In Vitro Propagation of Threatened Agave Species Using a Two-Stage System.

Agaves are plants with multiple possibilities of use and are naturally tolerant to low water availability conditions and high temperatures. This makes them species of great interest in the context of the necessary substitution of crops due to climate change. Unfortunately, the overexploitation of wild specimens has endangered many species of the genus that have not been domesticated or cultivated intensively. In vitro mass culture and propagation techniques have emerged as a very efficient option to produce agave plants that can be used without damage to the natural populations. A protocol is presented here for the in vitro micropropagation of agaves in a two-stage process. In the first step, clusters of slightly differentiated shoots are generated from stem segments cultivated on a semisolid medium added with cytokinin. In a second step, these shoot clusters are cultured in temporary immersion bioreactors where they grow and complete their differentiation, and then the shoots are rooted and transferred to soil. This protocol has been successfully applied to several threatened species of the Agave genus.

Comprehensive elucidation of the differential physiological kale response to cytokinins under in vitro conditions.

BACKGROUND: Kale, a versatile cruciferous crop, valued for its pro-health benefits, stress resistance, and potential applications in forage and cosmetics, holds promise for further enhancement of its bioactive compounds through in vitro cultivation methods. Micropropagation techniques use cytokinins (CKs) which are characterized by various proliferative efficiency. Despite the extensive knowledge regarding CKs, there remains a gap in understanding their role in the physiological mechanisms. That is why, here we investigated the effects of three CKs - kinetin (Kin), 6-benzylaminopurine (BAP), and 2-isopentenyladenine (2iP) - on kale physiology, antioxidant status, steroidal metabolism, and membrane integrity under in vitro cultivation. RESULTS: Our study revealed that while BAP and 2iP stimulated shoot proliferation, they concurrently diminished pigment levels and photosynthetic efficiency. Heightened metabolic activity in response to all CKs was reflected by increased respiratory rate. Despite the differential burst of ROS, the antioxidant properties of kale were associated with the upregulation of guaiacol peroxidase and the scavenging properties of ascorbate rather than glutathione. Notably, CKs fostered the synthesis of sterols, particularly sitosterol, pivotal for cell proliferation and structure of membranes which are strongly disrupted under the action of BAP and 2iP possibly via pathway related to phospholipase D and lipoxygenase which were upregulated. Intriguingly, both CKs treatment spurred the accumulation of sitostenone, known for its ROS scavenging and therapeutic potential. The differential effects of CKs on brassicasterol levels and brassinosteroid (BRs) receptor suggest potential interactions between CKs and BRs. CONCLUSION: Based on the presented results we conclude that the effect evoked by BAP and 2iP in vitro can improve the industrial significance of kale because this treatment makes possible to control proliferation and/or biosynthesis routes of valuable beneficial compounds. Our work offers significant insights into the nuanced effects of CKs on kale physiology and metabolism, illuminating potential avenues for their application in plant biotechnology and medicinal research.

AZGs: a new family of cytokinin transporters.

Cytokinins (CKs) are phytohormones structurally similar to purines that play important roles in various aspects of plant physiology and development. The local and long-distance distribution of CKs is very important to control their action throughout the plant body. Over the past decade, several novel CK transporters have been described, many of which have been linked to a physiological function rather than simply their ability to transport the hormone in vitro. Purine permeases, equilibrative nucleotide transporters and ATP-binding cassette transporters are involved in the local and long-range distribution of CK. In addition, members of the Arabidopsis AZA-GUANINE RESISTANT (AZG) protein family, AZG1 and AZG2, have recently been shown to mediate CK uptake at the plasma membrane and endoplasmic reticulum. Despite sharing ∼50% homology, AZG1 and AZG2 have unique transport mechanisms, tissue-specific expression patterns, and subcellular localizations that underlie their distinct physiological functions. AZG2 is expressed in a small group of cells in the overlying tissue around the lateral root primordia, where its expression is induced by auxins and it is involved in the regulation of lateral root growth. AZG1 is ubiquitously expressed, with high levels in the division zone of the root apical meristem. Here, it binds and stabilises the auxin efflux carrier PIN1, thereby shaping root architecture, particularly under salt stress. This review highlights the latest findings on the protein properties, transport mechanisms and cellular functions of this new family of CK transporters and discusses perspectives for future research in this field.

