Catalase (CAT) is one of the key enzymes in plant development and in the regulation of nitro-oxidative stress associated with the uncontrolled overproduction of reactive oxygen (ROS) and nitrogen species (RNS). Despite its well-established role in aerial organs, CAT functions in the root system remain underexplored. Using Arabidopsis thaliana cat2-1, defective in CAT2 and exhibiting reduced CAT activity, we demonstrated that CAT2 is directly involved in defining root architecture, particularly lateral root (LR) development under optimal growth conditions. Primary root (PR) and LR development were analysed in cat2-1 through morpho-cyto-histological investigations, nitro-oxidative assays, and metabolomic analyses. To further investigate CAT2's role in root architecture, we exposed cat2-1 to nitro-oxidative stress using 60 mu M cadmium sulphate (CdSO4). Results show that CAT2 is the main CAT isozyme in Arabidopsis root system. cat2-1 showed inhibition of LR development and PR elongation, exacerbated by Cd. These defects were highlighted by cyto-histological analyses, revealing alterations in the division/differentiation patterns of pericycle cells competent for LR initiation as well as in meristematic cells of PR and LRs. Cat2-1 root alterations were associated with an accumulation of trans-zeatintype cytokinin and with changes in ROS- and, to a lesser extent, RNS-homeostasis. Loss of CAT2 function exacerbated some of the Cd-related alterations, particularly the disorganisation of cell division and differentiation patterns within root meristems, and ROS accumulation, supporting a positive role of CAT2 in root architecture. Our results demonstrate a new role for CAT2 per se in Arabidopsis root system development, beyond its involvement in regulating stress response processes.

Sorghum is generally considered a moderately salt-tolerant crop, although substantial variations in salt tolerance exist among grain sorghum genotypes. Moreover, the mechanisms underlying salt tolerance in this species remain poorly understood compared with those of other crops. Brassinosteroids (BRs) regulate numerous developmental processes and stress responses and their exogenous application, also via seed priming, has been shown to mitigate the negative effects of salinity in several plant species, including sorghum. However, the role of BR seed priming in root development and the biochemical, cellular, and molecular responses to salt stress remains unclear. Seeds of the salt-tolerant sorghum genotype Bianca and the salt-sensitive genotype Tonkawa were primed with 1μM of 24-epibrassinolide and then cultured in vitro in the presence or absence of 150 mM NaCl. Root system morphology, lipid peroxidation levels, Raman spectral profiles associated with membrane and cell wall components, cytosolic K+ levels and vacuolar Na+ compartmentalization were evaluated. BR seed priming enhanced root system architecture under salt stress, promoting adventitious and lateral root formation, and reducing lipid peroxidation, particularly in the salt-sensitive Tonkawa. In this genotype, BR seed priming had a long-lasting effect, inducing Na+ sequestration in root cell vacuoles, modulating membrane and cell wall components, and improving root system branching, thereby ensuring adequate water uptake as a protective mechanism against high soil salinity. Overall, the results demonstrate the efficacy of BR seed priming in enhancing salt tolerance in sorghum, highlighting its potential as a sustainable strategy for improving crop performance under saline conditions.

Solanum tuberosum (potato) is the third most important food crop in the world. It is an autopolyploid heterozygous crop with high inbreeding depression. Potato tubers are used in biotechnology for crop enhancement because their buds easily adapt to in-vitro culture. Potato is prone to biotic stress, including soil and tuber-borne diseases. Biotechnological approaches improve potato output and quality for disease eradication and for reducing inbreeding depression. Clonal propagation by in vitro culture preserves heterozygosity in interesting cultivars, including “Agata”. Exogenously applied benzyl-adenine (BAP) and indole-3-butyric acid (IBA) favor different phases of the sprouting-to-tuberization process, but a role for exogenous brassinosteroids (24-epibrassinolide, eBL) has been also suggested. Using sequential applications of BAP, IBA, and eBL, at specific concentrations, the process from bud-sprouting to tuberization was morphologically/cyto-histologically analyzed. Tuber-bud sprouting was favored by 9 μM BAP and the subsequent application of eBL (0.1 μM) to stem mini cuttings favored shoot elongation, whereas that of IBA (1 μM) increased rhizogenesis. Tuberization occurred during ex vitro acclimation. Tuber microbiological analyses verified the absence of contamination due to pathogenic microorganisms. Altogether results represent a biotechnology advancement in cell culture applications of potato and show a method for producing healthy plants while maintaining their heterozygous features.

