Involvement of auxin and LTP proteins in the regulation of root nodule formation in Medicago truncatula - Sinorhizobium meliloti Symbiosis

Questo progetto di dottorato ha avuto come obiettivi: i) valutazione del ruolo del\u2019auxina di derivazione batterica nella simbiosi rizobio-leguminosa, che d\ue0 origine a noduli di tipo indeterminato, ii) lo studio funzionale di MtN5, una proteina di tipo \u201cPathogenesis Related\u201d, che viene indotta precocemente durante la nodulazione e che presenta omologie di sequenza con membri della famiglia delle Lipid Transfer Protein vegetali. L\u2019auxina (acido indol-3-acetico, IAA) \ue8 un ormone vegetale implicato in molti aspetti che riguardano la vita e lo sviluppo delle piante; un suo coinvolgimento nello sviluppo del nodulo radicale era stato ipotizzato gi\ue0 all\u2019inizio del secolo scorso. Studi successivi hanno dimostrato un\u2019inibizione del trasporto acropeto di IAA nella radice a seguito dell\u2019infezione con rizobi, con un conseguente accumulo di fitormone a livello del sito di infezione. E\u2019 stato dimostrato che la maggior parte dei batteri della rizosfera che producono effetti di promozione sulla crescita della pianta, rizobi inclusi, possiedono vie biosintetiche per IAA. I rizobi sono in grado di sintetizzare auxina in coltura liquida e, molto probabilmente, mantengono questa capacit\ue0 anche durante la nodulazione. Ad oggi, per\uf2, i dati riguardanti il ruolo dell\u2019auxina batterica nello sviluppo dei noduli sono ancora controversi; sono stati infatti documentati sia effetti stimolatori che inibitori. Molti degli eventi che stanno alla base dell\u2019associazione simbiotica tra rizobi e leguminose devono ancora essere chiariti. Ad esempio, la natura e la funzione dei segnali ormonali scambiati tra ospite e simbionte non sono ancora stati compresi nel dettaglio, cos\uec come le differenze e i parallelismi nella risposta delle leguminose verso il simbionte e verso i patogeni della radice. A tal riguardo, recenti osservazioni hanno dimostrato che la repressione della via di segnalazione intracellulare dell\u2019auxina risulta in una maggiore resistenza innata delle piante verso microrganismi patogeni. Piante di Medicago truncatula, specie modello per le leguminose che producono noduli di tipo indeterminato, e Medicago sativa (erba medica), specie correlata di interesse agronomico, sono state nodulate sia con rizobi wild-type e che con rizobi in grado di iper-produrre IAA (S. meliloti IAA). I risultati ottenuti hanno dimostrato che piante nodulate con S. meliloti IAA producevano un numero maggiore di noduli (aumento del 50% in M. sativa e aumento del 100% in M. truncatula) e un apparato radicale pi\uf9 ramificato. Inoltre il contenuto di auxina nei noduli prodotti da rizobi IAA \ue8 mediamente 10 volte superiore alla concentrazione dei noduli prodotti da rizobi wild-type. I livelli di espressione dei geni responsabili del trasporto di auxina \ue8 stato valutato mediante RT-PCR quantitativa (qRT-PCR) e il carrier di efflusso MtPIN2 \ue8 risultato significativamente pi\uf9 espresso (circa 2 volte) nel tessuto radicale di piante nodulate con rizobi IAA rispetto alle radici infettate con il rizobio di controllo. Questi risultati suggeriscono che l\u2019effetto di promozione osservato sulla nodulazione e sull\u2019accrescimento della radice laterale siano dovuti alla produzione di IAA nel nodulo e ad una sua redistribuzione all\u2019interno dell\u2019apparato radicale. E\u2019 stato ampiamente dimostrato che l\u2019ossido nitrico (NO) agisce come secondo messaggero nell\u2019induzione di radici laterali e avventizie stimolata da auxina. Considerando la comune organogenesi tra radici laterali e avventizie e noduli indeterminati, in questo lavoro abbiamo dimostrato che esiste un collegamento tra auxina e NO nella via di segnalazione che porta all\u2019induzione del nodulo. Per mezzo di uno screening preliminare, condotto mediante qRT-PCR e volto ad individuare geni differenzialmente espressi in piante nodulate con rizobi IAA e piante nodulate con rizobi wild-type, fu osservato che il gene MtN5 era pi\uf9 espresso negli apparati radicali di piante infettate con rizobi iperproduttori di auxina. Il prodotto genico di MtN5 \ue8 stato annotato come una Lipid Transfer Protein (LTP) putativa. Le LTP vegetali sono caratterizzate dalla capacit\ue0 sia di legare lipidi in vitro che di inibire la crescita di microrganismi. In questo progetto di tesi \ue8 stato dimostrato che MtN5 possiede la capacit\ue0 di legare lisolipidi e che, come molti altri membri di questa famiglia di proteine, possiede attivit\ue0 antimicrobica in vitro, in particolare contro Fusarium semitectum, Xanthomonas campestris e S. meliloti. Lo studio del profilo di espressione conferma che MtN5 viene precocemente indotta durante la nodulazione e che \ue8 specificamente localizzata all\u2019interno del nodulo radicale. Inoltre, l\u2019infezione di piante con F. semitectum provoca l\u2019accumulo di MtN5 nel tessuto radicale. La funzione di MtN5 nella nodulazione \ue8 stata studiata mediante la generazione di radici avventizie transgeniche, sia overesprimenti che silenziante per il gene di interesse. Le radici silenziate per MtN5 sviluppano circa la met\ue0 dei noduli rispetto a radici di controllo, mentre in radici transgeniche over-esprimenti MtN5 il numero di noduli \ue8 risultato incrrementato del 300% rispetto al controllo. I risultati ottenuti