the reproductive success.A complex regulatory network governs this transition through integrating flowering pathways in response to multiple exogenous and endogenous cues.Phytohormones are essential for proper plant developmental regulation and have been extensively studied for their involvement in the floral transition.Among various phytohormones,gibberellin(GA)plays a major role in affecting flowering in the model plant Arabidopsis thaliana.The GA pathway interact with other flowering genetic pathways and phytohormone signaling pathways through either DELLA proteins or mediating GA homeostasis.In this review,we summarize the recent advances in understanding the mechanisms of DELLA-mediated GA pathway in flowering time control in Arabidopsis,and discuss its possible link with other phytohormone pathways during the floral transition.     

Cytokinins: metabolism and function in plant adaptation to environmental stresses

In plants, the cytokinin (CK) phytohormones regulate numerous biological processes, including responses to environmental stresses, via a complex network of CK signaling. By an unknown mechanism, stress signals are perceived and transmitted through the His-Asp phosphorelay, an important component of the CK signal transduction pathway, triggering CK-responsive genes. Because of the intensive crosstalk between CKs and abscisic acid (ABA), modulation of CK levels and their signal transduction affects both ABA-dependent and ABA-independent pathways, enabling plant adaptation to adverse conditions. This review presents our current understanding of the functions of CKs and CK signaling in the regulation of plant adaptation to stress. Biotechnological strategies based on the modulation of CK levels have been examined with the aim of stabilizing agriculture yields.     

Cytokinin biosynthesis: A black box?

The biosynthetic origin of cytokinins (CKs) in plants has been debated ever since the recognition of CKs as plant hormones. Although several possible biosynthetic pathways have been suggested, none of the data published to date are conclusive. The enzymes involved in CK biosynthesis have not yet been purified and characterized in detail, nor have plant genes encoding CK biosynthetic enzymes been identified.     

Microscale magnetic microparticle‐based immunopurification of cytokinins from Arabidopsis root apex

Cytokinins (CKs) are pivotal plant hormones that have crucial roles in plant growth and development. However, their isolation and quantification are usually challenging because of their extremely low levels in plant tissues (pmol g^?1 fresh weight). We have developed a simple microscale magnetic immunoaffinity‐based method for selective one‐step isolation of CKs from very small amounts of plant tissue (less than 0.1 mg fresh weight). The capacity of the immunosorbent and the effect of the complex plant matrix on the yield of the rapid one‐step purification were tested using a wide range of CK concentrations. The total recovery range of the new microscale isolation procedure was found to be 30–80% depending on individual CKs. Immunoaffinity extraction using group‐specific monoclonal CK antibodies immobilized onto magnetic microparticles was combined with a highly sensitive ultrafast mass spectrometry‐based method with a detection limit close to one attomole. This combined approach allowed metabolic profiling of a wide range of naturally occurring CKs (bases, ribosides and N⁹‐glucosides) in 1.0‐mm sections of the Arabidopsis thaliana root meristematic zone. The magnetic immunoaffinity separation method was shown to be a simple and extremely fast procedure requiring minimal amounts of plant tissue.     

Ustilago maydis Produces Cytokinins and Abscisic Acid for Potential Regulation of Tumor Formation in Maize

The infection of maize (Zea mays) by the basidiomycete fungus Ustilago maydis leads to common smut of corn characterized by the production of tumors in susceptible aboveground plant tissues. LC-(ES)MS/MS profiles of abscisic acid (ABA) and 12 different cytokinins (CKs) were determined for infected and uninfected maize tissues over a time course following fungal exposure. Samples were taken at points corresponding to the appearance of disease symptoms. Axenic cultures of haploid and dikaryon forms of U. maydis were also profiled. This study confirmed the capability of Ustilago maydis to synthesize CKs, ABA, and auxin (IAA). It also provided evidence for the involvement of CK and ABA in the U. maydis-maize infection process. Significant quantities of CKs and ABA were detected from axenic cultures of U. maydis as was IAA. CKs and ABA levels were elevated in leaves and stems of maize after infection; notable was the high level of cis-zeatin 9-riboside. Variation among hormone profiles of maize tissues was observed at different time points during infection and between infections with nonpathogenic haploid and pathogenic dikaryon strains. This suggested that CKs and ABA accumulate and are likely metabolized in maize tissue infected with U. maydis. Because U. maydis produced these phytohormones at significant levels, it is possible that the fungal pathogen is a source of these compounds in infected tissue. This is the first study to confirm the production of CKs and document the production of ABA by U. maydis. This study also established an involvement of these phytohormones and a possible functional role for ABA in U. maydis infection of maize.     

