Chemistry
AgriculturalAndFoodSciences,1970-2015-Agricultural And Food Sciences-2005.jsonl.gz.gz
Agricultural And Food Sciences
416a21302e9108cae0f0a151082bfa1d637a3b0e
Zinc Uptake and Shoot Partitioning Between Zinc Efficient and Inefficient Exacum Genotypes

transporter ABSTRACT. Interspecifi c hybrids of exacum (Exacum L.) display variable responses to zinc nutrition. Our research compared two genotypes with contrasting zinc effi ciency phenotypes in terms of root cation exchange capacity (CEC), whole plant 65 Zn uptake, and the effects of Cu +2 and Mg +2 on 65 Zn uptake and partitioning to shoot tissues. Results show that the zinc effi cient and ineffi cient genotypes had signifi cantly different root CEC (27.2 and 16.9 cmol(+)·kg -1 root dry weight (DW), respectively) and whole plant 65 Zn uptake rates (0.048 and 0.026 μmol·h -1 ·g -1 DW, respectively). In equi- molar concentrations to Zn +2 , Cu +2 reduced Zn +2 uptake by approximately 50% in both genotypes while supplemental Mg +2 enhanced Zn +2 uptake. In addition, Mg +2 facilitated a larger proportion of absorbed 65

Exacum (Gentianaceae) contains ≈65 species (Klackenberg, 1985) that are hypothesized to comprise a polyploid series of species (Darlington and Wylie, 1955). Of these species, only E. affi ne L. has gained economic importance as an ornamental crop with this speciesʼ commercial history dating back to 1904 (Riseman, 2005). However, descriptions of other exacum species indicate the presence of valuable horticultural traits that include large fl owers (>2.5 cm) that range in color from light blue to dark violet, diverse growth habits (upright to procumbent), and attractive glossy green foliage. Based on these descriptions, several species native to Sri Lanka were collected and formed the basis of an interspecifi c breeding program designed to develop this germplasm for horticultural use.
During production of the interspecifi c populations, a significant number of genotypes developed Zn +2 defi ciency symptoms severe enough to cause concern for commercial production while other genotypes remained healthy and symptom free. We categorized our germplasm into effi cient and ineffi cient genotypes based on both the phenotypic expression of classic Zn +2 defi ciency symptoms (i.e., chlorotic and stunted growth due to shortening of internodes leading to "rosetting" and a drastic decrease in leaf size) and overall biomass production (i.e., Zn +2 ineffi cient genotypes produced approximately one-third the biomass of Zn +2 effi cient genotypes) when grown under identical conditions. Nutrient analyses identifi ed Zn +2 as the only element that differed signifi cantly between these phenotypes with the effi cient and ineffi cient genotypes containing 35 and 8 μg·g -1 DW shoot tissue, respectively (Riseman, 1997). In addition, the zinc defi ciency phenotype was correlated with specifi c interspecifi c pedigrees so we concluded the presence of a signifi cant genetic component originating from the native species used in the primary interspecifi c hybridizations (Riseman, 1997). The species used in these hybridizations displayed signifi cant variation in morphology, cytology, and isozyme profi les (Sumanasinghe, 1986) but were never observed with Zn +2 defi ciency symptoms in their native habitats (Riseman, 1997). In addition to these plant-based characters, these Sri Lankan species have been associated with distinct edaphic conditions found in their native habitats (Riseman, 1997). Signifi cant differences were found among soil samples for many edaphic variables including Zn +2 content (0.28 to 2.91 kg·ha -1 ), Cu +2 content (0.18 to 6.50 kg·ha -1 ), Mg +2 content (0.33 to 5.04 kg·ha -1 ), Cu +2 :Zn +2 ratio (0.5 to 6.0), and Mg +2 :Zn +2 ratio (0.28 to 4.5) (Riseman, 1997). There exists the possibility these disparate edaphic conditions acted as a selection pressure in the evolution of distinct taxa. It has been shown that variation in edaphic conditions can affect the evolution and development of ecotypes with genetically distinct nutrient uptake and translocation mechanisms (Rajakaruna et al., 2003).
