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.     

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis.     

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.     

The nitrate transporter AtNRT1.1 (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis

The movement of guard cells in stomatal complexes controls water loss and CO(2) uptake in plants. Examination of the dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) revealed that it is expressed and functions in Arabidopsis guard cells. CHL1 promoter-beta-glucuronidase and CHL1 promoter-green fluorescent protein constructs showed strong expression in guard cells, and immunolocalization experiments with anti-CHL1 antibody confirmed these results. To assess CHL1 function, chl1 mutant plants grown in the presence of nitrate were examined. Compared with wild-type plants, chl1 mutants had reduced stomatal opening and reduced transpiration rates in the light or when deprived of CO(2) in the dark. These effects result in enhanced drought tolerance in chl1 mutants. At the cellular level, chl1 mutants showed reduced nitrate accumulation in guard cells during stomatal opening and failed to show nitrate-induced depolarization of guard cells. In wild-type guard cells, nitrate induced depolarization, and nitrate concentrations increased threefold during stomatal opening. These results identify an anion transporter that functions in stomatal opening and demonstrate that CHL1 supports stomatal function in the presence of nitrate.     

Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation

To counteract fluctuating nutrient environments, plants have evolved high- and low-affinity uptake systems. These two systems were traditionally thought to be genetically distinct, but, recently, two Arabidopsis transporters, AtKUP1 and CHL1, were shown to have dual affinities. However, little is known about how a dual-affinity transporter works and the advantages of having a dual-affinity transporter. This study demonstrates that, in the case of CHL1, switching between the two modes of action is regulated by phosphorylation at threonine residue 101; when phosphorylated, CHL1 functions as a high-affinity nitrate transporter, whereas, when dephosphorylated, it functions as a low-affinity nitrate transporter. This regulatory mechanism allows plants to change rapidly between high- and low-affinity nitrate uptake, which may be critical when competing for limited nitrogen. These results demonstrate yet another regulatory role of phosphorylation in plant physiology.     

Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRT1, from rice

Elucidating how rice (Oryza sativa) takes up nitrate at the molecular level could help improve the low recovery rate (<50%) of nitrogen fertilizer in rice paddies. As a first step toward that goal, we have cloned a nitrate transporter gene from rice called OsNRT1. OsNRT1 is a new member of a growing transporter family called PTR, which consists not only of nitrate transporters from higher plants that are homologs of the Arabidopsis CHL1 (AtNRT1) protein, but also peptide transporters from a wide variety of genera including animals, plants, fungi, and bacteria. However, despite the fact that OsNRT1 shares a higher degree of sequence identity with the two peptide transporters from plants (approximately 50%) than with the nitrate transporters (approximately 40%) of the PTR family, no peptide transport activity was observed when OsNRT1 was expressed in either Xenopus oocytes or yeast. Furthermore, contrasting the dual-affinity nitrate transport activity of CHL1, OsNRT1 displayed only low-affinity nitrate transport activity in Xenopus oocytes, with a K(m) value of approximately 9 mM. Northern-blot and in situ hybridization analysis indicated that OsNRT1 is constitutively expressed in the most external layer of the root, epidermis and root hair. These data strongly indicate that OsNRT1 encodes a constitutive component of a low-affinity nitrate uptake system for rice.     

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.     

Identification of the Arabidopsis CHL3 gene as the nitrate reductase structural gene NIA2.

Chlorate, the chlorine analog of nitrate, is a herbicide that has been used to select mutants impaired in the process of nitrate assimilation. In Arabidopsis thaliana, mutations at any one of eight distinct loci confer resistance to chlorate. The molecular identities of the genes at these loci are not known; however, one of these loci--chl3--maps very near the nitrate reductase structural gene NIA2. Through the isolation, characterization, and genetic analysis of new chlorate-resistant mutants generated by gamma irradiation, we have been able to demonstrate that the CHL3 gene and the NIA2 gene are identical. Three new chlorate-resistant mutants were identified that had deletions of the entire NIA2 gene. These nia2 null mutants were viable and still retained 10% of wild-type nitrate reductase activity in the leaves of the plants. All three deletion mutations were found to be new alleles of chl3. Introduction of the NIA2 gene back into these chl3 mutants by Agrobacterium-mediated transformation partially complemented their mutant phenotype. From these data, we conclude that Arabidopsis has at least two functional nitrate reductase genes and that the NIA2 gene product accounts for the majority of the leaf nitrate reductase activity and chlorate sensitivity of Arabidopsis plants.     

NO3- and ClO3- fluxes in the chl1-5 mutant of Arabidopsis thaliana. Does the CHL1-5 gene encode a low-affinity NO3- transporter?