Soil salinity is threatening the cultivation of major cereal crops worldwide. Sorghum is a promising alternative to other cereals for both human and animal nutrition. This is due to the nutritional characteristics of its caryopses and its natural ability to grow in marginal environments, characterized by various stresses, including salinity. However, stress tolerance varies among sorghum genotypes, highlighting the need for technologies to enhance salinity resilience. Phytohormone seed priming is a cost-effective and eco-friendly approach to improve horticultural crops environmental stress tolerance as well as plant growth and vigour. Among phytohormones, brassinosteroids (BRs), when used as primed seed agents, have been shown to enhance morpho-anatomical and physiological defences against abiotic stress in various crops. However, further research is needed to understand their role as seed priming agent in restoring photosynthesis affected by salt stress, especially in salt-sensitive sorghum genotypes. Thus, this study evaluated the effects of seed priming with 24-epibrassinolide (24-eBL), a bioactive BR precursor on Bianca and Tonkawa, salt-tolerant and salt-sensitive genotypes, respectively, grown for 44 days in pots under 150 mM NaCl. The results demonstrate that seed priming with 1 µM 24-eBL for 8 h restores growth in Tonkawa but not in Bianca, by inducing anatomical leaf adaptations, such as restoration of mesophyll and substomatal air spaces, regulation of bulliform cell area, and modifications in cuticle thickness without altering its chemical composition. Overall, 24-eBL seed priming mitigates salt stress effects in the salt-sensitive genotype by improving leaf anatomy, which in turn improves photosynthetic efficiency and ultimately promotes biomass recovery.

Arsenic, a toxic metalloid, predominantly exists in soil as inorganic arsenate (AsV) and arsenite (AsIII). Upon root uptake, AsV is extensively reduced to AsIII in the plant. The Arabidopsis root system comprises primary, lateral and adventitious roots. It is unclear whether the inorganic arsenic-form and concentration affect specific components of the Arabidopsis root system. Synergistic and antagonistic interactions of brassinosteroids and jasmonates regulate plant development under stress, as shown by treatments with epibrassinolide (eBL) or methyl jasmonate (MeJA). However, the role of these phytohormones in root response to inorganic arsenic has been poorly studied. This research aimed to determine whether Arabidopsis roots of different type respond similarly or differently to AsIII and AsV, administered as NaAsO2 and Na2HAsO4·7 H2O respectively, and whether these responses are modulated by the application of eBL and/or MeJA. The results demonstrated that AsIII inhibited primary root elongation, in contrast to AsV, but promoted lateral and adventitious root formation, especially when combined with eBL. AsIII, more than AsV, induced irregular cell divisions in the quiescent center and the stem cell niche of root apices, mainly of lateral roots. The AsIII negative impact on lateral/adventitious roots was counteracted by eBL which favoured root formation. Xylogenesis was induced by periclinal divisions in the pericycle of the basal hypocotyl and was promoted by MeJA as a mechanical defense barrier against AsV. Collectively, results suggest that Arabidopsis responds to As by strengthening its root system and applications of eBL and MeJA ameliorate root development in specific ways, depending on the As-form.

