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First published online May 16, 2003; 10.1105/tpc.010256 American Society of Plant Biologists Role of Nicotianamine in the Intracellular Delivery of Metals and Plant Reproductive Development
a Laboratory of Plant Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 1 To whom correspondence should be addressed. E-mail annaoko{at}mail.ecc.u-tokyo.ac.jp; fax 81-3-6801-9557
Nicotianamine (NA), a chelator of metals, is ubiquitously present in higher plants. Nicotianamine aminotransferase (NAAT) catalyzes the amino group transfer of NA in the biosynthetic pathway of phytosiderophores and is essential for iron acquisition in graminaceous plants. The gene that encodes NAAT from barley was introduced into the nongraminaceous plant tobacco, which produces NA but not phytosiderophores. Transgenic tobacco plants (naat tobacco) that constitutively expressed the NAAT gene had young leaves with interveinal chlorosis and flowers that were abnormally shaped and sterile. Endogenous NA was consumed as a result of NAAT overproduction in naat tobacco. The resulting NA shortage caused disorders in internal metal transport, leading to these abnormal phenotypes. In addition to its role in long-distance metal transport, NA may be involved in the regulation of metal transfer within the cells. These results suggest that a shortage of NA impaired the functions of metal-requiring proteins, including transcription factors.
Metal ions, such as Fe, Mn, Zn, and Cu, are essential for normal plant growth. However, under aerobic conditions in the physiological pH range, these ions (particularly Fe) are sparingly soluble in the soil solution and are not readily available to plants. Therefore, plants have developed sophisticated and tightly regulated mechanisms to acquire Fe from the soil.
Mugineic acid family phytosiderophores (MAs) are natural Fe chelators that graminaceous plants secrete from their roots to solubilize Fe in the soil (Takagi, 1976
In tobacco and graminaceous plants, NA is formed by the trimerization of three molecules of S-adenosyl Met, a reaction catalyzed by nicotianamine synthase (NAS) (Shojima et al., 1989a  , 1990; Higuchi et al., 1995). By contrast, NAAT activity has not been detected in tobacco. Therefore, of the enzymes in the MAs biosynthetic pathway, NAS and S-adenosyl Met synthetase are ubiquitous in plants but NAAT is specific to graminaceous plants.
In a previous study, NAAT was purified from Fe-deficient barley roots (Takahashi et al., 1999 To investigate the consequences of ectopic NAAT production in nongraminaceous plants, tobacco was transformed with a construct containing the 35S promoter of Cauliflower mosaic virus combined with the coding region of the hvnaat-A gene. Here, we describe the phenotype of transgenic tobacco plants that overexpressed barley NAAT and consumed NA in situ. The transgenic tobacco had metal transport disorders and consequently developed a striking interveinal chlorosis in young leaves. In addition, flowers were abnormally shaped and sterile. These findings demonstrate the essential role of NA in growth, flower development, and fertility in plants.
Integration and Expression of a Barley naat-A in Tobacco hvnaat-A cDNA was inserted behind the 35S promoter of Cauliflower mosaic virus for constitutive expression in place of the  -glucuronidase (GUS) gene in the binary vector pIG121Hm. The plasmid was used to transform tobacco plants (Figure 2A). DNA gel blot analysis confirmed that naat-A was integrated into the tobacco genome (Figure 2B). RNA gel blot analysis showed that naat-A mRNA accumulated in transgenic tobacco (Figure 2C). NAAT activity was measured in transgenic tobacco leaves to ensure that NAAT had been translated correctly. NAAT activity was detected in transgenic tobacco but not in control tobacco (Figure 2D).
Tobacco Plants Overexpressing hvnaat-A Showed Interveinal Chlorosis naat tobacco plants grown under nutrient-sufficient conditions showed noticeable interveinal chlorosis in their young leaves (Figure 3B), a finding not observed in control tobacco plants (Figure 3A). This interveinal chlorosis disappeared gradually as the leaves grew older. Chlorotic leaves were examined using light and electron microscopy (Figures 3C to 3E). General leaf anatomy was not modified in naat tobacco, even within chlorotic regions. However, marked differences were observed within the vacuoles. A number of dark-stained, globular structures were present only in the vacuoles of naat tobacco plants (Figure 3D). These structures appeared in epidermal and mesophyll cells and were not restricted to specific tissues. In an effort to understand the detailed structure of these particles, we analyzed the ultrastructure of naat tobacco leaves by electron microscopy (Figure 3E). Unusual electron-dense globular structures were observed in almost all vacuoles in the epidermis and in mesophyll cells. The electron-dense globular structures came in a range of sizes, with the largest reaching  10 µm in diameter.