The CHL1 gene is considered to encode a low-affinity transport system (LATS) for NO3- in Arabidopsis thaliana (Y.-F. Tsay, J.I. Schroeder, K.A. Feldmann, N.M. Crawford [1993] Cell 72: 705-713). However, the anticipated reduced NO3- uptake by the LATS associated with loss of CHL1 gene activity in chl1-5 deletion mutants was evident only when plants were grown on NH4NO3. When KNO3 was the sole N source, NO3- accumulation and short-term tracer influx (using 13NO3- and 15NO3-) in leaves and roots of wild-type and mutant plants were essentially identical. Nevertheless, root uptake of 36CIO3- by the LATS and CIO3- accumulation in roots and shoots of mutant plants were significantly lower than in wild-type plants when grown on KNO3. One explanation for these results is that a second LATS is able to compensate for the chl1-5 deficiency in KNO3-grown plants. Growth on NH4NO3 may down-regulate the second LATS enough that the anticipated difference in NO3- uptake becomes apparent.     

Functional characterization of the Arabidopsis thaliana nitrate transporter CHL1 in the yeast Hansenula polymorpha

CHL1 (AtNRT1.1) is a dual-affinity nitrate transporter of Arabidopsis thaliana, in which phosphorylation at Thr 101 switches CHL1 from low to high nitrate affinity. CHL1 expressed in a Hansenula polymorpha high-affinity nitrate-transporter deficient mutant (Deltaynt1) restores nitrate uptake and growth. These events take place at nitrate concentrations as low as 500 muM, suggesting that CHL1 has a high-affinity for nitrate in yeast. Accordingly, CHL1 expressed in H. polymorpha presents a K (m) for nitrate of about 125 muM. The absence of nitrate, the CHL1 gene inducer, showed the high turnover rate of CHL1 expressed in yeast, which is counteracted by nitrate CHL1 induction. Furthermore, H. polymorpha strains expressing CHL1 become sensitive to 250 muM chlorate, as expected for CHL1 high-affinity behaviour. Given that CHL1 presented high affinity by nitrate, we study the role of CHL1 Thr101 in yeast. Strains producing CHL1Thr101Ala, unable to undergo phosphorylation, and CHL1Thr101Asp, where CHL1 phosphorylation is constitutively mimicked, were used. Yeast strains expressing CHL1Thr101Ala, CHL1Thr101Asp and CHL1 at the same rate showed that Deltaynt1CHL1Thr101Ala is strikingly unable to transport nitrate and contains a very low amount of CHL1 protein; however, Deltaynt1CHL1Thr101Asp restores nitrate uptake and growth, although no significant changes in nitrate affinity were observed. Our results show that CHL1-Thr101 is involved in regulating the levels of CHL1 expressed in yeast and suggest that the phosphorylation of this residue could be involved in targeting this nitrate transporter to the plasma membrane. The functional expression of CHL1 in H. polymorpha reveals that this yeast is a suitable tool for evaluating the real nitrate transport capacity of plant putative nitrate transporters belonging to different families and study their regulation and structure function relationship.     

BcNRT1, a plasma membrane-localized nitrate transporter from non-heading Chinese cabbage

A nitrate transporter, BcNRT1, was isolated from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) cultivar 'Suzhouqing'. The full-length cDNA was obtained using the rapid amplification of cDNA ends technique and contains an open reading frame of 1,770?bp that predicts a protein of 589 acid residues that possesses 12 putative transmembrane domains. Using the GUS marker gene driven by the BcNRT1 promoter, we found BcNRT1 expression to be concentrated in primary and lateral root tips and in shoots of transgenic Arabidopsis plants. The YFP fused to BcNRT1 and transformed into cabbage protoplasts indicated that BcNRT1 was localized to the plasma membrane. The expression of BcNRT1 in roots was induced by exposure to 25?mM nitrate, and the BcNRT1 cRNA heterologously expressed in Xenopus laevis oocytes showed nitrate conductance when nitrate was included in the medium. Moreover, mutant chl1-5 plants harboring 35S::BcNRT1 showed sensitivity to chlorate treatment and exhibited restored nitrate uptake. In conclusion, the results indicate that BcNRT1 functions as a low affinity nitrate transporter in non-heading Chinese cabbage.     