Knowledge on salt tolerance requires further investigation, particularly in plants of agro-food interest. Sorghum is a potentially useful plant because it is a emerging food species that combines high levels of salt tolerance with interesting nutritional characteristics. In sorghum different genotypes respond differently to saline stress and the early events characterizing the salt stress tolerance are not yet fully understood. Moreover, the number of salt resistant genotypes needs to be extended. The genotypes Bianca and Tonkawa are two possible candidates for extending sorghum cultivation to soils characterized by high levels of salinity. The root is the first organ that responds to soil conditions, especially during the initial stages of plant developmental. The research aim was to analyse the root system responses to salt stress (NaCl) of Bianca and Tonkawa genotypes to identify the morpho-functional and metabolic changes that occur during the initial stages of the root system development and to use them as discriminating parameters for assessing the different plant’s susceptibility to the salt. The results showed that salt stress negatively affected many morphological and cyto-histological root parameters, from seed germination to root system establishment. The salt altered the root meristem organization and quiescent centre (QC) definition, but similarly in both genotypes. By contrast, it reduced primary root (PR) length and induced a more extended oxidative stress in the adventitious roots (ARs) and lateral root primordia (LRPs) of Tonkawa in comparison with Bianca.The stele area and the number of protoxylem and phloem elements in the ARs were more reduced in 150mM NaCl-treated Tonkawa seedlings in comparison with those of Bianca. Moreover, the salt enhanced lignin deposition in protoxylem, early metaxylem and endodermis and changed the root metabolic profiles significantly increasing the levels of leucine, isoleucine, alanine, proline, trigonelline, allantoin and glutamine in Bianca compared to Tonkawa. Altogether, specific morpho-anatomical and metabolic differences between the genotypes were identified as discriminating markers of genotype salt susceptibility.

Although Boraginaceae have been classified as good sources of nectar for many insects, little is still known about their nectar and nectaries. Thus, in the present contribution, we investigated the nectar production dynamics and chemistry in Borago officinalis L. (borage or starflower), together with its potential interaction capacity with pollinators. A peak of nectar secretion (∼5.1 µL per flower) was recorded at anthesis, to decrease linearly during the following 9 days. In addition, TEM and SEM analyses were performed to understand ultrastructure and morphological changes occurring in borage nectary before and after anthesis, but also after its secretory phase. Evidence suggested that nectar was transported by the apoplastic route (mainly from parenchyma to epidermis) and then released essentially by exocytotic processes, that is a granulocrine secretion. This theory was corroborated by monitoring the signal of complex polysaccharides and calcium, respectively, via Thiéry staining and ESI/EELS technique. After the secretory phase, nectary underwent degeneration, probably through autophagic events and/or senescence induction. Furthermore, nectar (Nec) and other flower structures (i.e., sepals, gynoecia with nectaries, and petals) from borage were characterized by spectrophotometry and HPLC-DAD, in terms of plant secondary metabolites, both at early (E-) and late (L-) phase from anthesis. The content of phytochemicals was quantified and discussed for all samples, highlighting potential biological roles of these compounds in the borage flower (e.g., antimicrobial, antioxidant, staining effects). Surprisingly, a high significant accumulation of flavonoids was registered in L-Nec, with respect to E-Nec, indicating that this phenomenon might be functional and able to hide molecular (e.g., defence against pathogens) and/or ecological (e.g., last call for pollinators) purposes. Indeed, it is known that these plant metabolites influence nectar palatability, encouraging the approach of specialist pollinators, deterring nectar robbers, and altering the behaviour of insects.

In land plants plastid type differentiation occurs concomitantly with cellular differentiation and the transition from one type to another is under developmental and environmental control. Plastid dynamism is based on a bilateral communication between plastids and nucleus through anterograde and retrograde signaling. Signaling occurs through the interaction with specific phytohormones (abscisic acid, strigolactones, jasmonates, gibberellins, brassinosteroids, ethylene, salicylic acid, cytokinin and auxin). The review is focused on the modulation of plastid capabilities at both transcriptional and post-translational levels at the crossroad between development and stress, with a particular attention to the chloroplast, because the most studied plastid type. The role of plastid-encoded and nuclear-encoded proteins for plastid development and stress responses, and the changes of plastid fate through the activity of stromules and plastoglobules, are discussed. Examples of plastid dynamism in response to soil stress agents (salinity, lead, cadmium, arsenic, and chromium) are described. Albinism and root greening are described based on the modulation activities of auxin and cytokinin. The physiological and functional responses of the sensory epidermal and vascular plastids to abiotic and biotic stresses along with their specific roles in stress sensing are described together with their potential modulation of retrograde signaling pathways. Future research perspectives include an in-depth study of sensory plastids to explore their potential for establishing a transgenerational memory to stress. Suggestions about anterograde and retrograde pathways acting at interspecific level and on the lipids of plastoglobules as a novel class of plastid morphogenic agents are provided.