The effect of constitutive root isoprene emission on root phenotype and physiology under control and salt stress conditions.

Isoprene, a volatile hydrocarbon, is typically emitted from the leaves of many plant species. Given its well-known function in plant growth and defense aboveground, we examined its effects on root physiology. We used isoprene-emitting (IE) lines and a non-emitting (NE) line of Arabidopsis and investigated their performance by analyzing root phenotype, hormone levels, transcriptome, and metabolite profiles under both normal and salt stress conditions. We show that IE lines emitted tiny amounts of isoprene from roots and showed an increased root/shoot ratio compared with NE line. Isoprene emission exerted a noteworthy influence on hormone profiles related to plant growth and stress response, promoting root development and salt-stress resistance. Methyl erythritol 4-phosphate pathway metabolites, precursors of isoprene and hormones, were higher in the roots of IE lines than in the NE line. Transcriptome data indicated that the presence of isoprene increased the expression of key genes involved in hormone metabolism/signaling. Our findings reveal that constitutive root isoprene emission sustains root growth under saline conditions by regulating and/or priming hormone biosynthesis and signaling mechanisms and expression of key genes relevant to salt stress defense.

Cytokinins enhance the metabolic activity of in vitro-grown catmint (Nepeta nuda L.).

The phytohormones cytokinins are essential mediators of developmental and environmental signaling, primarily during cell division and endophytic interactions, among other processes. Considering the limited understanding of the regulatory mechanisms that affect the growth and bioactivity of the medicinal plant Nepeta nuda (Lamiaceae), our study aimed to explore how cytokinins influence the plant's metabolic status. Exogenous administration of active cytokinin forms on in vitro N. nuda internodes stimulated intensive callus formation and de novo shoot regeneration, leading to a marked increase in biomass. This process involved an accumulation of oxidants, which were scavenged by peroxidases using phenolics as substrates. The callus tissue formed upon the addition of the cytokinin 6-benzylaminopurine (BAP) acted as a sink for sugars and phenolics during the allocation of nutrients between the culture medium and regenerated plants. In accordance, the cytokinin significantly enhanced the content of polar metabolites and their respective in vitro biological activities compared to untreated in vitro and wild-grown plants. The BAP-mediated accumulation of major phenolic metabolites, rosmarinic acid (RA) and caffeic acid (CA), corresponded with variations in the expression levels of genes involved in their biosynthesis. In contrast, the accumulation of iridoids and the expression of corresponding biosynthetic genes were not significantly affected. In conclusion, our study elucidated the mechanism of cytokinin action in N. nuda in vitro culture and demonstrated its potential in stimulating the production of bioactive compounds. This knowledge could serve as a basis for further investigations of the environmental impact on plant productivity.

Cytokinins Reduce Viral Replication and Alter Plaque Morphology of Frog Virus 3 In Vitro.

Cytokinins (CKs) are a group of N6-substituted signaling molecules whose biosynthesis and metabolism have been documented in all kingdoms of life, including vertebrates. While their biological relevance in vertebrate systems continues to be elucidated, they have broadly been documented with therapeutic effects in exogenous applications. In this study, we evaluated the virostatic potential of four types of CKs including, N6-isopentenyladenine (iP), N6-isopentenyladenosine (iPR), N6-isopentenyladenosine-5'monophosphate (iPMP), and 2-methylthiol-N6-isopentenyladenosine (2MeSiPR) against the ranavirus type species, frog virus 3 (FV3). Following concurrent treatment and infection, iP and iPR reduced viral replication by 33.8% and 59.6%, respectively, in plaque formation assays. A decrease in viral replication was also observed when CK exposure was limited to 12 h prior to infection, where iP and iPR reduced viral replication by 31% and 23.75%, respectively. Treatment with iP and iPR was also marked by 48% and 60% decreases in viral load over 72 h, respectively, as measured in single step growth curves. Plaque morphology was altered in vitro, as iP and iPR treatment increased plaque area by 83% and 112% with lytic zone formation also becoming more prevalent in corresponding treatments. Treatment with iPMP and 2MeSiPR resulted in no effect on viral kinetics in vitro. The results of this study are the first to provide evidence of CK antiviral activity against a DNA virus and highlight the importance of their structure for therapeutic investigations.

