The role of strigolactones in photomorphogenesis of pea is limited to adventitious rooting

The recently discovered group of plant hormones, the strigolactones, have been implicated in regulating photomorphogenesis. We examined this extensively in our strigolactone synthesis and response mutants and could find no evidence to support a major role for strigolactone signaling in classic seedling photomorphogenesis (e.g. elongation and leaf expansion) in pea (Pisum sativum), consistent with two recent independent reports in Arabidopsis. However, we did find a novel effect of strigolactones on adventitious rooting in darkness. Strigolactone‐deficient mutants, Psccd8 and Psccd7, produced significantly fewer adventitious roots than comparable wild‐type seedlings when grown in the dark, but not when grown in the light. This observation in dark‐grown plants did not appear to be due to indirect effects of other factors (e.g. humidity) as the constitutively de‐etiolated mutant, lip1, also displayed reduced rooting in the dark. This role for strigolactones did not involve the MAX2 F‐Box strigolactone response pathway as Psmax2 f‐box mutants did not show a reduction in adventitious rooting in the dark compared with wild‐type plants. The auxin‐deficient mutant bushy also reduced adventitious rooting in the dark, as did decapitation of wild‐type plants. Rooting was restored by the application of indole‐3‐acetic acid (IAA) to decapitated plants, suggesting a role for auxin in the rooting response. However, auxin measurements showed no accumulation of IAA in the epicotyls of wild‐type plants compared with the strigolactone synthesis mutant Psccd8, suggesting that changes in the gross auxin level in the epicotyl are not mediating this response to strigolactone deficiency.     

Gibberellins inhibit adventitious rooting in hybrid aspen and Arabidopsis by affecting auxin transport

Knowledge of processes involved in adventitious rooting is important to improve both fundamental understanding of plant physiology and the propagation of numerous plants. Hybrid aspen (Populus tremula × tremuloïdes) plants overexpressing a key gibberellin (GA) biosynthesis gene (AtGA20ox1) grow rapidly but have poor rooting efficiency, which restricts their clonal propagation. Therefore, we investigated the molecular basis of adventitious rooting in Populus and the model plant Arabidopsis. The production of adventitious roots (ARs) in tree cuttings is initiated from the basal stem region, and involves the interplay of several endogenous and exogenous factors. The roles of several hormones in this process have been characterized, but the effects of GAs have not been fully investigated. Here, we show that a GA treatment negatively affects the numbers of ARs produced by wild‐type hybrid aspen cuttings. Furthermore, both hybrid aspen plants and intact Arabidopsis seedlings overexpressing AtGA20ox1, PttGID1.1 or PttGID1.3 genes (with a 35S promoter) produce few ARs, although ARs develop from the basal stem region of hybrid aspen and the hypocotyl of Arabidopsis. In Arabidopsis, auxin and strigolactones are known to affect AR formation. Our data show that the inhibitory effect of GA treatment on adventitious rooting is not mediated by perturbation of the auxin signalling pathway, or of the strigolactone biosynthetic and signalling pathways. Instead, GAs appear to act by perturbing polar auxin transport, in particular auxin efflux in hybrid aspen, and both efflux and influx in Arabidopsis.     

Abscisic acid promotes root system development in birch tissue culture: a comparison to aspen culture and conventional rooting‐related growth regulators

The research aim was to assess the effects of the plant hormone abscisic acid (ABA) and the growth regulator paclobutrazol (PBZ) on root system development during the in vitro culture of different birch and aspen genotypes. The studied genotypes involved two aspen (Populus tremula and Populus tremuloides × P. tremula) and two silver birch (Betula pendula) trees, with one of the birches characterized by its inability to root in vitro. For experiments, apical shoot segments were cultured on nutrient medium enriched with either ABA or PBZ. Additionally, the analysis of the endogenous hormones in shoots developed on hormone‐free medium was conducted by high‐performance liquid chromatography. The endogenous concentration of auxin indole‐3‐acetic acid was much higher in the aspens than that in the birches, while the highest concentration of ABA was found in the root‐forming birch. The culturing of this birch genotype on medium enriched with ABA resulted in an increased root length and a higher number of lateral roots without any negative effect on either shoot growth or adventitious root (AR) formation, although these two processes were largely inhibited by ABA in the aspens. Meanwhile, PBZ promoted AR formation in both aspen and birch cultures but impaired secondary root formation and shoot growth in birches. These results suggest the use of ABA for the in vitro rooting of birches and PBZ for the rooting of aspens.     

