Ethylene and auxin interaction in the control of adventitious rooting in Arabidopsis thaliana

Adventitious roots (ARs) are post-embryonic roots essential for plant survival and propagation. Indole-3-acetic acid (IAA) is the auxin that controls AR formation; however, its precursor indole-3-butyric acid (IBA) is known to enhance it. Ethylene affects many auxin-dependent processes by affecting IAA synthesis, transport and/or signaling, but its role in AR formation has not been elucidated. This research investigated the role of ethylene in AR formation in dark-grown Arabidopsis thaliana seedlings, and its interaction with IAA/IBA. A number of mutants/transgenic lines were exposed to various treatments, and mRNA in situ hybridizations were carried out and hormones were quantified In the wild-type, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) at 0.1 μM enhanced AR formation when combined with IBA (10 μM), but reduced it when applied alone; this effect did not occur in the ein3eil1 ethylene-insensitive mutant. ACC inhibited the expression of the IAA-biosynthetic genes WEI2, WEI7, and YUC6, but enhanced IBA-to-IAA conversion, as shown by the response of the ech2ibr10 mutant and an increase in the endogenous levels of IAA. The ethylene effect was independent of auxin-signaling by TIR1-AFB2 and IBA-efflux by ABCG carriers, but it was dependent on IAA-influx by AUX1/LAX3. Taken together, the results demonstrate that a crosstalk involving ethylene signaling, IAA-influx, and IBA-to-IAA conversion exists between ethylene and IAA in the control of AR formation

Jasmonate promotes auxin-induced adventitious rooting in dark-grown Arabidopsis thaliana seedlings and stem thin cell layers by a cross-talk with ethylene signalling and a modulation of xylogenesis

Background: Adventitious roots (ARs) are often necessary for plant survival, and essential for successful micropropagation. In Arabidopsis thaliana dark-grown seedlings AR-formation occurs from the hypocotyl and is enhanced by application of indole-3-butyric acid (IBA) combined with kinetin (Kin). The same IBA + Kin-treatment induces AR-formation in thin cell layers (TCLs). Auxin is the main inducer of AR-formation and xylogenesis in numerous species and experimental systems. Xylogenesis is competitive to AR-formation in Arabidopsis hypocotyls and TCLs. Jasmonates (JAs) negatively affect AR-formation in de-etiolated Arabidopsis seedlings, but positively affect both AR-formation and xylogenesis in tobacco dark-grown IBA + Kin TCLs. In Arabidopsis the interplay between JAs and auxin in AR-formation vs xylogenesis needs investigation. In de-etiolated Arabidopsis seedlings, the Auxin Response Factors ARF6 and ARF8 positively regulate AR-formation and ARF17 negatively affects the process, but their role in xylogenesis is unknown. The cross-talk between auxin and ethylene (ET) is also important for AR-formation and xylogenesis, occurring through EIN3/EIL1 signalling pathway. EIN3/EIL1 is the direct link for JA and ET-signalling. The research investigated JA role on AR-formation and xylogenesis in Arabidopsis dark-grown seedlings and TCLs, and the relationship with ET and auxin. The JA-donor methyl-jasmonate (MeJA), and/or the ET precursor 1-aminocyclopropane-1-carboxylic acid were applied, and the response of mutants in JA-synthesis and -signalling, and ET-signalling investigated. Endogenous levels of auxin, JA and JA-related compounds, and ARF6, ARF8 and ARF17 expression were monitored. Results: MeJA, at 0.01 μM, enhances AR-formation, when combined with IBA + Kin, and the response of the early-JA-biosynthesis mutant dde2–2 and the JA-signalling mutant coi1–16 confirmed this result. JA levels early change during TCL-culture, and JA/JA-Ile is immunolocalized in AR-tips and xylogenic cells. The high AR-response of the late JA-biosynthesis mutant opr3 suggests a positive action also of 12-oxophytodienoic acid on AR-formation. The crosstalk between JA and ET-signalling by EIN3/EIL1 is critical for AR-formation, and involves a competitive modulation of xylogenesis. Xylogenesis is enhanced by a MeJA concentration repressing AR-formation, and is positively related to ARF17 expression. Conclusions: The JA concentration-dependent role on AR-formation and xylogenesis, and the interaction with ET opens the way to applications in the micropropagation of recalcitrant species

