Plant biostimulants, such as PGPR (plant growth-promoting rhizobacteria), are important tools of an integrated crop management system, that may help agriculture become more sustainable and resilient. The mechanisms of their beneficial effect may include enhanced nutrient uptake, improvement of biotic/abiotic stress resilience by activation of growth regulators and stress-responsive hormones, oxidative stress reduction and induction of plant defence mechanisms (for review see Hamid et al. 2021).
Two PGPR used in the present study were already demonstrated to have beneficial effect on crop production; Azospirillum brasilense (AB) Ab-V5 increased nitrogen use efficiency, improved maize tolerance to stress (Zeffa et al. 2019), while Paraburkholderia graminis (PG) increased the leaf chlorophyll content in three different processing tomato genotypes (Caradonia et al. 2019).
In a previous study in greenhouse and open field experiments using grafted tomato plants and different biostimolants, the treatment with A. brasiliensis induced flowering stage and increased the number of flowers in greenhouse experiment, while for both AB and PG treatments increased marketable and total yields with higher fruit dry weight, leaf dry weight, plant total dry weight were observed in open field (Caradonia et al. 2020).
In order to understand the molecular mechanisms underlying these interactions, the aim of this study was to profile, for the first time, to the author knowledge, the transcriptomic changes induced in leaves of grafted tomato seedlings by root inoculations of beneficial microorganisms P. graminis C4D1M and A. brasiliensis sp.245. Interactions between root beneficial microorganisms and plants can improve crop performances inducing molecular and physiologic changes also in organs distant from the point of inoculation such as shoots, leaves and fruits (Zouari et al. 2014; Cervantes-Gámez et al. 2016; Fiorilli et al. 2018). Leaves were chosen for this experiment as they play an important role in growth and development of crops, are involved in many processes such as photosynthesis and response to drought stress and represent the first barrier of defence that foliar pathogens meet, during infection of plants. In the present study, grafted tomato was used as a host, since currently, the commercial tomato grafting is widely adopted in the main cropping areas. Data on interactions between A. brasiliensis sp. 245 or P. graminis C4D1M and not grafted plants would not be informative for interactions between these beneficial microorganisms and widely used grafted tomato plants since a recent study has revealed that grafting modifies tomato transcriptome (Spanò et al. 2020) and studies on maize and rice demonstrated that the effects of Azospirillum spp. inoculations differed on the strain/cultivar combinations (Chamam et al. 2013; Walker et al. 2011). Moreover, RNA trascriptomic profiling studies available in literature cooncetrated mainly on effects of microorganisms assessed under a biotic (Hu et al. 2018; Zhou et al. 2021) or under an abiotic stress factor (Balestrini et al. 2019). Conversely, our stategy was to compare the influence of two beneficial microorganisms on tomato seedlings grown in optimal conditions that may allow to better undestand the molecular mechanisms influenced by AB and PG treatments regardless of whether the plants are under stress or not.
The number of DEGs detected after PG and AB treatments was 512 and 427, respectively, and is consistent with those obtained in other studies evaluating the effects of microorganism-plant interactions on gene expression in leaves (Cervantes-Gámez et al. 2016; Liu et al. 2007).
Only few DEGs were modulated in both treatments, suggesting that the interactions between tomato and microorganisms are species-specific, and affect the activity of specific sets of genes, that however activate and/or involve similar pathways and mechanisms. As it could be expected for PGRP-plant interaction, the shared DEGs included genes involved in nutrients metabolism or activated in response to nutrient starvation, such as Solyc06g062540.3.1, encoding Inorganic pyrophosphatase 1-like, and Solyc06g007180.31 encoding asparagine synthetase [glutamine -hydrolizing]. Asparagine synthetase is responsible for the biosynthesis of asparagine, an ammino acid used for protein production and nitrogen assimilation, and a key molecule involved in recycling, transport and storage of nitrogen in all plant organs (Gaufichon et al. 2010). An up-regulation of some inorganic pyrophosphatase genes in both roots and leaves of chickpea plants under nutritional stress has been recently reported (Nasr Esfahani et al. 2021).
Functional annotation of the DEGs revelead that they coded mainly for proteins involved in water transport, regulation of transcription and hormones synthesis and singnalling pathways and those activated in response to oxidative or biotic and abiotic stresses.