The major‐effect quantitative trait locus CsARN6.1 encodes an AAA ATPase domain‐containing protein that is associated with waterlogging stress tolerance by promoting adventitious root formation

In plants, the formation of hypocotyl‐derived adventitious roots (ARs) is an important morphological acclimation to waterlogging stress; however, its genetic basis remains fragmentary. Here, through combined use of bulked segregant analysis‐based whole‐genome sequencing, SNP haplotyping and fine genetic mapping, we identified a candidate gene for a major‐effect QTL, ARN6.1, that was responsible for waterlogging tolerance due to increased AR formation in the cucumber line Zaoer‐N. Through multiple lines of evidence, we show that CsARN6.1 is the most possible candidate for ARN6.1 which encodes an AAA ATPase. The increased formation of ARs under waterlogging in Zaoer‐N could be attributed to a non‐synonymous SNP in the coiled‐coil domain region of this gene. CsARN6.1 increases the number of ARs via its ATPase activity. Ectopic expression of CsARN6.1 in Arabidopsis resulted in better rooting ability and lateral root development in transgenic plants. Transgenic cucumber expressing the CsARN6.1^Asp allele from Zaoer‐N exhibited a significant increase in number of ARs compared with the wild type expressing the allele from Pepino under waterlogging conditions. Taken together, these data support that the AAA ATPase gene CsARN6.1 has an important role in increasing cucumber AR formation and waterlogging tolerance.     

High-frequency induction of adventitious shoots from hypocotyl segments ofLiquidambar styracifiua L. by thidiazuron

The effects of thidiazuron (TDZ) on adventitious bud and shoot formation from hypocotyl segments of sweetgum (Liquidambar styracifiua) were tested alone and in combination with 2,4-dichlorophenoxyacetic acid (2,4-D). The combination of 1 mg/1 TDZ with 0.01 mg/l 2,4-D resulted in the highest frequency of bud production. Lower concentrations of TDZ stimulated shoot production, generating the most shoots at 0.1 mg/1 TDZ with 0.01 mg/1 of 2,4-D. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to a shoot proliferation medium lacking TDZ or containing naphthaleneacetic acid and benzyladenine in addition to TDZ. Shoot production in liquid culture was significantly greater than that in solid culture. Comparisons of in vitro and ex vitro rooting of the adventitious shoots demonstrated that ex vitro rooting produced plants with faster growth rates and more extensive root systems.Abbreviations BA Benzyladenine - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyacetic acid     

Phytotoxic effects of Heterothalamus psiadioides (Asteraceae) essential oil on adventitious rooting

Essential oils have many functions in plant development; among them, they take part in plant–plant interactions, such as allelopathy. The phytotoxicity of the essential oil of Heterothalamus psiadioides was evaluated on Arabidopsis thaliana adventitious rooting. This development process has demonstrated great sensitivity to phytotoxic essential oils and has been primarily studied by our research group. The essential oil of H. psiadioides showed highly negative effects on A. thaliana wild type (WT) adventitious rooting in very small amounts. As the hormone auxin has an important role on adventitious rooting, A. thaliana mutant superroot (sur1) that overproduces indole-3-acetic acid was tested to verify if it could better respond to the oil than WT. The mutant had a WT-like rooting response and the expression of its phenotype was not evident in treatments that had oil application. The essential oil also induced the generation of H₂O₂, a reactive oxygen species in high levels and trying to recover these effects with the aid of the antioxidant Trolox^® was unsuccessful. Increasing levels of H₂O₂ may affect leaf pigmentation of WT microcuttings and auxin levels of both WT and sur1 microcuttings. Since the expression of auxin-responsive genes is decreased by H₂O₂ treatment via mitogen-activated protein kinase activation, microcuttings growth and rooting are impaired causing altered development pattern.     