Modulating ascorbic acid levels to optimize somatic embryogenesis in Picea abies (L.) H. Karst. Insights into oxidative stress and endogenous phytohormones regulation.

Global warming has adversely affected Picea abies (L.) H. Karst. forests in Europe, prompting the need for innovative forest-breeding strategies. Somatic embryogenesis (SE) offers promise but requires protocol refinement. Understanding the molecular mechanisms governing somatic embryo development is essential, as oxidative stress plays a crucial role in SE regulation. Ascorbic acid (ASA), is a vital antioxidant that can potentially control oxidative stress. In the present study, we normalized ASA concentrations in induction and proliferation media to enhance embryogenic tissue (ET) regeneration and proliferation capacity of mature explants. The media were supplemented with ASA at 0 mg l-1, 25 mg l-1, 50 mg l-1, 100 mg l-1, and 200 mg l-1. The accumulation of hydrogen peroxide (H2O2) and endogenous phytohormones, including auxins, cytokinins, brassinosteroids, abscisic acid, and gibberellin, was measured in non-embryonic calli and ET. Subsequently, their impact on ET induction and multiplication was analyzed. Our results demonstrate that application of ASA at concentrations of 25 mg l-1 and 200 mg l-1 led to increased H2O2 levels, potentially inducing oxidative stress while simultaneously reducing the levels of all endohormone groups. Notably, the highest ET induction frequency (approximately 70%) was observed for ASA at 50 mg l-1. These findings will enhance SE induction procedures, particularly in more resistant explants, underscoring the significance of ASA application to culture media.

Zonal Chemical Signal Pathways Mediating Floral Induction in Apple.

Phytohormones that trigger or repress flower meristem development in apple buds are thought to be locally emitted from adjacent plant tissues, including leaves and fruitlets. The presence of fruitlets is known to inhibit adjacent buds from forming flowers and thus fruits. The resulting absence of fruitlets the following season restores flower-promoting signalling to the new buds. The cycle can lead to a biennial bearing behaviour of alternating crop loads in a branch or tree. The hormonal stimuli that elicit flowering is typically referred to as the floral induction (FI) phase in bud meristem development. To determine the metabolic pathways activated in FI, young trees of the cultivar 'Ruby Matilda' were subjected to zonal crop load treatments imposed to two leaders of bi-axis trees in the 2020/2021 season. Buds were collected over the expected FI phase, which is within 60 DAFB. Metabolomics profiling was undertaken to determine the differentially expressed pathways and key signalling molecules associated with FI in the leader and at tree level. Pronounced metabolic differences were observed in trees and leaders with high return bloom with significant increases in compounds belonging to the cytokinin, abscisic acid (ABA), phenylpropanoid and flavanol chemical classes. The presence of cytokinins, namely adenosine, inosine and related derivatives, as well as ABA phytohormones, provides further insight into the chemical intervention opportunities for future crop load management strategies via plant growth regulators.

Delayed senescence and crop performance under stress: always a functional couple?

Exposure to abiotic stresses accelerates leaf senescence in most crop plant species, thereby reducing photosynthesis and other assimilatory processes. In some cases, genotypes with delayed leaf senescence (i.e., "stay-greens") show stress resistance, particularly in cases of water deficit, and this has led to the proposal that senescence delay improves crop performance under some abiotic stresses. In this review, we summarize the evidence for increased resistance to abiotic stress, mostly water deficit, in genotypes with delayed senescence, and specifically focus on the physiological mechanisms and agronomic conditions under which the stay-green trait may ameliorate grain yield under stress.

Hormonal profiles in dormant turions of 22 aquatic plant species: do they reflect functional or taxonomic traits?

