植物根系对氮胁迫的形态学响应

NO3-不仅是植物营养的主要N源,而且也是调节植物新陈代谢和生长发育的信号。植物根系对N供给的形态学适应表现在4个方面:(1)植物侧根(LR)响应体外硝酸盐的局部刺激而伸长,AtNRT1.1在侧根响应体外硝酸盐的局部刺激中,作用于ANR1 MADS box基因上游;(2)植物LR分裂组织活动受到组织中高浓度的硝酸盐抑制,RNA结合蛋白FCA是高硝酸盐/ABA诱导侧根发育抑制的信号传输途径成分;(3)植物侧根的发生受到体外高C∶N比抑制,AtNRT2.1参与高C∶N比抑制侧根发生;(4)植物根响应体外L-谷氨酸盐的刺激而分枝,初生根的生长受到体外L-谷氨酸盐抑制,植物根系对L-谷氨酸盐的响应可能与一种同源于哺乳动物离子型谷氨酸盐受体的植物蛋白的感知作用有关。植物根形态对N供给的响应具有重要生理和生态意义。     

Regulation of Arabidopsis root development by nitrate availability

When the root systems of many plant species are exposed to a localized source of nitrate (NO3- they respond by proliferating their lateral roots to colonize the nutrient-rich zone. This study reviews recent work with Arabidopsis thaliana in which molecular genetic approaches are being used to try to understand the physiological and genetic basis for this response. These studies have led to the conclusion that there are two distinct pathways by which NO3- modulates root branching in Arabidopsis. On the one hand, meristematic activity in lateral root tips is stimulated by direct contact with an enriched source of NO3- (the localized stimulatory effect). On the other, a critical stage in the development of the lateral root (just after its emergence from the primary root) is highly susceptible to inhibition by a systemic signal that is related to the amount of NO3- absorbed by the plant (the systemic inhibitory effect). Evidence has been obtained that the localized stimulatory effect is a direct effect of the NO3- ion itself rather than a nutritional effect. A NO3(-)-inducible MADS-box gene (ANR1) has been identified which encodes a component of the signal transduction pathway linking the external NO3- supply to the increased rate of lateral root elongation. Experiments using auxin-resistant mutants have provided evidence for an overlap between the auxin and NO3- response pathways in the control of lateral root elongation. The systemic inhibitory effect, which does not affect lateral root initiation but delays the activation of the lateral root meristem, appears to be positively correlated with the N status of the plant and is postulated to involve a phloem-mediated signal from the shoot.     

Nitrate Signaling and the Two Component High Affinity Uptake System in Arabidopsis

Nitrate transporters are important for nitrogen acquisition by plants and in algae some require two gene products, NRT2 and NAR2, for function. The NRT2 family was already described and the recent identification of a family of the NAR2-type genes in higher plants showed that there was a homologue in Arabidopsis, AtNAR2.1. Using heterologous expression in yeast and oocytes we showed that the two Arabidopsis AtNRT2.1 and AtNAR2.1 proteins interacted to give a functional high affinity nitrate transport system (HATS). The gene knock out mutant atnar2.1-1 is deficient specifically for HATS activity and the resulting growth phenotype on low nitrate concentration is more severe than for the atnrt2.1-1 knock out mutant. Physiological characterisation of the plant N status and gene expression revealed a pattern that was characteristic of severe nitrogen deficiency. Consistent with the down regulation of AtNRT2.1 expression, the atnar2.1-1 plants also displayed the same phenotype as atnrt2.1 mutants in lateral root (LR) response to low nitrate supply. Using atnar2.1-1 plants constitutively expressing the NpNRT2.1 gene, we now show a specific role for AtNAR2.1 in LR response to low nitrate supply. AtNAR2.1 is also involved in the repression of LR initiation in response to high ratios of sucrose to nitrogen in the medium. Therefore the two component system itself is likely to be involved in the signaling pathway integrating nutritional cues for LR architecture regulation. Using a green fluorescent protein-NRT2.1 protein fusion we show the essential role of AtNAR2.1 for the presence of AtNRT2.1 to the plasma membrane.Key Words: high affinity nitrate transport, nitrate transporter, nitrate signalling, root growth     

