Spatiotemporal Regulation of Lateral Root Organogenesis in Arabidopsis by Cytokinin

The architecture of a plant’s root system, established postembryonically, results from both coordinated root growth and lateral root branching. The plant hormones auxin and cytokinin are central endogenous signaling molecules that regulate lateral root organogenesis positively and negatively, respectively. Tight control and mutual balance of their antagonistic activities are particularly important during the early phases of lateral root organogenesis to ensure continuous lateral root initiation (LRI) and proper development of lateral root primordia (LRP). Here, we show that the early phases of lateral root organogenesis, including priming and initiation, take place in root zones with a repressed cytokinin response. Accordingly, ectopic overproduction of cytokinin in the root basal meristem most efficiently inhibits LRI. Enhanced cytokinin responses in pericycle cells between existing LRP might restrict LRI near existing LRP and, when compromised, ectopic LRI occurs. Furthermore, our results demonstrate that young LRP are more sensitive to perturbations in the cytokinin activity than are developmentally more advanced primordia. We hypothesize that the effect of cytokinin on the development of primordia possibly depends on the robustness and stability of the auxin gradient.     

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.     

Lateral root stimulation in the early interaction between Arabidopsis thaliana and the ectomycorrhizal fungus Laccaria bicolor: Is fungal auxin the trigger?

Lateral root (LR) stimulation during early signal exchange between plant roots and ectomycorrhizal (ECM) fungi has recently been shown to be achieved by modulation of auxin gradients. We suggested that this modulation could occur through altered polar auxin transport (PAT) and through activation of auxin signalling pathways in the root. However, it remains unclear, which fungal molecules alter auxin pathways inside the plant partner. It has been suggested in previous studies that auxin released by the fungus could trigger observed plant responses during early signal exchange and later on during root colonization. Here we focus on the early interaction and we provide evidence for an alternative mechanism. Indeed, LR stimulation by the fungus in Arabidopsis thaliana followed a totally different timing than with exogenously applied auxin. Furthermore, experimental conditions that excluded the exchange of soluble molecules while allowing exchange of volatile(s) between the plant and the fungus were sufficient for LR induction, therefore questioning the role of secreted fungal auxin. These data suggest that volatiles released by the fungus and sensed by the plant may act upstream of altered auxin signaling in the plant.Key words: mycorrhiza, ectomycorrhiza, lateral root, auxin, volatiles, ethylene, jasmonic acidInteractions of plant roots with symbiotic, ectomycorrhizal soil fungi lead to lateral root (LR) stimulation during the very early interaction phase.¹ This LR stimulation has recently been shown to be independent of root colonization and to occur as well in non-mycorrhizal plants, such as Arabidopsis suggesting that fungal signals have a broad perception spectrum.^1,2 However, little is known about the type of signals exchanged between fungi and their plant partners during this early interaction phase. Several studies have proposed a role for the phytohormone auxin produced and secreted by ECM fungi as the signalling molecule during ECM fungus/plant signaling.^2–7 Recently we studied changes in auxin response and auxin transport in poplar and Arabidopsis thaliana roots during contact with the ECM fungus Laccaria bicolor.¹ We demonstrated that the presence of the fungus enhances the auxin response and distribution at the root apex and that this, as well as LR stimulation, is reliant on polar auxin transport through AtPIN2 and probably through PtPIN9 in poplar. Here, using Arabidopsis thaliana, whose LR stimulation by Laccaria bicolor has been demonstrated, we propose that not yet identified fungal volatiles may regulate auxin homeostasis in the plant, questioning the contribution of the auxin released by the fungus on the induction of LR.     