Rice is a worldwide cultivated crop that serves as an important source of food for the human population, but it is also the simplest route for arsenic (As) contamination of the food chain. The As inorganic forms, arsenate [As(V)] and arsenite [As(III)], are the highly toxic As species found in the soil and the most easily absorbed by the roots. The absorption of As(V) prevails in aerobic soils while that of As(III) is favored in anaerobic soils. As(V) is converted to As(III) in the roots, although small amounts of As(V) also remain in the plant organs. The root system is the first target of the action of both As forms. The mechanisms of action of As(V) and As(III) are still widely unknown. Understanding them is essential for selecting rice genotypes with a lower capacity of As uptake and transport to the caryopses, thus improving food safety. Auxin is the phytohormone necessary for the development and plasticity of the root system, and its action is modulated by endogenous/exogenous brassinosteroids (BRs), mainly under stress conditions. The research aim was to deepen the knowledge of the mechanisms triggered by As(III) or As(V) in rice roots with particular attention to the role played by the interaction between auxin transport and BRs. We show that As(III) is the main As species present in rice roots regardless of the As(III) or As(V) forms supplied to the growing medium. Arsenic alters auxin distribution in both adventitious and lateral roots, but strongly in the latter ones. The application of an exogenous BR, the 24-epibrassinolide (eBL), combined with As(III) or As(V) strongly increases the expression of the OsPIN2 and OsAUX1 genes involved in auxin transport, thus contributing to restore the correct auxin distribution altered by As, and mainly by As(III), with higher effects on the LRs. Moreover, eBL increases the antioxidant activity in the roots in the presence of As, but only when combined with As(V).

In land plants, plastids acquired different functions and structures in parallel with the increasing genetic, developmental, and morphological diversity attained by the plant tissues. There are transition dynamics among the morpho-functional features of the different plastid types. This review is focused on plastid structure and interconversion with a focus on recent findings and a special attention to plastid types that are less known than chloroplasts. The morpho-physiological and biochemical differences, which explain the multiple functions of each plastid type and the transcriptional and post-translational modulation of plastid capabilities are here described. The structural dynamism of plastids is also discussed through their ability to produce protrusions called stromules and the activity of lipoprotein particles known as plastoglobules. As a consequence of the proteome differences among plastid types, the conversion from one type of plastid to another requires an organellar proteome reorganization with a turnover of plastid proteins, but also a differentially regulated import of nuclear-encoded proteins. Plastid degradation by macroautophagy and microautophagy pathways is also described. Taken together, all this information allows us to interpret plastids as sensors in development and plastid interconversion as a way that the plant uses to modify its growth.

Brassinosteroids (BRs), an emerging class of phytohormones, affect numerous plant physiological and metabolic processes and can improve plant defense systems to counteract metalloid phytotoxicity. Nitric oxide (NO), a reactive nitrogen species (RNS), behaves as a signalling molecule activating plant cellular responses to various environmental conditions. Brassinosteroids induce NO synthesis through nitrate reductase (NR) and NO synthase (NOS) activities. Arsenite and arsenate, inorganic forms of the metalloid arsenic (As), cause both soil pollution and many disorders in numerous plants, including important crops like rice, due to the oxidative stress generated by the imbalance between RNS and reactive oxygen species (ROS). Rice is very susceptible to As toxicity because both As availability and solubility are high in flooded paddy fields in many cultivated areas. The research aims to investigate the effects of BRs on the rice root systems exposed to 10(-4) M Na(2)HAsO40.7 H2O [As-(V)] or 2.5 x 10(-5) M NaAsO2 [(AsI)-I-(II)], highlighting the induced cyto-histological events and dissecting the NO role in the root response. A specific concentration (10(-7) M) of 24-epibrassinolide (24-eBL), an exogenously applied BR, increases lateral root (LR) formation of more than similar to 50% in the presence of As-(III) or As-(V). In addition, eBL attenuates the thickening of the cell walls induced by As in the outermost root cortical layers of LRs and in the adventitious roots (ARs) by reducing of. 50% the lignin deposition, while it restores the As-(v)-altered NO levels by increasing OsNOS1 expression and the cellular NO distribution.