Concentration and Distribution of Metals in Leaves Interveinal chlorosis in young leaves is a common symptom of metal deficiency. Therefore, we measured the concentrations of four metals—Cu, Fe, Zn, and Mn—in the young leaves of control and naat tobacco plants by inductively coupled plasma emission spectrometry. The concentrations of all four metals in young leaves of naat tobacco were lower than those in control tobacco leaves (Figure 4A). The concentrations of Fe and Cu, in particular, were markedly lower in naat tobacco. The percentage decrease in concentration (relative to the control) was of the following order: Mn < Zn < Fe < Cu.
Because chlorosis was observed in the interveinal area of young leaves, the distributions of K, Zn, Mn, and Fe were analyzed by synchrotron radiation–induced x-ray fluorescence spectrometry (Figure 4B). There were similar distributions of K, Zn, and Mn in the leaf in naat tobacco and control tobacco; levels were high in the vein and low in the interveinal area in the leaves of both plants. By contrast, there was a marked difference in the distribution of Fe in the leaves of naat tobacco and control tobacco. The Fe concentration in the veins and especially the interveinal areas was much lower in naat tobacco leaves than in control tobacco leaves. Although the concentrations of Zn, Fe, and Cu all were reduced in naat tobacco leaves (Figure 4A), this finding suggests that the interveinal chlorosis (Figure 3B) was attributable to the decreased Fe concentration in the interveinal area.
NA Concentration in naat Tobacco
Interveinal Chlorosis Was Reversed after Treatment with NA-Fe To confirm that interveinal chlorosis resulted from the NA shortage in naat tobacco, Fe was supplied to naat tobacco either with or without NA. Excised naat tobacco axillary buds were treated with one of five solutions: (1) FeCl3, (2) FeSO4, (3) Fe(III) citrate, (4) FeCl3 plus NA, or (5) FeSO4 plus NA. The naat tobacco plants treated with a combination of NA and Fe (solutions 4 and 5) recovered completely from interveinal chlorosis after 110 h (Figures 6A, right, and 6B). Indeed, some recovery was evident within 48 h (data not shown). On the other hand, solutions without NA (solutions 1, 2, and 3) did not induce recovery from chlorosis (Figures 6A, left, and 6B), although Fe(III) citrate enhanced greening to some extent (Figure 6B). These results clearly show that a supply of NA-Fe to young naat tobacco leaves led to recovery from interveinal chlorosis. Interestingly, although the young leaves recovered from chlorosis with the NA-Fe treatments, the electron-dense globular structures observed in the vacuoles of naat tobacco were not eliminated (Figure 6C). Therefore, these unusual structures do not appear to be a direct cause of interveinal chlorosis. In naat tobacco, the 3''-oxo intermediate (Figure 1) synthesized from NA by NAAT was not converted to deoxymugineic acid. Therefore, the electron-dense globular structures observed in the vacuoles of naat tobacco might be an accumulation of the 3''-oxo intermediate or overproduced NAAT itself.
Figure 6D shows the SPAD values and metal concentrations in young leaves of treated axillary buds (see Methods). The SPAD values of young leaves treated with a solution of NA plus FeSO4 or FeCl3 were higher than those of young leaves treated with Fe(III) citrate and FeSO4 or FeCl3 without NA. This finding was consistent with the reversal of interveinal chlorosis evident in Figure 6B. Young naat tobacco leaves treated with solutions 3, 4, and 5 had higher concentrations of Fe than those treated with solutions 1 and 2. This finding indicates that both NA and citrate promote Fe transport to young leaves. However, the SPAD values of young leaves treated with Fe(III) citrate were the same as those of leaves treated with FeSO4. In addition, the SPAD values of young leaves treated with solutions containing NA were higher than those of young leaves treated with Fe(III) citrate (Figure 6D). These results show that although both NA and citrate promote Fe transport to young leaves, only NA promotes Fe transport to the interveinal area. The similar SPAD values of young leaves treated with Fe(III) citrate and FeSO4, which were significantly higher than those of leaves treated with FeCl3, suggest poor transport of Fe(III) in the absence of a chelate (NA or citrate). Moreover, young leaves treated with a solution containing NA also had high concentrations of Cu and Zn, demonstrating that NA also promotes the transport of these metals.