dimostrano che MtN5 facilita l\u2019interazione simbiotica tra M. truncatula e S. meliloti, agendo probabilmente negli stadi precoci dell\u2019infezione, e suggeriscono che MtN5 potrebbe avere un ruolo nella protezione dei noduli verso patogeni della radice. Ulteriori studi sono comunque necessari per ottenere una immagine pi\uf9 chiara del ruolo di MtN5 sia nella simbiosi che nella risposta verso i patogeni.The present thesis has had two main focuses: i) the evaluation of the role of bacteria-derived auxin in the symbiosis between rhizobia and legumes that bear indeterminate nodules, ii) the functional study of MtN5, a pathogenesis related protein which presents sequence homology with the members of the plant Lipid Transfer Proteins (LTP) family and is precociously induced during nodulation. Auxin (indol-3-acetic acid, IAA) is a phytohormone involved in many aspects of plants growth and development; The role of auxin in the development of the rhizobia-legumes symbiosis was first hypothesised at the beginning of the twentieth century. More recent studies have demonstrated that auxin is accumulated at the site of infection as a consequence of the inhibition of the acropetal auxin transport in roots upon rhizobia inoculation. The production of IAA has also been documented in plant-associated rhizobacteria, including rhizobia, that have promoting effects on plants growth. When grown in liquid media, rhizobia can synthesize auxin and most likely they retain the same capability also during the nodule development. However, up to date, the data concerning the role of bacteria-derived auxin in the establishment of the symbiotic association are still contradictory, since both stimulatory and inhibitory effects have been documented. Thus, there are still open questions in the understanding of the events that result in the establishment of the symbiosis. First of all the nature and the function of the hormonal signal(s) exchanged between the host and the symbiont are not thoroughly unfolded, as well as the parallelisms and the differences in the responses of legumes against root pathogens and root symbiont. In these regards, recent findings have pointed out that plants innate immunity results, at least in part, from the down-regulation of the auxin signalling pathway. Medicago truncatula and Medicago sativa plants were nodulated with both wild-type and auxin hyper-synthesising rhizobia (Sinorhizobium meliloti IAA). The results obtained showed that plants nodulated with S. meliloti IAA produced a higher number of root nodules (50% more nodules in M. sativa and 100% more nodules in M. truncatula) and a more branched root apparatus. The root nodules elicited by S. meliloti IAA had a higher IAA content (at least 10-fold) when compared to control nodules. The expression levels of the auxin carriers were evaluated and the efflux facilitator MtPIN2 resulted more abundant (about 2-fold) in the root tissue of IAA plants when compared to wild-type plants These data suggested that such promoting effects on nodulation and lateral root growth might be due to the increased auxin content detected in IAA nodule produced by auxin hyper-synthesising rhizobia, as well as to a redistribution of the phytohormone in the root tissue. It has been largely demonstrated that nitric oxide (NO) acts as second messenger in the auxin-induced pathway that leads to formation of lateral and adventitious roots. Since root nodules have the same organogenetic origin of lateral and adventitious roots, the possible connection between NO and root nodule induction was investigated and we demonstrated that NO participate in the signalling pathway for root nodule induction. During a preliminary screening carried out by means of qRT-PCR, it has been found that N5 gene of M. truncatula was more abundantly expressed in roots nodulated with S. meliloti IAA with respect to roots infected by wild-type rhizobia. The gene product of MtN5 was annotated as putative Lipid Transfer Protein (LTP). LTPs are characterized by their ability to bind lipids in vitro and the majority of them exhibits antimicrobial activity. In this thesis, it has been demonstrated that the recombinant MtN5 protein is able to bind lysolipids and possesses inhibitory activity against Fusarium semitectum, Xanthomonas campestris and S. meliloti. The studies of the expression pattern of both MtN5 transcript and MtN5 protein confirmed that it is precociously induced during nodulation and revealed that it is specifically localized in the root nodule. In addition, when M. truncatula plants are infected with the root pathogenic fungus F. semitectum, MtN5 protein is accumulated in the root apparatus. The function of MtN5 in nodulation has been studied through the generation of transgenic adventitious roots, both over-expressed and silenced for the gene of interest. MtN5-silenced roots developed approximately 50% fewer nodules as compared to control roots, whereas in hairy roots over-expressing MtN5 the nodule number was increased by about 300% with respect to control roots. Collectively the data indicate that MtN5 facilitates the symbiotic interaction between M. truncatula and S. meliloti, probably acting in the early stages of rhizobia infection, and suggest that it might have a role in the protection of nodules against root pathogen. However, further studies are needed to have a clear picture of the role played by MtN5 in both symbiosis and defence response against pathogens