In evaluating nutrient effi ciencies, Rengel (2001) outlines several generalizations relating to genotypic differences: 1) effi ciency mechanisms vary among crop species and genotypes, 2) more than one mechanism is often responsible for effi ciency in a particular genotype, 3) increased effi ciency of one genotype in comparison to the other is due to the involvement of additional mechanisms not present (or expressed) in the less effi cient genotype, and 4) expression of more than one effi ciency mechanism is likely to result in an additive effect. In addition, the mechanisms of nutrient effi ciency may operate at various levels of plant organization (i.e., molecular, physiological, structural, or developmental). Many individual processes have been cited in the literature as possible sources of Zn +2 effi ciency. These include differences in nutrient uptake kinetics where individual genotypes differ in their ability to transport Zn +2 into the symplasm (Bowen, 1987;Graham and Rengel, 1993); differences in root cation exchange capacity where genotypes possess different apoplastic binding affi nities for Zn +2 that may provide a resource or pool of Zn +2 available for uptake mechanisms to access (Sakal et al., 1988); differences in translocation rates where absorbed Zn +2 is differentially translocated within the plant (Graham and Rengel, 1993); and differences in the effects of competing ions where elemental interactions positively or negatively affect the uptake or translocation of Zn +2 (Amber and Brown, 1969;Brown and McDaniel, 1978;Jolley and Brown, 1991;Parker et al., 1992;Pedler et al., 2004).
This research was conducted to identify factors which contribute to zinc effi ciency and were suitable for use in germplasm evaluations designed to identify zinc effi cient genotypes. In addition, we feel this information helps to elucidate mechanisms that may have affected the evolution of exacum taxa. Previously, zinc effi ciency in our germplasm was determined to be primarily root-based (Riseman, 1997) and highly correlated with Zn +2 uptake per unit of root length (Riseman and Craig, 2000). However, additional root-based traits have been associated with Zn +2 effi ciency. In this research, we compared Zn +2 effi cient and ineffi cient genotypes with respect to 1) root cation exchange capacity (CEC) ; 2) whole plant Zn +2 uptake rates; 3) the competitive effects of Cu +2 and Mg +2 on Zn +2 uptake rates; and 4) the effects of Cu +2 and Mg +2 on Zn +2 partitioning to shoot tissues.
ROOT CEC EXPERIMENTS. Root CEC was measured according to the method of Helmy and Elgabaly (1958). Five-gram fresh weight root samples were acidifi ed through fi ve rinses of 0.1 N HCL. Samples were then rinsed fi ve times in deionized water followed by back titration to pH 8 with a 0.04 N KOH solution. Titration time was approximately 2 min. Extra care was paid to titration time to equalize the effect of carbonic acid formation by CO 2 diffusion into the solution. After titration, tissues were dried at 80 °C in a forced air oven for 24 h. Root CEC is expressed as cmol(+)·kg -1 dry tissue. ZN +2 DESORPTION, UPTAKE, AND TISSUE PARTITIONING EXPERI-MENTS. Preliminary trials were conducted to empirically determine an adequate desorption period following 6 h of exposure to 65 Znlabeled uptake solution. Plants were removed from the uptake solution (see 65 Zn uptake solution section below), blotted free of surface solution and placed in ice cold desorption solution consisting of 0.5 mM CaCl 2 . The plant roots and solution were agitated by gentle manual manipulation. Desorption solutions were sampled at 1, 2, 3, 4, 5, 7, 10, 12, 15, 20, and 25 min after root contact. At each sample interval, a 0.5-mL aliquot was collected and analyzed for radioactivity. A fi nal desorption period was determined when no additional radioactivity was recorded in three consecutive aliquots. This time period was used for all subsequent trials and for both genotypes. Based on these trials, a 15-min desorption period was used for all subsequent experiments.
Upon initiation of the 65 Zn uptake experiments, plants were moved from the glasshouse to the laboratory where the roots were washed free of sand with deionized water. Roots were easily removed from the sand without any perceivable damage. Plants were then placed into 1-L plastic containers with holes drilled through the lid to provide support for the shoot while allowing the roots to be immersed in the uptake solution. Two pretreatment solutions were used consecutively. The fi rst solution consisted of full-strength solution (Jonson et al., 1957) composed of the following salts and concentrations: KNO 3 (6 mM), Ca(NO 3 ) 2 (6 mM), NH 4 H 2 PO 4 (2 mM), MgSO 4 (225 μM), KCl (50 μM), H 3 BO 3 (25 μM), MnSO 4 (2 μM), CuSO 4 (0.5 μM), (NH 4 ) 6 Mo 7 O 24 (0.5 μM), FeSO 4 (2 μM), ZnSO 4 (2 μM). All solutions were constantly aerated. Plants remained exposed to the fi rst pretreatment solution for 24 h. The second solution consisted of only CaCl 2 (0.5 mM) and ZnCl 2 (2 μM). Plants remained exposed to the second pretreatment solution for an additional 24 h. All pretreatments and uptake experiments were used a water bath to maintain solution temperatures at 30 °C. Irradiance levels of 450 μmol·m -2 ·s -1 PAR were produced by one 1000-W metal halide lamp positioned 1 m above the plants.
