Interactions between jasmonates and ethylene in the regulation of root hair development in Arabidopsis

Root hair formation is an important model with which to study cell patterning and differentiation in higher plants. Ethylene and auxin are critical regulators of root hair development. The role of jasmonates (JAs) was examined in Arabidopsis root hair development as well as their interactions with ethylene in this process. The results have shown that both methyl jasmonate (MeJA) and jasmonic acid (JA) have a pronounced effect on promoting root hair formation. However, the effect of MeJA and JA on root hair formation was blocked by ethylene inhibitors Ag+ or aminoethoxyvinylglycine (AVG). The stimulatory effects of MeJA and JA were also diminished in ethylene-insensitive mutants etr1-1 and etr1-3. Furthermore, the JA biosynthesis inhibitors ibuprofen and salicylhydroxamic acid (SHAM) suppressed 1-aminocyclopropane-1-carboxylic acid (ACC)-induced root hair formation, and decreased the root hairs in seedlings of the ethylene over-producing mutant eto1-1. These results suggested that JAs promote root hair formation, through an interaction with ethylene.     

生长素和乙烯互作调控硝酸铵诱导的根毛分叉

以野生型拟南芥(WT)及其生长素和乙烯不敏感型突变体(aux1-7、axr1-3、etr1-1和etr1-3)为实验材料,采用固体培养法研究了高浓度硝酸铵对根毛发育的影响,以揭示其调控根毛发育的机制。结果表明:(1)随着外源硝酸铵浓度的逐渐增加,拟南芥根毛伸长受阻,产生大量的分叉根毛。(2)高浓度硝酸铵条件下,外源活性氧或活性氧产生抑制剂二苯基氯化碘(DPI)的添加能抑制高浓度硝酸铵诱导的分叉根毛产生。(3)高浓度硝酸铵条件下,外源生长素或乙烯合成前体物质1-氨基-环丙烷-1-羧酸(ACC)处理能恢复根毛的正常生长,解除高浓度硝酸铵诱导根毛分叉现象。(4)高浓度硝酸铵条件下,外源生长素处理乙烯不敏感型突变体或ACC处理生长素不敏感型突变体均能抑制突变体分叉根毛的形成。研究表明,活性氧、生长素和乙烯都参与了高浓度硝酸铵对根毛发育的过程调控;在硝酸铵诱导的根毛分叉中生长素和乙烯存在相互作用,在缺乏生长素信号通路时,乙烯能够发挥补充作用抑制分叉根毛的产生;在缺乏乙烯信号通路时,生长素也可以弥补缺失乙烯的作用抑制根毛的分叉,但是需要更高浓度的生长素才能充分抑制分叉根毛的产生。     

Root growth inhibition by NH4+ in Arabidopsis is mediated by the root tip and is linked to NH4+ efflux and GMPase activity

Root growth in higher plants is sensitive to excess ammonium (NH₄⁺). Our study shows that contact of NH₄⁺ with the primary root tip is both necessary and sufficient to the development of arrested root growth under NH₄⁺ nutrition in Arabidopsis. We show that cell elongation and not cell division is the principal target in the NH₄⁺ inhibition of primary root growth. Mutant and expression analyses using DR5:GUS revealed that the growth inhibition is furthermore independent of auxin and ethylene signalling. NH₄⁺ fluxes along the primary root, measured using the Scanning Ion‐selective Electrode Technique, revealed a significant stimulation of NH₄⁺ efflux at the elongation zone following treatment with elevated NH₄⁺, coincident with the inhibition of root elongation. Stimulation of NH₄⁺ efflux and inhibition of cell expansion were significantly more pronounced in the NH₄⁺‐hypersensitive mutant vtc1‐1, deficient in the enzyme GDP‐mannose pyrophosphorylase (GMPase). We conclude that both restricted transmembrane NH₄⁺ fluxes and proper functioning of GMPase in roots are critical to minimizing the severity of the NH₄⁺ toxicity response in Arabidopsis.     

