Analysis of gibberellins as free acids by ultra performance liquid chromatography–tandem mass spectrometry

AbstractA robust, reliable and high-throughput method for extraction and purification of gibberellins (GAs), a group of tetracyclic diterpenoid carboxylic acids that include endogenous growth hormones, from plant material was developed. The procedure consists of two solid-phase extraction steps (Oasis® MCX-HLB and Oasis® MAX) and gives selective enrichment and efficient clean-up of these compounds from complex plant extracts. The method was tested with plant extracts of Brassica napus and Arabidopsis thaliana, from which total recovery of internal standards of about 72% was achieved. A rapid baseline chromatographic separation of 20 non-derivatised GAs by ultra performance liquid chromatography is also presented where a reversed-phase chromatographic column Acquity CSH® and a mobile phase consisting of methanol and aqueous 10mM-ammonium formate is used. This method enables sensitive and precise quantitation of GAs by MS/MS in multiple-reaction monitoring mode (MRM) by a standard isotope dilution method. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. All studied GAs were determined as free acids giving dominant quasi-molecular ions of [M–H]− with limits of detection ranging between 0.08 and 10fmol and linear ranges over four orders of magnitude. Taking advantage of highly effective chromatographic separation of 20GAs and very sensitive mass spectrometric detection, the presented bioanalytical method serves as a useful tool for plant biologists studying the physiological roles of these hormones in plant development

Phytohormones in Sweet Cherry Buds During Winter Rest and Bud Development

This (two-season) study was undertaken to assess the involvement of gibberellins (GAs), cytokinins (CKs), and auxins (AX) in dormancy of the sweet cherry buds ‘Summit’. Our hypothesis consisted in the assumption that representatives of these hormone groups are able to mark the transition between different dormancy phases. Changes in the transition between endo- and ecodormancy and the stages of ontogenetic development were not recognizable by bioactive GA1, GA5, GA7. The transient increase of GA3 during ecodormancy might be interpreted as an indication of the preservation of ecodormancy. The content of the biological active bases tZ, cZ, and DHZ was equal between endo- and ecodormancy. However, the content increased significantly in the first phase of ontogenetic development. The summation of the representatives of the various CKs (total iP-type, total tZ-type, total CK bases, total CK ribosides, total CK nucleotides, total O-glucosides, total N-glucosides, and total CKs) showed no differences regarding their levels during endo- and ecodormancy. These values increased markedly in the subsequent phase. AX increased after ecodormancy. By contrast, from side green until open cluster no differences occurred. As shown for AX, the content of oxIAA increased after ecodormancy. The content of IAAsp was low during endodormancy and increased transiently during ecodormancy and early ontogenetic development. This study revealed that no changes in the content of different bioactive GAs (exception GA3), CKs, and AX occurred during winter rest, and more precisely, during endo- and ecodormancy. These metabolites, therefore, are not suitable to differentiate between these dormancy phases. The ontogenesis is accompanied by specific changes in the content of bioactive molecules, precursors, and conjugation products.Peer Reviewe

The dynamics of cytokinin changes after grafting of vegetative apices on flowering rapeseed plants

Despite numerous studies, the role of hormones in the induction of shoot apical meristem leading to reproductive development, especially regarding thermoperiodic plants, is still not fully understood. The key problem is separating the effects of the low temperature required for vernalization from those responsible for low temperature stress. An earlier experiment demonstrated the correlation between an increase of cytokinin level in the apical parts of winter rapeseed and the transition time into their reproductive phase during vernalization, i.e., low temperature treatment. From data obtained from the presented experiments, this study aims to contribute to the understanding the role of cytokinins in the induction of flowering based on the grafting of vegetative apical parts of winter rapeseed (scion) on the reproductive (stock) winter and spring genotypes. On the basis of analyses carried out using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry in combination with microscopic observation of changes at the apical meristem, it was indicated that the increase in the amount of trans-zeatin and trans- and cis-zeatin-O-glucoside derivatives appeared in the early stages of apex floral differentiation. During further development, the content of all investigated cytokinins passed through the maximum level followed by their decrease. The final level in reproductive apices was found to be higher than that in vegetative ones

Contents of endogenous brassinosteroids and the response to drought and/or exogenously applied 24-epibrassinolide in two different maize leaves