59Fe Transport in naat Tobacco After treatment with NA-59Fe, a significantly higher level of 59Fe was transported to the veins and interveinal areas in the oldest leaves (Figure 6E, leaf 3'). After only 1 h of treatment with NA-59Fe, 59Fe was distributed throughout the leaves (data not shown). In plants treated with 59Fe alone, however, 59Fe was transported only to the veins and not to the interveinal area (Figure 6E, leaves 1, 2, and 3). Furthermore, treatment with 59Fe alone resulted in no appreciable transport of 59Fe to the veins (Figure 6E, leaf 1). These results demonstrate again that in young naat tobacco leaves, NA promotes Fe transport not only in the veins but also from the veins to the interveinal areas.
Abnormally Shaped Flowers and Sterility
Type-I flower buds had an aberrant shape (Figure 7C), the first bud of naat tobacco being the most aberrant, and type-I flowers had bigger and thicker sepals than those of the wild type. Type-I flowers were classified into the following five groups, some flowers having a combination of several elements. (1) Projected petal. Projected petals were observed (Figures 7D and 7E) and filamentous petals often were observed (Figure 7E). (2) Chimeric flower organ. Petaloid sepals (Figures 7F, 7G, 7H, 7K, 7M, 7O, 7R, and 7S) and petaloid filaments (Figures 7J, 7L, and 7N) were observed; staminoid petals, such as those with the stamen fused to the petal, were common (Figures 7H, 7Q, and 7S). (3) Dehiscent flower. Dehiscent flowers were observed (Figures 7H, 7I, and 7S), with the corolla split open. (4) Abnormal number of flower organs. This type of flower showed supernumerary stamens and petals with multiple carpels (Figures 7M to 7S) or a decreased number of petals and stamens (Figure 7N). (5) Fused flower. This type of flower appeared as if two flowers had fused but had only one set of sepals (Figure 7T). Flowers with supernumerary flower organs could belong to this group (Figures 7M to 7S). The type-II flowers that developed in naat tobacco (Figures 8B and 8E) were distinct from type-I flowers (Figures 7B to 7T), although there were some common features. Also, type-II flowers had some similarities with flowers of wild-type tobacco under severe Fe deficiency (Figures 8C and 8F). The sepals, like those of type-I flowers, were large and thick but had a normal shape, even those that developed during the transition from type-I to type-II flowers. The sepals, stamen filaments, and petals were similar in color, and the anthers produced little pollen. Wild-type flowers under Fe deficiency did not have any type-I flowers. In addition, axillary buds exposed to Fe- or Zn-deficient conditions did not produce flowers that were the same as type-I flowers (data not shown). In naat tobacco, both types of flowers produced only small quantities of pollen, which was released several days after the flowers opened. Pollen grains were not filled in the anthers, which were shaped like arrowheads (Figure 8I), and the stamen filament was pink instead of white. Transverse sectioning of an anther in a naat tobacco bud revealed a small quantity of pollen that was poorly developed (Figures 8P and 8Q) compared with wild-type pollen (Figures 8M and 8N). In addition, aberrant pistils that were either too long or too short, and an irregularly shaped stigma, were observed frequently (Figure 8K). All naat tobacco plants with leaves exhibiting interveinal chlorosis were sterile. Wild-type tobacco and naat tobacco were crossed to determine which reproductive organ was responsible for the sterility. Wild-type pollen was applied to the stigmas of naat tobacco, or naat tobacco pollen was applied to the stigmas of wild-type tobacco (Table 1). Seeds matured in all seed pods produced by crossing naat tobacco pollen and wild-type pistils, although relatively few seeds were produced. By contrast, wild-type pollen placed on naat tobacco pistils produced a few seeds in only 1 of 11 seed pods. The seeds were small and light brown in color. This finding indicates that both the pollen and pistils of naat tobacco have the ability to complete fertilization. However, the number of pollen grains in naat tobacco was not sufficient for complete fertility, and the pistil of naat tobacco could not produce mature seeds because of the shortage of NA metal supply. Therefore, the sterility of naat tobacco appeared to be the result of (1) poor pollen production, (2) late anther dehiscence, and (3) poor ovary maturation after fertilization.