Nitrogen regulation of root branching

BACKGROUND: Many plant species can modify their root architecture to enable them to forage for heterogeneously distributed nutrients in the soil. The foraging response normally involves increased proliferation of lateral roots within nutrient-rich soil patches, but much remains to be understood about the signalling mechanisms that enable roots to sense variations in the external concentrations of different mineral nutrients and to modify their patterns of growth and development accordingly. SCOPE: In this review we consider different aspects of the way in which the nitrogen supply can modify root branching, focusing on Arabidopsis thaliana. Our current understanding of the mechanism of nitrate stimulation of lateral root growth and the role of the ANR1 gene are summarized. In addition, evidence supporting the possible role of auxin in regulating the systemic inhibition of early lateral root development by high rates of nitrate supply is presented. Finally, we examine recent evidence that an amino acid, L-glutamate, can act as an external signal to elicit complex changes in root growth and development. CONCLUSIONS: It is clear that plants have evolved sophisticated pathways for sensing and responding to changes in different components of the external nitrogen supply as well as their own internal nitrogen status. We speculate on the possibility that the effects elicited by external L-glutamate represent a novel form of foraging response that could potentially enhance a plant's ability to compete with its neighbours and micro-organisms for localized sources of organic nitrogen.     

Multiple roles of nitrate transport accessory protein NAR2 in plants

Two component high affinity nitrate transport system, NAR2/NRT2, has been defined in several plant species. In Arabidopsis, AtNAR2.1 has a role in the targeting of AtNRT2.1 to the plasma membrane. The gene knock out mutant atnar2.1 lacks inducible high-affinity transport system (IHATS) activity, it also shows the same inhibition of lateral root (LR) initiation on the newly developed primary roots as the atnrt2.1 mutant in response to low nitrate supply. In rice, OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a to provide nitrate uptake over high and low concentration ranges. In rice roots OsNAR2.1 and its partner NRT2s show some expression differences in both tissue specificity and abundance. It can be predicted that NAR2 plays multiple roles in addition to being an IHATS component in plants.Key words: NAR2, NRT2, nitrate transporter, root     

The Arabidopsis nitrate transporter AtNRT2.1 is targeted to the root plasma membrane.

Arabidopsis AtNRT2.1 protein is the best characterized high affinity nitrate transporter in higher plants. However, nothing is known about its sub-cellular localization. In this work, we used GFP imaging to follow the targeting of the AtNRT2.1 protein to the different cell membranes. A polyclonal antibody was also raised against a peptide derived from the AtNRT2.1 sequence. Comparison of wild type and mutant plant extracts showed that this antibody recognized specifically the AtNRT2.1 protein. Microsomal membranes were fractionated on sucrose gradients and immunological detections were performed on the different fractions. Altogether, our results demonstrate that the AtNRT2.1 protein is located in the plasma membrane of the root cells.     

Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis. Physiology and protein-protein interaction

The identification of a family of NAR2-type genes in higher plants showed that there was a homolog in Arabidopsis (Arabidopsis thaliana), AtNAR2.1. These genes encode part of a two-component nitrate high-affinity transport system (HATS). As the Arabidopsis NRT2 gene family of nitrate transporters has been characterized, we tested the idea that AtNAR2.1 and AtNRT2.1 are partners in a two-component HATS. Results using the yeast split-ubiquitin system and Xenopus oocyte expression showed that the two proteins interacted to give a functional HATS. The growth and nitrogen (N) physiology of two Arabidopsis gene knockout mutants, atnrt2.1-1 and atnar2.1-1, one for each partner protein, were compared. Both types of plants had lost HATS activity at 0.2 mm nitrate, but the effect was more severe in atnar2.1-1 plants. The relationship between plant N status and nitrate transporter expression revealed a pattern that was characteristic of N deficiency that was again stronger in atnar2.1-1. Plants resulting from a cross between both mutants (atnrt2.1-1 x atnar2.1-1) showed a phenotype like that of the atnar2.1-1 mutant when grown in 0.5 mm nitrate. Lateral root assays also revealed growth differences between the two mutants, confirming that atnar2.1-1 had a stronger phenotype. To show that the impaired HATS did not result from the decreased expression of AtNRT2.1, we tested if constitutive root expression of a tobacco (Nicotiana plumbaginifolia) gene, NpNRT2.1, previously been shown to complement atnrt2.1-1, can restore HATS to the atnar2.1-1 mutant. These plants did not recover wild-type nitrate HATS. Taken together, these results show that AtNAR2.1 is essential for HATS of nitrate in Arabidopsis.     