High Diversity of Indigenous Populations of Dalmatian Sage (Salvia officinalis L.) in Essential‐Oil Composition

Essential oils of 25 indigenous populations of Dalmatian sage (Salvia officinalis L.) that represent nearly half of native distribution area of the species were analyzed. Plantlets collected from wild populations were grown in the same field under the same environmental conditions and then sampled for essential‐oil analysis. The yield of essential oil ranged from 1.93 to 3.70% with average of 2.83%. Among the 62 compounds detected, eight (cis‐thujone, camphor, trans‐thujone, 1,8‐cineole, β‐pinene, camphene, borneol, and bornyl acetate) formed 78.13–87.33% of essential oils of individual populations. Strong positive correlations were observed between camphor and β‐pinene, β‐pinene and borneol, as well as between borneol and bornyl acetate. The strongest negative correlation was detected between camphor and trans‐thujone. Principal component analysis (PCA) on the basis of eight main compounds showed that first main component separated populations with high thujone content, from those rich in camphor, while the second component separated populations rich in cis‐thujone from those rich in trans‐thujone. Cluster analysis (CA) led to the identification of three chemotypes of S. officinalis populations: cis‐thujone; trans‐tujone, and camphor/β‐pinene/borneol/bornyl acetate. We propose that differences in essential oils of 25 populations are mostly genetically controlled, since potential environmental factors were controlled in this study.     

Micropropagation with a novel pattern of adventitious rooting in American sweetgum (Liquidambar styraciflua L.)

Liquidambar styraciflua L. has great potential not only as an ornamental, but also for its commercial importance in pulp and paper production or biomass energy. This study was designed to evaluate the influence of plant growth regulators on adventitious shoot multiplication from shoot tips, and in vitro adventitious rooting. The morphogenic capacity of intact leaves grown in vitro was also assayed for adventitious shoot formation and aerial root development. The highest shoot multiplication rate of 5.9 shoots per explant was achieved with 0.7 mg l⁻¹ 6-benzylaminopurine plus 0.01 mg l⁻¹ indole-3-butyric acid. Thidiazuron, alone or in combination with 6-benzylaminopurine, did not significantly support higher shoot multiplication rates. The organogenic ability of the in vitro grown leaves was significantly lower and slower in comparison with shoot tips. Microshoots rooted readily after transfer to a half-strength woody plant medium supplemented with 0.5–0.7 mg l⁻¹ 1-naphthaleneacetic acid, and were then successfully acclimatised to an ex vitro environment. A novel pattern of adventitious rooting was observed from the aerial parts of microshoots which were not in contact with the medium, including the parenchyma cells of the leaf blades as well as stem nodes and internodes. The regenerated plants established in soil did not show any detectable morphological variation.     

Do strigolactones contribute to plant defence?

Strigolactones are multifunctional molecules involved in several processes outside and within the plant. As signalling molecules in the rhizosphere, they favour the establishment of arbuscular mycorrhizal symbiosis, but they also act as host detection cues for root parasitic plants. As phytohormones, they are involved in the regulation of plant architecture, adventitious rooting, secondary growth and reproductive development, and novel roles are emerging continuously. In the present study, the possible involvement of strigolactones in plant defence responses was investigated. For this purpose, the resistance/susceptibility of the strigolactone‐deficient tomato mutant Slccd8 against the foliar fungal pathogens Botrytis cinerea and Alternaria alternata was assessed. Slccd8 was more susceptible to both pathogens, pointing to a new role for strigolactones in plant defence. A reduction in the content of the defence‐related hormones jasmonic acid, salicylic acid and abscisic acid was detected by high‐performance liquid chromatography coupled to tandem mass spectrometry in the Slccd8 mutant, suggesting that hormone homeostasis is altered in the mutant. Moreover, the expression level of the jasmonate‐dependent gene PinII, involved in the resistance of tomato to B. cinerea, was lower than in the corresponding wild‐type. We propose here that strigolactones play a role in the regulation of plant defences through their interaction with other defence‐related hormones, especially with the jasmonic acid signalling pathway.     

侧柏扦插不定根发生模式研究

该研究以侧柏一年生硬枝插穗为实验材料,利用连续组织切片技术观察插穗不定根发生发育过程中的组织结构变化,分析插穗外部形态变化、不定根原基起源和不定根的形成过程,探讨侧柏插穗不定根发生模式和不定根的组织学起源。结果显示:侧柏扦插后可由愈伤组织、皮部诱导产生不定根,出现皮部生根、愈伤组织生根、愈伤组织兼具皮部生根3种类型;侧柏插穗中存在少量潜伏根原基,但插穗生根类型以诱导生根为主;不定根原基诱导产生于愈伤组织、木质部、形成层及次生韧皮部等部位。研究认为侧柏扦插生根属于多位点发生模式,不定根原基的组织学起源是愈伤组织、髓射线、射线原始细胞、尚未分化成熟的木质部细胞,通过人工诱导同时激活这些不定根起源位点能够显著提高生根率和生根质量。     

