Distribution of Endogenous NO Regulates Early Gravitropic Response and PIN2 Localization in Arabidopsis Roots.

High-resolution and automated image analysis of individual roots demonstrated that endogenous nitric oxide (NO) contribute significantly to gravitropism of Arabidopsis roots. Lowering of endogenous NO concentrations strongly reduced and even reversed gravitropism, resulting in upward bending, without affecting root growth rate. Notably, the asymmetric accumulation of NO along the upper and lower sides of roots correlated with a positive gravitropic response. Detection of NO by the specific DAF-FM DA fluorescent probe revealed that NO was higher at the lower side of horizontally-oriented roots returning to initial values 2 h after the onset of gravistimulation. We demonstrate that NO promotes plasma membrane re-localization of PIN2 in epidermal cells, which is required during the early root gravitropic response. The dynamic and asymmetric localization of both auxin and NO is critical to regulate auxin polar transport during gravitropism. Our results collectively suggest that, although auxin and NO crosstalk occurs at different levels of regulation, they converge in the regulation of PIN2 membrane trafficking in gravistimulated roots, supporting the notion that a temporally and spatially coordinated network of signal molecules could participate in the early phases of auxin polar transport during gravitropism.

Decoding plant responses to iron deficiency: Is nitric oxide a central player?

Plants respond to iron deprivation by inducing a series of physiological and morphological responses to counteract the nutrient deficiency. These responses include: (i) the acidification of the extracellular medium, (ii) the reduction of ferric ion and (iii) the increased transport of ferrous ion inside of root cells. This iron transport system is present in strategy I plants and is strictly regulated; at low iron concentration the responses are induced whereas upon iron supply they are repressed. The mechanisms related with this process has been extensively studied, however, the specific cellular effectors involved in sensing iron deficiency, the cascade of components participating in signal transduction, and the way iron is metabolized and delivered, are yet poorly understood. Recently, it has been proposed nitric oxide (NO) as a signaling molecule required for plant responses to iron deficiency. NO is produced rapidly in the root epidermis of tomato plants that are growing under iron deficient conditions. Furthermore, it was demonstrated that NO is required for the expression and activity of iron uptake components in roots during iron deprivation. Here we propose and discuss a working hypothesis to understand the way NO is acting in plants responses to iron deficiency. We specifically highlight the cross talk between NO and plant hormones, and the interaction between NO, iron and glutathione for the formation of dinitrosyl iron complexes (DNICs). Finally, a potential role of DNICs in iron mobilization is proposed.

Nitric oxide accumulation is required for molecular and physiological responses to iron deficiency in tomato roots.

Iron is an essential and commonly limited nutrient for plants. To increase the uptake of iron during times of low iron supply, plants, except the grasses, activate a set of physiological and morphological responses in their roots that include iron reduction, soil acidification, Fe(II) transport and proliferation of root hairs. It is not known how root cells sense and transduce the changes that occur after the onset of iron deficiency. This work presents evidence that nitric oxide (NO) is produced rapidly in the root epidermis of tomato plants (Solanum lycopersicum) that are grown in iron-deficient conditions. The scavenging of NO prevented iron-deficiency-induced upregulation of the basic helix-loop-helix transcription factor FER, the ferric-chelate reductase LeFRO1 and the Fe(II) transporter LeIRT1 genes. On the other hand, exogenous application of the NO donor S-nitrosoglutathione enhanced the accumulation of FER, LeFRO1 and LeIRT1 mRNA in roots of iron-deficient plants. The activity of the root ferric-chelate reductase and the proliferation of root hairs induced by iron deficiency were stimulated by NO supplementation and suppressed by NO scavenging. Nitric oxide was ineffective in inducing iron-deficiency responses in the tomato fer mutant, which indicates that the FER protein is necessary to mediate the action of NO. Furthermore, NO supplementation improved plant growth under low iron supply, which suggests that NO is a key component of the regulatory mechanisms that control iron uptake and homeostasis in plants. In summary, the results of this investigation indicate that an increase in NO production is an early response of roots to iron deprivation that contributes to the improvement of iron availability by (i) modulating the expression of iron uptake-related genes and (ii) regulating the physiological and morphological adaptive responses of roots to iron-deficient conditions.

Nitric oxide modulates the expression of cell cycle regulatory genes during lateral root formation in tomato.