Effect of Biosaf in dairy buffalo cows

Biosaf is a thermostable live yeast concentrate (Saccharomyces cerevisiae Strain Sc47). The trial was carried out on 35 buffalo cows subdivided in 3 groups: Group A (Control with farm standard diet); Group B (with the same diet, added with 6g Biosaf/day); Group C (with the same diet, added with 60g Biosaf/day). The trial started within the first month from calving (1st month of lactation) up to the 8th month of lactation. The mean values for milk production during 8 months of trial are 8.95, 8.95 and 8.91 liters/d, respectively in Group A, B and C, without significant differences among groups. The protein percentage increased in groups receiving Biosaf. The effect was significant (P<0.05) in Group B and C at the 4th, 5th and 8th month of lactation in comparison to Control Group and in Group B at 4th month of lactation in comparison to Control Group: this indicates that Biosaf integration really increases milk protein and consequently mozzarella yield

Auxin controls quiescent centre formation in the adventitious root apex of Arabidopsis, and the switching between adventitious rooting and xylogenesis

Adventitious root (ARs) are essential for the survival of numerous plant species, because they contribute to anchorage, water-use efficiency and extraction of nutrients from the soil. Moreover, the regulation of AR induction is crucial for successful micropropagation in vitro and breeding programs. In Arabidopsis, ARs are formed by the hypocotyl pericycle, in planta, and by the stem endodermis in in vitro cultured thin cell layers (TCLs) (Falasca et al., 2004). The indeterminate growth of primary root (PR) and lateral roots (LRs) is supported by the quiescent centre (CQ), which co-ordinates the activity of the surrounding stem cells, with a central role in establishment, maintenance and elaboration of patterns in the apical meristem, with an involvement of both auxin and cytokinin (Jang and Feldman, 2005). In contrast, the organization and activity of the QC in the ARs still needs investigation, the same as the possible roles of auxin and cytokinin on its establishment and maintenance. The transcription factors SHORT ROOT (SHR) and SCARECROW (SCR) are key regulators jointly controlling PR and LR stem cell definition/maintenance, and radial patterning (Helariutta et al., 2000; Sabatini et al., 2003), but their involvement in AR-formation is uncertain. AUXIN RESISTANT1 (AUX1) and LIKE AUXIN RESISTANT3 (LAX3) are auxin influx carriers contributing to PR and LR development. LAX3 expression is known to be regulated by SHR (Sozzani et al., 2010), and SCR is known to be under auxin control. Moreover, the QC marker WOX5 is controlled by the SHR/SCR complex. The interaction of SHR/SCR in AR-identity acquisition in unknown, as the role of the complex on the auxin-influx carriers and QC markers of the ARs. Xylogenesis is known to be induced by auxin, but the genes involved are largely unknown. However, the SHR-SCR complex affects PR vascular differentiation regulating the microRNA165/6. The first aim of this research was to investigate whether the QC is established in Arabidopsis ARs, in planta and in TCLs, and whether its establishment and maintenance are under auxin-control. To this aim, the activity of PR/LR QC markers (QC25::GUS, pAGL42::GFP, pWOX5::GFP), the expression patterns of DR5::GUS line, of PIN1 and LAX3 auxin carriers expression, and YUCCA6 transcription were monitored. Subsequently, the research was aimed to determine SHR/SCR activities on AR-formation, and their relationship with the auxin-influx carriers AUX1 and LAX3 in AR-formation, but also in xylogenesis, in hypocotyls and stem TCLs of Arabidopsis. Results show that the QC is established in the ARs. Independently of the founder cells, auxin accumulation and WOX5 expression characterize the early derivative cells involved in AR-formation. By the activities of auxin influx/efflux carriers, an auxin-maximum is determined at the AR-tip, where WOX5 expression is restricted, positioning the QC. An auxin biosynthesis by YUCCA6 gene is required in the apex for QC maintenance. The SHR/SCR complex is early active during AR-formation, and AUX1, mediating the auxin influx in the AR-initiating cells, seems related to the priming activity of the complex. Interestingly, AR-formation and xylogenesis are developmental programmes inversely related, with SHR, SCR and AUX1 controlling the fine-tuning between the two

Ethylene role in adventitious root formation in Arabidopsis thaliana thin cell layers