Evaluating the function of putative hormone transporters

Hormones typically serve as long distance signaling molecules. To reach their site of action, hormones need to be transported from the sites of synthesis. Many plant hormones are mobile, thus requiring specific transport systems for the export from their source cells as well as subsequent import into target cells. Hormone transport in general is still poorly understood. Auxin is probably the most intensively studied plant hormone concerning transport in the moment. To advance our understanding of hormone transport we need two principal data sets: information on the properties of the transport systems including substrate specificity and kinetics, and we need to identify candidate genes for the respective transporters. Physiological transport data can provide an important basis for identifying and characterizing candidate transporters and to define their in vivo role. A recent publication in Plant Physiology highlights how kinetic and specificity studies may help to identify cytokinin transporters.¹Key words: kinetin, zeatin, adenine, phytohormone, transportBy definition, hormones are compounds that interact at low concentrations with cellular receptors to modulate signal transduction pathways. A comparison of the chemical structures of animal and plant hormones suggests potential common origins. Peptide hormones are found in both kingdoms and share common processing mechanisms (e.g., TRH, vasopressin and kinins in animals; systemins, phytosulfokines, self incompatibility peptides in plants).^2,3 Steroid hormones derived from cholesterol such as testosterone, cortisol and calcitriol regulate development in mammals; the steroid hormone brassinolide is essential for plant development.⁴ Glutamate can serve as metabolite and signal in both plants and animals.^5,6 Finally, lipid and phospholipid-derived signaling compounds such as linoleic acid and arachidonic acid also function in both plants and animals; with phospholipid-derived prostaglandins and eicosanoids bearing similarities to the plant defense compound jasmonic acid.⁷Other signaling compounds present in animals have yet to be shown to function in plants, e.g., glycoprotein hormones such as luteinizing hormone, follicle-stimulating hormone or thyroid-stimulating hormone have been not been described to exist in plants.⁸ Compounds structurally similar to animal amine-derived hormones derived from tyrosine and tryptophan (such as catecholamines and thyroxine) are also present in plants, but appear to function primarily in herbivore defense.⁹The best characterized, and arguably most important plant hormones, bear little similarity to animal hormones and are mechanistically distinct. These include auxins, cytokinins, gibberellins, abscisic acid, ethylene and an apparent carotenoid-derivative, the MAX-dependent regulator of auxin signaling.^10,11 Arguably, the stress response compound salicylic acid, which functions in stress, wounding and defense responses could also be considered a plant hormone.¹²Hormonal signaling mechanisms can be categorized as autocrine (acting at the site of biosynthesis), paracrine (acting in adjacent or proximal cells), and endocrine (acting in cells distal to the site of production). In both, plants and animals, paracrine and endocrine hormone action is mediated and influenced by multiple long distance delivery systems. Hormones move primarily through the circulatory system in animals, but, in plants, are mobilized by transpiration and source-sink flows, which can be directed by chemisomotically-driven cellular uptake and efflux. However, the mechanisms driving uptake and efflux at the cellular level, as well as the proteins that mediate this movement, are surprisingly similar in plants and animals, despite the dissimilarities of plant and animal cell structure (central vacuoles, cell walls and H⁺ versus K⁺/Na²⁺ in/out gradients).Surprisingly little is known about plant hormone transport. Most hormones have autocrine activity, but in order to act at a distance or to even act on adjacent cells they must be transported across membranes. The existence of cellular export and import mechanisms are suggested by the presence of multiple hormones in the phloem sap^13,14 and the well documented polar long distance movement of auxin.¹⁵ Brassinosteroid receptors have been demonstrated as integral plasma membrane proteins which receive the hormone signal from outside the cell.