Ruolo dell'etilene nella rizogenesi avventizia di Arabidopsis thaliana L. (Heynh)

La rizogenesi avventizia è controllata da molti fattori, in particolare genetici ed ormonali. Arabidopsis thaliana, di norma, differenzia in planta una o due radici avventizie alla base dell’ipocotile. Nonostante questa capacità e l’ampia informazione disponibile per questa pianta sul ruolo dei fitormoni in diversi aspetti dello sviluppo, le conoscenze sul ruolo dell’etilene nella radicazione avventizia sono molto scarse. Inoltre, i dati disponibili, inerenti anche altre specie, non offrono una chiara ed univoca interpretazione, sia se si considerino gli effetti dell’etilene sulla rizogenesi avventizia in planta, sia se si prendano in esame quelli sulla formazione di radici avventizie che in espianti coltivati in vitro (Biondi et al, 1990; Pan et al, 2002; Li et al, 2009). Informazioni sono, al contrario, disponibili sulla relazione tra etilene e radicazione laterale, l’altro tipo di radicazione post-embrionale, con cui quella avventizia condivide gli eventi di sviluppo (Malamy e Benfey, 1997, Della Rovere et al, 2013). Studi recenti dimostrato che l’ACC (acido 1-amminociclopropan-1-carbossilico), diretto precursore dell’etilene, inibisce il processo di radicazione laterale in A. thaliana e in pomodoro (Negi et al, 2008, 2010). Tuttavia, concentrazioni molto basse del composto (10-8M) sono state riportate stimolare il processo (Ivanchenko et al, 2008). È noto che l’auxina ha un ruolo essenziale nella genesi e sviluppo delle radici avventizie in planta e in vitro (Falasca e Altamura, 2003; Ludwig-Müller et al, 2005). È possibile che il ruolo dell’etilene nella rizogenesi avventizia sia mediato dal signaling dell’auxina e/o dal suo trasporto (come avviene per la crescita della radice primaria; Růžička et al, 2007) e/o dalla biosintesi auxinica (come riscontrato sempre per la radice primaria, Stepanova et al, 2005). Lo scopo della Ricerca è indagare il ruolo dell’etilene nella radicazione avventizia in A. thaliana, in planta ed in espianti costituiti da tessuti superficiali caulinari (strati cellulari sottili, TCL) coltivati in vitro. I TCL sono stati coltivati in presenza di auxina (IBA), combinata o meno con diverse concentrazioni di ACC. Per lo studio in planta sono state esaminate plantule cresciute in assenza di ormoni esogeni oppure in presenza di solo IBA, alla concentrazione usata per i TCL, o di solo ACC a diverse concentrazioni. Per gli esperimenti sono stati utilizzati il mutante ein2-1 ed il suo wild type Col-0. Nel mutante non è funzionale la proteina EIN2-1, implicata in tutte le risposte all’etilene sinora note in quanto coinvolta in un passaggio critico nella trasduzione del segnale di questo ormone (Qiao et al, 2009). Nei TCL di entrambi i genotipi si è osservato che l’IBA (10 µM) è necessario per indurre il processo rizogenico. Trattando i campioni wild-type con diverse concentrazioni di ACC (0; 0,01;0,04;0,1 µM), combinate con IBA (10 µM), si è osservata inibizione della rizogenesi avventizia da parte della dose più elevata di ACC, al contrario, gli espianti di ein2-1 sono risultati insensibili al trattamento con ACC. In planta l’IBA ha stimolato la radicazione avventizia nel wild type. In presenza di solo ACC, alle stesse concentrazioni utilizzate per i TCL, si è osservato un effetto simile a quello riscontrato negli espianti, con 0,1 µM di ACC riducente nel wild-type la rizogenesi rispetto al trattamento senza ACC. I dati finora ottenuti emerge in Arabidopsis thaliana un ruolo inibitorio dell’etilene sulla rizogenesi avventizia in planta ed in TCL

The switch in cell-identity acquisition leading to either adventitious rooting or xylogenesis is controlled by SHR and SCR, and involves AUX1, in Arabidopsis thaliana hypocotyls and stem thin cell layers.