The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) is regulated by auxin in both shoots and roots

The AtNRT1.1 (CHL1) gene of Arabidopsis encodes a dual-affinity nitrate transporter and contributes to both low and high affinity nitrate uptake. Localization studies have shown that CHL1 expression is preferentially targeted to nascent organs and growing regions of roots and shoots in Arabidopsis. In roots, CHL1 expression is concentrated in the tips of primary and lateral roots and is activated during lateral root initiation. In shoots, strong CHL1 expression is found in young leaves and developing flower buds. These findings suggest that CHL1 expression might be regulated by a growth signal such as the phytohormone auxin. To test this, auxin regulation of CHL1 was examined. Using transgenic Arabidopsis plants containing CHL1::GUS/GFP DNA constructs, it was found that treatment with exogenous auxin or introduction of the auxin overproducing mutations (yucca and rooty) resulted in a strong increase in CHL1::GUS/GFP signals in roots and leaves. When mature roots were treated with auxin to induce lateral root formation, CHL1::GFP signals were dramatically enhanced in dividing pericycle cells and throughout primordia development. RNA blot analysis showed that CHL1 mRNA levels in whole seedlings increase within 30 min of auxin treatment. The distribution of CHL1 expression in Arabidopsis roots and shoots was found to be similar to that of DR5::GUS, a synthetic, auxin-responsive gene. These results indicate that auxin acts as an important signal regulating CHL1 expression and contributes to the targeting of CHL1 expression to nascent organs and root tips in Arabidopsis.     

Disruption of the nitrate transporter genes<Emphasis Type="Italic"> AtNRT2.1</Emphasis> and<Emphasis Type="Italic"> AtNRT2.2</Emphasis> restricts growth at low external nitrate concentration

The high-affinity transport systems in Arabidopsis thaliana (L.) Heynh. involve potentially seven genes. Among these, the AtNRT2.1 and/or AtNRT2.2 genes have been shown to play a major role in the inducible component of this transport system. The physiological impact of a disruption of AtNRT2.1 and AtNRT2.2 on plant growth and N-metabolism was investigated. The reduced nitrate uptake in the mutant under a limiting N-regime was found to correlate with a significant difference in shoot/root ratio between wild type and mutant and a drastically reduced nitrate level in the shoot of the mutant. Carbohydrate analyses of plants under a low nitrate supply revealed a slight increase in glucose and fructose in the mutant shoots as well as an increase in sucrose and starch contents in mutant shoots. Interestingly, the AtNRT2.4 and AtNRT2.5 genes were over-expressed in the mutant growing in reduced N-conditions, without any compensation by root nitrate influx. These results are discussed in the context of the putative role of the different NRT2 genes.Abbreviations DW Dry weight - FW Fresh weight - HATS High-affinity transport system - LATS Low-affinity transport system - NRT Nitrate transporter - WT Wild type     

Characterization of a dual-affinity nitrate transporter MtNRT1.3 in the model legume Medicago truncatula

Primary root growth in the absence or presence of exogenous NO(3)(-) was studied by a quantitative genetic approach in a recombinant inbred line (RIL) population of Medicago truncatula. A quantitative trait locus (QTL) on chromosome 5 appeared to be particularly relevant because it was seen in both N-free medium (LOD score 5.7; R(2)=13.7) and medium supplied with NO(3)(-) (LOD score, 9.5; R(2)=21.1) which indicates that it would be independent of the general nutritional status. Due to its localization exactly at the peak of this QTL, the putative NRT1-NO(3)(-) transporter (Medtr5g093170.1), closely related to Arabidopsis AtNRT1.3, a putative low-affinity nitrate transporter, appeared to be a significant candidate involved in the control of primary root growth and NO(3)(-) sensing. Functional characterization in Xenopus oocytes using both electrophysiological and (15)NO(3)(-) uptake approaches showed that Medtr5g093170.1, named MtNRT1.3, encodes a dual-affinity NO(3)(-) transporter similar to the AtNRT1.1 'transceptor' in Arabidopsis. MtNRT1.3 expression is developmentally regulated in roots, with increasing expression after completion of germination in N-free medium. In contrast to members of the NRT1 superfamily characterized so far, MtNRT1.3 is environmentally up-regulated by the absence of NO(3)(-) and down-regulated by the addition of the ion to the roots. Split-root experiments showed that the increased expression stimulated by the absence of NO(3)(-) was not the result of a systemic signalling of plant N status. The results suggest that MtNRT1.3 is involved in the response to N limitation, which increases the ability of the plant to acquire NO(3)(-) under N-limiting conditions.     