¹⁶ This suggests a need for the hormone to first move into the apoplasm after biosynthesis. However, until recently, only the cellular auxin transport mechanisms mediated by the AUX/LAX, PIN and AtABCB/PGP proteins has been well characterized (reviewed in ref. ¹⁷).The study of these transporters has benefited from the use of plant, yeast and animal expression systems to characterize the proteins involved. Analyses of auxin transport proteins have capitalized on earlier suppression cloning and radiotracer uptake studies used successfully to characterize ion and metabolite transporters in yeast.^18–21 In cases where yeast systems have proven intractable for analysis of auxin transport proteins, heterologous systems based on mammalian cell systems have proven to be highly effective for radiotracer uptake studies.^18–23 Xenopus oocyte expression has been successfully utilized to characterize the AUX/LAX family of auxin influx symporters.^24,25 Plant cell culture systems have also been used to characterize transport proteins. This can however be problematic when endogenous substrates are metabolized by the cells, as is the case with IAA in tobacco BY-2 and Arabidopsis cell cultures.¹⁹ It is also difficult to assess the function of plant proteins in undifferentiated cell cultures, which may differ from the native function in phloem or xylem parenchyma cells.A recent article describes the use of a heterologous expression system based on the fission yeast S. pombe to express and characterize the PIN1 auxin efflux protein after knock-out of the endogenous yeast PIN-like gene AEL1.²¹ Previously, PIN1 had only been functionally expressed in plant cell systems and was nonfunctional when expressed in baker''s yeast or mammalian cells.^19,22 This report suggests that PIN1, interacts synergistically with the AtABCB19/PGP19 auxin efflux transporter, but appears to also mediate auxin efflux on its own, consistent with the distant phylogenetic similarity of the auxin efflux transporter protein family to major facilitator proteins.Subsequent work in the Murphy lab has shown that S. pombe can be used for comparisons of all known auxin transporters in a single system in which all ABC transporters and a solitary AUX1-like gene had been knocked out (Yang and Murphy, unpublished). This system also allows for the more detailed analyses of substrate specificity, transport kinetics and coupling mechanisms (primary and secondary active transport, uniport, cotransport antiport) necessary for functional assignment of auxin transport proteins. This system may also provide an attractive alternative to baker''s yeast when functional expression of a plant protein in Saccharomyces cerevisiae proves unsuccessful.Similar efforts are required for characterizing the transport of all other plant hormones including cytokinin. Arabidopsis transporters mediating both trans-zeatin and adenine uptake had been identified using yeast as an expression system.²⁶ Recently, the Schulz and Frommer labs provided a reference data set for trans-zeatin uptake by characterizing radiolabeled trans-zeatin uptake in Arabidopsis cell cultures.¹ The data show that the uptake kinetics of trans-zeatin are multiphasic, indicating the presence of both low- and high-affinity transport systems. The protonophore CCCP is an effective inhibitor of cytokinin uptake, consistent with H⁺-mediated uptake. Other physiologically active cytokinins such as isopentenyladenine and benzylaminopurine are effective competitors of trans-zeatin uptake, whereas allantoin had no inhibitory effect. Adenine competes for zeatin uptake indicating that degradation products of cytokinin oxidases can be transported by the same systems. Comparison of adenine and trans-zeatin uptake in Arabidopsis seedlings reveals similar uptake kinetics. Kinetic properties as well as substrate specificity determined in cell cultures are compatible with the hypothesis that members of the plant-specific PUP transporter family may play a role in adenine transport to scavenge extracellular adenine. In addition, the findings are also compatible with the hypothesis that this class of transporters may be involved at least in low affinity (µM range) cytokinin uptake. PUPs are encoded by a large gene family of 21 members, so it is conceivable that other members of the family may be involved in high affinity transport. Systematic analyses of single knock outs in Arabidopsis and combinations thereof my help to shed more light on the role of PUPs in cytokinin transport.     

