| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Biological and Environmental Engineering, Cornell Univ., Ithaca, NY 14853
* Corresponding author (baa7{at}cornell.edu)
Received for publication June 25, 2002.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Abbreviations: EDTA, ethylenediaminetetraacetic acid • FTH, flow-through hydroponic
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
One of the principal responses of plants to Cd exposure is to synthesize phytochelatins. Phytochelatins are peptides of the general structure (
–glu–cys)n–gly, where n ranges from 2 up to 11 (Grill et al., 1985), though in some plants the terminal glycine may be absent or replaced by an alternate amino acid (see review by Rauser, 1995). On exposure to Cd, phytochelatins are synthesized rapidly by the constitutive enzyme phytochelatin synthase, which catalyzes the transfer of –glu–cys from glutathione to other glutathione molecules to form phytochelatin n = 2 or to (–glu–cys)n–gly polymers to form phytochelatin chains of length n + 1 (Grill et al., 1989). Recent work by Vatamaniuk et al. (2000) using the purified recombinant phytochelatin synthase from Arabidopsis thaliana has shown that the Cd–bis–glutathione complex is a necessary substrate along with free glutathione, which explains the metal dependence of the reaction mechanism. Phytochelatins are also induced in plants, some yeast, and algae on exposure to many other metals including Cu, Ag, Hg, Pb, and Zn, and even the metalloids As and Se (Rauser, 1995).
Phytochelatin detoxifies metals by forming a metal–thiol complex in the cytosol, which then may be sequestered in the cell vacuole. Transport of the Cd complex across the tonoplast membrane has been shown to be an ATP-dependent process in membrane vesicles prepared from oat (Avena sativa L.) roots (Salt and Rauser, 1995). Phytochelatin production is an important component of constitutive cadmium tolerance in plants (Howden et al., 1995), and even in the absence of significant vacuolar transport, phytochelatin synthase expression confers Cd tolerance in yeast (Clemens et al., 1999).
Though there are many complicating factors, the direct relationship between metal stress and phytochelatin synthesis yields a potential biochemical indicator of metal exposure (Verkleij, 1993; Gawel et al., 1996; Keltjens and van Beusichem, 1998a,b). For this reason, among others, many investigators have examined the relationship between Cd exposure and phytochelatin production, but what has been missing in most of these studies is a quantitative evaluation of phytochelatin concentrations in trace metal–defined medium and at low environmentally relevant Cd concentrations (see review by Sanità di Toppi and Gabbrielli, 1999). In this paper we have examined whether a synthetic chelator, EDTA, may be used to fix defined bioavailable Cd concentrations in a hydroponic growth medium to stimulate phytochelatin and glutathione production in lettuce. The range of Cd concentrations used in our experiments falls within the free Cd ion concentrations (10-10 to 2 x 10-7 M) measured in soils by Sauvé et al. (2000).
A flow-through hydroponic (FTH) system with a support bed and discontinuous nutrient delivery was developed to simulate a soil environment while retaining the benefits of an aqueous growth medium. This system was used to relate chronic Cd exposure to trace metal uptake and translocation and phytochelatin production in romaine lettuce, a vegetable known to accumulate high levels of metals in its edible aboveground biomass compared with other crops (Brown et al., 1996). A comparison study was performed using standard hydroponic methods to evaluate the effects of changing total Cd–EDTA concentrations (with a fixed [Cd2+]). Phytochelatin and glutathione concentrations were quantified by high performance liquid chromatography (HPLC) followed by fluorescence detection.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Hydroponic System and Growth Conditions
The flow-through hydroponic system (FTH) consisted of four troughs constructed from polycarbonate sheets (2286 x 88.9 x 76.2 mm or 90 x 3.5 x 3 in) housed in a Plexiglas box (2.43 x 0.76 x 0.30 m or 8 x 2.5 x 1 ft) to contain spills. Small fans circulated air within the box. Troughs were filled with clear acrylic pellets (2.54-mm [0.1-in] diameter) that leached insignificant amounts of trace metals in an acid extraction test. Nutrient solution was pumped from 10-L polycarbonate carboys through tubing connected to the head of each trough. The Masterflex pump (Cole-Parmer Instrument Co., Vernon Hills, IL) was regulated by a programmable timer (ChronTrol Corporation, San Diego, CA) and was used to circulate medium through the system for 10 to 15 min every 1 to 2 h depending on the needs of the plants. Solution saturated the substrate and drained by gravity from the troughs through tubing and back into the carboys. In the interval between wetting cycles, the solution remaining in the troughs diminished from evapotranspiration until the substrate was dry, but plants did not wilt. The troughs rested on a slight decline (approximately 3°) in the growth box to facilitate drainage. Exhausted solution in carboys was replaced weekly with fresh medium. As plants grew and the evapotranspiration rate of the system increased, it became necessary to replace the solution every 3 to 4 d. Plants (up to 18 per trough) were held in place at 76.2-mm (3-in) intervals by stiff black acrylic strips that covered the troughs.