Exogenously applied brassinosteroids (BRs) improve plant response to drought. However, many important aspects of this process, such as the potential differences caused by different developmental stages of analyzed organs at the beginning of drought, or by BR application before or during drought, remain still unexplored. The same applies for the response of different endogenous BRs belonging to the C27, C28-and C29- structural groups to drought and/or exogenous BRs. This study examines the physiological response of two different leaves (younger and older) of maize plants exposed to drought and treated with 24-epibrassinolide (epiBL), together with the contents of several C27, C28-and C29-BRs. Two timepoints of epiBL application (prior to and during drought) were utilized to ascertain how this could affect plant drought response and the contents of endogenous BRs. Marked differences in the contents of individual BRs between younger and older maize leaves were found: the younger leaves diverted their BR biosynthesis from C28-BRs to C29-BRs, probably at the very early biosynthetic steps, as the levels of C28-BR precursors were very low in these leaves, whereas C29-BR levels vere extremely high. Drought also apparently negatively affected contents of C28-BRs (particularly in the older leaves) and C29-BRs (particularly in the younger leaves) but not C27-BRs. The response of these two types of leaves to the combination of drought exposure and the application of exogenous epiBL differed in some aspects. The older leaves showed accelerated senescence under such conditions reflected in their reduced chlorophyll content and diminished efficiency of the primary photosynthetic processes. In contrast, the younger leaves of well-watered plants showed at first a reduction of proline levels in response to epiBL treatment, whereas in drought-stressed, epiBL pre-treated plants they were subsequently characterized by elevated amounts of proline. The contents of C29- and C27-BRs in plants treated with exogenous epiBL depended on the length of time between this treatment and the BR analysis regardless of plant water supply; they were more pronounced in plants subjected to the later epiBL treatment. The application of epiBL before or during drought did not result in any differences of plant response to this stressor

Analysis of plant growth-promoting properties of Bacillus amyloliquefaciens UCMB5113 using Arabidopsis thaliana as host plant

This study showed that Bacillus amyloliquefaciens UCMB5113 colonizing Arabidopsis roots changed root structure and promoted growth implying the usability of this strain as a novel tool to support sustainable crop production.Root architecture plays a crucial role for plants to ensure uptake of water, minerals and nutrients and to provide anchorage in the soil. The root is a dynamic structure with plastic growth and branching depending on the continuous integration of internal and environmental factors. The rhizosphere contains a complex microbiota, where some microbes can colonize plant roots and support growth and stress tolerance. Here, we report that the rhizobacterium Bacillus amyloliquefaciens subsp. plantarum UCMB5113 stimulated the growth of Arabidopsis thaliana Col-0 by increased lateral root outgrowth and elongation and root-hair formation, although primary root elongation was inhibited. In addition, the growth of the above ground tissues was stimulated by UCMB5113. Specific hormone reporter gene lines were tested which suggested a role for at least auxin and cytokinin signaling during rhizobacterial modulation of Arabidopsis root architecture. UCMB5113 produced cytokinins and indole-3-acetic acid, and the formation of the latter was stimulated by root exudates and tryptophan. The plant growth promotion effect by UCMB5113 did not appear to depend on jasmonic acid in contrast to the disease suppression effect in plants. UCMB5113 exudates inhibited primary root growth, while a semi-purified lipopeptide fraction did not and resulted in the overall growth promotion indicating an interplay of many different bacterial compounds that affect the root growth of the host plant. This study illustrates that beneficial microbes interact with plants in root development via classic and novel signals

Gibberellin–Abscisic Acid Balances during Arbuscular Mycorrhiza Formation in Tomato

Plant hormones have become appropriate candidates for driving functional plant mycorrhization programs, including the processes that regulate the formation of arbuscules in arbuscular mycorrhizal (AM) symbiosis. Here, we examine the role played by ABA/GA interactions regulating the formation of AM in tomato. We report differences in ABA and GA metabolism between control and mycorrhizal roots. Active synthesis and catabolism of ABA occur in AM roots. GAs level increases as a consequence of a symbiosis-induced mechanism that requires functional arbuscules which in turn is dependent on a functional ABA pathway. A negative interaction in their metabolism has been demonstrated. ABA attenuates GA-biosynthetic and increases GA-catabolic gene expression leading to a reduction in bioactive GAs. Vice versa, GA activated ABA catabolism mainly in mycorrhizal roots. The negative impact of GA3 on arbuscule abundance in wild-type plants is partially offset by treatment with ABA and the application of a GA biosynthesis inhibitor rescued the arbuscule abundance in the ABA-deficient sitiens mutant. These findings, coupled with the evidence that ABA application leads to reduce bioactive GA1, support the hypothesis that ABA could act modifying bioactive GA level to regulate AM. Taken together, our results suggest that these hormones perform essential functions and antagonize each other by oppositely regulating AM formation in tomato roots.This study was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT) and Fondos Europeos de Desarrollo Regional (FEDER) through the Ministerio de Economía y Competitividad in Spain (AGL2008-00742; AGL2011-25930) as well as the Junta de Andalucía (Research Group BIO 260). JM-R was supported by a research fellowship from the FPU-MICINN program. The work on hormone measurements was funded by a Grant Agency of the Czech Republic (grant no. 14-34792S) and by the Ministry of Education, Youth and Sports of the Czech Republic through the National Program for Sustainability I (Nr. LO1204).Peer reviewedPeer Reviewe