Insert Adventitious roots (ARs) contribute to propagation of crops/forest species. SHORT ROOT (SHR) and SCARECROW (SCR) transcription factors affect primary (PR)/lateral (LR) root formation and radial patterning. In Arabidopsis PR, SHR is expressed in the stele, its protein moves into the adjacent layer to control SCR transcription and endodermis specification, and SHR and SCR jointly regulate microRNAs involved in vascular differentiation. There is also evidence of SHR- and SCR-like genes involvement in AR formation in cultured woody cuttings. Arabidopsis stem thin cell layers (TCLs) are without vasculature and need auxin and cytokinin for AR formation. AR-induction occurs in TCL endodermis and seedling hypocotyl pericycle, however AtSHR and AtSCR involvement in adventitious-rooting-identity acquisition is unknown in both cases. AUX1 and LAX3 are auxin-influx carriers. LAX3 contributes to AR-tip auxin-maximum in planta and TCLs, and AUX1 is involved in organ identification in vitro. The interaction of the carriers with SHR and/or SCR in AR-identity acquisition is unknown. Xylogenesis in vitro and in planta is auxin-induced. Arabidopsis shows xylogenesis in addition to AR-formation under the same exogenous hormones in planta and TCLs, but how xylogenic identity is acquired is unknown. The research was aimed to determine SHR, SCR, AUX1 and LAX3 involvement in Arabidopsis AR- and xylogenic-identity-acquisition, and the relationship between the morphogenic programmes. To the aim gene expression and auxin localization were monitored, and seedlings/TCLs of single/double mutants of all the genes, grown with the same auxin and cytokinin concentrations, were analyzed. Results show that AR formation is inhibited from the first divisions in all null mutants, except lax3, showing ARprimordia in both seedlings and TCLs. All mutants also exhibited enhanced xylogenesis in planta and TCLs, however lax3 response was lower than in the other mutants. In conclusion, the same genes, under the same hormonal input, affect AR-induction and xylogenesis at the same time but in an opposite way, highlighting that the programmes are subjected to a reciprocal fine-tuning involving SHR, SCR, and AUX1 in planta and in vitro. The results open the way to understand the genetic basis of AR-recalcitrance in woody species in which the AR-formation-block is associated to enhanced xylogenesis

Adventitious rooting: what happens between ethylene and auxins?

Adventitious roots (ARs) are roots that develop from non-root tissues, mostly from aerial plant parts (1). It is an important adaptive response to stress. Several protocols for AR induction have been developed in Arabidopsis thaliana, in planta and in in vitro systems, e.g., stem thin cell layers (TCLs). The latter system improves the knowledge of the process because it allows the study of AR formation in a limited cell context and starting in tissues different from those usually involved in planta. ARs are controlled by multiple endogenous and environmental factors, and auxin, described as the rooting hormone, is one of the major control factors in planta, and is essential for ARs in TCLs (2). The auxin indole-3-acetic acid (IAA) is a potent growth regulator, but it is known that different auxins have a differential root-inducing ability. In accordance, recent studies have demonstrated the importance of the natural auxin-precursor indole-3-butyric acid (IBA), because IBA-derived IAA is a significant part of the auxin necessary for a lot of the processes related to seedling development (3). Moreover, when applied exogenously, IBA exhibits a greater ability to promote ARs compared with IAA (4). Ethylene (ET) could be another hormone involved in AR formation, because it affects a variety of processes during the plant lifetime, including adaptive stress responses. In particular, ET influences many features of auxin-dependent plant growth by altering auxin signaling, synthesis and/or transport (5). However, in A.thaliana, there is still limited information about ET roles on AR formation, and data, about other species are in contrast (6). Instead, there is information about lateral roots (LRs), i.e., the other post-embryonic rooting of the plant, showing developmental stages similar to ARs (2). In fact, in A.thaliana, studies in planta show an inhibitory effect of 1-aminocyclopropane-1-carboxylic acid (ACC), the direct ET precursor, on LR formation (7). However, this effect seems concentration dependent, because concentrations lower than 10-7M stimulate the process (8). The present research studied ET effects on AR formation in seedlings and TCLs of A. thaliana investigating the possible relationship of ET with IBA and IAA. For this reason, after a preliminary screening of ACC concentrations, AR density was evaluated with/without ACC (0.1μM), and with/without IBA or IAA (10 μM) in both systems, by the use of wild type (wt) and ET/auxin mutants. The presence of both auxins was detected in wt seedlings grown without exogenous hormones (HF). Contrariwise the TCLs showed no significant level of these hormones under HF treatment (9). For this reason, TCLs were cultured in the presence of either IAA or IBA. Only the latter auxin induced a high AR response. As consequence, the IBA treatment was chosen as the AR control treatment for TCLs, to compare with the HF treatment of the seedlings in planta. In both systems, the presence of ACC caused a significant reduction in AR density. Because IBA acts only through its conversion to IAA (10), AR production, with/without ACC, was evaluated in knockout mutants of ET and IAA, i.e. the ET insensitive mutants ein2-1 and ein3eil1, the IAA biosynthetic double mutant wei2wei7 and the IAA partially insensitive double mutant tir1afb2. The AR response showed that no change in AR density per seedling/AR number per TCL occurred in these mutants with/without ACC, suggesting that ET uses for AR formation the same reception pathways of all the other ET-dependent processes (11). ET exhibited an indirect action, i.e. modulated IAA biosynthesis and reception, in accordance with the endogenous auxin levels detected. All together, results show that in A. thaliana ET affects AR formation in planta and TCLs, through a regulation of IAA biosynthesis and reception. The possible ET role on the conversion of IBA into IAA is under study