Auxin,gibberellins and the gravitropic response of grass leaf sheath pulvini

The role of hormones in mediating tropic responses has been a central question in plant biology. Another key issue concerns how interactions between hormones regulate plant responses. In the September 2007 issue of Physiologia Plantarum, we published a paper relevant to both these questions.¹ This paper focuses on gravitropism in the barley leaf sheath pulvinus. The results support the Cholodny-Went theory on hormones and tropic responses, and highlight how an environmental factor (gravity) appears to first affect auxin content and consequently that of bioactive gibberellins (GAs). It appears that while GAs do not actually trigger the gravitropic bending of barley pulvini, they do act to magnify the bending response.Key words: auxin, gibberellin, Cholodny-Went theory, barley, pulvinus, gravitropism, mutant     

Expression of AMIDASE1 (AMI1) is suppressed during the first two days after germination

The regulation of cellular auxin levels is a critical factor in determining plant growth and architecture, as indole-3-acetic acid (IAA) gradients along the plant axis and local IAA maxima are known to initiate numerous plant growth responses. The regulation of auxin homeostasis is mediated in part by transport, conjugation and deconjugation, as well as by de novo biosynthesis. However, the pathways of IAA biosynthesis are yet not entirely characterized at the molecular and biochemical level. It is suggested that several biosynthetic routes for the formation of IAA have evolved. One such pathway proceeds via the intermediate indole-3-acetamide (IAM), which is converted into IAA by the activity of specific IAM hydrolases, such as Arabidopsis AMIDASE1 (AMI1). In this article we present evidence to support the argument that AMI1-dependent IAA synthesis is likely not to be used during the first two days of seedling development.Key words: Arabidopsis thaliana, auxin biosynthesis, AMIDASE1, indole-3-acetic acid, indole-3-acetamide, LEAFY COTYLEDON1, seed developmentAuxins are versatile plant hormones that play diverse roles in regulating many aspects of plant growth and development.¹ To enable auxins to develop their activity, a tight spatiotemporal control of cellular indole-3-acetic acid (IAA) contents is absolutely necessary since it is well-documented that auxin action is dose dependent, and that high IAA levels can have inhibitory effects on plant growth.² To achieve this goal, plants have evolved a set of different mechanisms to control cellular hormone levels. On the one hand, plants possess several pathways that contribute to the de novo synthesis of IAA. This multiplicity of biosynthetic routes presumably facilitates fine-tuning of the IAA production. On the other hand, plants are equipped with a variety of enzymes that are used to conjugate free auxin to either sugars, amino acids or peptides and small proteins, respectively, or on the contrary, that act as IAA-conjugate hydrolases, releasing free IAA from corresponding conjugates. IAA-conjugates serve as a physiologically inactive storage form of IAA from which the active hormone can be quickly released on demand. Alternatively, conjugation of IAA can mark the first step of IAA catabolism. In general, conjugation and deconjugation of free IAA are ways to positively or negatively affect active hormone levels, which adds another level of complexity to the system. Additionally, IAA can be transported from cell to cell in a polar manner, which is dependent on the action of several transport proteins. All together, these means are used to form auxin gradients and local maxima that are essential to initiate plant growth processes, such as root or leaf primordia formation.³     

Salt Stress Reduces Root Meristem Size by Nitric Oxide-Mediated Modulation of Auxin Accumulation and Signaling in Arabidopsis