Auxin and nitric oxide control indeterminate nodule formation

<p>Abstract</p> <p>Background</p> <p>Rhizobia symbionts elicit root nodule formation in leguminous plants. Nodule development requires local accumulation of auxin. Both plants and rhizobia synthesise auxin. We have addressed the effects of bacterial auxin (IAA) on nodulation by using <it>Sinorhizobium meliloti </it>and <it>Rhizobium leguminosarum </it>bacteria genetically engineered for increased auxin synthesis.</p> <p>Results</p> <p>IAA-overproducing <it>S. meliloti </it>increased nodulation in <it>Medicago </it>species, whilst the increased auxin synthesis of <it>R. leguminosarum </it>had no effect on nodulation in <it>Phaseolus vulgaris</it>, a legume bearing determinate nodules. Indeterminate legumes (<it>Medicago </it>species) bearing IAA-overproducing nodules showed an enhanced lateral root development, a process known to be regulated by both IAA and nitric oxide (NO). Higher NO levels were detected in indeterminate nodules of <it>Medicago </it>plants formed by the IAA-overproducing rhizobia. The specific NO scavenger cPTIO markedly reduced nodulation induced by wild type and IAA-overproducing strains.</p> <p>Conclusion</p> <p>The data hereby presented demonstrate that auxin synthesised by rhizobia and nitric oxide positively affect indeterminate nodule formation and, together with the observation of increased expression of an auxin efflux carrier in roots bearing nodules with higher IAA and NO content, support a model of nodule formation that involves auxin transport regulation and NO synthesis.</p

The involvement of root-specific LTPs in the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti

Lipid transfer proteins (LTPs) are small basic proteins that constitute a large family characterized by the ability to transfer phospholipids between a donor and an acceptor membrane and can have many different roles in vivo. Recently it has been demonstrated that MtN5, a non specific LTP (ns-LTP) classified as type III (Wang et al., 2012), is involved in the symbiotic interaction between legumes and rhizobia (Pii et al. 2009, Pii et al., 2012). MtN5 is a nod factor responsive gene expressed at a very early phase of rhizobial symbiosis in the epidermis and root hairs and later in primordia and nodules. There are evidences that MtN5 positively regulates the nodulation process. Interestingly, two other putative type III ns-LTPs (Medtr3g055250 and Medtr7g052640) have been identified in Medicago truncatula genome. The aim of this study is to shed light on the role of these ns-LTPs in the symbiotic interaction between M. truncatula and Sinorhizobium meliloti