The B‐box protein BBX19 suppresses seed germination via induction of ABI5

Seed germination is a fundamental process in the plant life cycle and is regulated by functionally opposing internal and external inputs. Here we explored the role of a negative regulator of photomorphogenesis, a B‐box‐containing protein (BBX19), as a molecular link between the inhibitory action of the phytohormone abscisic acid (ABA) and the promoting role of light in germination. We show that seeds of BBX19‐overexpressing lines, in contrast to those of BBX19 RNA interference lines, display ABA hypersensitivity, albeit independently of elongated hypocotyl 5 (HY5). Moreover, we establish that BBX19 functions neither via perturbation of GA signaling, the ABA antagonistic phytohormone, nor through interference with the DELLA protein germination repressors. Rather, BBX19 functions as an inducer of ABA INSENSITIVE5 (ABI5) by binding to the light‐responsive GT1 motifs in the gene promoter. In summary, we identify BBX19 as a regulatory checkpoint, directing diverse developmental processes and tailoring adaptive responses to distinct endogenous and exogenous signals.     

Comprehensive transcriptome analysis of phytohormone biosynthesis and signaling genes in microspore/pollen and tapetum of rice

To investigate the involvement of phytohormones during rice microspore/pollen (MS/POL) development, endogenous levels of IAA, gibberellins (GAs), cytokinins (CKs) and abscisic acid (ABA) in the mature anther were analyzed. We also analyzed the global expression profiles of genes related to seven phytohormones, namely auxin, GAs, CKs, brassinosteroids, ethylene, ABA and jasmonic acids, in MS/POL and tapetum (TAP) using a 44K microarray combined with a laser microdissection technique (LM-array analysis). IAA and GA(4) accumulated in a much higher amount in the mature anther compared with the other tissues, while CKs and ABA did not. LM-array analysis revealed that sets of genes required for IAA and GA synthesis were coordinately expressed during the later stages of MS/POL development, suggesting that these genes are responsible for the massive accumulation of IAA and GA(4) in the mature anther. In contrast, genes for GA signaling were preferentially expressed during the early developmental stages of MS/POL and throughout TAP development, while their expression was down-regulated at the later stages of MS/POL development. In the case of auxin signaling genes, such mirror-imaged expression observed in GA synthesis and signaling genes was not observed. IAA receptor genes were mostly expressed during the late stages of MS/POL development, and various sets of AUX/IAA and ARF genes were expressed during the different stages of MS/POL or TAP development. Such cell type-specific expression profiles of phytohormone biosynthesis and signaling genes demonstrate the validity and importance of analyzing the expression of phytohormone-related genes in individual cell types independently of other cells/tissues.     

Plant organ senescence – regulation by manifold pathways

Senescence is the final stage of plant ontogeny before death. Senescence may occur naturally because of age or may be induced by various endogenous and exogenous factors. Despite its destructive character, senescence is a precisely controlled process that follows a well‐defined order. It is often inseparable from programmed cell death (PCD), and a correlation between these processes has been confirmed during the senescence of leaves and petals. Despite suggestions that senescence and PCD are two separate processes, with PCD occurring after senescence, cell death responsible for senescence is accompanied by numerous changes at the cytological, physiological and molecular levels, similar to other types of PCD. Independent of the plant organ analysed, these changes are focused on initiating the processes of cellular structural degradation via fluctuations in phytohormone levels and the activation of specific genes. Cellular structural degradation is genetically programmed and dependent on autophagy. Phytohormones/plant regulators are heavily involved in regulating the senescence of plant organs and can either promote [ethylene, abscisic acid (ABA), jasmonic acid (JA), and polyamines (PAs)] or inhibit [cytokinins (CKs)] this process. Auxins and carbohydrates have been assigned a dual role in the regulation of senescence, and can both inhibit and stimulate the senescence process. In this review, we introduce the basic pathways that regulate senescence in plants and identify mechanisms involved in controlling senescence in ephemeral plant organs. Moreover, we demonstrate a universal nature of this process in different plant organs; despite this process occurring in organs that have completely different functions, it is very similar. Progress in this area is providing opportunities to revisit how, when and which way senescence is coordinated or decoupled by plant regulators in different organs and will provide a powerful tool for plant physiology research.     