Plants (in triplicate) were also grown in 400-mL polypropylene beakers filled with nutrient solution and housed in the Plexiglas box described above. Each plant was supported by a black acrylic plate that covered the mouth of the beaker, while its roots dangled free in solution through a hole in the center of the plate. The nutrient solution was replenished at least every other day, so that roots remained submerged. Beakers were emptied completely and refilled with fresh solution regularly to avoid significant changes in medium composition.
All materials used to grow the lettuce were acid washed with 1 M HCl and rinsed with Milli-Q water (Millipore, Bedford, MA) to remove trace metal contaminants. Black plastic was used to shade carboys, troughs, tubing, and beakers to minimize algal growth in the nutrient medium.
Growth Media
All growth solutions in this study shared the same macronutrient composition, which was derived from the medium used by Both et al. (1997). The nutrient solution contained KNO3 (2.5 mM), NH4NO3 (0.4 mM), Ca(NO3)2 (2.0 mM), KH2PO4 (0.6 mM), MgSO4 (1 mM), H3BO4 (12.5 µM), and (NH4)6 Mo7O24·4H2O (0.1 µM). The pH was unbuffered at 5.5 to 6. Total EDTA and trace metal concentrations were varied in the experiments as described below. Trace metal speciation was calculated using the chemical equilibrium program MINEQL (Westall et al., 1976). The free ion concentrations of all the trace metals (besides Cd) were held constant and were the same as those used by Norvell and Welch (1993). Free ion concentrations are reported as -log[Me2+] (or pMe) and were as follows: pFe(III) = 17.6, pCo(II) = 10.2, pCu(II) = 12.0, pMn(II) = 6.8, and pZn = 9.7. In medium containing 50 µM EDTA, total metal concentrations in the three Cd-containing medium (in order of decreasing Cd) and the control were 1.0, 7.0, 20.0, 23.5 µM (FeCl3), 0.01, 0.09, 0.20, 0.23 µM (CoCl2), 0.07, 0.47, 1.0, 1.1 µM (CuSO4), 0.37, 1.5, 3.0, 3.4 µM (MnCl2), and 0.07, 0.46, 1.0, 1.2 µM (ZnSO4), respectively. The total CdCl2 added for pCd = 9.6 was 7.0 µM, for pCd = 8.6 was 32.1 µM, and for pCd = 7.6 was 47.4 µM. Notice that total metal concentrations decreased as Cd increased since Cd and other metals are nearly 100% chelated by EDTA. The first set of plants cultured at pCd = 9.6 for the time course experiments contained total trace metal concentrations identical to the pCd = 9.6 medium described above, except that the total CdCl2 added was 10 µM and the medium contained an additional 10 µM Na2EDTA. Free metal ion concentrations were within 0.2 log units of those listed above.