The development of the plant root system is highly plastic, which allows the plant to adapt to various environmental stresses. Salt stress inhibits root elongation by reducing the size of the root meristem. However, the mechanism underlying this process remains unclear. In this study, we explored whether and how auxin and nitric oxide (NO) are involved in salt-mediated inhibition of root meristem growth in Arabidopsis (Arabidopsis thaliana) using physiological, pharmacological, and genetic approaches. We found that salt stress significantly reduced root meristem size by down-regulating the expression of PINFORMED (PIN) genes, thereby reducing auxin levels. In addition, salt stress promoted AUXIN RESISTANT3 (AXR3)/INDOLE-3-ACETIC ACID17 (IAA17) stabilization, which repressed auxin signaling during this process. Furthermore, salt stress stimulated NO accumulation, whereas blocking NO production with the inhibitor N^ω-nitro-l-arginine-methylester compromised the salt-mediated reduction of root meristem size, PIN down-regulation, and stabilization of AXR3/IAA17, indicating that NO is involved in salt-mediated inhibition of root meristem growth. Taken together, these findings suggest that salt stress inhibits root meristem growth by repressing PIN expression (thereby reducing auxin levels) and stabilizing IAA17 (thereby repressing auxin signaling) via increasing NO levels.Due to agricultural practices and climate change, soil salinity has become a serious factor limiting the productivity and quality of agricultural crops (Zhu, 2007). Worldwide, high salinity in the soil damages approximately 20% of total irrigated lands and takes 1.5 million ha out of production each year (Munns and Tester, 2008). In general, high salinity affects plant growth and development by reducing plant water potential, altering nutrient uptake, and increasing the accumulation of toxic ions (Hasegawa et al., 2000; Munns, 2002; Zhang and Shi, 2013). Together, these effects severely reduce plant growth and survival.Because the root is the first organ to sense high salinity, salt stress plays a direct, important role in modulating root system architecture (Wang et al., 2009). For instance, salt stress negatively regulates root hair formation and gravitropism (Sun et al., 2008; Wang et al., 2008). The role of salt in lateral root formation depends on the NaCl concentration. While high NaCl levels inhibit lateral root formation, lower NaCl levels stimulate lateral root formation in an auxin-dependent manner (Zolla et al., 2010; Ji et al., 2013). The root meristem plays an essential role in sustaining root growth (Perilli et al., 2012). Salt stress inhibits primary root elongation by suppressing root meristem activity (West et al., 2004). However, how this inhibition occurs remains largely unclear.Plant hormones are important intermediary signaling compounds that function downstream of environmental stimuli. Among plant hormones, indole-3-acetic acid (IAA) is thought to play a fundamental role in root system architecture by regulating cell division, expansion, and differentiation. In Arabidopsis (Arabidopsis thaliana) root tips, a distal auxin maximum is formed and maintained by polar auxin transport (PAT), which determines the orientation and extent of cell division in the root meristem as well as root pattern formation (Sabatini et al., 1999). PINFORMED (PIN) proteins, which are components of the auxin efflux machinery, regulate primary root elongation and root meristem size (Blilou et al., 2005; Dello Ioio et al., 2008; Yuan et al., 2013, 2014). The auxin signal transduction pathway is activated by direct binding of auxin to its receptor protein, TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN SIGNALING F-BOX (AFB), promoting the degradation of Aux/IAA proteins, releasing auxin response factors (ARFs), and activating the expression of auxin-responsive genes (Gray et al., 2001; Dharmasiri et al., 2005a; Kepinski and Leyser, 2005). Aux/IAA proteins are short-lived, nuclear-localized proteins that play key roles in auxin signal activation and root growth modulation (Rouse et al., 1998). Other hormones and stresses often regulate auxin signaling by affecting Aux/IAA protein stability (Lim and Kunkel, 2004; Nemhauser et al., 2004; Wang et al., 2007; Kushwah and Laxmi, 2014).Nitric oxide (NO) is a signaling molecule with diverse biological functions in plants (He et al., 2004; Fernández-Marcos et al., 2011; Shi et al., 2012), including important roles in the regulation of root growth and development. NO functions downstream of auxin during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002). Exogenous auxin-induced NO biosynthesis is associated with nitrate reductase activity during lateral root formation, and NO is necessary for auxin-induced lateral root and root hair development (Pagnussat et al., 2002; Lombardo et al., 2006). Pharmacological and genetic analyses in Arabidopsis indicate that NO suppresses primary root growth and root meristem activity (Fernández-Marcos et al., 2011). Additionally, both exogenous application of the NO donor sodium nitroprusside (SNP) and overaccumulation of NO in the mutant chlorophyll a/b binding protein underexpressed1 (cue1)/nitric oxide overproducer1 (nox1) result in reduced PIN1 expression and auxin accumulation in root tips. The auxin receptors protein TIR1 is S-nitrosylated by NO, suggesting that this protein is a direct target of NO in the regulation of root development (Terrile et al., 2012).Because NO is a free radical, NO levels are dynamically regulated by endogenous and environmental cues. Many phytohormones, including abscisic acid, auxin, cytokinin, salicylic acid, jasmonic acid, and ethylene, induce NO biosynthesis (Zottini et al., 2007; Kolbert et al., 2008; Tun et al., 2008; García et al., 2011). In addition, many abiotic and biotic stresses or stimuli, such as cold, heat, salt, drought, heavy metals, and pathogens/elicitors, also stimulate NO biosynthesis (Zhao et al., 2009; Mandal et al., 2012). Salt stress stimulates NO and ONOO^– accumulation in roots (Corpas et al., 2009), but the contribution of NO to root meristem growth under salinity stress has yet to be examined in detail.In this study, we found that salt stress significantly down-regulated the expression of PIN genes and promoted AUXIN RESISTANT3 (AXR3)/IAA17 stabilization. Furthermore, salt stress stimulated NO accumulation, and pharmacological inhibition of NO biosynthesis compromised the salt-mediated reduction in root meristem size. Our results support a model in which salt stress reduces root meristem size by increasing NO accumulation, which represses PIN expression and stabilizes IAA17, thereby reducing auxin levels and repressing auxin signaling.     