Nitric oxide (NO) is a bioactive molecule involved in diverse physiological functions in plants. It has previously been reported that the NO donor sodium nitroprusside (SNP) applied to germinated tomato seeds was able to induce lateral root (LR) formation in the same way that auxin treatment does. In this paper, it is shown that NO modulates the expression of cell cycle regulatory genes in tomato pericycle cells and leads, in turn, to induced LR formation. The addition of the NO scavenger CPTIO at different time points during auxin-mediated LR development indicates that NO is required for LR primordia formation and not for LR emergence. The SNP-mediated LR promotion could be prevented by the cell cycle inhibitor olomoucine, suggesting that NO is involved in cell cycle regulation. A system was developed in which the formation of LRs was synchronized. It was based on the control of NO availability in roots by treatment with the NO scavenger CPTIO. The expression of the cell cycle regulatory genes encoding CYCA2;1, CYCA3;1, CYCD3;1, CDKA1, and the Kip-Related Protein KRP2 was studied using RT-PCR analysis in roots with synchronized and non-synchronized LR formation. NO mediates the induction of the CYCD3;1 gene and the repression of the CDK inhibitor KRP2 gene at the beginning of LR primordia formation. In addition, auxin-dependent cell cycle gene regulation was dependent on NO.

Nitric oxide functions as a positive regulator of root hair development.

THE ROOT EPIDERMIS IS COMPOSED OF TWO CELL TYPES: trichoblasts (or hair cells) and atrichoblasts (or non-hair cells). In lettuce (Lactuca sativa cv. Grand Rapids var. Rapidmor oscura) plants grown hydroponically in water, the root epidermis did not form root hairs. The addition of 10 microM sodium nitroprusside (SNP), a nitric oxide (NO) donor, resulted in almost all rhizodermal cells differentiated into root hairs. Treatment with the synthetic auxin 1-naphthyl acetic acid (NAA) displayed a significant increase of root hair formation (RHF) that was prevented by the specific NO scavenger carboxy-PTIO (cPTIO). In Arabidopsis, two mutants have been shown to be defective in NO production and to display altered phenotypes in which NO is implicated. Arabidopsis nos1 has a mutation in an NO synthase structural gene (NOS1), and the nia1 nia2 double mutant is null for nitrate reductase (NR) activity. We observed that both mutants were affected in their capacity of developing root hairs. Root hair elongation was significantly reduced in nos1 and nia1 nia2 mutants as well as in cPTIO-treated wild type plants. A correlation was found between endogenous NO level in roots detected by the fluorescent probe DAF-FM DA and RHF. In Arabidopsis, as well as in lettuce, cPTIO blocked the NAA-induced root hair elongation. Taken together, these results indicate that: (1) NO is a critical molecule in the process leading to RHF and (2) NO is involved in the auxin-signaling cascade leading to RHF.

Nitric oxide is involved in the Azospirillum brasilense-induced lateral root formation in tomato.

Azospirillum spp. is a well known plant-growth-promoting rhizobacterium. Azospirillum-inoculated plants have shown to display enhanced lateral root and root hair development. These promoting effects have been attributed mainly to the production of hormone-like substances. Nitric oxide (NO) has recently been described to act as a signal molecule in the hormonal cascade leading to root formation. However, data on the possible role of NO in free-living diazotrophs associated to plant roots, is unavailable. In this work, NO production by Azospirillum brasilense Sp245 was detected by electron paramagnetic resonance (6.4 nmol. g-1 of bacteria) and confirmed by the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA). The observed green fluorescence was significantly diminished by the addition of the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Azospirillum-inoculated and noninoculated tomato (Lycopersicon esculentum L.) roots were incubated with DAF-2 DA and examined by epifluorescence microscopy. Azospirillum-inoculated roots displayed higher fluorescence intensity which was located mainly at the vascular tissues and subepidermal cells of roots. The Azospirillum-mediated induction of lateral root formation (LRF) appears to be NO-dependent since it was completely blocked by treatment with cPTIO, whereas the addition of the NO donor sodium nitroprusside partially reverted the inhibitory effect of cPTIO. Overall, the results strongly support the participation of NO in the Azospirillum-promoted LRF in tomato seedlings.

Nitric oxide and iron in plants: an emerging and converging story.