In the field experiment of our previous study (Caradonia et al. 2020), all the investigated treatments reduced the number of fruits affected by blossom-end rot (BER), a physiological disorder that causes important economic losses Although BER is mainly associated to the soil concentration of calcium available, it may be influenced also by reduced nutrient and water uptake (Hagassou et al. 2019; Caradonia et al. 2020). In the present study, the transcription profiling enabled identification of three acquaporins as differentially expressed: Solyc06g075650.3.1 in both treatments, and Solyc06g060760.3.1 and Solyc06g011350.3.1 only in AB treatment. Aquaporins, known as water channel proteins, help plants in the transport of water and other solutes such as glycerol and urea. Aquaporins also regulate the opening and closure of stomata, that are crucial processes for the temperature regulation of leaves and the evaporation of water (Chaumont et al. 2005). Therefore, the induction of aquaporin-coding genes can improve plant performance, especially under drought stress (Reuscher et al. 2013). Other proteins important for drought stress response are dehydrins, in this work Solyc02g062390.3.1 gene that codes for Dehydrin DHN2 was observed to be induced in both treatments. Dehydrins with an hydrophilic nature can improve hydratation and reduce water loss in plants (Roychoudhury and Nayek 2014). A study on pepper plants inoculated with Bacillus licheniformis K11 showed an induced expression of dehydrin-like protein gene and an higher number of survived plants, compared with the not inoculated control under drought stress (Lim and Kim 2013).
Many studies documented that plant growth promoting rhizobacteria can trigger a wide variety of defence mechanisms in plants (e.g. oxidative burst, production of antimicrobial compounds and expression of defence-related genes) (Bari and Jones 2009; Robert-Seilaniantz et al. 2011). In this study, particularly AB treatment modulated the expression of many genes involved in responses to oxidative stress and defense response such as ascorbate oxidase, peroxydase, polyphenol oxidase, PRs proteins, etc. Oxidative stress occurs when the balance between ROS production and degradation is broken, leading to an increase of ROS concentration that damages nucleic acids, proteins, and lipids (Lushchak 2011). To respond to the harmful effects of ROS, plants have developed systems involving enzymes such as superoxide dismutase, catalase, ascorbate oxidase, the peroxidases, etc. (Hasanuzzaman et al. 2012). Ascorbate oxidase (AO) catalyses the oxidation of ascorbic acid to monodehydroascorbate, influencing the content of ascorbate and oxigen, and affecting the redox state. Furthermore, this enzyme has a role in the perception of environmental factors and stress responses (Pignocchi et al. 2006). In addition, AO was also proposed as relevant in the establishment of mutualistic plant-microbe interactions as its induction was during nodulation in Lotus japonicus and during the colonization by an arbuscular mycorrhizal (AM) fungus (De Tullio et al. 2013). Studies on trasgenic tomato plants reported that peroxidase (POX) and polyphenol oxidase (PPO) are induced by wounding and pathogen attacks (Li and Steffens 2002; Thipyapong et al. 2004). Moreover, induced defense responses of PGPR-treated tomato plants to Alternaria solani were shown to be associated with enhanced POX and PPO biosynthesis (Babu et al. 2015). In the present study, a significant activation of antioxidant peroxidase and polyphenol oxidase ezymes was observed in plants treated with plant growth promoting rhizobacteria (PGPR) without pathogen infections. This suggests that these enzyme may be involved also in a sort of defense priming.
Interestingly, the PG inoculation induced the expression of five genes (Solyc05g007630.3.1, Solyc07g049700.1.1, Solyc05g013260.3.1, Solyc09g098100.4.1, Solyc05g005130.3.1) coding for putative late blight resistance protein homolog, while Solyc10g008700.1 coding for MYB49 trascription factor, whose expression was reported to correlate with an increase of resistance of tomato plants to Phytophtora infestans, (Cui et al. 2018) was induced by AB treatment. These results may suggest that treatment with beneficial microorganisms AB and PG might induce reponses that share mechanisms with those involved in resistance response to P. infestans. In a previous study (Caradonia et al. 2020), the field high moisture conditions allowed the spread of the oomicete Phytophthora infestans that was only partially controlled by foliar spray application using copper treatments in the field. However, at harvest time, better results were obtained for PGPR-treated than for control plants for both morphological parameters and fruit quality traits, suggesting the treatment contrasted the effects of infection. Since only few plant protection products are authorized in Europe against this pathogen for both potato and tomato crops, the control of late blight is a challenge (Park et al. 2013) especially in organic farming, where only copper compounds can be applied. It would be thus very intestesting to investigate in futher field and greenhouse trials the use of these beneficial bacteria as alternative or to reduce the use of copper compounds in organic farming against P.infestans for development of sustainable tomato management with low external inputs.