Knotted1-like homeobox (KNOX) transcription factors are involved in plant development, playing complex roles in aerial organs. As Prunus species include important fruit tree crops of Italy, an exhaustive investigation of KNOX genes was performed using genomic and RNA-seq meta-analyses. Micropropagation is an essential technology for rootstock multiplication; hence, we investigated KNOX transcriptional behavior upon increasing 6-benzylaminopurine (BA) doses and the effects on GF677 propagules. Moreover, gene function in Prunus spp. was assessed by Gisela 6 rootstock transformation using fluorescence and peach KNOX transgenes. Based on ten Prunus spp., KNOX proteins fit into I-II-M classes named after Arabidopsis. Gene number, class member distribution, and chromosome positions were maintained, and exceptions supported the diversification of Prunus from Cerasus subgenera, and that of Armeniaca from the other sections within Prunus. Cytokinin (CK) cis-elements occurred in peach and almond KNOX promoters, suggesting a BA regulatory role in GF677 shoot multiplication as confirmed by KNOX expression variation dependent on dose, time, and interaction. The tripled BA concentration exacerbated stress, altered CK perception genes, and modified KNOX transcriptions, which are proposed to concur in in vitro anomalies. Finally, Gisela 6 transformation efficiency varied (2.6–0.6%) with the genetic construct, with 35S:GFP being more stable than 35S:KNOPE1 lines, which showed leaf modification typical of KNOX overexpression.

The root system is formed by the primary root (PR), which forms lateral roots (LRs) and, in some cases, adventitious roots (ARs), which in turn may produce their own LRs. The formation of ARs is also essential for vegetative propagation in planta and in vitro and for breeding programs. Root formation and branching is coordinated by a complex developmental network, which maximizes the plant's ability to cope with abiotic stress. Rooting is also a response caused in a cutting by wounding and disconnection from the donor plant. Brassinosteroids (BRs) are steroid molecules perceived at the cell surface. They act as plant-growth-regulators (PGRs) and modulate plant development to provide stress tolerance. BRs and auxins control the formation of LRs and ARs. The auxin/BR interaction involves other PGRs and compounds, such as nitric oxide (NO), strigolactones (SLs), and sphingolipids (SPLs). The roles of these interactions in root formation and plasticity are still to be discovered. SLs are carotenoid derived PGRs. SLs enhance/reduce LR/AR formation depending on species and culture conditions. These PGRs possibly crosstalk with BRs. SPLs form domains with sterols within cellular membranes. Both SLs and SPLs participate in plant development and stress responses. SPLs are determinant for auxin cell-trafficking, which is essential for the formation of LRs/ARs in planta and in in vitro systems. Although little is known about the transport, trafficking, and signaling of SPLs, they seem to interact with BRs and SLs in regulating root-system growth. Here, we review the literature on BRs as modulators of LR and AR formation, as well as their crosstalk with SLs and SPLs through NO signaling. Knowledge on the control of rooting by these non-classical PGRs can help in improving crop productivity and enhancing AR-response from cuttings.

The heavy metal cadmium (Cd) affects root system development and quiescent center (QC)-definition in Arabidopsis root-apices. The brassinosteroids-(BRs)-mediated tolerance to heavy metals has been reported to occur by a modulation of nitric oxide (NO) and root auxin-localization. However, how BRs counteract Cd-action in different root types is unknown. This research aimed to find correlations between BRs and NO in response to Cd in Arabidopsis's root system, monitoring their effects on QC-definition and auxin localization in root-apices. To this aim, root system developmental changes induced by low levels of 24-epibrassinolide (eBL) or by the BR-biosynthesis inhibitor brassinazole (Brz), combined or not with CdSO4, and/or with the NO-donor nitroprusside (SNP), were investigated using morpho-anatomical and NO-epifluorescence analyses, and monitoring auxin-localization by the DR5::GUS system. Results show that eBL, alone or combined with Cd, enhances lateral (LR) and adventitious (AR) root formation and counteracts QC-disruption and auxin-delocalization caused by Cd in primary root/LR/AR apices. Exogenous NO enhances LR and AR formation in Cd-presence, without synergism with eBL. The NO-signal is positively affected by eBL, but not in Cd-presence, and BR-biosynthesis inhibition does not change the low NO-signal caused by Cd. Collectively, results show that BRs ameliorate Cd-effects on all root types acting independently from NO.