Although iron is plentiful, it exists primarily in its insoluble form and is therefore not freely available to plants. Thus, complex strategies involving chelators, production of reductive agents, reductase activities, proton-mediated processes, specialized storage proteins, and others, act in concert to mobilize iron from the environment into the plant and within the plant. Because of its fundamental role in plant productivity and ultimately in human nutrition, several unsolved and central questions concerning sensing, trafficking, homeostasis and delivery of iron in plants are currently a matter of intense debate. Here, we discuss some recent studies focusing on iron nutrition in plants as well as evidence from iron homeostasis in animals and propose a new scenario involving the formation of nitric oxide and iron-nitrosyl complexes as part of the dynamic network that governs plant iron homeostasis.

Nitric oxide plays a central role in determining lateral root development in tomato.

Nitric oxide (NO) is a bioactive molecule that functions in numerous physiological processes in plants, most of them involving cross-talk with traditional phytohormones. Auxin is the main hormone that regulates root system architecture. In this communication we report that NO promotes lateral root (LR) development, an auxin-dependent process. Application of the NO donor sodium nitroprusside (SNP) to tomato ( Lycopersicon esculentum Mill.) seedlings induced LR emergence and elongation in a dose-dependent manner, while primary root (PR) growth was diminished. The effect is specific for NO since the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) blocked the action of SNP. Depletion of endogenous NO with CPTIO resulted in the complete abolition of LR emergence and a 40% increase in PR length, confirming a physiological role for NO in the regulation of root system growth and development. Detection of endogenous NO by the specific probe 4,5-diaminofluorescein diacetate (DAF-2 DA) revealed that the NO signal was specifically located in LR primordia during all stages of their development. In another set of experiments, SNP was able to promote LR development in auxin-depleted seedlings treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Moreover, it was found that LR formation induced by the synthetic auxin 1-naphthylacetic acid (NAA) was prevented by CPTIO in a dose-dependent manner. All together, these results suggest a novel role for NO in the regulation of LR development, probably operating in the auxin signaling transduction pathway.

Nitric oxide: the versatility of an extensive signal molecule.

Nitric oxide (NO) is a small highly diffusible gas and a ubiquitous bioactive molecule. Its chemical properties make NO a versatile signal molecule that functions through interactions with cellular targets via either redox or additive chemistry. In plants, NO plays a role in a broad spectrum of pathophysiological and developmental processes. Although nitric oxide synthase (NOS)-dependent NO production has been reported in plants, no gene, cDNA, or protein has been isolated to date. In parallel, precise and regulated NO production can be measured from the activity of the ubiquitous enzyme nitrate reductase (NR). In addition to endogenous NO formation, high NO emissions are observed from fertilized soils, but their effects on the physiology of plants are largely unknown. Many environmental and hormonal stimuli are transmitted either directly or indirectly by NO signaling cascades. The ability of NO to act simultaneously on several unrelated biochemical nodes and its redox homeostatic properties suggest that it might be a synchronizing molecule in plants.

Nitric oxide improves internal iron availability in plants.

Iron deficiency impairs chlorophyll biosynthesis and chloroplast development. In leaves, most of the iron must cross several biological membranes to reach the chloroplast. The components involved in the complex internal iron transport are largely unknown. Nitric oxide (NO), a bioactive free radical, can react with transition metals to form metal-nitrosyl complexes. Sodium nitroprusside, an NO donor, completely prevented leaf interveinal chlorosis in maize (Zea mays) plants growing with an iron concentration as low as 10 microM Fe-EDTA in the nutrient solution. S-Nitroso-N-acetylpenicillamine, another NO donor, as well as gaseous NO supply in a translucent chamber were also able to revert the iron deficiency symptoms. A specific NO scavenger, 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, blocked the effect of the NO donors. The effect of NO treatment on the photosynthetic apparatus of iron-deficient plants was also studied. Electron micrographs of mesophyll cells from iron-deficient maize plants revealed plastids with few photosynthetic lamellae and rudimentary grana. In contrast, in NO-treated maize plants, mesophyll chloroplast appeared completely developed. NO treatment did not increase iron content in plant organs, when expressed in a fresh matter basis, suggesting that root iron uptake was not enhanced. NO scavengers 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and methylene blue promoted interveinal chlorosis in iron-replete maize plants (growing in 250 microM Fe-EDTA). Even though results support a role for endogenous NO in iron nutrition, experiments did not establish an essential role. NO was also able to revert the chlorotic phenotype of the iron-inefficient maize mutants yellow stripe1 and yellow stripe3, both impaired in the iron uptake mechanisms. All together, these results support a biological action of NO on the availability and/or delivery of metabolically active iron within the plant.