Adventitious roots (ARs) are post-embryonic roots arising from non-pericycle tissues in primary root (PR) and various tissues in aerial organs. ARs are essential for successful vegetative propagation. Several protocols for AR induction were developed in Arabidopsis thaliana, in planta and in in vitro systems, e.g. stem thin cell layer (TCL) culture, which allow to study AR formation in a limited cell context. ARs are controlled by multiple factors, and auxin is a central player. Exogenous auxin is essential for ARs in TCLs. Ethylene could be another hormone involved, but presently there are many unresolved questions concerning its role, and in A. thaliana available data are in contrast. There is information about lateral roots (LRs), because an inhibitory effect of ACC (1- aminocyclopropane-1-carboxylic acid), direct ethylene precursor, on their formation was reported, but low concentrations stimulate the process (Negi et al, 2008; Ivanchenko et al, 2008). The Research studied ethylene effects, and the relationship with auxin, on AR formation in TCLs. Wild type TCLs were treated with various ACC concentrations. Significant reduction in AR number per TCL only occurred with 0.1μM, showing that specific ethylene levels might regulate AR formation. Because a relationship between auxin biosynthesis and ethylene was reported for the PR (Stepanova et al, 2005), TCLs of the double mutant wei2wei7, blocked in auxin biosynthesis, were grown with/without 0.1μM ACC. The number of ARs/TCL in this mutant was further reduced without ACC in comparison with the wt, demonstrating that endogenous auxin biosynthesis cooperates with exogenous auxin input on the AR formation. In this mutant no significant change in AR number was caused by ACC addition, showing that the effect of ethylene biosynthesis on AR response depends on auxin biosynthesis. TCLs were also excised from the ethylene insensitive mutants ein2-1 and ein3eil1 and cultured with/without 0.1μM ACC to verify whether ethylene signalling was also involved. In both mutants, no change in AR number/TCL occurred with/without ACC addition, and in both treatments ARs strongly elongated and showed a lot of LRs. All together results suggest that specific ethylene levels are involved in AR formation from TCLs possibly through a regulation of auxin biosynthesis, and ethylene perception also affects AR development and branching

Indole-3-butyric acid promotes adventitious rooting in Arabidopsis thaliana thin cell layers by conversion into indole-3-acetic acid and stimulation of anthranilate synthase activity

Abstract Background Indole-3-acetic acid (IAA), and its precursor indole-3-butyric acid (IBA), control adventitious root (AR) formation in planta. Adventitious roots are also crucial for propagation via cuttings. However, IBA role(s) is/are still far to be elucidated. In Arabidopsis thaliana stem cuttings, 10 μM IBA is more AR-inductive than 10 μM IAA, and, in thin cell layers (TCLs), IBA induces ARs when combined with 0.1 μM kinetin (Kin). It is unknown whether arabidopsis TCLs produce ARs under IBA alone (10 μM) or IAA alone (10 μM), and whether they contain endogenous IAA/IBA at culture onset, possibly interfering with the exogenous IBA/IAA input. Moreover, it is unknown whether an IBA-to-IAA conversion is active in TCLs, and positively affects AR formation, possibly through the activity of the nitric oxide (NO) deriving from the conversion process. Results Revealed undetectable levels of both auxins at culture onset, showing that arabidopsis TCLs were optimal for investigating AR-formation under the total control of exogenous auxins. The AR-response of TCLs from various ecotypes, transgenic lines and knockout mutants was analyzed under different treatments. It was shown that ARs are better induced by IBA than IAA and IBA + Kin. IBA induced IAA-efflux (PIN1) and IAA-influx (AUX1/LAX3) genes, IAA-influx carriers activities, and expression of ANTHRANILATE SYNTHASE -alpha1 (ASA1), a gene involved in IAA-biosynthesis. ASA1 and ANTHRANILATE SYNTHASE -beta1 (ASB1), the other subunit of the same enzyme, positively affected AR-formation in the presence of exogenous IBA, because the AR-response in the TCLs of their mutant wei2wei7 was highly reduced. The AR-response of IBA-treated TCLs from ech2ibr10 mutant, blocked into IBA-to-IAA-conversion, was also strongly reduced. Nitric oxide, an IAA downstream signal and a by-product of IBA-to-IAA conversion, was early detected in IAA- and IBA-treated TCLs, but at higher levels in the latter explants. Conclusions Altogether, results showed that IBA induced AR-formation by conversion into IAA involving NO activity, and by a positive action on IAA-transport and ASA1/ASB1-mediated IAA-biosynthesis. Results are important for applications aimed to overcome rooting recalcitrance in species of economic value, but mainly for helping to understand IBA involvement in the natural process of adventitious rooting