Four additional media (two controls and two with pCd = 8.6) were designed to examine the effectiveness of the EDTA buffer. One set was designed with a lower EDTA concentration (10 µM) and contained the following trace metal composition with and without Cd addition: 1.44, 3.95 µM (FeCl3), 0.02, 0.05 µM (CoCl2), 0.09, 0.27 µM (CuSO4), 0.44, 0.87 µM (MnCl2), and 0.09, 0.27 µM (ZnSO4). The other set was designed with a higher EDTA concentration (100 µM) and contained 10-fold high total metal concentrations compared with the low EDTA medium with and without Cd. The total Cd added to the low and high EDTA medium was 6.4 µM and 64 µM, respectively.
Sample Collection and Preparation
In the initial set of experiments, two treatments (control and pCd = 9.6; new leaves only) were sampled over time: on Days 5, 7, 13, and 17 following transplantation into the experimental medium. For the rest of the experiments plants were grown in the troughs or beakers for 2 to 3 wk following transplantation and then sampled over the course of 1 wk or longer as needed for adequate replication of data points. In general, duplicate samples were taken from several plants selected at random from within the trough.
Samples of lettuce old leaf, new leaf, and root tissue (0.1–0.2 g fresh weight) were harvested and processed immediately for analysis of thiols, and other tissue samples were set aside for analysis of metal content. "Old" leaves were selected from the whorls of mature leaves at the base of the plant, while "new" leaves were harvested from younger tissue near the apical meristem. The large central veins of leaves were removed before analysis to minimize variability in metal or thiol content associated with the inclusion of vascular tissue. Samples were immersed in 2.5 mL of 10 mM methanesulfonic acid (MSA) in 5-mL grinding tubes, heated in a 70°C water bath for 2 min to denature degradative enzymes in the sample, and immediately cooled in an ice bath. To facilitate grinding, tissues were finely chopped with a razor blade and then transferred back to the grinding tube and homogenized with a Teflon pestle by an overhead stirrer (Wheaton Science Products, Millville, NJ) for 2 to 4 min on ice. The slurry was centrifuged for 10 min at 13 000 rpm at 4°C (Biofuge Fresco; Heraeus Instruments, Hanau, Germany). The supernatant was retained for derivatization.
The lettuce tissue extract was then diluted, if necessary, with 10 mM MSA and derivatized at room temperature with monobromobimane (mBBr; Molecular Probes, Eugene, OR), a fluorescent probe that selectively reacts with sulfhydryl groups, generally following the protocol described in Ahner et al. (1995), which was originally based on the method of Newton et al. (1981). Changes from the method published by Ahner et al. (1995) include 10-fold lower probe and reductant concentrations as well as the addition of excess dithiothreitol after the reaction to utilize excess unreacted probe.
Measurements were also made on duplicate leaf tissues that had been placed in cryovials in liquid N2. Frozen tissue samples always contained less phytochelatin (48% on average) and nearly always contained less glutathione (16% on average) as compared with fresh tissues and the relative decrease was highly variable (Fig. 1) . Numerous attempts to vary freezing techniques (including rapid freezing in liquid N2 slush) and derivatization protocols did not result in better yields (data not shown).
|
–glu–cys = 2(n = 2) + 3(n = 3).
Analysis of Metal Content
Roots were rinsed with Milli-Q water, and leaf and root samples were dried at 100°C overnight. Samples were dry-ashed at 450°C and analyzed for metal content by inductively coupled plasma atomic emission spectrometry at the Cornell Fruit and Vegetable Science ICP Laboratory, Ithaca, NY.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cadmium-treated beaker lettuce (pCd = 8.6) also showed signs of physiological toxicity in all three EDTA treatments by harvest time. In the low and medium EDTA solutions, plants were chlorotic with brown patches on the tips of leaves. Symptoms were more severe in plants grown at the lowest EDTA concentration. The lettuce grown in the highest EDTA solution was not chlorotic, but Cd-treated plants were noticeably smaller than controls, which appeared healthy at all EDTA levels.