Ethylene and auxin interaction in adventitious rooting in Arabidopsis thaliana

Adventitious roots (ARs) are roots that develop from non-root tissues, mostly from aerial plant parts (Verstraeten et al, 2014). ARs are controlled by multiple endogenous and environmental factors, with auxin exhibiting a central role in the process (Pacurar et al, 2014). The auxin indole-3-acetic acid (IAA) is a potent growth regulator, but recent studies have demonstrated the importance of the natural auxin-precursor IBA (indole-3-butyric acid) because IBA-derived IAA is a significant part of the auxin necessary for seedling development (Strader and Bartel, 2011). Ethylene could be another hormone involved in AR-formation, because it affects a variety of processes during the plant lifetime, and influences many features of auxin-dependent plant growth by altering auxin signaling, synthesis and/or transport (Muday et al, 2012). However, in A.thaliana, presently there is limited information, and contrasting data are available for other species, highlighting many unresolved questions concerning the possible role(s) of ethylene in AR-formation (Li et al, 2009). Instead, there is information about lateral roots (LRs), i.e., the other post-embryonic roots sharing developmental stages with ARs (Della Rovere et al, 2013). In A.thaliana, studies in planta show an inhibitory effect of ACC (1-aminocyclopropane-1-carboxylic acid), the direct ethylene precursor, on LR-formation (Negi et al, 2008). However, this effect seems related to the concentration (about 10-8M), because lower concentrations stimulate the process (Ivanchenko et al, 2008). The present research investigated ethylene effects on AR-formation in seedlings of A. thaliana, and the possible relationship of ethylene with auxins (IBA and IAA). For this reason, AR-density in the hypocotyl was evaluated with/without ACC (0.1µM), and with/without IBA (10 µM), by the use of wild type (wt) seedlings, mutants and transgenic GUS lines. Wt seedlings showed a significant reduction in AR-density in the presence of ACC, whereas in the presence of IBA, the same ACC concentration caused a synergistic effect in AR proliferation compared to IBA alone, suggesting a possible opposite effect of ACC on endogenous IAA and exogenous IBA. Investigation on DR5::GUS seedlings supported the hypothesis, because a weaker signal was shown in the presence of ACC compared to the hormone-free condition, and a stronger signal in the presence of IBA plus ACC compared with IBA alone. To better investigate the hypothesis of a contrasting effect of ethylene with the two auxins, AR-production was evaluated in knockout mutants under the same treatments, i.e. the ethylene insensitive mutants ein2-1 and ein3eil1, the IAA-biosynthetic double mutant wei2wei7, the IAA partially insensitive double mutant tir1afb2, and the double mutant blocked in IAA-influx lax3aux1. The AR-response of these mutants showed that ethylene acts on the AR-process by modulating IAA biosynthesis and reception. The hormone does not affect auxin influx, as shown by lax3aux1 response and confirmed by LAX3::GUS and AUX1::GUS lines showing a similar signal expression with/without ACC. All together, results suggest that ethylene perception affects AR-formation in seedlings, possibly through a regulation of IAA biosynthesis and perception, but does not act on auxin influx. Moreover, ethylene seems to interact positively with exogenous IBA possibly favouring its conversion into IAA, thus promoting AR-rooting