Analysis the role of arabidopsis <Emphasis Type="Italic">CKRC6/ASA1</Emphasis> in auxin and cytokinin biosynthesis

The crosstalk between auxin and cytokinin (CK) is important for plant growth and development, although the underlying molecular mechanisms remain unclear. Here, we describe the isolation and characterization of a mutant of Arabidopsis Cytokinin-induced Root Curling 6 (CKRC6), an allele of ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) that encodes the á-subunit of AS in tryptophan (Trp) biosynthesis. The ckrc6 mutant exhibits root gravitropic defects and insensitivity to both CK and the ethylene precursor 1-aminocyclopropane-1-carboxylicacid (ACC) in primary root growth. These defects can be rescued by exogenous indole-3-acetic acid (IAA) or tryptophan (Trp) supplementation. Furthermore, our results suggest that the ckrc6 mutant has decreased IAA content, differential expression patterns of auxin biosynthesis genes and CK biosynthesis isopentenyl transferase (IPT) genes in comparison to wild type. Collectively, our study shows that auxin controls CK biosynthesis based on that CK sensitivity is altered in most auxin-resistant mutants and that CKs promote auxin biosynthesis but inhibit auxin transport and response. Our results also suggest that CKRC6/ASA1 may be located at an intersection of auxin, CK and ethylene metabolism and/or signaling.     

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.     

Light Quality Regulates Lateral Root Development in Tobacco Seedlings by Shifting Auxin Distributions

Light is an important environmental regulator of diverse growth and developmental processes in plants. However, the mechanisms by which light quality regulates root growth are poorly understood. We analyzed lateral root (LR) growth of tobacco seedlings in response to three kinds of light qualities (red, white, and blue). Primary (1°) LR number and secondary (2°) LR density were elevated under red light (on days 9 and 12 of treatment) in comparison with white and blue lights. Higher IAA concentrations measured in roots and lower in leaves of plants treated with red light suggest that red light accelerated auxin transport from the leaves to roots (in comparison with other light qualities). Corroborative evidence for this suggestion was provided by elevated DR5::GUS expression levels at the shoot/root junction and in the 2° LR region. Applications of N-1-naphthylphthalamic acid (NPA) to red light-treated seedlings reduced both 1° LR number and 2° LR density to levels similar to those measured under white light; DR5::GUS expression levels were also similar between these light qualities after NPA application. Results were similar following exogenous auxin (NAA) application to blue light-treated seedlings. Direct [³H]IAA transport measurement indicated that the polar auxin transport from shoot to root was increased by red light. Red light promoted PIN3 expression levels and blue light reduced PIN1, 3–4 expression levels in the shoot/root junction and in the root, indicating that these genes play key roles in auxin transport regulation by red and blue lights. Overall, our findings suggest that three kinds of light qualities regulate LR formation in tobacco seedlings through modification of auxin polar transport.     