Phytochelatin, Glutathione, and Cadmium in Plants in Response to Variable Cadmium
The FTH experiments were designed to examine the production of phytochelatin in romaine lettuce grown in medium containing a defined range of free Cd2+ concentrations. Initial experiments (run with a control trough and only the lowest Cd concentration, pCd = 9.6) were performed to examine how phytochelatin changed as a function of time in the troughs. Differences in phytochelatin concentration between Cd-treated and control plants were already apparent in new leaves of plants after 5 d and concentrations remained constant over a period of 2 wk (Fig. 2)
. The average Cd concentration (measured at the end of the time course) in new leaves collected from control plants was 0.48 ± 0.2 mg kg-1 dry wt. (n = 3), whereas in the Cd-treated plants the average concentration was 5.8 ± 2.5 mg kg-1 dry wt. (n = 4).
|
|
|
Phytochelatin, Glutathione, and Cadmium in Plants Grown with Variable EDTA
In these experiments plants were grown hydroponically in beakers to test whether an EDTA buffer controls the bioavailability or uptake of Cd. Free ion concentrations of Cd and other trace metals were constant across treatments, while total metal concentrations varied with the concentration of EDTA in each treatment solution (10, 50, and 100 µM EDTA).
Following at least 14 d in treatment medium, the same differences in phytochelatin concentration between control and Cd treated plants were observed in these experiments in each of the three tissue types, whereas glutathione did not uniformly increase in tissues in response to Cd addition (Fig. 5) . Increases in total Cd concentration as EDTA was increased only appeared to increase thiols in roots, with phytochelatin increasing at the two higher EDTA concentrations and glutathione only increasing at the highest EDTA concentration. In Cd-treated new leaves, a slight decrease in phytochelatin concentrations is observed at the highest EDTA concentration (Fig. 5b). Interestingly, glutathione concentrations of new leaves from plants grown at 10 µM EDTA (both Cd and control) are higher than the other treatments (Fig. 5a). This may reflect a nutrient deficiency or stress at the lower EDTA concentration.
|
|
Comparison of Flow-Through Hydroponic and Hydroponic Plants
Direct comparisons may be made between plants grown hydroponically in beakers in the 50 µM EDTA medium and their FTH counterparts, since these two sets of plants were raised in identical nutrient medium. Generally, Cd concentrations (as well as most other metals; data not shown) were greater in plants grown in the FTH system than in the beakers (60, 50, and 30% more in roots, new leaves, and old leaves, respectively). By providing a substrate and delivering nutrients intermittently to the rhizosphere, the FTH system simulates field conditions more closely than traditional hydroponic methods, in which plant roots are continually immersed in solution, as in our beaker system. We hypothesized that wetting the root zone at intervals would encourage root hair formation (Graves, 1992), thereby increasing the surface area of the roots and thus the uptake of metals into the plants. A crude examination of lettuce roots by light microscopy showed an increase in root hairs in the FTH plants over the beaker plants. Clearly, both the physical environment of the root zone and the nutrient medium composition are essential factors determining trace metal accumulation in plant tissues and must be considered when relating hydroponic studies to each other and to field situations.
Although Cd levels were higher in FTH plants, phytochelatin and glutathione concentrations were fairly similar in these two sets of plants (Fig. 3 and 5), except for significantly higher glutathione concentrations in all control tissues from beaker plants and higher (though highly variable) phytochelatin in roots of Cd-treated beaker plants. While it is possible that these differences are due to age differences in the plants (beaker plant tissues were on average from younger plants), it is more likely that physiological differences resulted from differences in nutrient solution delivery.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
While there is evidence that phytochelatins may be functionally important to plants even under conditions of low metal stress (Thumann et al., 1991) and the enzyme for synthesis is constitutively expressed (Grill et al., 1989; Ha et al., 1999), there have been few reports of "background" phytochelatin concentrations in plant tissues. In our study, concentrations measured in control plants (up to 25 µmol kg-1 fresh wt.) are similar to those measured in other plants. Keltjens and van Beusichem (1998b) reported 50 and 20 µmol kg-1 fresh wt. (estimated from graphs and converted from dry weight to fresh weight) in the shoots of maize (Zea mays L.) and wheat (Triticum aestivum L.), respectively. Tukendorf (1993) reported a value of 3 µmol kg-1 fresh wt. in the roots of spinach (Spinacia oleracea L.) plants but did not detect phytochelatin in the leaves. Earlier analyses of partially purified Cd complexes isolated from lettuce roots (Inouhe et al., 1994) and leaves (McKenna and Chaney, 1995) did not yield ligands identical to phytochelatin. While we have not shown that phytochelatin is binding Cd in lettuce, it is clearly present and probably an important component of metal detoxification. Both groups reported storing plant tissues at -20°C before analysis and, as we have reported, even storing lettuce tissue in liquid N2 significantly reduces our ability to measure glutathione and phytochelatin (Fig. 1). Thus, Cd speciation in vivo may be altered by freezing and storage.