BACKGROUND AND AIMS: Turions are vegetative, dormant overwintering organs formed in aquatic plants in response to unfavourable ecological conditions. Contents of cytokinin (CK), auxin metabolites and abscisic acid (ABA) as main growth and development regulators were compared in innately dormant autumnal turions of 22 aquatic plant species of different functional ecological or taxonomic groups with those in non-dormant winter apices in three aquatic species and with those in spring turions of four species after their overwintering. METHODS: The hormones were analysed in miniature turion samples using ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry. KEY RESULTS: In innately dormant turions, the total contents of each of the four main CK types, biologically active forms and total CKs differed by two to three orders of magnitude across 22 species; the proportion of active CK forms was 0.18-67 %. Similarly, the content of four auxin forms was extremely variable and the IAA proportion as the active form was 0.014-99 %. The ABA content varied from almost zero to 54 µmol kg-1 dry weight and after overwintering it usually significantly decreased. Of all functional traits studied, hormone profiles depended most on the place of turion sprouting (surface vs bottom) and we suggest that this trait is crucial for turion ecophysiology. CONCLUSIONS: The key role of ABA in regulating turion dormancy was confirmed. However, the highly variable pattern of the ABA content in innately dormant and in overwintered turions indicates that the hormonal mechanism regulating the innate dormancy and its breaking in turions is not uniform within aquatic plants.

The Long-Distance Transport of Some Plant Hormones and Possible Involvement of Lipid-Binding and Transfer Proteins in Hormonal Transport.

Adaptation to changes in the environment depends, in part, on signaling between plant organs to integrate adaptive response at the level of the whole organism. Changes in the delivery of hormones from one organ to another through the vascular system strongly suggest that hormone transport is involved in the transmission of signals over long distances. However, there is evidence that, alternatively, systemic responses may be brought about by other kinds of signals (e.g., hydraulic or electrical) capable of inducing changes in hormone metabolism in distant organs. Long-distance transport of hormones is therefore a matter of debate. This review summarizes arguments for and against the involvement of the long-distance transport of cytokinins in signaling mineral nutrient availability from roots to the shoot. It also assesses the evidence for the role of abscisic acid (ABA) and jasmonates in long-distance signaling of water deficiency and the possibility that Lipid-Binding and Transfer Proteins (LBTPs) facilitate the long-distance transport of hormones. It is assumed that proteins of this type raise the solubility of hydrophobic substances such as ABA and jasmonates in hydrophilic spaces, thereby enabling their movement in solution throughout the plant. This review collates evidence that LBTPs bind to cytokinins, ABA, and jasmonates and that cytokinins, ABA, and LBTPs are present in xylem and phloem sap and co-localize at sites of loading into vascular tissues and at sites of unloading from the phloem. The available evidence indicates a functional interaction between LBTPs and these hormones.

High- or Low-Yielding F2 Progeny of Wheat Is Result of Specific TaCKX Gene Coexpression Patterns in Association with Grain Yield in Paternal Parent.

Members of the TaCKX gene family (GFM) encode oxidase/dehydrogenase cytokinin degrading enzymes (CKX), which play an important role in the homeostasis of phytohormones, affecting wheat development and productivity. Therefore, the objective of this investigation was to test how the expression patterns of the yield-related TaCKX genes and TaNAC2-5A (NAC2) measured in 7 days after pollination (DAP) spikes and the seedling roots of parents are inherited to apply this knowledge in the breeding process. The expression patterns of these genes were compared between parents and their F2 progeny in crosses of one mother with different paterns of awnless cultivars and reciprocal crosses of awned and awnless lines. We showed that most of the genes tested in the 7 DAP spikes and seedling roots of the F2 progeny showed paternal expression patterns in crosses of awnless cultivars as well as reciprocal crosses of awned and awnless lines. Consequently, the values of grain yield in the F2 progeny were similar to the pater; however, the values of seedling root mass were similar to the mother or both parents. The correlation analysis of TaCKX GFMs and NAC2 in spikes and spikes per seedling roots reveals that the genes correlate with each other specifically with the pater and the F2 progeny or the mother and the F2 progeny, which shape phenotypic traits. The numbers of spikes and semi-empty spikes are mainly correlated with the specific coexpression of the TaCKX and NAC2 genes expressed in spikes or spikes per roots of the pater and F2 progeny. Variable regression analysis of grain yield and root mass with TaCKX GFMs and NAC2 expressed in the tested tissues of five crosses revealed a significant dependency of these parameters on the mother and F2 and/or the pater and F2 progeny. We showed that the inheritance of yield-related traits depends on the specific cooperative expression of some TaCKX GFMs, in some crosses coupled with NAC2, and is strongly dependent on the genotypes used for the crosses. Indications for parental selection in the breeding of high-yielding lines are discussed.

Plant Regeneration via Adventitious Shoot Formation from Immature Zygotic Embryo Explants of Camelina.