Plant growth-promoting bacteria and nitrate availability: impacts on root development and nitrate uptake

Plant growth-promoting bacteria (PGPB) and NO-3 availability both affect NO-3 uptake and root architecture. The presence of external NO-3 induces the expression of NO-3 transporter genes and elicits lateral root elongation in the part of the root system exposed to the NO-3 supply. By contrast, an increase in NO-3 supply leads to a higher plant N status (low N demand), which represses both the NO-3 transporters and lateral root development. The effects of PGPB on NO-3 uptake and root development are similar to those of low NO-3 availability (concomitant stimulation of NO-3 uptake rate and lateral root development). The mechanisms responsible for the localized and long-distance regulation of NO-3 uptake and root development by NO-3 availability are beginning to be elucidated. By contrast, the signalling and transduction pathways elicited by the rhizobacteria remain totally unknown. This review will compare the effects of NO-3 availability and PGPB on root morphogenesis and NO-3 uptake, in order to determine whether interactions exist between the NO-3-dependent and the PGPB-dependent regulatory pathways.     

High-affinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1

The NAR2 protein of Chlamydomonas reinhardtii has no known transport activity yet it is required for high-affinity nitrate uptake. Arabidopsis (Arabidopsis thaliana) possesses two genes, AtNRT3.1 and AtNRT3.2, that are similar to the C. reinhardtii NAR2 gene. AtNRT3.1 accounts for greater than 99% of NRT3 mRNA and is induced 6-fold by nitrate. AtNRT3.2 was expressed constitutively at a very low level and did not compensate for the loss of AtNRT3.1 in two Atnrt3.1 mutants. Nitrate uptake by roots and nitrate induction of gene expression were analyzed in two T-DNA mutants, Atnrt3.1-1 and Atnrt3.1-2, disrupted in the AtNRT3.1 promoter and coding regions, respectively, in 5-week-old plants. Nitrate induction of the nitrate transporter genes AtNRT1.1 and AtNRT2.1 was reduced in Atnrt3.1 mutant plants, and this reduced expression was correlated with reduced nitrate concentrations in the tissues. Constitutive high-affinity influx was reduced by 34% and 89%, respectively, in Atnrt3.1-1 and Atnrt3.1-2 mutant plants, while high-affinity nitrate-inducible influx was reduced by 92% and 96%, respectively, following induction with 1 mm KNO(3) after 7 d of nitrogen deprivation. By contrast, low-affinity influx appeared to be unaffected. Thus, the constitutive high-affinity influx and nitrate-inducible high-affinity influx (but not the low-affinity influx) of higher plant roots require a functional AtNRT3 (NAR2) gene.     

The Arabidopsis nitrate transporter NRT2.4 plays a double role in roots and shoots of nitrogen-starved plants

Plants have evolved a variety of mechanisms to adapt to N starvation. NITRATE TRANSPORTER2.4 (NRT2.4) is one of seven NRT2 family genes in Arabidopsis thaliana, and NRT2.4 expression is induced under N starvation. Green fluorescent protein and β-glucuronidase reporter analyses revealed that NRT2.4 is a plasma membrane transporter expressed in the epidermis of lateral roots and in or close to the shoot phloem. The spatiotemporal expression pattern of NRT2.4 in roots is complementary with that of the major high-affinity nitrate transporter NTR2.1. Functional analysis in Xenopus laevis oocytes and in planta showed that NRT2.4 is a nitrate transporter functioning in the high-affinity range. In N-starved nrt2.4 mutants, nitrate uptake under low external supply and nitrate content in shoot phloem exudates was decreased. In the absence of NRT2.1 and NRT2.2, loss of function of NRT2.4 (triple mutants) has an impact on biomass production under low nitrate supply. Together, our results demonstrate that NRT2.4 is a nitrate transporter that has a role in both roots and shoots under N starvation.     

Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2.1

? Interactions between the Arabidopsis NitRate Transporter (AtNRT2.1) and Nitrate Assimilation Related protein (AtNAR2.1, also known as AtNRT3.1) have been well documented, and confirmed by the demonstration that AtNRT2.1 and AtNAR2.1 form a 150-kDa plasma membrane complex, thought to constitute the high-affinity nitrate transporter of Arabidopsis thaliana roots. Here, we have investigated interactions between the remaining AtNRT2 family members (AtNRT2.2 to AtNRT2.7) and AtNAR2.1, and their capacity for nitrate transport. ? Three different systems were used to examine possible interactions with AtNAR2.1: membrane yeast split-ubiquitin, bimolecular fluorescence complementation in A. thaliana protoplasts and nitrate uptake in Xenopus oocytes. ? All NRT2s, except for AtNRT2.7, restored growth and β-galactosidase activity in the yeast split-ubiquitin system, and split-YFP fluorescence in A. thaliana protoplasts only when co-expressed with AtNAR2.1. Thus, except for AtNRT2.7, all other NRT2 transporters interact strongly with AtNAR2.1. ? Co-injection into Xenopus oocytes of cRNA of all NRT2 genes together with cRNA of AtNAR2.1 resulted in statistically significant increases of uptake over and above that resulting from single cRNA injections.     