The expression of many transcription factor genes beloging to MYB and WRKY families, known to be modulated in response to different abiotic and biotic stresses (Huang et al. 2012; Wang et al. 2020), was modulated by AB and PG treatments in the present study as well. The unique nomenclature for tomato MYB and WRKY transcription factors family adopted and reviewed by Zhao et al. 2014 and Huang et al. 2012 respectively, was followed. AB treatment induced SlMYB71 (Solyc05g053150.2.1) together with SlMYB49 (Solyc10g008700.3.1) (already mentioned above), known to be also up-regulated along with the fruit development (Zhao et al. 2014), SlMYB41 (Solyc07g054840.4.1), that was reported to affects root architecture and improves tolerance to salinity in tomato plants (Campobenedetto et al. 2021), and SlMYB63 (Solyc10g005550.3.1), a root-specific transcription factor functioning as a node of convergence in the induced systemic resistance and Fe starvation signalling pathways (Buoso et al. 2019). Among WRKY transcription factors that were reported in literature to show significant induction under stresses of drought, salt and invasion of pathogen implying that these family members might be putative regulators in response to various biotic and abiotic stresses (Huang et al. 2012), we found SlWRKY43 (Solyc12g042590.2.1) and SlWRKY73 (Solyc03g113120.4.1) induced by AB treatments, SlWRKY41 (Solyc01g095630.3.1) and SlWRKY46 (Solyc08g067340.4.1) upregolated by PG, and SlWRKY6 (Solyc02g080890.3.1) induced by both treatments.
Many signalling molecules are involved in the cross-talks beetween crops and soil microorganisms. Phytohormones, such as auxins, gibberellins, ethylene, etc, are considered as the main signal molecules in plants (Puga-Freitas and Blouin 2015). In this study, genes involved in auxins, giberrellin, ethylene and abscisic acid metabolism and signaling were found to be induced in at least one of the two treatments. Auxins and gibberellins are involved in many aspects of plant growth and development. Mariotti et al. (2011) associated the positive modulation of indoleacetic acids (IAA), auxine responsive moelcules, and gibberellins with the fruit-set and early fruit growth in tomato. Recently, auxine responsive GH3.1 was proposed as a major player in balancing the auxin synthesis and metabolism, ensuring fruit set in pathenocarpic tomato in any conditions (Zhang et al. 2021). In the present study, Solyc01g107390 coding for GH3.1 was induced by AB treatment; it is worth noting that in the field experiment (Caradonia et al. 2020), higher number of fruits was reported for AB than for PG treatment, the latter anyway being higher than the control not inoculated.
Flower development, fruit ripening, organ senescence, abscission and responses to abiotic and biotic stresses are also modulated by ethylene (Iqbal et al. 2017; Liu et al. 2016). Ethylene signaling and response, in turn, are regulated by Ethylene Response Factors (ERFs) (Khan et al. 2017). The unique nomenclature for tomato ERF transcription factors family, adopted and reviewed by Liu et al. (2016), was followed for the genes identified in the present work. Numerous ERFs related to fruit ripening were modulated by PG treatments: SlERF.B4 (Solyc03g093540.1.1), SlERF.B5 (Solyc03g093550.1.1) and SlERF.D4 (Solyc10g050970.1.1) with preferential expression in young unripe fruits that declines at the onset of ripening and, SlERF.B1(Solyc05g052040.1.1) and SlERF.B13 (Solyc08g078190.2.1), known to be upregualated during ripening (Liu et al. 2016). Two other ERFs, SlERF.B2 (Solyc03g093560.1.1) and SlERF.F1 (Solyc10g006130.1.1), reported to be involved in salt and drought tolerance (Pan et al. 2012) and photosynthesis and growth regulation (Upadhyay et al. 2013), respectively, were induced by PG treatment as well.
AB induced expresion of a gene coding for SlERF.C4 (Solyc09g089930) reported in leterature to be involved in pathogen resistance and exhibiting high expression in roots, leaves, flowers, and immature fruits (Zhang et al. 2004).