Average glutathione concentrations in controls were generally an order of magnitude lower than values reported for the roots and leaves of spinach (Tukendorf, 1993) and tobacco (Nicotiana tabacum L.) (Vögeli-Lange and Wagner, 1996), which may be due to the higher water content of lettuce compared with these other plants. Cadmium clearly stimulated glutathione production in lettuce (Fig. 3), contrary to reports of glutathione depletion in plants treated with Cd for 4 d (Rüegsegger and Brunold, 1992) and 15 d (Tukendorf, 1993). The long-term and environmentally relevant levels of Cd exposure used in this study (as compared with the high concentrations used in earlier studies) may reveal a more realistic biochemical response to metal stress. Indeed, recent molecular studies have shown that Cd stimulates the expression of genes encoding enzymes involved in glutathione synthesis (Schäfer et al., 1998). Though Zhu et al. (1999) showed that overexpression of glutathione synthase in a plant could increase Cd tolerance, the relative amounts of glutathione and phytochelatin in our plants would suggest that glutathione synthesis is not limiting phytochelatin production at the exposure levels examined.
The complex chemistry of Cd in soils necessitates the use of hydroponic buffers to examine the bioavailability of various aqueous Cd species. While it is known that kinetically labile species such as the hydrated Cd2+ ion or inorganic complexes are bioavailable to plant tissues (Welch and Norvell, 1999), it is less clear whether Cd–EDTA complexes are. We observed greater Cd accumulation in roots and old leaves of plants exposed to higher Cd–EDTA concentrations. One possible explanation for this observation is that a larger pool of chelated metal facilitates metal uptake by replacing ions taken up by plant roots (Laurie et al., 1991) or it is possible that the Cd–EDTA complex is taken up directly. Chelate-bound metal uptake and translocation has been reported for Pb (e.g., Huang et al., 1997; Vassil et al., 1998), including detection of Pb–EDTA in xylem sap and recovery of Pb and ETDA in leaves of Indian mustard [Brassica juncea (L.) Czern.]. Collins et al. (2001) recently reported metal–EDTA complexes in xylem of barley (Hordeum vulgare L.) proportional to the composition of the soil solution. While apoplastic transport with entry points at discontinuous joints of endodermis has been invoked to explain such phenomena (at relatively low chelator concentrations; Bell et al., 1991), the mechanism whereby metal–EDTA complexes enter the xylem is not yet understood. At high chelator concentrations (such as with the Pb experiments), it is likely that membranes are damaged and the complexes are then able to pass nonselectively through open channels (Vassil et al., 1998).
In our experiments it is difficult to determine which of these processes was responsible for increased Cd accumulation. The higher phytochelatin concentration in roots (especially the higher n = 3) at the two higher EDTA concentrations points to the former explanation whereas the lack of a change in the phytochelatin and glutathione in the new leaves would suggest the latter. It is probably a combination of both processes.
In contrast to our own, other studies have shown that EDTA in nutrient media restricts the uptake of Cd and the chemically similar metal Zn, thus suggesting that there are species-specific differences. Checkai et al. (1987) reported that free Cd2+ concentrations determined the Cd content of tomato (Lycopersicon esculentum Mill.) plants when EDTA was added to a resin-buffered hydroponic system. Yang et al. (1994) tested both EDTA and DTPA as nutrient solution buffers and found that rice (Oryza sativa L.), while able to retrieve and translocate Zn–DTPA, took up little Zn–EDTA. In our study, of the metals examined, Zn concentrations in plant tissues were the least sensitive to the total Zn concentration in the growth medium (data not shown).