Inhibition of adventitious root development in apple rootstocks by cytokinin is based on its suppression of adventitious root primordia formation

Cytokinin (CK) inhibits adventitious root (AR) formation in stem cuttings. Little is known, however, about the mechanism underlying the inhibitory effect. In this study, 2 mg l^?1 of exogenous 6‐benzyl adenine (6‐BA) was administered to 3 and 7‐day‐old apple rootstocks ‘M.26’ cuttings (3 and 7 days 6‐BA) by transferring them from a rooting medium containing indole‐3‐butanoic acid to the medium containing 6‐BA. Anatomical and morphological observations revealed that the exogenous application of 6‐BA inhibited primordia formation in the 3 days 6‐BA but not the 7 days 6‐BA group. The concentration of auxin (IAA), the ratios of IAA/CK and IAA/abscisic acid were lower in 3 days 6‐BA than in 7 days 6‐BA. Expression analysis of genes known to be associated with AR formation was also analyzed. In the 3 days 6‐BA group, high level of CK inhibited the synthesis and transport of auxin, as a result, low endogenous auxin level suppressed the auxin signaling pathway genes, as were other AR development and cell cycle related genes; all of which had an inhibitory impact on AR primordium formation. On the contrary, low CK level in the 7 days 6‐BA, reduced the inhibitory impact on auxin levels, leading to an upregulated expression of genes known to promote AR primordia formation. Collectively, our data indicated that 3–7 days is the time period in which AR primordia formation occurs in cuttings of ‘M.26’ and that the inhibition of AR development by CK is due to the suppression of AR primordia development over 3–7 days period after culturing in rooting medium.     

Strigolactones suppress adventitious rooting in Arabidopsis and pea

Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting. CYCLIN B1 expression, an early marker for the initiation of adventitious root primordia in Arabidopsis, is enhanced in more axillary growth2 (max2), a strigolactone response mutant, suggesting that strigolactones restrain the number of adventitious roots by inhibiting the very first formative divisions of the founder cells. Strigolactones and cytokinins appear to act independently to suppress adventitious rooting, as cytokinin mutants are strigolactone responsive and strigolactone mutants are cytokinin responsive. In contrast, the interaction between the strigolactone and auxin signaling pathways in regulating adventitious rooting appears to be more complex. Strigolactone can at least partially revert the stimulatory effect of auxin on adventitious rooting, and auxin can further increase the number of adventitious roots in max mutants. We present a model depicting the interaction of strigolactones, cytokinins, and auxin in regulating adventitious root formation.     

Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.     

Distinct modes of adventitious rooting in Arabidopsis thaliana

The literature describes different rooting protocols for Arabidopsis thaliana as models to study adventitious rooting, and results are generally perceived as comparable. However, there is a lack of investigations focusing on the distinct features, advantages and limitations of each method in the study of adventitious rooting with both wild-type (WT) ecotypes and their respective mutants. This investigation was undertaken to evaluate the adventitious rooting process in three different experimental systems, all using A. thaliana, analysing the same rooting parameters after transient exposure to auxin (indole-3-acetic acid) and control conditions: excised leaves, de-rooted plants and etiolated seedlings. The founding tissues and sites of origin of roots differed depending on the system used, whereas all rooting patterns were of the direct type (i.e., without callus formation). None of the systems had an absolute requirement for exogenous auxin, although rooting was enhanced by this phytohormone, with the exception of de-rooted plants, which had adventitious rooting strongly inhibited by exogenous auxin. Root elongation was much favoured in isolated leaves. Auxin-overproducing mutants could not be used in the detached leaf system due to precocious senescence; in the de-rooted plant system, these mutants had a WT-like rooting response, whereas the expression of the 'rooty' phenotype was only evident in the etiolated seedling system. Adventitious rooting of etiolated WT seedlings in the presence of exogenous auxin was inhibited by exogenous flavonoids, which act as auxin transport inhibitors; surprisingly, the flavonoid-deficient mutant chs had a lower rooting response compared to WT. Although Arabidopsis is an excellent model system to study adventitious rooting, physiological and developmental responses differed significantly, underlining the importance of avoiding data generalisation on rooting responses derived from different experimental systems with this species.     