The Abnormal Morphology of Reproductive Organs Was Reversed by NA Treatment To determine whether the endogenous NA shortage caused the morphological abnormalities in the inflorescence, NA was supplied to naat tobacco using the same method described for Figure 6A, except that decreased concentrations of Fe and NA were used. Excised naat tobacco axillary buds were treated with one of five solutions (Table 2). Only a solution of 100 µM NA with 20 µM Fe(III) citrate reversed the morphological abnormalities (Table 2). After NA treatment, both the chlorosis in young leaves and the morphological abnormalities in flowers were reversed (Figure 9D), and plenty of pollen was produced (Figure 9B). A solution of 20 µM Fe(III) citrate without NA did not reverse the chlorosis in young leaves, nor did it reverse the flower abnormalities (Figures 9A and 9C). A solution of 20 µM NA was not sufficient to reverse the morphological abnormalities in flowers, and a solution of 20 µM NA with 20 µM FeCl3 reversed neither the chlorosis nor the morphological abnormalities. Although a solution of 20 µM NA with 20 µM FeSO4 reversed the chlorosis in young leaves, it did not reverse the abnormalities in flowers at all. A solution of 20 µM NA with 20 µM Fe(III) citrate restored 43% of the flowers from the excised axillary buds. These results indicate that NA and Fe are required for normal flower development. However, we cannot exclude the possibility that other metals transported by NA were responsible for the restoration of normal flower development.
Fertility Was Restored by Grafting To examine whether the endogenous NA shortage caused sterility, naat tobacco was grafted onto either wild-type tobacco or transgenic tobacco overexpressing the barley nicotianamine synthase gene (HvNAS1) (nas tobacco) (Figure 10A). Fertility was not restored after grafting onto wild-type tobacco, despite the recovery from interveinal chlorosis of the young leaves. Most anthers remained abnormal, and pollen did not develop (data not shown). Although a few anthers appeared partially normal, the seeds that were produced did not mature.
By contrast, grafting onto nas tobacco restored fertility to naat tobacco, and the leaves did not develop chlorosis (Figures 10B and 10C). In addition, most anthers were normal, the pinkish color of the stamen filament reverted to white (Figure 10D), pollen grains developed fully (Figures 10E and 10F), and seeds matured. However, many seeds did not develop fully, leading to shriveled seed pods (Figure 10H) and a reduced number of mature seeds (Figure 10I). Mature seeds obtained from grafted plants germinated normally, but the leaves of the seedlings had interveinal chlorosis and the inflorescences were morphologically abnormal and sterile (data not shown). The SPAD values of young leaves from nongrafted naat tobacco, grafted naat tobacco, and nas tobacco were as follows: 25.4 ± 2.4, 28.6 ± 6.0, and 29.2 ± 3.4, indicating the highest chlorophyll concentration in the nas tobacco.
Metal Concentrations in naat Tobacco Grafted onto nas Tobacco
Overexpression of HvNAS1 Increased Fe and Zn Concentrations in Young Leaves and Flowers Metal concentrations in the young leaves and flowers of nas tobacco also were analyzed (Figure 11). In young leaves of nas tobacco, the Zn concentration was 2.5 times higher than that of wild-type tobacco. The Fe concentration also was higher (1.9-fold). In flowers, there was a marked increase in Fe concentration of 2.4-fold in the pistil, 2.5-fold in the anther, 2.4-fold in the pollen, and 2.9-fold in the filament. The Zn concentration also was higher, except in the sepal, by the following amounts: pistil, 1.7-fold; anther, 1.4-fold; pollen, 1.9-fold; filament, 2.0-fold; and petal, 1.5-fold. The Mn concentration in young leaves was lower than that in the wild-type anther, pistil, and petal but higher than that in the whole flower, pollen, filament, and sepal. In contrast to these metals, the Cu concentration was similar to that in the wild type, except for the sepal, in which there was a 1.7-fold increase compared with the wild type. As for other metals in the sepal, the Fe concentration was 1.6-fold higher and the Zn and Mn concentrations were almost the same as in the wild type. Seeds of nas tobacco also had higher Fe and Zn concentrations (Fe, 2.3-fold; and Zn, 1.8-fold), but the Cu concentration was similar to that in the wild type. These results indicate that NA promoted the transport of Fe in particular and also of Zn to young leaves, seeds, and all flower organs except the sepal.