The fertilising potential of manure-based biogas fermentation residues: pelleted vs. liquid digestate

Spreading of manure on agricultural soils is a main source of ammonia emissions and/or nitrate leaching. It has been addressed by the European Union with the Directives 2001/81/EC and 91/676/EEC to protect the environment and the human health. The disposal of manure has therefore become an economic and environmental challenge for farmers. Thus, the conversion of manure via anaerobic digestion in a biogas plant could be a sustainable solution, having the byproducts (solid and liquid digestates) the potential to be used as fertilizers for crops. This work aimed at characterizing and assessing the effect of digestates obtained from a local biogas plant (Biogas Wipptal, Gmbh), either in the form of liquid fraction or as a solid pellet on: (i) the fertility of the soils during an incubation experiment; (ii) the plant growth and nutritional status of different species (maize and cucumber). Moreover, an extensive characterization of the pellet was performed via X-ray microanalytical techniques. The data obtained showed that both digestates exhibit a fertilizing potential for crops, depending on the plant species and the fertilizer dose: the liquid fraction increases the shoot fresh weight at low dose in cucumber, conversely, the solid pellet increases the shoot fresh weight at high dose in maize. The liquid digestate may have the advantage to release nutrients (i.e. nitrogen) more rapidly to plants, but its storage represents the main constraint (i.e. ammonia volatilization). Indeed, pelleting the digestates could improve the storability of the fertilizer besides enhancing plant nutrient availability (i.e. phosphate and potassium), plant biomass and soil biochemical quality (i.e. microbial biomass and activity). The physical structure and chemical composition of pellet digestates allow nutrients to be easily mobilized over time, representing a possible source of mineral nutrients also in long-term applications

Copper toxicity compromises root acquisition of nitrate in the high affinity range

The application of copper (Cu)-based fungicides for crop protection plans has led to a high accumulation of Cu in soils, especially in vineyards. Copper is indeed an essential micronutrient for plants, but relatively high concentrations in soil or other growth substrates may cause toxicity phenomena, such as alteration of the plant’s growth and disturbance in the acquisition of mineral nutrients. This last aspect might be particularly relevant in the case of nitrate (NO3−), whose acquisition in plants is finely regulated through the transcriptional regulation of NO3− transporters and plasma membrane H+-ATPase in response to the available concentration of the nutrient. In this study, cucumber plants were grown hydroponically and exposed to increasing concentrations of Cu (i.e., 0.2, 5, 20, 30, and 50 µM) to investigate their ability to respond to and acquire NO3−. To this end, the kinetics of substrate uptake and the transcriptional modulation of the molecular entities involved in the process have been assessed. Results showed that the inducibility of the high-affinity transport system was significantly affected by increasing Cu concentrations; at Cu levels higher than 20 µM, plants demonstrated either strongly reduced or abolished NO3− uptake activity. Nevertheless, the transcriptional modulation of both the nitrate transporter CsNRT2.1 and the accessory protein CsNRT3.1 was not coherent with the hindered NO3− uptake activity. On the contrary, CsHA2 was downregulated, thus suggesting that a possible impairment in the generation of the proton gradient across the root PM could be the cause of the abolishment of NO3− uptake

Selenium biofortification in fragaria 7 ananassa: Implications on strawberry fruits quality, content of bioactive health beneficial compounds and metabolomic profile

Selenium (Se) is an essential nutrient for humans, due to its antioxidant properties, whereas, to date, its essentiality to plants still remains to be demonstrated. Nevertheless, if added to the cultivation substrate, plants growth resulted enhanced. However, the concentration of Se in agricultural soils is very variable, ranging from 0.01 mg kg-1 up to 10 mg kg-1 in seleniferous areas. Therefore several studies have been performed aimed at bio-fortifying crops with Se and the approaches exploited were mainly based on the application of Se fertilizers. The aim of the present research was to assess the biofortification potential of Se in hydroponically grown strawberry fruits and its effects on qualitative parameters and nutraceutical compounds. The supplementation with Se did not negatively affect the growth and the yield of strawberries, and induced an accumulation of Se in fruits. Furthermore, the metabolomic analyses highlighted an increase in flavonoid and polyphenol compounds, which contributes to the organoleptic features and antioxidant capacity of fruits; in addition, an increase in the fruits sweetness also was detected in biofortified strawberries. In conclusion, based on our observations, strawberry plants seem a good target for Se biofortification, thus allowing the increase in the human intake of this essential micronutrient