The roles of three functional sulphate transporters involved in uptake and translocation of sulphate in Arabidopsis thaliana

To investigate the uptake and long-distance translocation of sulphate in plants, we have characterized three cell-type-specific sulphate transporters, Sultr1;1, Sultr2;1 and Sultr2;2 in Arabidopsis thaliana. Heterologous expression in the yeast sulphate transporter mutant indicated that Sultr1;1 encodes a high-affinity sulphate transporter (Km for sulphate 3.6 +/- 0.6 microM), whereas Sultr2;1 and Sultr2;2 encode low-affinity sulphate transporters (Km for sulphate 0.41 +/- 0.07 mM and >/= 1.2 mM, respectively). In Arabidopsis plants expressing the fusion gene construct of the Sultr1;1 promoter and green fluorescent protein (GFP), GFP was localized in the lateral root cap, root hairs, epidermis and cortex of roots. beta-glucuronidase (GUS) expressed with the Sultr2;1 promoter was specifically accumulated in the xylem parenchyma cells of roots and leaves, and in the root pericycles and leaf phloem. Expression of the Sultr2;2 promoter-GFP fusion gene showed specific localization of GFP in the root phloem and leaf vascular bundle sheath cells. Plants continuously grown with low sulphate concentrations accumulated high levels of Sultr1;1 and Sultr2;1 mRNA in roots and Sultr2;2 mRNA in leaves. The abundance of Sultr1;1 and Sultr2;1 mRNA was increased remarkably in roots by short-term stress caused by withdrawal of sulphate. Addition of selenate in the sulphate-sufficient medium increased the sulphate uptake capacity, tissue sulphate content and the abundance of Sultr1;1 and Sultr2;1 mRNA in roots. Concomitant decrease of the tissue thiol content after selenate treatment was consistent with the suggested role of glutathione (GSH) as a repressive effector for the expression of sulphate transporter genes.     

Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport

Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-β-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect root-to-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step.     

Uptake of Nitrate by Mutants of Arabidopsis thaliana, Disturbed in Uptake or Reduction of Nitrate

A study of nitrate and chlorate uptake by Arabidopsis thaliana was made with a wildtype and two mutant types, both mutants having been selected by resistance to high chlorate concentrations. All plants were grown on a nutrient solution with nitrate and/or ammonium as the nitrogen source. Uptake was determined from depletion in the ambient solution. Nitrate and chlorate were able to induce their own uptake mechanisms. Plants grown on ammonium nitrate showed a higher subsequent uptake rate of nitrate and chlorate than plants grown on ammonium alone. Mutant B25, which has no nitrate reductase activity, showed higher rates of nitrate and chlorate uptake than the wildtype, when both types were grown on ammonium nitrate. Therefore, the uptake of nitrate is not dependent on the presence of nitrate reductase. Nitrate has a stimulating effect on nitrate and chlorate uptake, whereas some product of nitrate and ammonium assimilation inhibits uptake of both ions by negative feedback. Mutant B 1, which was supposed to have a low chlorate uptake rate, also has disturbed uptake characteristics for nitrate.     

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.     

Nitrate transporters and peptide transporters

In higher plants, two types of nitrate transporters, NRT1 and NRT2, have been identified. In Arabidopsis, there are 53 NRT1 genes and 7 NRT2 genes. NRT2 are high-affinity nitrate transporters, while most members of the NRT1 family are low-affinity nitrate transporters. The exception is CHL1 (AtNRT1.1), which is a dual-affinity nitrate transporter, its mode of action being switched by phosphorylation and dephosphorylation of threonine 101. Two of the NRT1 genes, CHL1 and AtNRT1.2, and two of the NRT2 genes, AtNRT2.1 and AtNRT2.2, are known to be involved in nitrate uptake. In addition, AtNRT1.4 is required for petiole nitrate storage. On the other hand, some members of the NRT1 family are dipeptide transporters, called PTRs, which transport a broad spectrum of di/tripeptides. In barley, HvPTR1, expressed in the plasma membrane of scutellar epithelial cells, is involved in mobilizing peptides, produced by hydrolysis of endosperm storage protein, to the developing embryo. In higher plants, there is another family of peptide transporters, called oligopeptide transporters (OPTs), which transport tetra/pentapeptides. In addition, some OPTs transport GSH, GSSH, GSH conjugates, phytochelatins, and metals.     

Identification of two tungstate-sensitive molybdenum cofactor mutants, chl2 and chl7, of Arabidopsis thaliana.