IBA induces adventitious rooting in Arabidopsis thaliana thin cell layers by conversion into IAA, involving nitric oxide formation, IAA transport, and IAA biosynthesis

Adventitious roots (AR) are essential for plant survival and propagation via cuttings. Indole-3-acetic acid (IAA), and its precursor indole-3-butyric acid (IBA), control the AR-process. In cuttings of numerous species, exogenous IBA is more AR-inductive than exogenous IAA, but the reason needs investigation. In Arabidopsis thaliana thin cell layers (TCLs), IBA induces ARs when combined with Kinetin (Falasca et al., Plant Cell Rep, 2004), but in dark-grown seedlings IBA is able alone to induce AR-formation (Veloccia et al., J Exp Bot, 2016). In cuttings as in planta, the endogenous IAA/IBA contents are determinant for the AR-process, and differences in response to exogenous IAA/IBA may depend on differences in endogenous contents. It is unknown whether Arabidopsis TCLs contain endogenous IAA/IBA at culture onset. Results showed that IAA and IBA were at undetectable levels at culture onset, and this was an optimal premise to investigate AR-formation under the total control of exogenous auxin, revealing possible differences between IAA and IBA. The AR-response of TCLs from various ecotypes, transgenic lines and knockout mutants showed that IBA was an AR-inducer better than IAA. IBA positively affected IAA cellular efflux and influx, and expression of ANTHRANILATE SYNTHASE-alpha1 (ASA1), a gene of the tryptophan-dependent IAA biosynthesis. Moreover, ASA1 and ANTHRANILATE SYNTHASE-beta1 (ASB1), the other subunit of the enzyme, positively affected AR-formation in the presence of exogenous IBA. The AR-response of IBA-treated TCLs from ech2ibr10 mutant, blocked into IBA-to-IAA conversion, was strongly reduced, showing that IBA acted mainly by conversion into IAA. Nitric oxide (NO), a downstream signal of IAA, but also a by-product of the conversion process, was early detected in IAA- and IBA-treated TCLs, but at higher levels in the latter ones. Altogether results showed that exogenous IBA induced AR-formation in TCLs by conversion into IAA involving NO, IAA transport, and ASA1/ASB1-mediated IAA biosynthesis

Indole-3-butyric acid promotes adventitious rooting in Arabidopsis thin cell layers