The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) is activated and functions in nascent organ development during vegetative and reproductive growth

The AtNRT1.1 (CHL1) transporter provides a primary mechanism for nitrate uptake in Arabidopsis and is expected to localize to the epidermis and cortex of the mature root, where the bulk of nitrate uptake occurs. Using fusions to GFP/GUS marker genes, we found CHL1 expression concentrated in the tips of primary and lateral roots, with very low signals in the epidermis and cortex. A time-course study showed that CHL1 is activated in the primary root tip early in seedling development and at the earliest stages of lateral root formation. Strong CHL1 expression also was found in shoots, concentrated in young leaves and developing flower buds but not in the shoot meristem. These expression patterns were confirmed by immunolocalization and led us to examine CHL1 function specifically in the growth of developing organs. chl1 mutants showed a reduction in the growth of nascent roots, stems, leaves, and flower buds. The growth of nascent primary roots was inhibited in the mutants even in the absence of added nitrate, whereas elongation of lateral root primordia was inhibited specifically at low nitrate and acidic pH. Interestingly, chl1 mutants also displayed a late-flowering phenotype. These results indicate that CHL1 is activated and functions in the growth of nascent organs in both shoots and roots during vegetative and reproductive growth.     

Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1;3-dependent manner

Root development is strongly affected by the plant's nutritional status and the external availability of nutrients. Employing split-root systems, we show here that local ammonium supply to Arabidopsis thaliana plants increases lateral root initiation and higher-order lateral root branching, whereas the elongation of lateral roots is stimulated mainly by nitrate. Ammonium-stimulated lateral root number or density decreased after ammonium or Gln supply to a separate root fraction and did not correlate with cumulative uptake of (15)N-labeled ammonium, suggesting that lateral root branching was not purely due to a nutritional effect but most likely is a response to a sensing event. Ammonium-induced lateral root branching was almost absent in a quadruple AMMONIUM TRANSPORTER (qko, the amt1;1 amt1;2 amt1;3 amt2;1 mutant) insertion line and significantly lower in the amt1;3-1 mutant than in the wild type. Reconstitution of AMT1;3 expression in the amt1;3-1 or in the qko background restored higher-order lateral root development. By contrast, AMT1;1, which shares similar transport properties with AMT1;3, did not confer significant higher-order lateral root proliferation. These results show that ammonium is complementary to nitrate in shaping lateral root development and that stimulation of lateral root branching by ammonium occurs in an AMT1;3-dependent manner.     

Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake.

The Arabidopsis CHL1 (AtNRT1) gene encodes an inducible component of low-affinity nitrate uptake, which necessitates a "two-component" model to account for the constitutive low-affinity uptake observed in physiological studies. Here, we report the cloning and characterization of a CHL1 homolog, AtNRT1:2 (originally named NTL1), with data to indicate that this gene encodes a constitutive component of low-affinity nitrate uptake. Transgenic plants expressing antisense AtNRT1:2 exhibited reduced nitrate-induced membrane depolarization and nitrate uptake activities in assays with 10 mM nitrate. Furthermore, transgenic plants expressing antisense AtNRT1:2 in the chl1-5 background exhibited an enhanced resistance to chlorate (7 mM as opposed to 2 mM for the chl1-5 mutant). Kinetic analysis of AtNRT1:2-injected Xenopus oocytes yielded a K(m) for nitrate of approximately 5.9 mM. In contrast to CHL1, AtNRT1:2 was constitutively expressed before and after nitrate exposure (it was repressed transiently only when the level of CHL1 mRNA started to increase significantly), and its mRNA was found primarily in root hairs and the epidermis in both young (root tips) and mature regions of roots. We conclude that low-affinity systems of nitrate uptake, like high-affinity systems, are composed of inducible and constitutive components and that with their distinct functions, they are part of an elaborate nitrate uptake network in Arabidopsis.