Ectopic xylary element (EXE) formation in planta is a poorly investigated process, and it is unknown if it occurs as a response to the soil pollutant Cadmium (Cd). The pericycle cells of Arabidopsis thaliana hypocotyl give rise to EXEs under specific hormonal inputs. Cadmium triggers pericycle responses, but its role in EXE formation is unknown. Brassinosteroids (BRs) affect numerous developmental events, including xylogenesis in vitro, and their exogenous application by 24-epibrassinolide (eBL) helps to alleviate Cd-stress by increasing lateral/adventitious rooting. Epibrassinolide's effects on EXEs in planta are unknown, as well as its relationship with Cd in the control of the process. The research aims to establish an eBL role in pericycle EXE formation, a Cd role in the same process, and the possible interaction between the two. Results show that 1 nM eBL causes an identity reversal between the metaxylem and protoxylem within the stele, and its combination with Cd reduces the event. All eBL concentrations increase EXEs, also affecting xylary identity by changing from protoxylem to metaxylem in a concentration-dependent manner. Cadmium does not affect EXE identity but increases EXEs when combined with eBL. The results suggest that eBL produces EXEs to form a mechanical barrier against the pollutant.

Developmental and environmental signaling networks often converge during plant growth in response to changing conditions. Stress-induced hormones, such as jasmonates (JAs), can influence growth by crosstalk with other signals like brassinosteroids (BRs) and ethylene (ET). Nevertheless, it is unclear how avoidance of an abiotic stress triggers local changes in development as a response. It is known that stress hormones like JAs/ET and BRs can regulate the division rate of cells from the first asymmetric cell divisions (ACDs) in meristems, suggesting that stem cell activation may take part in developmental changes as a stress-avoidance-induced response. The root system is a prime responder to stress conditions in soil. Together with the primary root and lateral roots (LRs), adventitious roots (ARs) are necessary for survival in numerous plant species. AR and LR formation is affected by soil pollution, causing substantial root architecture changes by either depressing or enhancing rooting as a stress avoidance/survival response. Here, a detailed overview of the crosstalk between JAs, ET, BRs, and the stress mediator nitric oxide (NO) in auxin-induced AR and LR formation, with/without cadmium and arsenic, is presented. Interactions essential in achieving a balance between growth and adaptation to Cd and As soil pollution to ensure survival are reviewed here in the model species Arabidopsis and rice.

Peroxisomes are important in plant physiological functions and stress responses. Through the production of reactive oxygen and nitrogen species (ROS and RNS), and antioxidant defense enzymes, peroxisomes control cellular redox homeostasis. Peroxin (PEX) proteins, such as PEX7 and PEX5, recognize peroxisome targeting signals (PTS1/PTS2) important for transporting proteins from cytosol to peroxisomal matrix. pex7-1 mutant displays reduced PTS2 protein import and altered peroxisomal metabolism. In this research we analyzed the role of PEX7 in the Arabidopsis thaliana root system exposed to 30 or 60 μM CdSO4. Cd uptake and translocation, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) levels, and reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels and catalase activity were analyzed in pex7-1 mutant primary and lateral roots in comparison with the wild type (wt). The peroxisomal defect due to PEX7 mutation did not reduce Cd-uptake but reduced its translocation to the shoot and the root cell peroxisomal signal detected by 8-(4-Nitrophenyl) Bodipy (N-BODIPY) probe. The trend of nitric oxide (NO) and peroxynitrite in pex7-1 roots, exposed/not exposed to Cd, was as in wt, with the higher Cd-concentration inducing higher levels of these RNS. By contrast, PEX7 mutation caused changes in Cd-induced hydrogen peroxide (H2O2) and superoxide anion (O2●−) levels in the roots, delaying ROS-scavenging. Results show that PEX7 is involved in counteracting Cd toxicity in Arabidopsis root system by controlling ROS metabolism and affecting auxin levels. These results add further information to the important role of peroxisomes in plant responses to Cd.

Nitric oxide (NO) has signalling roles in plant stress responses. Cadmium (Cd) and arsenic (As) soil pollutants alter plant development, mainly the root-system, by increasing NO-content, triggering reactive oxygen species (ROS), and forming peroxynitrite by NO-reaction with the superoxide anion. Interactions of NO with ROS and peroxynitrite seem important for plant tolerance to heavy metal(oid)s, but the mechanisms underlying this process remain unclear. Our goal was to investigate NO-involvement in rice (Oryza sativa L.) root-system after exposure to Cd or As, to highlight possible differences in NO-behaviour between the two pollutants. To the aim, morpho-histological, chemical and epifluorescence analyses were carried out on roots of different origin in the root-system, under exposure to Cd or As, combined or not with sodium nitroprusside (SNP), a NO-donor compound. Results show that increased intracellular NO levels alleviate the root-system alterations induced by Cd, i.e., inhibition of adventitious root elongation and lateral root formation, increment in lignin deposition in the sclerenchyma/endodermis cell-walls, but, even if reducing As-induced endodermis lignification, do not recover the majority of the As-damages, i.e., enhancement of AR-elongation, reduction of LR-formation, anomalous tissue-proliferation. However, NO decreases both Cd and As uptake, without affecting the pollutants translocation-capability from roots to shoots. Moreover, NO reduces the Cd-induced, but not the As-induced, ROS levels by triggering peroxynitrite production. Altogether, results highlight a different behaviour of NO in modulating rice root-system response to the toxicity of the heavy metal Cd and the metalloid As, which depends by the NO-interaction with the specific pollutant.

Oryza sativa L. is a worldwide food-crop frequently growing in cadmium (Cd)/arsenic (As) polluted soils, with its root-system as the first target of the pollutants. Root-system development involves the establishment of optimal indole-3-acetic acid (IAA) levels, also requiring the conversion of the IAA natural precursor indole-3-butyric acid (IBA) into IAA, causing nitric oxide (NO) formation. Nitric oxide is a stress-signaling molecule. In rice, a negative interaction of Cd or As with endogenous auxin has been demonstrated, as some NO protective effects. However, a synergism between the natural auxins (IAA and/or IBA) and NO was not yet determined and might be important for ameliorating rice metal(oid)-tolerance. With this aim, the stress caused by Cd/As toxicity in the root cells and the possible recovery by either NO or auxins (IAA/IBA) were evaluated after Cd or As (arsenate) exposure, combined or not with the NO-donor compound sodium-nitroprusside (SNP). Root fresh weight, membrane electrolyte leakage, and H2O2 production were also measured. Moreover, endogenous IAA/IBA contents, transcription-levels of OsYUCCA1 and OsASA2 IAA-biosynthetic-genes, and expression of the IAA-influx-carrier OsAUX1 and the IAA-responsive DR5::GUS construct were analyzed, and NO-epifluorescence levels were measured. Results showed that membrane injury by enhanced electrolyte leakage occurred under both pollutants and was reduced by the treatment with SNP only in Cd-presence. By contrast, no membrane injury was caused by either exogenous NO or IAA or IBA. Cd- and As-toxicity also resulted into a decreased root fresh weight, mitigated by the combination of each pollutant with either IAA or IBA. Cd and As decreased the endogenous NO-content, increased H2O2 formation, and altered auxin biosynthesis, levels and distribution in both adventitious (ARs) and mainly lateral roots (LRs). The SNP-formed NO counteracted the pollutants’ effects on auxin distribution/levels, reduced H2O2 formation in Cd-presence, and enhanced AUX1-expression, mainly in As-presence. Each exogenous auxin, but mainly IBA, combined with Cd or As at 10 µM, mitigated the pollutants’ effects by increasing LR-production and by increasing NO-content in the case of Cd. Altogether, results demonstrate that NO and auxin(s) work together in the rice root system to counteract the specific toxic-effects of each pollutant.