Camelina is an oil seed crop that is enjoying increasing interest because it has a particularly valuable fatty acid profile, is modest regarding its water and nutrient requirements, and is comparatively resilient to abiotic and biotic stress factors. The regeneration of plants from cells accessible to genetic manipulation is an essential prerequisite for the generation of genetically engineered plants, be it by transgenesis or genome editing. Here, immature embryos were used on the assumption that their incomplete differentiation was associated with totipotency. In culture, regenerative structures appeared adventitiously at the embryos' hypocotyls. For this, the application of auxin- or cytokinin-type growth regulators was essential. The formation of regenerative structures was most efficient when indole-3-acetic acid was added to the induction medium at 1 mg/L, zygotic embryos of the medium walking stick stage were used, and their hypocotyls were stimulated by pricking to a wound response. Histological examinations revealed that the formation of adventitious shoots was initiated by locally activated cell division and proliferation in the epidermis and the outer cortex of the hypocotyl. While the regeneration of plants was established in principle using the experimental line Cam139, the method proved to be similarly applicable to the current cultivar Ligena, and hence it constitutes a vital basis for future genetic engineering approaches.

New Hybrid Ethylenediurea (EDU) Derivatives and Their Phytoactivity.

Natural and synthetic phytohormones are widely used in agriculture. The synthetic cytokinin ethylenediurea (EDU) induces protection in plants against ozone phytotoxicity. In our study, new hybrid derivatives of EDU were synthesized and tested for phytoactivity. The germination potential (Gp), germination of seeds (G), and relative water content in leaves (RWC), characterizing the drought resistance of plants, were determined. The results of laboratory studies showed that EDU and its hybrid derivatives have a positive effect on root length, the growth and development of shoots, as well as the ability of plants to tolerate stress caused by a lack of water.

Natural Products Biosynthesis by Streptomyces netropsis IMV Ac-5025 under Exogenous Sterol Action.

Streptomycetes are known as producers of bioactive substances, particularly antibiotics. Streptomyces netropsis IMV Ac-5025 simultaneously produces different classes of antibiotics, including polyene compounds, phytohormones, and sterols, but the metabolic pathways involved in their biosynthesis are largely understudied. The aim of this work was to explore the biosynthesis of polyene antibiotics, sterols, and phytohormones when the producer is cultivated in a nutrient medium supplemented with exogenous ß-sitosterol. Gas chromatography and high-performance liquid chromatography were applied to analyze the spectrum of bioactive compounds. The obtained results demonstrated not only an increase in the accumulation of biomass but also polyene antibiotics, intracellular sterols, auxins, and cytokinins, when cultivating S. netropsis IMV Ac-5025 in a liquid medium with the addition of ß-sitosterol. The amount of biomass raised 1.5-2-fold, whilst the sum of polyene antibiotics increased 4.5-fold, sterols' sum (ergosterol, cholesterol, stigmasterol, ß-sitosterol, and 24-epibrassinolide) by 2.9-fold, auxins' sum (indole-3-acetic acid, indole-3-acetic acid hydrazide, indole-3-carbinol, indole-3-butyric acid, indole-3-carboxaldehyde, and indole-3-carboxylic acid) by 6-fold, and cytokinins' sum (zeatin, isopentyladenine, zeatin riboside, and isopentenyladenosine) by 11-fold. Thus, we put forward the hypothesis that ß-sitosterol plays a regulatory role in the network of biosynthetic reactions of S. netropsis IMV Ac-5025.

Fungal-Mediated Biotransformation of the Plant Growth Regulator Forchlorfenuron by Cunninghamella elegans.