γ‐Aminobutyric acid addition alleviates ammonium toxicity by limiting ammonium accumulation in rice (Oryza sativa) seedlings

Excessive use of nitrogen (N) fertilizer has increased ammonium (NH₄⁺) accumulation in many paddy soils to levels that reduce rice vegetative biomass and yield. Based on studies of NH₄⁺ toxicity in rice (Oryza sativa, Nanjing 44) seedlings cultured in agar medium, we found that NH₄⁺ concentrations above 0.75 mM inhibited the growth of rice and caused NH₄⁺ accumulation in both shoots and roots. Use of excessive NH₄⁺ also induced rhizosphere acidification and inhibited the absorption of K, Ca, Mg, Fe and Zn in rice seedlings. Under excessive NH₄⁺ conditions, exogenous γ‐aminobutyric acid (GABA) treatment limited NH₄⁺ accumulation in rice seedlings, reduced NH₄⁺ toxicity symptoms and promoted plant growth. GABA addition also reduced rhizosphere acidification and alleviated the inhibition of Ca, Mg, Fe and Zn absorption caused by excessive NH₄⁺. Furthermore, we found that the activity of glutamine synthetase/NADH‐glutamate synthase (GS; EC 6.3.1.2/NADH‐GOGAT; EC1.4.1.14) in root increased gradually as the NH₄⁺ concentration increased. However, when the concentration of NH₄⁺ is more than 3 mM, GABA treatment inhibited NH₄⁺‐induced increases in GS/NADH‐GOGAT activity. The inhibition of ammonium assimilation may restore the elongation of seminal rice roots repressed by high NH₄⁺. These results suggest that mitigation of ammonium accumulation and assimilation is essential for GABA‐dependent alleviation of ammonium toxicity in rice seedlings.     

The roles of jasmonate signalling in nitrogen uptake and allocation in rice (Oryza sativa L.)

Herbivore damage by chewing insects activates jasmonate (JA) signalling that can elicit systemic defense responses in rice. Few details are known, however, concerning the mechanism, whereby JA signalling modulates nutrient status in rice in response to herbivory. (¹⁵NH₄)₂SO₄ labelling experiments, proteomic surveys, and RT‐qPCR analyses were used to identify the roles of JA signalling in nitrogen (N) uptake and allocation in rice plants. Exogenous applications of methyl jasmonate (MeJA) to rice seedlings led to significantly reduced N uptake in roots and reduced translocation of recently‐absorbed ¹⁵N from roots to leaves, likely occurring as a result of down‐regulation of glutamine synthetase cytosolic isozyme 1–2 and ferredoxin–nitrite reductase. Shoot MeJA treatment resulted in a remobilization of endogenous unlabelled ¹⁴N from leaves to roots, and root MeJA treatment also increased ¹⁴N accumulation in roots but did not affect ¹⁴N accumulation in leaves of rice. Additionally, proteomic and RT‐qPCR experiments showed that JA‐mediated plastid disassembly and dehydrogenases GDH2 up‐regulation contribute to N release in leaves to support production of defensive proteins/compounds under N‐limited condition. Collectively, our results indicate that JA signalling mediates large‐scale systemic changes in N uptake and allocation in rice plants.     

Regulation by fixed nitrogen of host-symbiont recognition in the Rhizobium-clover symbiosis

Either NO₃⁻ (16 millimolar) or NH₄⁺ (1 millimolar) completely inhibited infection and nodulation of white clover seedlings (Trifoliin repens) inoculated with Rhizobium trifolii. The binding of R. trifolii to root hairs and the immunologically detectable levels of the plant lectin, trifoliin, on the root hair surface had parallel declining slopes as the concentration of either NO₃⁻ or NH₄⁺ was increased in the rooting medium. This supports the role of trifoliin in binding R. trifolii to clover root hairs. Agglutination of R. trifolii by trifoliin from seeds was not inhibited by these levels of NO₃⁻ or NH₄⁺. The results suggest that these fixed N ions may play important roles in regulating an early recognition process in the Rhizobium-clover symbiosis, namely the accumulation of high numbers of infective R. trifolii cells on clover root hairs.     