Arabidopsis SHR and SCR transcription factors and AUX1 auxin-influx carrier control the switch between adventitious rooting and xylogenesis in planta and in in-vitro-cultured thin cell layers

Background and Aims Adventitious roots (ARs) are essential for vegetative propagation. The Arabidopsis thaliana transcription factors SHORT ROOT (SHR) and SCARECROW (SCR) affect primary/lateral root development, but their involvement in AR formation is uncertain. LAX3 and AUX1 auxin influx carriers contribute to primary/ lateral root development. LAX3 expression is regulated by SHR, and LAX3 contributes to AR tip auxin maximum. In contrast, AUX1 involvement in AR development is unknown. Xylogenesis is induced by auxin plus cytokinin as is AR formation, but the genes involved are largely unknown. Stem thin cell layers (TCLs) form ARs and undergo xylogenesis under the same auxin plus cytokinin input. The aim of this research was to investigate SHR, SCR, AUX1 and LAX3 involvement in AR formation and xylogenesis in intact hypocotyls and stem TCLs in arabidopsis. Methods Hypocotyls of scr-1, shr-1, lax3, aux1-21 and lax3/aux1-21 Arabidopsis thaliana null mutant seedlings grown with or without auxin plus cytokinin were examined histologically, as were stem TCLs cultured with auxin plus cytokinin. SCR and AUX1 expression was monitored using pSCR::GFP and AUX1::GUS lines, and LAX3 expression and auxin localization during xylogenesis were monitored by using LAX3::GUS and DR5::GUS lines. Key Results AR formation was inhibited in all mutants, except lax3. SCR was expressed in pericycle anticlinally derived AR-forming cells of intact hypocotyls, and in cell clumps forming AR meristemoids of TCLs. The apex was anomalous in shr and scr ARs. In all mutant hypocotyls, the pericycle divided periclinally to produce xylogenesis. Xylary element maturation was favoured by auxin plus cytokinin in shr and aux1-21. Xylogenesis was enhanced in TCLs, and in aux1-21 and shr in particular. AUX1 was expressed before LAX3, i.e. in the early derivatives leading to either ARs or xylogenesis. Conclusions AR formation and xylogenesis are developmental programmes that are inversely related, but they involve fine-tuning by the same proteins, namely SHR, SCR and AUX1. Pericycle activity is central for the equilibrium between xylary development and AR formation in the hypocotyl, with a role for AUX1 in switching between, and balancing of, the two developmental programmes

Growth parameters, hormonal balance and thiol-peptide compound metabolism in Arabidopsis thaliana seedlings growing under excess zinc

To date, almost no information is available in roots and shoots of the model plant Arabidopsis thaliana (L.) Heynh. about the hierarchic relationship between metal accumulation, phytohormone levels, and glutathione/phytochelatin content, and how this relation affects root and shoot development. For this purpose, specific concentrations of zinc, alone or in triple combination with cadmium and copper, were supplied for two weeks to seedlings growing in a hydroponic system and using Petri dishes with a gradient of distances between germinating seeds and metal-contaminated agarized medium. Zinc accumulation was determined by anodic stripping voltammetry in plant tissues and digested agar samples, and a significant competition in metal uptake was observed. Microscopic and high-resolution scanning analyses revealed that root morphology was affected by metal exposure, with increases in root system total length and surface mainly due to the higher branching and number of lateral roots, accompanied by higher average root diameter. The confocal microscopy analysis of auxin accumulation and influx in the cells by the use of transgenic Arabidopsis lines (DR5:GUS, LAX3:GUS and AUX1:GUS) and the mass spectrometry of plant tissues revealed significant changes in auxin levels and accumulation in the seedling exposed to zinc alone or in combination. Real time quantitave PCR analysis of some genes involved in auxin and cytokinin synthesis showed on average a metal upregulated transcription. The production of thiol-peptides was induced by zinc alone or in combination, but the expression of the genes involved in thiol-peptide synthesis was not stimulated by the metals, suggesting a full post-transcriptional control. Results show that the Cd/Cu/Zn-induced changes in root morphology are caused by a hormonal unbalance, mainly governed by the auxin/cytokinin ratio. The remodeling of the root architecture in response to zinc could be a pollution ‘escaping strategy’ aimed at seeking metal-free areas. The methods used and the results obtained by this model plant could be transferred to species with bioremediation or agronomic importance