Summary The characterization of mutants that are resistant to the herbicide chlorate has greatly increased our understanding of the structure and function of the genes required for the assimilation of nitrate. Hundreds of chlorate-resistant mutants have been identified in plants, and almost all have been found to be defective in nitrate reduction due to mutations in either nitrate reductase (NR) structural genes or genes required for the synthesis of the NR cofactor molybdenum-pterin (MoCo). The chlorate-resistant mutant ofArabidopsis thaliana, ch12, is also impaired in nitrate reduction, but the defect responsible for this phenotype has yet to be explained.chl2 plants have low levels of NR activity, yet the map position of thechl2 mutation is clearly distinct from that of the two NR structural genes that have been identified inArabidopsis. In addition,chl2 plants are not thought to be defective in MoCo, as they have near wild-type levels of xanthine dehydrogenase activity, which has been used as a measure of MoCo in other organisms. These results suggest thatchl2 may be a NR regulatory mutant. We have examinedchl2 plants and have found that they have as much NR (NIA2) mRNA as wild type a variable but often reduced level of NR protein, and one-eighth the NR activity of wild-type plants. It is difficult to explain these results by a simple regulatory model; therefore, we reexamined the MoCo levels inchl2 plants using a sensitive, specific assay for MoCo: complementation ofNeurospora MoCo mutant extracts. We found thatchl2 has low levels of MoCo — about one-eighth the wild-type level and less than the level in anotherArabidopsis MoCo mutantchl6 (B73). To confirm this result we developed a new diagnostic assay for MoCo mutants, growth inhibition by tungstate. Bothchl2 andchl6 are sensitive to tungstate at concentrations that have no effect on wildtype plants. The tungstate sensitivity as well as the chlorate resistance, low NR activity and low MoCo levels all cosegregate, indicating that all are due to a single mutation that maps to thechl2 locus, 10 centimorgans fromerecta on chromosome 2. We also report on the isolation of a new chlorate-resistant mutant ofArabidopsis, ch17, which is a MoCo mutant with the same phenotypes aschl2 andchl6.     

Flavonoids are differentially taken up and transported long distances in Arabidopsis

Flavonoids are synthesized in response to developmental and environmental signals and perform many functions in plants. Arabidopsis (Arabidopsis thaliana) roots grown in complete darkness do not accumulate flavonoids since the expression of genes encoding enzymes of flavonoid biosynthesis is light dependent. Yet, flavonoids accumulate in root tips of plants with light-grown shoots and light-shielded roots, consistent with shoot-to-root flavonoid movement. Using fluorescence microscopy, a selective flavonoid stain, and localized aglycone application to transparent testa mutants, we showed that flavonoids accumulated in tissues distal to the application site, indicating uptake and movement systems. This was confirmed by time-course fluorescence experiments and high-performance liquid chromatography. Flavonoid applications to root tips resulted in basipetal movement in epidermal layers, with subsequent fluorescence detected 1 cm from application sites after 1 h. Flavonoid application to midroot or cotyledons showed movement of flavonoids toward the root tip mainly in vascular tissue. Naringenin, dihydrokaempferol, and dihydroquercetin were taken up at the root tip, midroot, or cotyledons and traveled long distances via cell-to-cell movement to distal tissues, followed by conversion to quercetin and kaempferol. In contrast, kaempferol and quercetin were only taken up at the root tip. Using ATP-binding cassette (ABC) transporter and H(+)-ATPase inhibitors suggested that a multidrug resistance-associated protein ABCC transporter facilitated flavonoid movement away from the application site.     

Root-to-shoot transport of sulfate in Arabidopsis. Evidence for the role of SULTR3;5 as a component of low-affinity sulfate transport system in the root vasculature

Xylem transport of sulfate regulates distribution of sulfur in vascular plants. Here, we describe SULTR3;5 as an essential component of the sulfate transport system that facilitates the root-to-shoot transport of sulfate in the vasculature. In Arabidopsis (Arabidopsis thaliana), SULTR3;5 was colocalized with the SULTR2;1 low-affinity sulfate transporter in xylem parenchyma and pericycle cells in roots. In a yeast (Saccharomyces cerevisiae) expression system, sulfate uptake was hardly detectable with SULTR3;5 expression alone; however, cells coexpressing both SULTR3;5 and SULTR2;1 showed substantial uptake activity that was considerably higher than with SULTR2;1 expression alone. The V(max) value of sulfate uptake activity with SULTR3;5-SULTR2;1 coexpression was approximately 3 times higher than with SULTR2;1 alone. In Arabidopsis, the root-to-shoot transport of sulfate was restricted in the sultr3;5 mutants, under conditions of high SULTR2;1 expression in the roots after sulfur limitation. These results suggested that SULTR3;5 is constitutively expressed in the root vasculature, but its function to reinforce the capacity of the SULTR2;1 low-affinity transporter is only essential when SULTR2;1 mRNA is induced by sulfur limitation. Consequently, coexpression of SULTR3;5 and SULTR2;1 provides maximum capacity of sulfate transport activity, which facilitates retrieval of apoplastic sulfate to the xylem parenchyma cells in the vasculature of Arabidopsis roots and may contribute to the root-to-shoot transport of sulfate.