马尾松组培苗对氮素形态的生长响应

马尾松属高氮需求树种,然而在苗木培育中马尾松对氮素,尤其是不同形态氮素的需求尚不明确。该文以马尾松组培苗为试验材料,采用基质培养方法,针对硝态氮、铵态氮两种氮素形态均分别设置了2、4、8、16 mmol·L~(-1)4个处理,以不添加氮素为对照,对苗木的高径生长、根构型参数(总根长、总表面积、总体积、平均直径和根尖数)以及生物量的变化进行了分析。结果表明:(1)在2～8 mmol·L~(-1)硝态氮处理下,除根冠比外,苗高、地径、根构型参数、生物量均不低于对照,其中以2 mmol·L~(-1)水平下苗木生长效果最好,苗高、地径、根构型参数、生物量均高于对照;在16 mmol·L~(-1)硝态氮处理下,苗高、总根长与根尖数低于对照。(2)在2～16 mmol·L~(-1)供试范围内,铵态氮处理下的苗木根冠比小于对照,但其苗高、地径、根构型参数、生物量均不低于对照,整体上以4 mmol·L~(-1)处理下的苗木生长表现最佳。(3)在任一供氮水平,除根冠比和2mmol·L~(-1)处理下的根总表面积与根尖数在两种氮素形态间无明显差异外,铵态氮处理下的苗木生长情况显著优于硝态氮处理,这说明马尾松组培苗偏好于吸收和利用铵态氮。综上结果表明,外施硝态氮、铵态氮均能促进马尾松组培苗生长,但需控制在适宜浓度范围内,其中以2 mmol·L~(-1)硝态氮和4 mmol·L~(-1)铵态氮处理效果较佳。高浓度硝态氮会抑制苗木高度及根系发育,且在相同施肥水平下,对苗木生长的促进效果大多弱于铵态氮。因此,今后为达到培育优质壮苗和提高肥效、减少肥害的目的,可考虑使用铵态氮肥。     

Simultaneous measurement of ammonium,nitrate and proton fluxes along the length of eucalypt roots

Knowledge of the preferred source of N for Eucalyptus nitens will lead to improved fertiliser management practices in plantations. Ion selective microelectrodes were used non-invasively to measure simultaneously net fluxes of NH₄ ⁺, NO₃ ^– and H⁺ along the tap root of solution-cultured E. nitens. Measurements were conducted in solutions containing 100 m NH₄NO₃. The pattern of fluxes was such that there was a large influx of NH₄ ⁺, a smaller influx of NO₃ ^– and large H⁺ efflux. The ratio of these fluxes was constant, according to the ratio 3:1:–6 (NH₄ ⁺:NO₃ ^–:H⁺). Within the region 20–60 mm from the root apex of E. nitens seedlings there was spatial and temporal variation in fluxes but flux patterns remained constant. Root hair density did not affect fluxes nor did proximity to lateral roots. Variation was less than that found in previous studies of localised root fluxes using similar high-resolution measurement techniques. It was concluded that small-scale spatial variation in fluxes may have confounded previous studies. There were associations between fluxes of all three ions, the strongest associations being between NH₄ ⁺ and H⁺, and NH₄ ⁺ and NO₃ ^–. Overall, these results are consistent with NH₄ ⁺ being the preferred source N for E. nitens.     

Nitrogen limitation in mycorrhizal Norway spruce (Picea abies) seedlings induced mycelial foraging for ammonium: implications for Ca and Mg uptake

Although many studies support the importance of the external mycelium for nutrient acquisition of ectomycorrhizal plants, direct evidence for a significant contribution to host nitrogen nutrition is still scarce. We grew nonmycorrhizal seedlings and seedlings mycorrhizal with Paxillus involutus (Batsch) Fr. in a sand culture system with two compartments separated by a 45-m Nylon mesh. Hyphae, but not roots, can penetrate this net. Nutrient solutions were designed to limit seedling growth by nitrogen. Hyphal density in the hyphal compartment, host N status and shoot growth of mycorrhizal seedlings significantly increased in response to NH₄ ⁺ addition to the hyphal compartment. Labeling the compartment only accessible to hyphae with ¹⁵NH₄ ⁺ showed that the increase in N uptake in the mycorrhizal seedlings was a result of hyphal N acquisition from the hyphal compartment. These results indicate that hyphae of P. involutus may actively forage into N-rich patches and improve host N status and growth. In the mycorrhizal seedlings, which received additional NH₄ ⁺ via their external mycelium, the increase in NH₄ ⁺ supply less negatively affected Ca and Mg uptake than in nonmycorrhizal seedlings, where the additional NH₄ ⁺ was directly supplied to the roots. This was most likely due to the close link of NH₄ ⁺ uptake and H⁺ extrusion, which, in the nonmycorrhizal seedlings, lead to a strong acidification in the root compartment, and subsequently reduced Ca and Mg uptake, whereas in the mycorrhizal seedlings the site of intensive NH₄ ⁺ uptake and acidification was in the hyphal and not in the root compartment. Our data support the idea that the ectomycorrhizal mycelium connected to an N-deficient host may actively forage for N. The mycelium may also be important as a biological buffer system ameliorating negative influence of high NH₄ ⁺ supply on cation uptake.     