NtNAS Expression
Transgenic tobacco, constitutively expressing a barley naat-A gene (naat tobacco), showed distinct NAAT activity (Figure 2D) that led to NA consumption in situ. Thus, endogenous NA was not detected in naat tobacco but was present in control plants (Figure 5). This finding suggested that the naat tobacco phenotype is mainly the result of a lack of endogenous NA (Figures 3, 7, and 8).
NA Is Essential for Fe Transport in Veins and from Veins to the Interveinal Area Treatment of naat tobacco with Fe alone did not reverse interveinal chlorosis. Fe(III) citrate increased the Fe concentration in young leaves but did not reverse interveinal chlorosis. This result suggests that citrate transported Fe(III) to veins in the young leaves but not into the interveinal area. On the other hand, treatment with Fe in combination with NA increased the Fe concentration and reversed chlorosis (Figures 6A, right, 6B, and 6D, Table 2). In addition, exogenously supplied 59Fe, when given in combination with NA, was transported immediately to the veins and interveinal areas in young naat tobacco leaves (Figure 6E). Thus, NA enhanced Fe transport not only to the veins in young leaves but also to the interveinal areas.
Young leaves treated with a solution containing NA also had a higher concentration of Cu and Zn (Figure 6D), indicating that NA also promotes the transport of these metals to the young leaf. NA chelates the four metals (Fe, Zn, Mn, and Cu) analyzed in this study (Benes et al., 1983
The interveinal chlorosis phenotype in young leaves also has been reported in the tomato mutant chloronerva, which lacks endogenous NA (Böhme and Scholz, 1960
NA Is Essential for Reproductive Growth and Fertility If NA was not constitutively consumed by NAAT in situ, a NA concentration of <100 µM would be sufficient to reverse the morphological abnormalities in flowers. It is conceivable, therefore, that NA affects flower morphogenesis at very low concentrations, suggesting a number of important conclusions. (1) A higher NA concentration is required for normal flower development than for normal leaf development. (2) Citrate cannot be substituted for NA at the reproductive stage, nor can Fe supplied as Fe(III) citrate be transported from veins to interveinal areas of young leaves. (3) Both NA and Fe are required for normal flower development, and the combination of NA with Fe(III) citrate is effective for normal flower development. (4) Cu and Zn also participate in normal flower development (Figure 6D). Grafting of naat tobacco onto wild-type tobacco reversed interveinal chlorosis and aberrant flower shape but did not reverse sterility. Indeed, many anthers remained abnormal, and even flowers with restored anthers did not have any mature seeds. On the other hand, grafting of naat tobacco onto nas tobacco reversed interveinal chlorosis in young leaves, aberrant flower shape, pollen maturation defects, late anther dehiscence, and sterility. Mature seeds of naat tobacco grafted onto wild-type tobacco were produced, but they were few in number (data not shown). Because wild-type tobacco produced less NA than nas tobacco (data not shown), it is likely that the amount of NA produced by the wild type was not sufficient to compensate for the consumption of endogenous NA by NAAT, resulting in overall insufficient NA to mature seeds. Grafting onto nas tobacco increased the concentrations of Fe, Cu, and Zn in whole naat tobacco flowers (Figure 11), and the flowers of nas tobacco had higher concentrations of Fe, Cu, and Zn than did wild-type flowers. Interestingly, the Cu concentration was not as high in nas tobacco, suggesting that NA may be involved in Cu homeostasis. The results from the grafting experiment suggest that seed maturation requires more NA than do normal leaf and flower development and that NA affects metal concentrations in flowers. These results suggest that NA is essential for normal inflorescence formation, for the production of normal pollen, and for seed maturation.
Importance of Metals at the Reproductive Stage
Microsporogenesis requires a great deal of energy, which might impose stress on anther development, and many researchers have noted the importance of mitochondria in male gametophyte development (Warmke and Lee, 1978
Fe deficiency causes peroxidase activity depression and the accumulation of phenolic compounds (Sijmons et al., 1985
NA Functions at the Reproductive Stage
Metal Carrier NA transports metals to young leaves (Figure 13A), reproductive organs (Figures 13B and 13C), and seeds (Figure 13D). Grafting experiments and metal concentrations in nas tobacco show that NA promotes the transport of Fe, Cu, and Zn to each flower organ and to seeds. NtNAS expression in flower organs also indicates that NA promotes metal transport into the inflorescence. In particular, NtNAS expression in the stamen filament suggests that NA transports metals required for the maturation of pollen in the anther. In young leaf and flower, citrate could not substitute for NA. These results suggest that NA transports metals to cells of the reproductive organs through the specific transporter for NA-metal complexes in vascular bundles of young leaves, inflorescences, and seeds.
In the xylem, Fe is thought to be translocated mainly as Fe(III) citrate (Tiffin, 1966
Regulation of Intercellular Metals and Metal-Requiring Proteins
Zn Finger: Morphogenesis and Fertility
Zn plays an important role in key structural motifs in transcriptional regulatory proteins, including the Zn finger, Zn cluster, and RING finger domains. Furthermore, the transcriptional factors that participate in flower development include many Zn finger proteins (Takatsuji, 1999 The sterility of naat tobacco may be related to these Zn-finger proteins, because it is conceivable that Zn deficiency in the cell, resulting from the shortage of NA, affects the functions of Zn-finger proteins, leading to sterility. Moreover, if NA functions as an intercellular metal carrier, the absence of NA may inhibit the accurate transfer of Zn to the proteins or the removal of Zn from the proteins.
FIL (FILAMENTOUS FLOWER) is one of the Zn-finger proteins involved in morphogenesis; it has two Zn ions. Its binding to DNA or interaction with other transcription factors is regulated by the release of one Zn ion by EDTA (Kanaya et al., 2001
Fe-Requiring Proteins
NA also may affect dehiscence. A flower of an Fe-deficient axillary bud (Figure 8F) dehisced its anthers normally even though there was little pollen. By contrast, in naat tobacco, there was a delay in anther dehiscence, for which jasmonic acid is required. Because allene oxide synthase, a key enzyme for jasmonic acid biosynthesis, has heme-Fe (Kubigsteltig et al., 1999 In conclusion, we present here various roles for NA, especially at the reproductive stage. For a long time, it has been supposed that NA is necessary for the redistribution of Fe, Zn, and Mn via the phloem and that it is required for Cu transport in the xylem. This study showed that the role of NA in the transport of metals is required for the normal function of young leaves, for the development of reproductive organs, and for fertility. In addition, we have suggested that NA may be required for the intracellular regulation of metal binding proteins, such as Zn-finger proteins.
Binary Vector Construction hvnaat-A cDNA was isolated from Fe-deficient barley (Hordeum vulgare) roots (Takahashi et al., 1999 ) and cloned into the SmaI site of the pUC118 vector. pUC118-hvnaat-A was digested with XbaI and SacI and cloned into the pIG121Hm vector (Hiei et al., 1994).
Plant Material and Growth Conditions
DNA Gel Blot Analysis
RNA Gel Blot Analysis
Nicotianamine Aminotransferase Enzyme Assay
Microscopy
Determination of Metal Concentrations
Elemental Mapping
Extraction and Analysis of NA
Regreening Test for Interveinal Chlorosis
59Fe Uptake
Recovery Test for Morphological Flower Abnormality
Grafting of naat Tobacco onto nas Tobacco Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes.
Accession Number
We thank Kyoko Higuchi (Tokyo University of Agriculture) for providing the nas tobacco seeds, Kenzo Nakamura (Nagoya University) for the pIG121Hm vector, Takeshi Kitahara (University of Tokyo) for NA, Hirotaka Yamaguchi, Takashi Negishi, and Takuya Nakagawa for technical assistance with our experiments, and P. Blamey (University of Tokyo) for assistance with English expression.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010256. Received December 31, 2002; accepted March 31, 2003.
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Abstract
INTRODUCTION
2.8-fold), that of Cu increased 
NA) could occur during Fe transfer from xylem to phloem in the vein, and NA-Fe could be transported through a NA-metal complex–specific transporter. Fe could be supplied to the inflorescence and seed by NA via the phloem after transloading from the xylem elsewhere (e.g., the stalk). The NA-metal complex–specific transporter also could transport NA-Fe to each flower organ and seed. Recently, Vert et al. (2002)
-32P-ATP. The membrane was hybridized overnight at 65°C with the labeled probe in 0.5 M Church phosphate buffer (Church and Gilbert, 1984