Ratios of phytochelatin cysteinyl S to Cd reported by others are similar to those we observed in the new leaves of FTH plants and all tissues in beaker plants (Rauser and Meuwly, 1995; Vögeli-Lange and Wagner, 1996). The low ratio of sulfhydryl to Cd in roots may be explained by apoplastic binding of Cd in root tissues (Cataldo et al., 1983; Hart et al., 1998), but it is also likely that Cd storage in vacuoles (separating it from the cytosolic phytochelatin synthase) is responsible for the lower ratio in both roots and old leaves. Cadmium can be transported across the tonoplast into the vacuole as a phytochelatin complex (Salt and Rauser, 1995) and in the acidic environment of the vacuole, the Cd–phytochelatin complex probably dissociates, and the phytochelatin degrades or is reused by the plant (Grill et al., 1988). This recycling process would allow Cd to accumulate in tissues as they aged without a concomitant increase in phytochelatin concentrations. Alternatively, if Cd–EDTA was taken up, transported intact to the shoot, and preferentially deposited in old leaves, similarly low ratios could result, since when bound to EDTA, Cd is unable to chelate glutathione and hence serve as a substrate for phytochelatin synthase (Vatamaniuk et al., 2000). Since sulfhydryl to Cd ratios are much lower overall in FTH plants as compared with beaker plants, we can surmise that either more Cd–EDTA is taken up by these plants or, more likely, compartmentalization of Cd in roots and old leaves is more efficient.
It has been proposed that phytochelatin be monitored as a biomarker for heavy metal stress in plants, particularly in edible crops (Keltjens and van Beusichem, 1998b). This study demonstrates that while phytochelatin may provide some measure of Cd stress in lettuce, it is not an appropriate indicator of bioavailable Cd in the environment. The margin of error is often too large to readily distinguish between orders of magnitude of free Cd ion exposure due to variability in phytochelatin production among individual plants. Furthermore, phytochelatin and glutathione production did not increase universally in response to increases in tissue Cd concentrations and the relationship between these variables is strongly dependent on tissue type and even the age of the tissue.
In the soil environment, it is unknown whether metal–organic chelates are available for plant uptake. Sauvé et al. (2000) estimated that the fraction of dissolved Cd in contaminated soil solutions bound to soluble organic matter often exceeds that in the form of the free ion, especially at higher pH values. Ascertaining the bioavailability of this organic fraction, and elucidating the transport mechanisms involved, are both promising areas for future research.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
–glutamylcysteine dipeptide transpeptidase (phytochelatin synthase). Proc. Natl. Acad. Sci. USA 86:6838–6842.-glutamylcysteine synthesis in maize seedlings. Plant Physiol. 99:428–433.-glutamylcysteine synthetase isoform. Plant Mol. Biol. 37:87–97.[ISI][Medline]Related articles in JEQ:
This article has been cited by other articles:
|
|
 |
|
  R. L. Chaney, J. S. Angle, C. L. Broadhurst, C. A. Peters, R. V. Tappero, and D. L. Sparks Improved Understanding of Hyperaccumulation Yields Commercial Phytoextraction and Phytomining Technologies J. Environ. Qual., August 31, 2007; 36(5): 1429 - 1443. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Singla-Pareek, S. K. Yadav, A. Pareek, M.K. Reddy, and S.K. Sopory Transgenic Tobacco Overexpressing Glyoxalase Pathway Enzymes Grow and Set Viable Seeds in Zinc-Spiked Soils Plant Physiology, February 1, 2006; 140(2): 613 - 623. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| The SCI Journals | Agronomy Journal | Crop Science | |||
| Journal of Natural Resources and Life Sciences Education |
Vadose Zone Journal | ||||
| Soil Science Society of America Journal | Journal of Plant Registrations | The Plant Genome | |||