Regulation of isoflavone production in hydroponically grown Pueraria montana (kudzu) by cork pieces, XAD-4, and methyl jasmonate

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g⁻¹ dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.     

Evidence that exogenous urea acts as a potent cue to alleviate ammonium‐inhibition of root system growth of cotton plant (Gossypium hirsutum)

Many plants grown with low‐millimolar concentration of NH₄⁺ as a sole nitrogen source develop NH₄⁺‐toxicity symptoms. To date, crucial molecular identities and a practical approach involved in the improvement of plant NH₄⁺‐tolerance remain largely unknown. By phenotyping of upland cotton grown on varied nitrogen forms, we came across a phenomenon that caused sub‐millimolar concentrations of urea (e.g., up 50 μM) to repress the growth inhibition of roots and whole plant cultivated in a NH₄⁺‐containing nutrient solution. A growth‐recovery assay revealed that the relief in NH₄⁺‐inhibited growth required only a short‐term exposure (≧12 h) of the roots to urea, implying that urea could elicit an internal signaling and be involved in antagonizing NH₄⁺‐sensitivity. Intriguingly, split‐root experiments demonstrated that low urea occurrence in one root‐half could efficaciously stimulate not only supplied root but also the root‐half grown in NH₄⁺‐solution without urea, indicating the existence of urea‐triggered local and systemic long‐distance signaling. In the split‐root experiment we also observed high arginase activity, strong arginine reduction and remarkable upregulation of polyamine biosynthesis‐related genes (ADC1/2, SPDS and SPMS). Therefore, we suggest that external urea might serve as an effective cue (signal molecule) in an arginine‐/polyamine‐related process for ameliorating NH₄⁺‐suppressed root growth, providing a novel aspect for deeper exploring and understanding plant NH₄⁺‐tolerance.     

Induction of adventitious roots and analysis of ginsenoside content and the genes involved in triterpene biosynthesis inPanax ginseng

Adventitious roots were produced directly from root segments ofPanax ginseng seedlings when cultured on an MS solid medium containing 3.0 mg L^-1 IBA. Omitting NH₄NO₃ from this medium greatly enhanced both the frequency of adventitious root formation and the number of roots per expiants. This frequency declined markedly with the age of the root, but could be increased through repeated sub-culturing events. A two-step procedure that included NH₄NO₃ free media for the first two weeks of culture, followed by transfer onto media containing NH₄NO₃ for another four weeks, greatly improved total fresh weights of these adventitious roots compared with a method of continuous culture over six weeks in media that always contained NH₄NO₃. Expression of the genes involved in triterpene biosynthesis was analyzed by RT-PCR. Ginsenoside contents were enhanced by the omission of NH₄NO₃ and were also greatly increased by treatment with methyl jasmonate.     

Arbuscular mycorrhiza maintains nodule function during external NH<Stack><Subscript>4</Subscript><Superscript>+</Superscript></Stack> supply in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> (L.)

The synergistic benefits of the dual inoculation of legumes with nodule bacteria and arbuscular mycorrhizae (AM) are well established, but the effect of an external NH₄⁺ supply on this tripartite relationship is less clear. This effect of NH₄⁺ supply was investigated with regards to the growth and function of the legume host and both symbionts. Nodulated Phaseolus vulgaris seedlings with and without AM, were grown in a sand medium with either 0 N, 1 mM or 3 mM NH₄⁺. Plants were harvested at 30 days after emergence and measurements were taken for biomass, N₂ fixation, photosynthesis, asparagine concentration, construction costs and N nutrition. The addition of NH₄⁺ led to a decline in the percentage AM colonization and nodule dry weights, although AM colonization was affected to a lesser extent. NH₄⁺ supply also resulted in a decrease in the reliance on biological nitrogen fixation (BNF); however, the AM roots maintained higher levels of NH₄⁺ uptake than their non-AM counterparts. Furthermore, the non-AM plants had a higher production of asparagine than the AM plants. The inhibitory effects of NH₄⁺ on nodule function can be reduced by the presence of AM at moderate levels of NH₄⁺ (1 mM), via improving nodule growth or relieving the asparagine-induced inhibition of BNF.     

Isolation and characterization of a novel ammonium overly sensitive mutant, <Emphasis Type="Italic">amos2</Emphasis>, in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Ammonium (NH₄ ⁺) toxicity is a significant agricultural problem globally, compromising crop growth and productivity in many areas. However, the molecular mechanisms of NH₄ ⁺ toxicity are still poorly understood, in part due to a lack of valuable genetic resources. Here, a novel Arabidopsis mutant, amos2 (ammonium overly sensitive 2), displaying hypersensitivity to NH₄ ⁺ in both shoots and roots, was isolated. The mutant exhibits the hallmarks of NH₄ ⁺ toxicity at significantly elevated levels: severely suppressed shoot biomass, increased leaf chlorosis, and inhibition of lateral root formation. Amos2 hypersensitivity is associated with excessive NH₄ ⁺ accumulation in shoots and a reduction in tissue potassium (K⁺), calcium (Ca²⁺), and magnesium (Mg²⁺). We show that the lesion is specific to the NH₄ ⁺ ion, is independent of NH₄ ⁺ metabolism, and can be partially rescued by elevated external K⁺. The amos2 lesion was mapped to a 16-cM interval on top of chromosome 1, where no similar mutation has been previously mapped. Our study identifies a novel locus controlling cation homeostasis under NH₄ ⁺ stress and provides a tool for the future identification of critical genes involved in the development of NH₄ ⁺ toxicity.     

Root growth as a function of ammonium and nitrate in the root zone

We examined the effect of soil NH₄⁺ and NO₃^? content upon the root systems of field-grown tomatoes, and the influence of constant, low concentrations of NH₄⁺ or NO₃^? upon root growth in solution culture. In two field experiments, few roots were present in soil zones with low extractable NH₄⁺ or NO₃^?; they increased to a maximum in zones having 2μg-N NO₃^? g^?1 soil and 6 μg-N NO₃⁼ g^?1 soil, but decreased in zones having higher NH₄⁺ or NO₃^? levels. Root branching was relatively insensitive to available mineral nitrogen. Plants maintained in solution culture at constant levels of NH₄⁺ or NO₃^?, had similar shoot biomass, but all root parameters – biomass, length, branching and area – were greater under NH4 nutrition than under NO₃^?. These results suggest that the size of root system depends on a functional equilibrium between roots and shoots (Brouwer 1967) and on the balance between soil NH₄⁺ and NO₃^?.     

The causes for barley root growth retardation in the presence of ammonium and glutamate

In barley (Hordeum vulgare L.) seedlings, the rate of root growth, osmotic pressure (Π), hydraulic conductance (L _p), and longitudinal (δl) and transverse (δD/D) extensibility of root cells were measured. The seedlings were grown on Knop solution with nitrate or without nitrate with addition of 5–10 mM NH₄⁺ or 0.5–1.0 mM glutamate. Root growth retardation on the 1st–4th days of exposure to NH₄⁺ was determined by a decrease in δl in the zone of elongation, whereas root thickening was evidently related to an increase in Π. Biphasic dynamics of δl in the presence of NH₄⁺ was imitated by medium acidification near the root surface to pH 3.7, which confirms a conclusion, we have done earlier, about a non-monotonous pH-dependence of longitudinal extensibility. Root growth retardation during the first day of exposure to Glu was also determined by a decrease in the δl, which was, however, accompanied by an increase in the δD/D and L _p. Fast Glu-induced changes of measured root parameters were imitated by root exposure to oryzalin, ionomycin, and inhibitors of the H⁺-pump. It was supposed that a decrease in δl in the presence of NH₄⁺ and Glu was related to cortical microtubule disorganization with the involvement of cytosolic calcium Ca_cyt²⁺. A decrease in the δD/D and L _p in the presence of NH₄⁺ was related to apoplast acidification and a high activity of the plasmalemmal H⁺-pump. An increase in the δD/D and L _p in the presence of Glu indicates the inhibition of the plasmalemmal H⁺-pump. On the 2nd–4th days of exposure to Glu, root growth ceased, as distinct from treatment with NH₄⁺. This complete root growth inhibition by Glu was possibly related to a rapid uptake of Ca²⁺ through Glu-sensitive Ca²⁺-channels, Ca²⁺-dependent inhibition of the plasmalemmal H⁺-pump, and a decrease in mitotic activity.