Distinct hormonal and morphological control of dormancy and germination in Chenopodium album dimorphic seeds

Dormancy and heteromorphism are innate seed properties that control germination timing through adaptation to the prevailing environment. The degree of variation in dormancy depth within a seed population differs considerably depending on the genotype and maternal environment. Dormancy is therefore a key trait of annual weeds to time seedling emergence across seasons. Seed heteromorphism, the production of distinct seed morphs (in color, mass or other morphological characteristics) on the same individual plant, is considered to be a bet-hedging strategy in unpredictable environments. Heteromorphic species evolved independently in several plant families and the distinct seed morphs provide an additional degree of variation. Here we conducted a comparative morphological and molecular analysis of the dimorphic seeds (black and brown) of the Amaranthaceae weed Chenopodium album. Freshly harvested black and brown seeds differed in their dormancy and germination responses to ambient temperature. The black seed morph of seedlot #1 was dormant and 2/3rd of the seed population had non-deep physiological dormancy which was released by after-ripening (AR) or gibberellin (GA) treatment. The deeper dormancy of the remaining 1/3rd non-germinating seeds required in addition ethylene and nitrate for its release. The black seeds of seedlot #2 and the brown seed morphs of both seedlots were non-dormant with 2/3rd of the seeds germinating in the fresh mature state. The dimorphic seeds and seedlots differed in testa (outer seed coat) thickness in that thick testas of black seeds of seedlot #1 conferred coat-imposed dormancy. The dimorphic seeds and seedlots differed in their abscisic acid (ABA) and GA contents in the dry state and during imbibition in that GA biosynthesis was highest in brown seeds and ABA degradation was faster in seedlot #2. Chenopodium genes for GA and ABA metabolism were identified and their distinct transcript expression patterns were quantified in dry and imbibed C. album seeds. Phylogenetic analyses of the Amaranthaceae sequences revealed a high proportion of expanded gene families within the Chenopodium genus. The identified hormonal, molecular and morphological mechanisms and dormancy variation of the dimorphic seeds of C. album and other Amaranthaceae are compared and discussed as adaptations to variable and stressful environments

Correlations between Phytohormones and Drought Tolerance in Selected Brassica Crops: Chinese Cabbage, White Cabbage and Kale

Drought is one of the major abiotic stresses affecting the productivity of Brassica crops. To understand the role of phytohormones in drought tolerance, we subjected Chinese cabbage (B. rapa ssp. pekinensis), white cabbage (B. oleracea var. capitata), and kale (B. oleracea var. acephala) to droughta nd examined the stress response on the physiological, biochemical and hormonal levels. The phytohormones abscisic acid (ABA), auxin indole-3-acetic acid (IAA), brassinosteroids (BRs), cytokinins (CKs), jasmonates (JAs), and salicylic acid (SA) were analyzed by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). Based on the physiological and biochemical markers the Chinese cabbage exhibited the lowest tolerance, followed by the white cabbage, while the kale appeared to be the most tolerant to drought. The drought tolerance of the kale correlated with increased levels of SA, ABA, IAA, CKs iP(R) and cZ(R), and typhasterol (TY), a precursor of active BRs. In contrast, the drought sensitivity of the Chinese cabbage correlated with a significant increase in ABA, JAs and the active BRs castasterol (CS) and brassinolide (BL). The moderately tolerant white cabbage, positioned between the kale and Chinese cabbage, showed more similarity in terms of the phytohormone patterns with the kale. We concluded that the drought tolerance in Brassicaceae is mostly determined by the increased endogenous levels of IAA, CKs, ABA and SA and the decreased levels of active BRs

The Arabidopsis RLCK VI_A2 Kinase Controls Seedling and Plant Growth in Parallel with Gibberellin

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation