Multiple signaling pathways control nitrogen-mediated root elongation in maize

Response of root system architecture to nutrient availability is an essential way for plants to adapt to soil environments. Nitrogen can affect root development either as a result of changes in the external concentration, or through changes in the internal nutrient status of the plant. Low soil N stimulates root elongation in maize. Recent evidence suggests that plant hormones auxin and cytokinin, as well as NO signaling pathway, are involved in the regulation of root elongation by low nitrogen nutrition.Key words: nitrogen, root growth, auxin, cytokinin, NONitrogen acquisition is determined by N demand for plant growth. At low N stress, N demand for maximum plant growth rate is not matched by plant N uptake. To acquire adequate N, plants may increase root length density to explore a larger soil volume and/or increase N uptake activity. High root density is also an important root trait for competition with soil microorganisms.¹ Since nitrate is a highly mobile, non-adsorbing ion, theoretic analysis predicts that its uptake is not limited by transport through soil, and a small root system is sufficient for nitrate acquisition.^2–4 In field conditions, however, genotypes that are efficient in N acquisition generally had a larger root system and higher root length density.^5,6 Under conditions of insufficient N supply, N mass flow to roots may not be adequate to meet the N demand for plant growth. Even in N-sufficient soils, various soil constraints (low water content, etc) may reduce the N mass flow rate. In these cases, large root size and high density will be very important for the utilization of the spatially distributed N, especially newly mineralized N, and the competition for organic N with soil microorganisms.^7,8The development of lateral roots in Arabidopsis in response to nitrate supply has been widely studied.⁹ Less attention has been paid to primary root growth in response to N, possibly because root elongtion is insensitive to increased N supply in Arabidopsis.^10,11 In maize, however, root elongation was sigificantly promoted by suboptimal N supply, and inhibited by overdose supply of N (Fig. 1).^12,13 Until recently less is known about the underlying physiological mechansms. It is well documented that cytokinin is a root-to-shoot signal communicating N availability in addition to nitrate itself.¹⁴ Exogenous cytokinin application suppresses the elongation of primary roots.¹⁵ Recent work in Arabidopsis overexpressing cytokinin synthase (IPT) demonstrate that long-term CK overproduction inhibited primary root elongation by reducing quantitative parameters of primary root meristem.¹⁶ By comparing two maize inbred lines whose root elongation had a differential response to low N stress, it was found that the change of cytokinin content in roots was closely related to low-N induced root elongation.¹³ In the N-sensitive genotype 478, cytokinin (Zeatin + Zeatin riboside) content was significantly lower at low N condition. While in N-insensitive genotype Wu312, cytokinin content was hardly affected at various N supplies. Higher N supply shortened the distance from root apex to the first visible lateral roots, a phenomenen similar to that caused by exogenous cytokinins. Furthermore, exogenous cytokinin 6-benzylaminopurine (6-BA) completely reversed the stimulatory effect of low nitrate on root elongation. All the data suggests that the inhibitory effect of high concentration of nitrate on root elongation is, at least in part, mediated by increased cytokinin level in roots.Open in a separate windowFigure 1Root elongation is inhibited at high nitrate supply.Auxin regulates many cellular responses crucial for plant development. Auxin plays a key role in establishing and elaborating patterns in root meristems.^17,18 Root elongation of Arabidopsis is enhanced by exogenous auxin at low concentrations, but is inhibited at high concentrations.¹⁹ In an earlier report, a high external nitrate supply (8 mM) did cause a 70% decrease in the auxin concentration of the root in soybean.²⁰ In maize, inhibition of root growth by high nitrate was found closely related to the reduction of IAA levels in roots and exogenous NAA and IAA restored primary root growth in high nitrate concentrations.²¹ Interesting, it was found that auxin concentrations in phloem exudates were reduced by a greater nitrate supply, suggesting that shoot-to-root auxin transport may be inhibited by high N supply. Considering the antagonism between auxin and cytokinin.²² it was possible that, by increasing the cytokinin level and decreasing the auxin level, high nitrate supply may have negative influences on root apex activity so that root apical dominance is weakened and, therefore, root elongation is suppressed and lateral roots grow closer to the root apex.Nitric oxide (NO) is emerging as an important messenger molecule associated with many biochemical and physiological processes in plants. The involvement of NO in IAA-induced adventitious root development has also been reported.²³ Given that nitrate is a substrate for NR-catalysed NO production, and root development and growth are closely related to NO, it is expected that NO may play a role in nitrate-dependent root growth. Surprisingly, endogenous levels of NO in the root apices of maize seedlings grown in high nitrate solution were much lower than those in apices grown in low nitrate. The nitrate-induced inhibition of root elongation in maize was markedly reversed by treatments of the roots with a NO donor (SNP) and IAA.²⁴ These data suggest that the arrest of root elongation by high levels of external nitrate concentrations may result from an alteration of endogenous NO levels in root apical cells. NR mediated NO production is unlikely to be involved in the nitrate-dependent NO production and root elongation because NR activity is lower at low N supply. A NO synthase (NOS) inhibitor reduced root elongation in maize plants grown in the low-nitrate medium, suggest that NOS activity may be inhibited in plants grown in high-nitrate solution, thus leading to a reduction of the endogenous NO levels.Taken together, high nitrogen supply increases cytokinin level, but decreases auxin and NO levels in roots of maize. Besides, it was well documented ethylene has a negative effect on root elongation of various plants.^25–27 Exogenous supply of cytokinin increase ethylene production (Stenlid 1982; Bertell et al., 1990). Recently, it was demonstrated in Arabidopsis that auxin transport from the root apex via the lateral root cap is required for ethylene-mediated inhibition of root growth.²⁸ Therefore, a complex multiple siganlling pathways may be involved in N-mediated root elongation (Fig. 2). Further study is required to understand how these pathways interact with each other to reduce root elongation in response to high nitrate supply.Open in a separate windowFigure 2A simplified model explaining nitogen-mediated root elongation in maize.     

Localized auxin biosynthesis and postembryonic root development in Arabidopsis

Auxin is a phytohormone essential for plant development. Due to the high redundancy in auxin biosynthesis, the role of auxin biosynthesis in embryogenesis and seedling development, vascular and flower development, shade avoidance and ethylene response were revealed only recently. We previously reported that a vitamin B₆ biosynthesis mutant pdx1 exhibits a short-root phenotype with reduced meristematic zone and short mature cells. By reciprocal grafting, we now have found that the pdx1 short root is caused by a root locally generated signal. The mutant root tips are defective in callus induction and have reduced DR5::GUS activity, but maintain relatively normal auxin response. Genetic analysis indicates that pdx1 mutant could suppress the root hair and root growth phenotypes of the auxin overproduction mutant yucca on medium supplemented with tryptophan (Trp), suggesting that the conversion from Trp to auxin is impaired in pdx1 roots. Here we present data showing that pdx1 mutant is more tolerant to 5-methyl anthranilate, an analogue of the Trp biosynthetic intermediate anthranilate, demonstrating that pdx1 is also defective in the conversion from anthranilate to auxin precursor tryptophan. Our data suggest that locally synthesized auxin may play an important role in the postembryonic root growth.Key words: auxin synthesis, root, PLP, PDX1The plant hormone auxin modulates many aspects of growth and development including cell division and cell expansion, leaf initiation, root development, embryo and fruit development, pattern formation, tropism, apical dominance and vascular tissue differentiation.^1–3 Indole-3-acetic acid (IAA) is the major naturally occurring auxin. IAA can be synthesized in cotyledons, leaves and roots, with young developing leaves having the highest capacity.^4,5Auxin most often acts in tissues or cells remote from its synthetic sites, and thus depends on non-polar phloem transport as well as a highly regulated intercellular polar transport system for its distribution.²The importance of local auxin biosynthesis in plant growth and development has been masked by observations that impaired long-distance auxin transport can result in severe growth or developmental defects.^3,6 Furthermore, a few mutants with reduced free IAA contents display phenotypes similar to those caused by impaired long-distance auxin transport. These phenotypes include defective vascular tissues and flower development, short primary roots and reduced apical dominance, or impaired shade avoidance and ethylene response.^7–15 Since these phenotypes most often could not be rescued by exogenous auxin application, it is difficult to attribute such defects to altered local auxin biosynthesis. By complementing double, triple or quadruple mutants of four Arabidopsis shoot-abundant auxin biosynthesis YUCCA genes with specific YUCCA promoters driven bacterial auxin biosynthesis iaaM gene, Cheng et al. provided unambiguous evidence that auxin biosynthesis is indispensable for embryo, flower and vascular tissue development.^8,13 Importantly, it is clear that auxin synthesized by YUCCAs is not functionally interchangeable among different organs, supporting the notion that auxin synthesized by YUCCAs mainly functions locally or in a short range.^6,8,13The central role of auxin in root meristem patterning and maintenance is well documented,^1,2,16 but the source of such IAA is still unclear. When ¹⁴C-labeled IAA was applied to the five-day-old pea apical bud, the radioactivity could be detected in lateral root primordia but not the apical region of primary roots.¹⁷ Moreover, removal of the shoot only slightly affected elongation of the primary root, and localized application of auxin polar transport inhibitor naphthylphthalamic acid (NPA) at the primary root tip exerted more profound inhibitory effect on root elongation than at any other site.¹⁸ These results suggest that auxin generated near the root tip may play a more important role in primary root growth than that transported from the shoot. In line with this notion, Arabidopsis roots have been shown to harbor multiple auxin biosynthesis sites including root tips and the region upward from the tip.⁴Many steps of tryptophan synthesis and its conversion to auxin involve transamination reactions, which require the vitamin B₆ pyridoxal 5-phosphate (PLP) as a cofactor. We previously reported that the Arabidopsis mutant pdx1 that is defective in vitamin B₆ biosynthesis displays dramatically reduced primary root growth with smaller meristematic zone and shorter mature cortical cells.¹⁹ In the current investigation, we found that the root tips of pdx1 have reduced cell division capability and reduced DR5::GUS activity, although the induction of this reporter gene by exogenous auxin was not changed. Reciprocal grafting indicates that the short-root phenotype of pdx1 is caused by a root local rather than shoot generated factor(s). Importantly, pdx1 suppresses yucca mutant, an auxin overproducer, in root hair proliferation although it fails to suppress the hypocotyl elongation phenotype.²⁰ Our work thus demonstrated that pdx1 has impaired root local auxin biosynthesis from tryptophan. To test whether the synthesis of tryptophan is also affected in pdx1 mutant, we planted pdx1 together with wild-type seeds on Murashige and Skoog (MS) medium supplemented with 5-mehtyl-anthranilate (5-MA), an analogue of the Trp biosynthetic intermediate anthranilate.²¹ Although pdx1 seedlings grew poorly under the control conditions, the growth of wild-type seedlings was more inhibited than that of the pdx1 seedlings on 10 µM 5-MA media (Fig. 1A–D). Compared with the elongated primary root on MS, wild-type seedlings showed very limited root growth on 5-MA (Fig. 1E). The relatively increased tolerance to 5-MA of pdx1 thus indicates that the pdx1 mutant may be defective in Trp biosynthesis, although amino acid analysis of the bulked seedlings did not find clear changes in Trp levels in the mutants (our unpublished data).Open in a separate windowFigure 1The pdx1 mutant seedlings are relatively less sensitive to toxic 5-methyl anthranilate (5-MA). (A and C) Five-day-old seedlings of the wild type (Col-0) (A) or pdx1 (C) on MS medium. (B and D) Five-day-old seedlings of the wild type (B) or pdx1 (D) on MS medium supplemented with 10 µM 5-MA. (E) Eight-day-old seedlings of the wild type or pdx1 on MS medium without or with 10 µM 5-MA supplement. Sterilized seeds were planted directly on the indicated medium and after two days of cold treatment, the plates were incubated under continuous light at 22–24°C before taking pictures.We reported that PDX1 is required for tolerance to oxidative stresses in Arabidopsis.¹⁹ Interestingly, redox homeostasis appears to play a critical role in Arabidopsis root development. The glutathione-deficient mutant root meristemless1 (rml1) and the vitamin C-deficient mutant vitamin C1 (vtc1) both have similar stunted roots.^22,23 Nonetheless, pdx1 is not rescued by either glutathione or vitamin C¹⁹ suggesting that the pdx1 short-root phenotype may not be resulted from a general reduction of antioxidative capacity. Interestingly, ascorbate oxidase is found to be highly expressed in the maize root quiescent center.²⁴ This enzyme can oxidatively decarboxylate auxin in vitro, suggesting that the quiescent center may be a site for metabolizing auxin to control its homeostasis.²⁵ It is therefore likely that the reduced auxin level in pdx1 root tips could be partially caused by increased auxin catabolism resulted from reduced vitamin B₆ level. We thus conducted experiments to test this possibility. A quiescent center-specific promoter WOX5 driven bacterial auxin biosynthetic gene iaaH²⁶ was introduced into pdx1 mutant. The transgenic seeds were planted on media supplemented with different concentrations of indoleacetamide (IAM), the substrate of iaaH protein. Although promotion of lateral root growth was observed at higher IAM concentrations, which indicates increased tryptophan-independent auxin production from the transgene, no change in root elongation was observed between pdx1 with or without the WOX5::iaaH transgene at any concentration of IAM tested (data not shown), suggesting that the pdx1 short-root phenotype may not be due to increased auxin catabolism.Taken together, in addition to auxin transport; temporally, spatially or developmentally coordinated local auxin biosynthesis defines the plant growth and its response to environmental changes.^8,14,15     

Lysigenous aerenchyma formation in maize root is confined to cortical cells by regulation of genes related to generation and scavenging of reactive oxygen species

To adapt to waterlogging, maize (Zea mays) forms lysigenous aerenchyma in root cortex as a result of ethylene-promoted programmed cell death (PCD). Respiratory burst oxidase homolog (RBOH) gene encodes a homolog of gp91^phox in NADPH oxidase, and has a role in the generation of reactive oxygen species (ROS). Recently, we found that during aerenchyma formation, RBOH was upregulated in all maize root tissues examined, whereas an ROS scavengingrelated metallothionein (MT) gene was downregulated specifically in cortical cells. Together these changes should lead to high accumulations of ROS in root cortex, thereby inducing PCD for aerenchyma formation. As further evidence of the involvement of ROS in root aerenchyma formation, the PCD was inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Based on these results, we propose a model of cortical cell-specific PCD for root aerenchyma formation.Key words: aerenchyma, ethylene, laser microdissection, maize (Zea mays), metallothionein, programmed cell death, reactive oxygen species, respiratory burst oxidase homologIn both wetland and non-wetland plants, lysigenous aerenchyma is formed in roots by creating gas spaces as a result of death and subsequent lysis of some cortical cells, and allows internal transport of oxygen from shoots to roots under waterlogged soil conditions.^1–3 In rice (Oryza sativa) and some other wetland plant species, lysigenous aerenchyma is constitutively formed under aerobic conditions, and is further enhanced under waterlogged conditions.⁴ On the other hand, in non-wetland plants, including maize (Zea mays), lysigenous aerenchyma does not normally form under well-drained soil conditions, but is induced by waterlogging.⁵ Ethylene is involved in lysigenous aerenchyma formation,^1–3,6,7 but the molecular mechanisms are unclear.We recently identified two reactive oxygen species (ROS)-related genes that were specifically regulated in maize root cortex by waterlogged conditions, but not in the presence of an ethylene perception inhibitor 1-methylcyclopropene (1-MCP).⁵ One was respiratory burst oxidase homolog (RBOH), which has a role in ROS generation and the other was metallothionein (MT), which has a role in ROS scavenging. These results suggest that ROS has a role in ethylene signaling in the PCD that occurs during lysigenous aerenchyma formation.     

Strigolactones' ability to regulate root development may be executed by induction of the ethylene pathway

The newly defined phytohormones strigolactones (SLs) were recently shown to act as regulators of root development. Their positive effect on root-hair (RH) elongation enabled examination of their cross talk with auxin and ethylene. Analysis of wild-type plants and hormone-signaling mutants combined with hormonal treatments suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs. The SL and auxin hormonal pathways were suggested to converge for regulation of RH elongation; this convergence was suggested to be mediated via the ethylene pathway, and to include regulation of auxin transport.Key words: strigolactone, auxin, ethylene, root, root hair, lateral rootStrigolactones (SLs) are newly identified phytohormones that act as long-distance shoot-branching inhibitors (reviewed in ref. ¹). In Arabidopsis, SLs have been shown to be regulators of root development and architecture, by modulating primary root elongation and lateral root formation.^2,3 In addition, they were shown to have a positive effect on root-hair (RH) elongation.² All of these effects are mediated via the MAX2 F-box.^2,3In addition to SLs, two other plant hormones, auxin and ethylene, have been shown to affect root development, including lateral root formation and RH elongation.^4–6 Since all three phytohormones (SLs, auxin and ethylene) were shown to have a positive effect on RH elongation, we examined the epistatic relations between them by examining RH length.⁷ Our results led to the conclusion that SLs and ethylene are in the same pathway regulating RH elongation, where ethylene may be epistatic to SLs.⁷ Moreover, auxin signaling was shown to be needed to some extent for the RH response to SLs: the auxin-insensitive mutant tir1-1,⁸ was less sensitive to SLs than the wild type under low SL concentrations.⁷On the one hand, ethylene has been shown to induce the auxin response,^9–12 auxin synthesis in the root apex,^11,12 and acropetal and basipetal auxin transport in the root.^4,13 On the other, ethylene has been shown to be epistatic to SLs in the SL-induced RH-elongation response.⁷ Therefore, it might be that at least for RH elongation, SLs are in direct cross talk with ethylene, whereas the cross talk between SL and auxin pathways may converge through that of ethylene.⁷ The reduced response to SLs in tir1-1 may be derived from its reduced ethylene sensitivity;^7,14 this is in line with the notion of the ethylene pathway being a mediator in the cross talk between the SL and auxin pathways.The suggested ethylene-mediated convergence of auxin and SLs may be extended also to lateral root formation, and may involve regulation of auxin transport. In the root, SLs have been suggested to affect auxin efflux,^3,15 whereas ethylene has been shown to have a positive effect on auxin transport.^4,13 Hence, it might be that in the root, the SLs'' effect on auxin flux is mediated, at least in part, via the ethylene pathway. Ethylene''s ability to increase auxin transport in roots was associated with its negative effect on lateral root formation: ethylene was suggested to enhance polar IAA transport, leading to alterations in the quantity of auxin that unloads into the tissues to drive lateral root formation.⁴ Under conditions of sufficient phosphate, SL''s effect was similar to that of ethylene: SLs reduced the appearance of lateral roots; this was explained by their ability to change auxin flux.³ Taken together, one possibility is that the SLs'' ability to affect auxin flux and thereby lateral root formation in the roots is mediated by induction of ethylene synthesis.To conclude, root development may be regulated by a network of auxin, SL and ethylene cross talk.⁷ The possibility that similar networks exist elsewhere in the SLs'' regulation of plant development, including shoot architecture, cannot be excluded.     

Root tips moving through soil: An intrinsic vulnerability

Root elongation occurs by the generation of new cells from meristematic tissue within the apical 1–2 mm region of root tips. Therefore penetration of the soil environment is carried out by newly synthesized plant tissue, whose cells are inherently vulnerable to invasion by pathogens. This conundrum, on its face, would seem to reflect an intolerable risk to the successful establishment of root systems needed for plant life. Yet root tip regions housing the meristematic tissues repeatedly have been found to be free of microbial infection and colonization. Even when spore germination, chemotaxis, and/or growth of pathogens are stimulated by signals from the root tip, the underlying root tissue can escape invasion. Recent insights into the functions of root border cells, and the regulation of their production by transient exposure to external signals, may shed light on long-standing observations.Key words: border cells, chemotaxis, zoospores, neutrophil extracellular traps (NETs)…The evidence suggests that there has evolved within plants, mechanisms for extremely rapid adjustment to changes in the soil environment. The logical conclusion is that plants can and do selectively manipulate the ecological balances within the rhizosphere to their own advantage.¹“Sloughed root cap cells” that detach from the root tip were long presumed to be moribund tissue serving to lubricate passage of the elongating root.² The discovery nearly a century ago that these cells from Zea mays L. and Pisum sativum L. can remain 100% viable for weeks after detachment into hydroponic culture did not alter this perception.³ In recent decades, studies have shown that the cells from root caps of most species are metabolically active and can survive even after detachment into the soil.⁴ Moreover, the cell populations express distinct patterns of gene expression reflecting tissue specialization and were therefore given the name root ‘border’ cells.⁵ Like ‘border towns’ that exist at the boundary of disparate countries and cultures, border cells are part of the plant and part of the soil, yet distinct from both.The soil is a dynamic environment whose pH, surface charge, water availability, texture and composition can range markedly on a large and small scale.^1,6,7 The concept of a ‘microniche’ emphasizes that the biological requirements for a particular soil microorganism may be met within one site but not another site only a micron away.⁸ Thus, the rhizosphere—the region adjacent to root surfaces—can support much higher levels of microorganisms than bulk soil a few millimeters distant.⁹ This phenomenon is recognized to be driven by an increased availability of nutrients released from plants into the external environment.¹⁰ Less well recognized is the dynamic variation that occurs along the root surface, and its significance in patterns of disease development. As roots emerge and the new tissue differentiates progressively through stages from root cap, root apical meristem, elongation zone, and finally mature roots with lignified cell walls, the material released into the environment also changes.^11–13 More than 90% of bulk carbon released from young roots of legumes is delivered by the root cap, a 1 mm zone at the apex.¹⁴ Some pathogens are attracted specifically to the root tip region, presumably in response to such exudates.^15,16 For example, instantaneous swarming occurs when a cotton root is placed into a suspension of Pythium dissotocum zoospores (Sup. Fig. 1). This host-specific attraction is specific to the root tip region where border cells are present (Sup. Fig. 2). Border cells remain attractive to zoospores when removed from the root (Sup. Fig. 3). The nature of the attractant is not known, but its impact is localized and transient (Sup. Fig. 4).Newly generated tissue is highly susceptible to infection by pathogens, in general, so elongating root tips would be predicted to be vulnerable to invasion. And yet, root apices repeatedly have been found to escape infection and colonization.^17–19 Recent discoveries about parallels between mammalian white blood cells and root border cells may provide new insight into this apparent conundrum.²⁰ Neutrophils, a type of white blood cell, are produced in response to infection. Neutrophil extracellular traps (NETs) then attract and kill the invader through a process that requires extracellular DNA (exDNA) and an array of extracellular proteins.^21,22 Border cell production, like that of neutrophils, also is induced in response to signals from pathogens and root tip resistance to infection requires exDNA and an array of extracellular proteins.^20,23 Root tip specific chemotaxis, like that seen with Pythium zoospores, has been presumed to involve steps in a process of pathogen invasion.^15,16 It may, instead, involve a process of extracellular trapping and killing by cells designed to protect root meristems from invasion, in a manner analogous to that which occurs in mammalian defense. If tests confirm this model, the mystery of how root tips escape infection by soilborne pathogens they attract could be resolved.     

EZ-Rhizo software: The gateway to root architecture analysis

Plants are sessile organisms that have to cope with the available nutritional resources and environmental constraints in the place where they germinate. To fully exploit their nearby resources, they have evolved a highly plastic and responsive root system. Adaptations to limited nutrients include a wide range of specific root responses, e.g., the emergence of new root types, root branching or specific growth of lateral roots. These root system architecture (RSA) features are of utmost importance when investigating the underlying mechanisms by forward, reverse or quantitative genetic approaches. The EZ-Rhizo software was developed to facilitate such root measurements in a fast, simple and accurate way. The performances of EZ-Rhizo in providing about 20 primary and derived RSA parameters were illustrated by looking at natural variability across 23 Arabidopsis accessions. The different RSA profiles obtained from plants grown in favorable condition illustrated the wide reservoir of natural genetic resources underlying specific features of root growth. This diversity was used here to correlate the RSA genetic variability with growth, development and environmental properties of accession origins.Key words: Arabidopsis thaliana, root architecture, image analysis, software, natural variationUptake of water and mineral nutrients from the soil is essential for plant life. The root system achieves these fundamental functions through its highly responsive and plastic morphology, which allows plants to exploit fully the soil physico-chemical resources. The geometry of different parts of the root system defines the so-called root system architecture. RSA is broadly determined by the genetic make-up of the plant and is subject to the abiotic and biotic environment of the root, as well as the physiological status of the plant.^1,2 Among environmental factors, the availability of nutrients such as nitrogen (N), phosphorus (P) and potassium (K) best illustrates the influence of the soil environment on RSA.^3–6 Specific root responses to nutrient and other abiotic stresses are routinely used to characterise mutants⁷ but the lack of an efficient means to capture RSA in a fast and effective way hampers forward genetic screens based on RSA other than the main root length. RSA has been used in quantitative trait locus (QTL) approaches to identify the molecular basis of intrinsic or environmental factors of RSA.^8–11 Again, only a small number of quantitative studies¹² have taken into account other RSA parameters than the main root length, mainly because of the lack of efficient tools to capture them comprehensively. To overcome this technical difficulty, the computer program EZ-Rhizo was developed and now allows fast and accurate measurement of RSA features from Arabidopsis plants growing on a vertical support.¹³ The software performs RSA analysis in a few user-supervised steps from a scanned Petri dish. The software measures 10 or more primary RSA parameters (Fig. 1) from which several other parameters are derived (e.g., lateral root density, branched zone length). The results are stored in a user-friendly database which can be queried to find specific data or exported for use in other programs such as Microsoft® Excel®.Open in a separate windowFigure 1Schematic representation of RSA parameters measured by EZ-Rhizo. Main root (MR) primary features: path length (dashed line), vector length (grey arrow), vector angle (α), number of lateral root (LR, black dots). MR derived features: straightness (path:vector lengths), MR root depth (d), basal zone (path length between the root collar and the position of the first LR, white box on the left side), branched zone (path length between the first and the last LR position, grey box on the left side) and apical zone (path length from the position of the last LR to the root tip, black box on the left side), LR density over the MR, LR density over the branched zone. Lateral root (1^st or n order) primary features: position on the MR (or on the LRn-1), path length, vector length, vector angle, number of LRn+1 (open dots). Derived LR features: straightness (path:vector lengths). Overall root feature: total root size (sum of all root path length). LR (or LRn) are numbered according to their position on the MR (or LRn-1). LR parameters are illustrated here only for LR1.Providing the EZ-Rhizo program as a freeware (http://EZ-Rhizo.psrg.org.uk) should therefore open the gate to fast and accurate RSA measurements and contribute to better progress in understanding RSA behavior. As an example to illustrate EZ-Rhizo performances, we used Arabidopsis natural variation to measure multiple RSA parameters across 23 Arabidopsis accessions grown on vertical Petri dishes in half-strength MS medium.¹³ The high natural RSA variation observed across these accessions was used in an attempt to find correlations between RSA and in vitro growth, development as well as the environment of accession origins (geography, climatology).     

Assessing fungal root colonization for plant improvement

Fungal endophytes display a broad range of symbiotic interactions with their host plants. Current studies on their biology, diversity and benefits are unravelling their high relevance on plant adaptation to environmental stresses. Implementation of such properties may open new perspectives in agriculture and forestry. We aim to exploit the endophytic capacities of the fungal species Fusarium equiseti, a naturally occurring root endophyte which has shown antagonism to plant pathogens, and Pochonia chlamydosporia, a nematophagous fungus with putative endophytic behavior, for plant protection and adaptation to biotic and abiotic stress. A real-time PCR protocol for quantification of the fungal population, together with Agrobacterium-mediated genetic transformation with the GFP gene for confocal microscopy analyses, were designed and applied to assess endophytic development of both these fungal species. Although quantification of both F. equiseti and P. chlamydosporia yielded similar degrees of root colonization, microscopical observations demonstrated differences in infection and development patterns. Furthermore, we found evidences of plant response against endophyte colonization, supporting a balanced antagonism between the endophyte virulence and the plant defenses. Optimization and application of the methodologies presented herein will allow elucidation of beneficial interactions among these endophytes and their host plants.Key words: endophytes, Fusarium equiseti, fungal detection, mutualism, Pochonia chlamydosporia, root colonizationTissues of nearly all plants in natural ecosystems appear to be colonized by endophytic fungi, whose importance in plant development and distribution seem to be crucial, but not yet clearly understood. Fungal endophytes may assist their host plants for adaptation to habitat, protection against biotic or abiotic stresses, plant growth promotion or soil nutrients uptake.^1–5 Recently, biology and function of fungal endophytes in nature has been extensively reviewed,⁶ and these have been grouped into four classes according to their differential interactions with host plants, which may range from mere neutralism to an active mutualism.Exploitation of beneficial properties of endophytes is of great relevance at an applied level, either to increase production yields of agricultural crops, control of plant diseases or pests, adapt plants to unsuitable growth conditions, or in reforestation activities. In this sense, we aim to exploit the endophytic capacities of selected fungi from two independent approaches: through the use of natural endophytes, which have shown to confer benefits to their host plants,⁵ or via a putative endophytic colonization of plant tissues by fungi with desirable properties. The latter strategy has been already introduced for several biological control agents such as nematophagous fungi colonizing roots,⁷ or entomopathogenic fungi in above-ground organs.^8,9 As representatives of each category, we selected two fungal species according to their endophytic capacities and antagonism to root pathogens.^10,11 First, Fusarium equiseti represents naturally occurring endophytes from natural vegetation growing under saline stress conditions.¹² The second, Pochonia chlamydosporia, is a nematode egg-parasite which behaves as a putative endophyte of roots.¹³ Both species have promising properties for application as biocontrol agents of either fungal and/or nematode plant parasites. The achievement of these objectives will depend on an exhaustive knowledge on the endophytic behavior and interactions with the host and other existing microbes, which starts with the assessment of the establishment of fungal populations within plant organs (e.g., roots). In many instances this is a complicated task. Fungal growth is characterized by an implicit irregular development due to the filamentous nature of mycelia, whose complexity increases in structured substrates, such as the interior of plant tissues.These difficulties are enhanced due to uneven distribution of nuclei (from zero to several thousands per cell), physiological activity or vitality of hyphae, and influence of competition among species.¹⁴ As a consequence of this, methods applied traditionally to estimate fungal occurrence within plant tissues (e.g., direct visualization, plating on culture media or immunolocalization^4,15,16) are biased or rather laborious and time consuming. Although applications of molecular methods have been directed to settle these problems, these may also present other weaknesses. Combination of quantitative and qualitative data achieved by both molecular and microscopy methods is probably the best choice to monitor fungi in plant roots, since the advantages of each technique may complement the drawbacks of the other.^14,17We recently optimized and applied both real-time PCR and microscopy techniques to exhaustively study the endophytic development of F. equiseti and P. chlamydosporia in barley roots.¹⁸ The first approach consisted in a specific quantification of nucleic acids from either fungus in roots using Molecular beacon probes.¹⁹ This allows an accurate monitoring of the respective amplicon generation over PCR cyclings, which can be correlated with the amount of fungal biomass.²⁰ Fungal populations detected in roots were statistically similar for both fungal species studied, with an overall colonization of roots which ranged from ca. 5 to 11 ng of fungal target DNA per 100 ng of total DNA. The endophytic proportion of these populations (assessed by surface sterilization of roots prior to DNA extraction) was represented by values between ca. 0.5 to 1 ng of fungal target DNA per 100 ng of total DNA.These quantitative data resulted in an increased sensitivity and dynamic range as compared with traditional culturing methods. Nevertheless, though quantification yielded good results under a gnotobiotic system, where only those fungi of interest are present within plant tissues, implementation for non-axenic semi-field or field experimentation should include modifications. These should cover corrections for presence of other colonizing microorganisms under non-axenic growth conditions-which may contribute to the amount of total DNA extracted from roots-, or variability on DNA extraction yields among samples.²¹ Inclusion of internal standards such as the simultaneous detection of fungal and host plant in a single reaction tube²² would settle these interferences. Multiplex PCR amplification of respective plant or fungal loci would permit detection of differential fluorescent signals emitted by Molecular beacons specific for either target DNA. We are currently optimizing primers and probes designed in Maciá-Vicente et al.¹⁸ for multiplexing with primers and Molecular beacons specific for the host (barley) ubiquitin gene (Fig. 1).Open in a separate windowFigure 1Amplification by conventional PCR of DNA from plant (h; barley), endophyte (e; P. chlamydosporia), or endophyte-colonized plants (h + e). For this, single locus detection with primers specific for the fungal alkaline serine protease p1,¹⁸ (Fp) and the plant ubiquitin (Pp) is shown, and multiplex PCR using both primer pairs (Fp + Pp) for one-tube simultaneous detection.In addition to real-time PCR assays, endophytic behavior of F. equiseti and P. chlamydosporia was assessed using live-cell microscopy. Aiming to develop techniques for further studies in semi-field experiments, we generated genetically transformed isolates for both F. equiseti and P. chlamydosporia, with constitutive expression of the fluorescent reporter protein GFP. For this purpose an Agrobacterium tumefaciens-mediated protocol was applied due to its efficiency in fungal transformation.²³ Observation of barley roots inoculated with GFP-tagged isolates under laser scanning confocal microscopy permitted a time-course qualitative monitoring of the infection processes by F. equiseti and P. chlamydosporia, which displayed different endophytic patterns. Loading studies with the endocytotic tracker FM4-64 allowed a discrimination of new traits of fungal colonization of roots. Fungal hyphae appeared tightly fitted in a plant membrane-derived sheath during the first invasive stages, in a similar manner to that which occurs during pathogenesis,²⁴ but also in mutualistic interactions,²⁵ indicating it may be (at least originarly) an unspecific barrier to fungal invasion. However, this membrane was lost with time, as a consequence of hyphal aggressiveness over plant cell infection.Differential wavelength emission between GFP and FM4-64 also allowed detection of a heterogeneous distribution of viable and non-viable hyphae within the root cortex, the latter linked to plant defense responses such as abundant production of papillae or vacuoles. This colonization pattern suggests that endophytic interaction established by both fungi is a “push and pull” balance between hyphal growth and the capacity of the plant to get rid of the invader. Yet we do not know whether remaining undegraded nucleic acids contents of within-cortex dead hyphae may contribute to fungal target DNA detection by real-time PCR. Although this fact may represent a bias for endophytes quantification, combination with microscopical analyses complements the information achieved.We are currently investigating practical and theoretical aspects of the root inoculation of fungi with endophytic capabilities and antagonistic potential to root pathogens (fungi and nematodes). This research also includes evaluating root colonization abilities for different host plants, both monocots and eudicots. As an example, root colonization efficiency by P. chlamydosporia dramatically changes between barley and tomato. In the latter, hyphae within roots are sparse and restricted to epidermal root cells,¹³ with no evident connection between infected cells (Fig. 2A). In spite of this restricted distribution, fungal chlamydospores (the propagules usually found in P. chlamydosporia-harboring soils) may be frequently found on the root surface. Their viability (according to GFP expression in the cytoplasm), irrespective of that of surrounding hyphae (Fig. 2B), could support that root colonization creates a stable source of inoculum to sustain the populations of the microorganism in the rhizospheric soil. Our final goal is to optimize biocontrol performance and crop growth promotion by endophytes under agricultural conditions.Open in a separate windowFigure 2Laser scanning confocal microscopy images of one-month-old tomato roots colonized by GFP-tagged P. chlamydosporia. (A) P. chlamydosporia hypha restricted to a single epidermal root cell. (B) Chlamydospore in the root surface. Note GFP fluorescence within cells (viable) in contrast to lack of fluorescence in peduncle (non-viable). Bars = 20 µm.     

Modulation of root branching by a coumarin derivative

A healthy root system is crucial to plant growth and survival. To maintain efficiency of root function, plants have to dynamically modulate root system architecture through various adaptive mechanisms such as lateral root formation to respond to a changing and diversified soil environment. Exogenous application of a coumarin derivative, 4-methylumbelliferone (4-MU), in Arabidopsis thaliana inhibits seed germination by mainly reducing primary root growth. UDP-glycosyltransferases play an integral role in the biochemical mechanism of 4-MU detoxification in plant roots.1 However, 4-MU treatment also dramatically led to increased lateral root initiation, elongation and density. Moreover, marked root bending at the root-hypocotyl junction and auxin redistribution appeared to contribute to the 4-MU-mediated lateral root formation. We propose that 4-MU would serve as a useful chemical tool to study auxin-mediated root branching.Plant roots are required for the acquisition of water and nutrients, for response to abiotic and biotic factors in the soil, and to anchor the plant in the ground.² To maintain efficiency of root function, plants have to dynamically modulate root system architecture (RSA) by regulation of primary root growth, lateral root (LR) formation and elongation and root hair increase.² Recent studies on root patterning have made significant progress toward understanding the molecular and physiological basis of RSA.³ For example, auxin synthesis, transport and distribution are required for LR initiation and primordium development.² However, determination of the underlying RSA patterning mechanism remains to be elucidated.Coumarins are a group of natural products in plants that originate from the general phenylpropanoid pathway.⁴ They are often found to accumulate in the root tissues⁵ and are involved in plant defense, root development and nitrogen uptake and metabolism.^1,6-8 Some coumarins also receive attention for their pharmacological properties. For example, 4-MU is a potent apoptotic agent with strong anti-invasive and antiangiogenic properties against prostate cancer cells.⁹We have demonstrated that exogenous 4-MU was accumulated in the root system in a concentration-dependent manner. After continuous exposure to 4-MU, growth of the primary roots exhibited a dosage-dependent inhibition of root length, whereas the growth of cotyledon and hypocotyls was not significantly changed. Moreover, 4-MU was found to be glycosylated to 4-methylumbelliferyl-β-D-glucoside (4-MU-Glc) by UDP-glycosyltransferases (UGTs) for detoxification.¹ Here we report that marked bending of the primary roots and auxin redistribution in root system contributes to 4-MU-induced root branching. After exposure to 125 µM 4-MU for 6 d, the primary root length was reduced by 25% compared with the untreated seedlings, but the first LR emerged at the root-hypocotyl junction 3 d earlier in the Arabidopsis DR5::GUS lines compared with untreated seedlings. The GUS activity and distribution in the primary roots of DR5::GUS seedlings were coordinately regulated in response to 4-MU treatment (Fig. 1A-D). Interestingly, primary root shape was also affected upon 4-MU treatment as evidenced by marked bending of the primary roots followed by emergence of lateral roots at the root-hypocotyl junctions. As the roots grew, the bend continued to develop and a hook formed at the root-hypocotyl junction (Fig. 1F). After exposure to 125 μM 4-MU for 22 d, abundant lateral roots formed from the bent region (Fig. 1F). We also observed that auxin accumulation in the bent region was significantly reduced after root branching was well established, compared with the untreated plants (Fig. 1E and F). It has been demonstrated that LR formation can be induced mechanically by either gravitropic curvature or by transient bending.^10,11 We suggest that 4-MU-induced LR proliferation is triggered by both mechanical bending of the primary roots at the root-hypocotyl junctions and the local auxin redistribution.Open in a separate windowFigure 1.Changes of auxin distribution in response to 4-MU as observed using DR5::GUS reporter fusion. (A) Auxin accumulation in root-hypocotyl junction after exposure to 125 µM 4-MU for 6 d. (B-D) Detection of 4-MU accumulation in root under UV (325 nm). (B) Brightfield; (C) UV channel (325 nm); (D) Merge of (B) and (C). (E) An untreated root system of 22-d-old DR5::GUS seedling. (F) A root system of 22-d-old DR5::GUS seedling in the presence of 125 µM 4-MU. Asterisks indicate the localization of auxin accumulation. It was noted that LR formation upon 4-MU treatment was closely associated with auxin distribution and 4-MU accumulation in roots.Our finding of 4-MU-dependent root patterning is intriguing in light of the important role of RSA in plant physiology. Given that LR initiation is stimulated by 4-MU and that this compound is effectively detoxified in plant roots by glycosylation, a new way to augment root function could be provided through applying 4-MU to modulate RSA. In addition, 4-MU could serve as a useful chemical tool for understanding auxin-mediated root branching, for example, by screening Arabidopsis mutants in the presence of this compound.Coumarins synthesis from phenylpropanoid precursors occurs with an especially high number of structural variations in higher plants via numerous possible modifications at specific positions of the benzene ring.^4,5 For example, hydroxylation of coumarins at 6-position catalyzed by a 2-oxoglutarate-dependant dioxygenase (F6''H1) is important for the biosynthesis of scopoletin.¹² Coumarin synthesis in Arabidopsis plants can result in the accumulation of umbelliferone and its derivative skimmin but not 4-MU⁵ in which 4-MU possesses a pivotal methyl group at the 4-position of the benzene ring. Our results suggest that 4-MU uptake does not benefit plant growth as it is a phytotoxic compound found to inhibit primary root growth and seed germination. This finding explains why Arabidopsis plants do not naturally accumulate 4-MU and its derivatives. Nevertheless, 4-MU has been found and isolated from other higher plants such as Dalbergia volubilis and Eupatorium pauciflorum, indicating the existence of a biosynthetic pathway leading to the formation of 4-MU in nature.¹³     

Creating drought- and salt-tolerant cotton by overexpressing a vacuolar pyrophosphatase gene

Increased expression of an Arabidopsis vacuolar pyrophosphatase gene, AVP1, leads to increased drought and salt tolerance in transgenic plants, which has been demonstrated in laboratory and field conditions. The molecular mechanism of AVP1-mediated drought resistance is likely due to increased proton pump activity of the vacuolar pyrophosphatase, which generates a higher proton electrochemical gradient across the vacuolar membrane, leading to lower water potential in the plant vacuole and higher secondary transporter activities that prevent ion accumulation to toxic levels in the cytoplasm. Additionally, overexpression of AVP1 appears to stimulate auxin polar transport, which in turn stimulates root development. The larger root system allows AVP1-overexpressing plants to absorb water more efficiently under drought and saline conditions, resulting in stress tolerance and increased yields. Multi-year field-trial data indicate that overexpression of AVP1 in cotton leads to at least 20% more fiber yield than wild-type control plants in dry-land conditions, which highlights the potential use of AVP1 in improving drought tolerance in crops in arid and semiarid areas of the world.Key words: drought tolerance, proton pump, salt tolerance, transgenic cotton, vacuolar membraneDrought and salinity are major environmental factors that limit agricultural productivity in most parts of the world.¹ Climate change will likely make many places worse in terms of water availability and soil salinization,² which will have negative impacts on food production in world agriculture. Yet, the demand for more food will continue to rise because of the growing world population that may reach 9 billon people by 2050.³ Therefore, the primary challenge we face during this century is the production of more food under the constraints of limited water and fertilizer on marginal soils.Many genes that respond to abiotic stresses have been identified in the model plant Arabidopsis,⁴ and some of them were shown to play important roles in protecting plants under abiotic stress conditions.⁵ The Arabidopsis vacuolar pyrophosphatase gene AVP1 appears to be one of the most promising genes that may be used to improve drought- and salt-tolerance in crops.⁶ Roberto Gaxiola''s group first demonstrated that overexpression of AVP1 could lead to significantly improved drought- and salt-tolerance in transgenic Arabidopsis plants.⁷ Later when this gene was introduced into tomato⁸ and rice,⁹ similar tolerance phenotypes were observed. Overexpression of AVP1 in cotton, not only improved drought- and salt-tolerance in greenhouse conditions, but also increased fiber yield in dryland field conditions.⁶ AVP1-expressing cotton plants produced larger root systems and bigger shoot biomass than controls when grown under hydroponic conditions in the presence of up to 200 mM NaCl.⁶ In the greenhouse, AVP1-expressing cotton plants also produced more root and shoot biomass than controls when grown under saline conditions or reduced irrigation.⁶ The increased yield by AVP1-expressing cotton plants is due to more bolls produced, which in turn is due to larger shoot system that AVP1-expressing cotton plants develop under saline or drought conditions.⁶The larger root systems of AVP1-expressing cotton plants under saline and water-deficit conditions allow transgenic plants access to more of the soil profile and available soil water resulting in increased biomass production and yield. Li et al. showed that the larger root systems of AVP1-overexpressing Arabidopsis is caused by increased auxin polar transport in the root, which stimulates root development in AVP1-overexpressing Arabidopsis plants.¹⁰ Furthermore, a recent comparative study of transgenic Arabidopsis lines that produce enlarged leaves showed that auxin levels were increased by 50% in AVP1-overexpressing plants.¹¹ To test if altered auxin level is responsible for the observed larger root systems in AVP1-expressing cotton plants, we germinated wild-type and AVP1-expressing cotton plants in the absence or presence of the auxin polar transport inhibitor Naphthylphthalamic acid (NPA). Both wild-type and AVP1-expressing cotton plants developed robust lateral root systems in the absence of NPA (Fig. 1A). The presence of 50 µM NPA resulted in nearly complete inhibition of lateral root development in wild-type plants, while lateral root development in AVP1-expressing plants was reduced, it was significantly greater than wild-type (Fig. 1B). These data indicate that AVP1-overexpression could overcome the inhibitory effects of NPA on root development in AVP1-expressing cotton plants, suggesting that either increased auxin transport or higher auxin concentration in the root systems of AVP1-expressing cotton plants is responsible for the observed larger root systems, and eventually for the increased boll numbers and fiber yields under dryland field conditions.Open in a separate windowFigure 1Root development of wild-type and AVP1-expressing cotton plants in the absence and presence of auxin transport inhibitor NPA. (A) Phenotype of cotton roots after 10 days of growth in the absence of NPA. WT, Wild-type; 1, 5, 9, three independent AVP1-overexpressing cotton lines. (B) Phenotype of cotton roots after 10 days of growth in the presence of 50 µm NPA.Many genes that may play important roles under water-deficit conditions have been tested in laboratory conditions,^4,5 but very few have been tested vigorously in field conditions. A bacterial cold shock protein gene was shown to improve drought tolerance in maize based on multi-year and multi-place field trial experiments,¹² and it appears that this gene will likely gain approval for commercial release and become the first genetically engineered product that demonstrates improved drought tolerance in a major crop in the U.S. Another example of increased drought tolerance supported by multiple field trial experiments is through downregulation of farnesylation in transgenic canola plants.¹³ Downregulation of farnesyltransferase by antisense or RNAi techniques in transgenic canola leads to increased sensitivity to abscisic acid, consequently resulting in smaller guard cell aperture under drought conditions. These transgenic canola plants lose less water through transpiration and are more drought resistant. Data from more than 5 years of field studies in Canada consistently proved that this approach can indeed increase drought tolerance in transgenic canola. Our study with AVP1-expressing cotton over the last several years in field conditions is another example that genetic engineering approach can be an efficient tool in generating drought-tolerant crops. AVP1-expressing cotton plants can establish a larger shoot mass in dryland conditions (Fig. 2), which results in increased boll numbers and fiber production. Our approach is likely applicable to other major crops as well.Open in a separate windowFigure 2Wild-type and AVP1-expressing cotton plants grown in the dryland field condition. Plants were planted in the middle of may 2009 and the picture was taken in the middle of July 2009 at the USDA experimental Farm in Lubbock, Texas.     

Involvement of nitrate reductase in auxin-induced NO synthesis

It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.¹ Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (N^G-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.^2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.^4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation.Key words: atnoa1, indole-3-butyric acid, nia1, nia2 double mutant, nitric oxideLateral roots are formed from root pericycle cells postembryonically which process is promoted by indole-acetic acid (IAA). It was recognized that IAA share common steps with NO in the signal transduction cascade towards the auxin induced adventitious and lateral root formation.^6–8 Previously it was suggested that besides IAA, indol-3-butyric (IBA) is a true endogenous auxin in Arabidopsis, which acts in adventious and lateral root development.^9,10 Our results showed that IBA induced LR initials emitted intensive NO fluorescence in Arabidopsis. This increased level of NO was present only in the LR initials in contrast to primary root (PR) sections where it remained at the control level.In plants NO can be produced by a number of enzyme systems and non-enzymatic ways. In roots, the most likely candidates of NO synthesis are NR enzymes (cytoplasmic and plasma membrane-bounded isoenzymes, cNR and PM-NR). Recently a new type of enzyme, the PM-bounded nitrite:NO reductase (Ni:NOR) was identified as a possible source of NO in roots.¹¹ Because of the several formation potentials of NO, the identification of its source in plant tissues under different conditions is complicated. Using diverse mutants proved to be a good opportunity to investigate the possible sources of NO. In our experiments wild-type (Col-1), Atnoa1 (nitric oxide synthase associated 1 deficient) and nia1, nia2 (NR deficient) seedlings were applied in order to determine the enzymatic source of NO induced by auxin. In roots of these plants, different NO levels were measured in their control state (i.e., without IBA treatment). The NO content in Atnoa1 roots was similar to that of wild-type, while nia1, nia2 showed lower NO fluorescence than the other groups of plants. This result suggests that NR activity is needed to NO synthesis in roots. Further on, it was demonstrated that IBA induced NO generation in both the wild type and Atnoa1 root primordia, but this induction failed in the NR-deficient mutant. This reveals that the NO accumulation in root primordia induced by auxin requires NR activity. These observations were evidenced also by biochemical manner. On the one part, we applied L-NMMA, which is a specific inhibitor of mammalian NOS, on the other part, the inhibitor of NR enzyme tungstate was used and we monitored NO fluorescence in wild-type roots. The NOS inhibitor displayed no effect on NO levels neither at control state nor during auxin treatment, while tungstate inhibited NO synthesis in lateral roots and primary roots of control plants. The effect of tungstate was similar in auxin-treated roots, since application of this NR enzyme inhibitor decreased NO levels in PRs and LRs (Fig. 1).Open in a separate windowFigure 1NO fluorescence in lateral roots (white columns) and primary roots (grey columns) of control, control + 1 mM tungstate, IBA and IBA + 1 mM tungstate-treated wild-type Arabidopsis thaliana. Vertical bars are standard errors.Some speculations can be made on these results. Although more efforts are needed to make the scene clear, now we can predict that auxin somehow may induce NR isoenzymes, which produce nitrite in root cells. From this point, two further scenarios are possible: as the result of accumulated nitrite, either the NO-producing activity of NR or Ni:NOR activity are promoted, hereby NO is generated from nitrite reduction. NO formed in these two possible ways modulates the expression of certain cell cycle regulatory genes contributing to division of pericycle cells in LR primordia, as was published in tomato.¹²Nowadays research in the “NO-world” of plants is running very actively. Nevertheless, lot of more work is needed to reveal all the unknown faces of this novel multipurpose signaling molecule.