Genetic approach towards the identification of auxin-cytokinin crosstalk components involved in root development

Phytohormones are important plant growth regulators that control many developmental processes, such as cell division, cell differentiation, organogenesis and morphogenesis. They regulate a multitude of apparently unrelated physiological processes, often with overlapping roles, and they mutually modulate their effects. These features imply important synergistic and antagonistic interactions between the various plant hormones. Auxin and cytokinin are central hormones involved in the regulation of plant growth and development, including processes determining root architecture, such as root pole establishment during early embryogenesis, root meristem maintenance and lateral root organogenesis. Thus, to control root development both pathways put special demands on the mechanisms that balance their activities and mediate their interactions. Here, we summarize recent knowledge on the role of auxin and cytokinin in the regulation of root architecture with special focus on lateral root organogenesis, discuss the latest findings on the molecular mechanisms of their interactions, and present forward genetic screen as a tool to identify novel molecular components of the auxin and cytokinin crosstalk.     

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways.     

Involvement of nitric oxide and auxin in signal transduction of copper-induced morphological responses in Arabidopsis seedlings

Background and Aims

Plants are able to adapt to the environment dynamically through regulation of their growth and development. Excess copper (Cu²⁺), a toxic heavy metal, induces morphological alterations in plant organs; however, the underlying mechanisms are still unclear. With this in mind, the multiple signalling functions of nitric oxide (NO) in plant cells and its possible regulatory role and relationship with auxin were examined during Cu²⁺-induced morphological responses.

Methods

Endogenous auxin distribution was determined by microscopic observation of X-Gluc-stained DR5::GUS arabidopsis, and the levels of NO, superoxide and peroxynitrite were detected by fluorescence microscopy. As well as wild-type, NO-overproducer (nox1) and -deficient (nia1nia2 and nia1nia2noa1-2) arabidopsis plants were used.

Key Results

Cu²⁺ at a concentration of 50 µm resulted in a large reduction in cotyledon area and hypocotyl and primary root lengths, accompanied by an increase in auxin levels. In cotyledons, a low Cu²⁺ concentration promoted NO accumulation, which was arrested by nitric oxide synthase or nitrate reductase inhibitors. The 5-μm Cu²⁺-induced NO synthesis was not detectable in nia1nia2 or nia1nia2noa1-2 plants. In roots, Cu²⁺ caused a decrease of the NO level which was not associated with superoxide and peroxynitrite formation. Inhibition of auxin transport resulted in an increase in NO levels, while exogenous application of an NO donor reduced DR5::GUS expression. The elongation processes of nox1 were not sensitive to Cu²⁺, but NO-deficient plants showed diverse growth responses.

Conclusions

In plant organs, Cu²⁺ excess results in severe morphological responses during which the endogenous hormonal balance and signal transduction are affected. Auxin and NO negatively regulate each other''s level and NO intensifies the metal-induced cotyledon expansion, but mitigates elongation processes under Cu²⁺ exposure.     

Activation of gibberellin 20-oxidase 2 undermines auxin-dependent root and root hair growth in NaCl-stressed <Emphasis Type="Italic">Arabidopsis</Emphasis> seedlings

Although salt stress mainly disturbs plant root growth by affecting the biosynthesis and signaling of phytohormones, such as gibberellin (GA) and auxin, the exact mechanisms of the crosstalk between these two hormones remain to be clarified. Indole-3-acetic acid (IAA) is a biologically active auxin molecule. In this study, we investigated the role of Arabidopsis GA20-oxidase 2 (GA20ox2), a final rate-limiting enzyme of active GA biosynthesis, in IAA-directed root growth under NaCl stress. Under the NaCl treatment, seedlings of a loss-of-function ga20ox2-1 mutant exhibited primary root and root hair elongation, altered GA₄ accumulation, and decreased root Na⁺ contents compared with the wild-type, transgenic GA20ox2-complementing, and GA20ox2-overexpression plant lines. Concurrently, ga20ox2-1 alleviated the tissue-specific inhibition of NaCl on IAA generation by YUCCAs, IAA transport by PIN1 and PIN2, and IAA accumulation in roots, thereby explaining how NaCl increased GA20ox2 expression in shoots but disrupted primary root and root hair growth in wild-type seedlings. In addition, a loss-of-function pin2 mutant impeded GA20ox2 expression, indicating that GA20ox2 function requires PIN2 activity. Thus, the activation of GA20ox2 retards IAA-directed primary root and root hair growth in response to NaCl stress.