Establishment of culturing conditions and assessment of antioxidant activity and somaclonal variation in the adventitious root suspension cultures of <Emphasis Type="Italic">Oplopanax elatus</Emphasis> Nakai

Oplopanax elatus Nakai, a plant traditionally used in folk medicine, is currently in population decline due to uncontrolled harvesting. In the present study, we investigated the factors affecting O. elatus adventitious root production, including hormones (alone or in combination), explant type, basal salt type and strength, sucrose concentration, pH, and temperature. Results revealed that adventitious root formation was optimal with root explants grown on 1/2 Murashige and Skoog (MS) medium containing 0.5 mg L^?1 Indole-3-butyric acid (IBA) (pH 5.8) at 25 °C. Chlorogenic acid concentration was highest in roots propagated in 1/2 MS medium containing 0.5 mg L^?1 IBA; vanillin, another phenolic compound, was also detected in cultures. Liquid media containing 3% sucrose exhibited the highest radical scavenging activity and total phenolic compound contents. X-ray diffraction revealed significant differences in the elemental intensity between adventitious root and field-grown plantlet extracts. Analysis of simple sequence repeats confirmed that adventitious roots regenerated in vitro were genetically similar to their mother plant. Thus, we identified the optimal conditions for proliferation of O. elatus adventitious roots in liquid culture, from which, secondary metabolites, particularly bioactive compounds associated with the medicinal use of this plant, can be mass produced without further population deterioration.     

Environment‐Related Variations of the Composition of the Essential Oils of Rosemary (Rosmarinus officinalis L.) in the Balkan Penninsula

Composition of the essential oils of Rosmarinus officinalis of ten populations from the Balkan Peninsula were determined by GC/FID and GC/MS. The main constituents were 1,8‐cineole, camphor, α‐pinene, and borneol. Multivariate statistical analysis (UPGMA cluster analysis and principal‐component analysis (PCA)) revealed two major types of rosemary oil, i.e., 1,8‐cineole and camphor‐type, and two intermediate types, i.e., camphor/1,8‐cineole/borneol type and 1,8‐cineole/camphor type. The regression analyses (simple linear regression and stepwise multiple regression) have shown that, with respect to basic geographic, orographic, and 19 bioclimatic characteristics of each population, bioclimatic factor temperature of habitat represented the dominant abiogenetic factor, which, in chemical sense, led to differentiation of populations in the studied region. Also, the regression analysis have shown that some constituents of essential oils are independent of any single bioclimatic factors. However, some constituents display statistically significant correlations with some abiotic factors.     

Histological analysis of adventitious rooting in Arabidopsis thaliana (L.) Heynh seedlings

ABSTRACT

Adventititous rooting is essential for the post-embryonic growth of the root apparatus in various species. In Arabidopsis thaliana, adventitious rooting has been reported in some mutants, and auxin seems to be the inducer of the process. The objective of the study was to identify the tissues involved in adventitious rooting in the most commonly used ecotypes for molecular and genetic studies (i.e. Columbia, Wassilewskija and Landsberg erecta) both in the presence and absence of exogenous auxin. Seedlings of the three ecotypes were grown under various conditions. When grown under 16 hours light/day for 11 days, all seedlings showed adventitious roots, both with and without auxin, however, both adventitious and lateral rooting were enhanced by exogenous auxin (2 µM naphthaleneacetic acid). Independently of the presence of auxin and of the ecotype, the hypocotyl pericycle produced adventitious roots directly (i.e., according to the same pattern of lateral root formation by the pericycle cells in the primary root). However, in the presence of auxin, roots of indirect origin also, and mainly, formed and their formation was preceded by the exfoliation of the tissues external to the stele. Exfoliation was caused by cell hypertrophy, separation, and disintegration, which mainly involved the endodermis. At the exfoliation site, the pericycle, with a minor contribution of a few endodermal cells, produced the callus from which indirect roots arose. The finding that adventitious rooting occurs in the absence of auxin (all ecotypes) indicates that this process is part of the normal root apparatus in Arabidopsis, with the hypocotyl pericycle as the target tissue of the process. Exogenous auxin alters adventitious rhizogenesis mainly affecting the endodermis response.