Adventitious roots (ARs) are post-embryonic roots formed in planta by tissues of the primary root in secondary vascular structure and by tissues of the aerial organs. Indole-3-acetic acid (IAA), and its natural precursor indole-3-butyric acid (IBA), control AR formation in planta and in vitro, however IBA roles have to be elucidated. Arabidopsis thin cell layers (TCL) consist of stem inflorescence tissue external to the vascular system and 10 microM IBA applied with 0.1microM Kinetin induce AR formation from stem TCL. In the Arabidopsis transversal stem cuttings, it has been hypothesized that the induction of AR formation by exogenous IBA occurs by an interaction with the endogenous IAA content, but there is no information about the interaction between the two auxins in the TCLs. In Arabidopsis seedlings it has been demonstrated that IBA is sufficient to stimulate IAA transport because PIN-FORMED1 (PIN1) IAA-efflux carrier, AUXIN RESISTANT1 (AUX1) and LIKE AUXIN RESISTANT3 (LAX3) IAA-influx carriers are active also in the presence of IBA alone. The WEAK ETHYLENE-INSENSITIVE2/ANTHRANILATE SYNTHASE alpha1 (WEI2/ASA1) and WEI7/ANTHRANILATE SYNTHASE beta1 (ASB1), are genes involved in IAA-biosynthesis and required for AR formation in Arabidopsis seedlings. It is unknown whether the same genes are involved in AR-formation by TCLs. The aim of the research was to determine the endogenous levels of IBA and IAA at the onset of the culture in Arabidopsis TCLs. Another aim was to understand whether IBA alone was able to induce AR formation in TCL, whether the IAA transport by PIN1, LAX3, and AUX1 was affected, whether an IBA conversion into IAA was needed, and whether an IAA biosynthesis by WEI2/ASA1 and WEI7/ASB1 was also involved. Results indicate that IBA induced AR-formation by conversion into IAA, with this process involving nitric oxide formation and activity, and by positively affecting IAA-transport and ASA1/ASB1-mediated IAA-biosynthesis

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

Adventitious roots (ARs) are roots arising from non-pericycle tissues in roots in primary structure, and from tissues of the aerial organs and of the roots in secondary structure. The ARs are necessary for survival in numerous plants, for vegetative propagation in planta and in vitro, and for breeding programs. In Arabidopsis thaliana, ARs originate from the pericycle of the hypocotyl of the seedling, exhibit the same developmental stages of lateral roots (LRs), and their formation is favoured by seedling growth under continuous darkness. Indole-3-acetic acid (IAA) is the natural auxin controlling AR-formation in planta. However, recent studies have demonstrated also the importance of the natural auxin-precursor indole-3-butyric acid (IBA), because IBA-derived IAA is a part of the auxin necessary for a lot of the processes related to seedling development. Moreover, when applied exogenously, IBA exhibits a greater ability to promote ARs compared with IAA, possibly because its higher stability. Ethylene could be another hormone involved in the AR-process, because it influences many features of auxin-dependent plant growth by altering auxin signaling, synthesis and/or transport. However, there are still many questions concerning its role in AR-formation. Moreover, it is still unknown whether ethylene affects AR-formation and LR-formation in the same way, being both post-embryonic organs. In A.thaliana, recent studies show an inhibitory effect of 1-aminocyclopropane-1-carboxylic acid (ACC), i.e., the direct ethylene precursor, on LR-formation, even if low concentrations stimulate the process. Our objective was to investigate the effect of ethylene on AR-formation in the model plant Arabidopsis thaliana, by the use of ACC, and the possible interaction of ethylene with the two main natural auxins, i.e., the active form IAA, and its natural precursor IBA. To the aim, numerous mutants and transgenic lines were exposed to different treatments, and mRNA in situ hybridizations, and hormone quantifications, were carried out. The optimal IBA concentration (10μM) for enhancing AR-formation by the seedlings was preliminarly established, and the ACC concentration with a physiological effect on AR-process in the wt detected. It was found that the concentration of ACC (0.1μM) caused an inhibition of AR-formation in the seedlings. Treatments with/without ACC and/or IBA, at the selected concentrations, were carried out to investigate the AR-response, firstly in the wt, and then in ethylene insensitive mutants, mutants of auxin biosynthesis, reception, and transport, and mutants blocked at the level of IBA-to-IAA conversion, and cellular efflux. It was observed that ethylene acts with an opposite effect on endogenous IAA and exogenous IBA. In fact, the application of ACC alone reduced AR-formation, whereas the combination of ACC and IBA enhanced it. In accordance, ACC alone inhibited IAA biosynthesis and favoured IBA-to-IAA conversion. Moreover, ACC affected ethylene signalling, but did not affect either IAA reception by TIR1 and AFB2, or transport by AUX1, LAX3, and PIN1. The evaluations of hormonal concentrations and the detection of IAA cellular localization by a DR5::GUS line sustained these results. Altogether, the research demonstrates that a crosstalk between ethylene and IAA exists in the control of AR-formation, and involves ethylene signalling and IBA-to-IAA conversion