The synthetic cytokinin forchlorfenuron (FCF), while seemingly presenting relatively low toxicity for mammalian organisms, has been the subject of renewed scrutiny in the past few years due to its increasing use in fruit crops and potential for bioaccumulation. Despite many toxicological properties of FCF being known, little research has been conducted on the toxicological effects of its secondary metabolites. Given this critical gap in the existing literature, understanding the formation of relevant FCF secondary metabolites and their association with mammalian metabolism is essential. To investigate the formation of FCF metabolites in sufficient quantities for toxicological studies, a panel of four fungi were screened for their ability to catalyze the biotransformation of FCF. Of the organisms screened, Cunninghamella elegans (ATCC 9245), a filamentous fungus, was found to convert FCF to 4-hydroxyphenyl-forchlorfenuron, the major FCF secondary metabolite identified in mammals, after 26 days. Following the optimization of biotransformation conditions using a solid support system, media screening, and inoculation with a solid pre-formed fungal mass of C. elegans, this conversion time was significantly reduced to 7 days-representing a 73% reduction in total reaction time as deduced from the biotransformation products and confirmed by LC-MS, NMR spectroscopic data, as well as a comparison with synthetically prepared metabolites. Our study provides the first report of the metabolism of FCF by C. elegans. These findings suggest that C. elegans can produce FCF secondary metabolites consistent with those produced via mammalian metabolism and could be used as a more efficient, cost-effective, and ethical alternative for producing those metabolites in useful quantities for toxicological studies.

MsMYB62-like as a negative regulator of anthocyanin biosynthesis in Malus spectabilis.

Crabapple is a valuable tree species in gardens due to its captivating array of flower and leaf colors, rendering it a favored choice in landscaping. The economic and ornamental values of Malus crabapple are closely associated with the biosynthesis of anthocyanin, a pigment responsible for its vibrant hues. The intricate regulation of anthocyanin biosynthesis involves the concerted activity of various genes. However, the specific mechanism governing this process in crabapple warrants in-depth exploration. In this study, we explored the inhibitory role of MsMYB62-like in anthocyanin biosynthesis. We identified MsDFR and MsANS as two downstream target genes of MsMYB62-like. These genes encode enzymes integral to the anthocyanin biosynthetic pathway. The findings demonstrate that MsMYB62-like directly binds to the promoters of MsDFR and MsANS, resulting in the downregulation of their expression levels. Additionally, our observations indicate that the plant hormone cytokinins exert a suppressive effect on the expression levels of MsMYB62-like, while concurrently upregulating MsDFR and MsANS. This study reveals that the MsMYB62-like-MsDFR/MsANS module plays an important role in governing anthocyanin levels in Malus crabapple. Notably, the regulatory interplay is modulated by the plant hormone cytokinins.

Epigenetics and plant hormone dynamics - a functional and methodological perspective.

Plant hormones, pivotal regulators of plant growth, development, and response to environmental cues, have recently emerged as central modulators of epigenetic processes governing gene expression and phenotypic plasticity. This review addresses the complex interplay between plant hormones and epigenetic mechanisms, highlighting the diverse methodologies that have been harnessed to decipher these intricate relationships. We present a comprehensive overview to understand how phytohormones orchestrate epigenetic modifications, shaping plant adaptation and survival strategies. Conversely, we explore how epigenetic regulators ensure hormonal balance and regulate the signalling pathways of key plant hormones. Furthermore, our investigation includes a search for novel genes that are regulated by plant hormones under the control of epigenetic processes. Our review offers a contemporary overview of the epigenetic-plant hormone crosstalk, emphasizing its significance in plant growth, development, and potential agronomical applications.

OsNAC103, an NAC transcription factor negatively regulates plant height in rice.

MAIN CONCLUSION: OsNAC103 negatively regulates rice plant height by influencing the cell cycle and crosstalk of phytohormones. Plant height is an important characteristic of rice farming and is directly related to agricultural yield. Although there has been great progress in research on plant growth regulation, numerous genes remain to be elucidated. NAC transcription factors are widespread in plants and have a vital function in plant growth. Here, we observed that the overexpression of OsNAC103 resulted in a dwarf phenotype, whereas RNA interference (RNAi) plants and osnac103 mutants showed no significant difference. Further investigation revealed that the cell length did not change, indicating that the dwarfing of plants was caused by a decrease in cell number due to cell cycle arrest. The content of the bioactive cytokinin N6-Δ2-isopentenyladenine (iP) decreased as a result of the cytokinin synthesis gene being downregulated and the enhanced degradation of cytokinin oxidase. OsNAC103 overexpression also inhibited cell cycle progression and regulated the activity of the cell cyclin OsCYCP2;1 to arrest the cell cycle. We propose that OsNAC103 may further influence rice development and gibberellin-cytokinin crosstalk by regulating the Oryza sativa homeobox 71 (OSH71). Collectively, these results offer novel perspectives on the role of OsNAC103 in controlling plant architecture.