Amplification of ABA biosynthesis and signaling through a positive feedback mechanism in seeds

Abscisic acid is an essential hormone for seed dormancy. Our previous study using the plant gene switch system, a chemically induced gene expression system, demonstrated that induction of 9‐cis‐epoxycarotenoid dioxygenase (NCED), a rate‐limiting ABA biosynthesis gene, was sufficient to suppress germination in imbibed Arabidopsis seeds. Here, we report development of an efficient experimental system that causes amplification of NCED expression during seed maturation. The system was created with a Triticum aestivum promoter containing ABA responsive elements (ABREs) and a Sorghum bicolor NCED to cause ABA‐stimulated ABA biosynthesis and signaling, through a positive feedback mechanism. The chimeric gene pABRE:NCED enhanced NCED and ABF (ABRE‐binding factor) expression in Arabidopsis Columbia‐0 seeds, which caused 9‐ to 73‐fold increases in ABA levels. The pABRE:NCED seeds exhibited unusually deep dormancy which lasted for more than 3 months. Interestingly, the amplified ABA pathways also caused enhanced expression of Arabidopsis NCED5, revealing the presence of positive feedback in the native system. These results demonstrated the robustness of positive feedback mechanisms and the significance of NCED expression, or single metabolic change, during seed maturation. The pABRE:NCED system provides an excellent experimental system producing dormant and non‐dormant seeds of the same maternal origin, which differ only in zygotic ABA. The pABRE:NCED seeds contain a GFP marker which enables seed sorting between transgenic and null segregants and are ideal for comparative analysis. In addition to its utility in basic research, the system can also be applied to prevention of pre‐harvest sprouting during crop production, and therefore contributes to translational biology.     

Exogenous phytohormones and the induction of plant galls by insects

The mechanism of gall induction by insects has remained elusive. Previous studies have met with limited success in attempting to induce galls by injection or application of chemical compounds. To determine whether an exogenous source of phytohormones plays a role in gall induction, we injected cytokinin (CK), auxin (IAA), gibberellic acid (GA), and abscisic acid (ABA) in various concentrations, ratios, and combinations into leaf petioles of Capsicum annuum L. cv. California Wonder (Solanaceae). We found that CK + IAA injections lead to gall-like growth in C. annuum. GA enhanced and ABA inhibited gall growth except in the presence of GA. Isopentenyl adenine (IP) was the most effective type of CK at inducing growth. Our work is consistent with the hypothesis that exogenous CK + IAA produced and supplied by insects leads to gall induction. We hypothesize that insects have obtained the capability to induce galls via acquisition of the biosynthetic pathways to produce IAA and trans-zeatin family CKs through microbial symbiosis or lateral gene transfer.     

Euglena gracilis (Euglenophyceae) produces abscisic acid and cytokinins and responds to their exogenous application singly and in combination with other growth regulators

The main objective of this study was to determine the optimal concentrations of a wide spectrum of exogenous phytohormones for effective stimulation of cell division and production of maximum cell yield in Euglena gracilis Klebs cultured in vitro. Results indicate that two hormones combined exert more effective growth stimulation than a single hormone or three, four or five different hormones combined. Specifically, trans-zeatin at 10^?7 M combined with abscisic acid at 10^?9 M produced optimal conditions for growth, yielding the maximum cell concentration. High concentrations of exogenous phytohormones were toxic to Euglena. The addition of trans-zeatin, N6-isopentenyladenine, and benzylaminopurine to Euglena cultures resulted in dense, dark green chloroplasts, suggesting that exogenous phytohormones increased the production of chlorophyll. Given the response to exogenous growth regulators, the study identified and quantified the types of endogenous cytokinins (CKs) and abscisic acid (ABA) synthesized in vitro by Euglena gracilis. HPLC-(ESI) MS/MS analysis revealed that the algal cells produced and released into the medium a mixture of CKs and ABA. The main CKs identified in the cell pellets and supernatant samples were from a t-RNA degradation pathway and included: cis-zeatin (cZ) derivatives cZR, cZNT, MeSZ and MeSZR, and to a lesser extent, the free base N6-isopentenyladenine (iP) and its derivatives iPR, iPNT, MeSiP and MeSiPA. A positive response to ABA, and the relatively high levels detected in E. gracilis, suggest that this hormone is important for alleviating stress conditions of in vitro culture that might otherwise restrict cell division.