Cultivating resilience: Harnessing pyoverdine-producing Pseudomonas to contrast iron deficiency in cucumber plants

Iron (Fe) deficiency in crops significantly reduces yield, impacting agricultural productivity worldwide. Synthetic Fe chelates are commonly applied as fertilizers to address this issue, but their synthetic nature and prolonged use poses environmental risks. Thus, inoculation of plant growth-promoting bacteria rises as an alternative to enhance Fe uptake in crops while minimizing reliance on synthetic chelates. This study aimed to examine the influence of Pseudomonas RMC4 inoculation and pyoverdine application on cucumber plants cultivated hydroponically under Fe deficiency conditions. Evaluations included the SPAD index, plant biomass, root morphology, Fe-chelate reductase activity, gene expression, and ionomic analysis. Following Fe deficiency, Pseudomonas RMC4 inoculation improved the SPAD index, increased dry weight, enhanced root development, and facilitated Fe acquisition mechanisms, thus, fostering the endogenous resilience of the plant to the limited Fe availability. This improvement was observed with bacterial inoculation or pyoverdine application alongside an insoluble Fe source (ferrihydrite). Overall, the results suggest the beneficial impact of Pseudomonas RMC4 inoculation in alleviating symptoms of Fe deficiency. Future studies will investigate bacterial application under field conditions to assess its potential in replacing synthetic Fe-chelates fertilizers in crop production in favor of more sustainable agricultureThis work was financially supported by the Italian Ministry of Education, University and Research (MIUR) through the project Cleopatra coded 2022AAATEA and by the Free University of Bolzano through the project COMPETITIVE. The authors also gratefully acknowledge the f inancial support of the Ministerio de Ciencia, Innovaci´ on y Universidades and the Agencia Estatal de Investigaci´ on through the project PID2022-141721OB-C2. Jos´ e María Lozano-Gonz´alez is the recipient of the FPI grant from the Ministerio de Ciencia e Innovaci´ on (PRE-2019091246

Time-Resolved Investigation of Molecular Components Involved in the Induction of NO3- High Affinity Transport System in Maize Roots

The induction, i.e., the rapid increase of nitrate ([Formula: see text]) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to [Formula: see text] treatment were characterized in terms of changes in [Formula: see text] uptake rate and plasma membrane (PM) H(+)-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3, and PM H(+)-ATPase gene families. The behavior of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a 3c150 kDa oligomer. The expression trend during the induction of the 11 identified PM H(+)-ATPase transcripts indicates that those mainly involved in the response to [Formula: see text] treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A non-denaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H(+)-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms

Recent achievements and new research opportunities for optimizing macronutrient availability, acquisition, and distribution for perennial fruit crops

CITATION: Kalcsits, Lee et al. 2020. Recent achievements and new research opportunities for optimizing macronutrient availability, acquisition, and distribution for perennial fruit crops. Agronomy, 10(11): 1738, doi.org/10.3390/agronomy10111738.The original publication is available at: https://www.mdpi.comTree responses to fertilizer management are complex and are influenced by the interactions between the environment, other organisms, and the combined genetics of composite trees. Increased consumer awareness of the environmental impact of agriculture has stimulated research toward increasing nutrient-use efficiency, improving environmental sustainability, and maximizing quality. Here, we highlight recent advancements and identify knowledge gaps in nutrient dynamics across the soil–rhizosphere–tree continuum for fruit crops. Beneficial soil management practices can enhance nutrient uptake and there has been significant progress in the understanding of how roots, microorganisms, and soil interact to enhance nutrient acquisition in the rhizosphere. Characterizing root architecture, in situ, still remains one of the greatest research challenges in perennial fruit research. However, the last decade has advanced the characterization of root nutrient uptake and transport in plants but studies in tree fruit crops have been limited. Calcium, and its balance relative to other macronutrients, has been a primary focus for mineral nutrient research because of its important contributions to the development of physiological disorders. However, annual elemental redistribution makes these interactions complex. The development of new approaches for measuring nutrient movement in soil and plant systems will be critical for achieving sustainable production of high-quality fruit in the future.Publisher's versio