At harvest, roots were blotted with paper towels to remove surface solution, desorbed for 15 min, partitioned by tissue category and dried at 80 °C for 48 h. Roots were cut into 10-mm segments and mixed to form homogenous subsamples. Shoots were partitioned into individual leaf pairs and internode sections. After drying, all samples were weighed and measured for radioactivity.
COMPETITION EXPERIMENTS. For ion competition experiments, a single radioactive CaCl 2 + ZnCl 2 solution, as described above, was made and divided into two equal aliquots. The fi rst aliquot remained unchanged and served as the control while the second aliquot was supplemented with either Mg +2 or Cu +2 . This arrangement allowed for a common specifi c activity to be used within each replication. Both Mg +2 and Cu +2 were used in equimolar concentrations to the Zn +2 concentration (2 μM) and evaluated individually in separate trials. Magnesium chloride and copper chloride were used as the source of the competing ions. Three complete replications were conducted for each ion. Plant tissues were divided into the following categories prior to analyses: "upper shoot," meaning all tissue distal to and including youngest fully expanded leaf pair; "mid shoot," meaning all tissue basal to upper shoot to the second oldest leaf pair; "lower shoot," meaning all tissue basal to the mid shoot to the oldest leaf pair; "lower stem," meaning all tissue basal to the oldest leaf pair; and "roots," meaning all root tissue. 65 Zn radioactivity was measured on a Gamma Scintillation Spectrometer (model 5230; Packard Instrumentation Co., Downers Grove, Ill.). The detection parameters were set at a lower limit of 450 MeV with a window of 210 MeV. All samples were counted for a minimum of either 20 min or 10,000 counts. Background radiation was accounted for by bracketing each sample with blanks and subtracting the average between them from the embedded sample. Specifi c activity was determined by counting an initial aliquot collected from the uptake solution prior to exposure to the plants and then converted to counts per minute (CPM)/μmol Zn +2 based on the initial Zn +2 concentration of 2 μmol·L -1 . Individual specifi c activity calculations were determined for each block and utilized in only that block.
EXPERIMENTAL DESIGN AND STATISTICS. For root CEC experiments, fi ve plants of each genotype were utilized as replicates. The roots of each plant were partitioned into three groups and considered subsamples. Statistics were performed by StatView SE + Graphics (Abacus Concepts, Berkeley, Calif.) statistical software.
For 65 Zn uptake and ion competition experiments, two plants of each genotype were placed in a single container; each container served as a replicate in the statistical analyses. A total of 10 replicates were utilized in each experiment. Statistical procedures (i.e., analysis of variance, mean separation and paired t tests of means) were performed with Systat for the Macintosh version 5.2 (Systat, Evanston, Ill.). For the ion competition studies, data are presented as both absolute values and as a percentage of control. For Zn +2 uptake and partitioning comparisons, plant tissues were grouped as described above.

ROOT CEC.
The effi cient genotype had signifi cantly greater root CEC than the ineffi cient genotype (Fig. 1). This difference is presumed to have affected the amount of Zn +2 released from the root tissues following 15 min of desorption. Following desorption, the two genotypes released signifi cantly different amounts of Zn +2 , with the effi cient genotype releasing 0.25 μmol·g -1 DW root while the ineffi cient genotype released only 0.16 μmol Zn +2 .
ZN +2 UPTAKE AND TISSUE PARTITIONING. After 1 h, there were no signifi cant genotype differences in 65 Zn concentration within any tissue category (Table 1). However, in each category, the zinc effi cient genotype contained a higher mean 65 Zn concentration than the ineffi cient genotype. After 6 h of exposure to the uptake solution, whole-plant Zn +2 uptake rates were signifi cantly greater in the effi cient genotype (188.2 pmol·h -1 ·g -1 DW) than in the ineffi cient genotype (94.8 pmol·h -1 ·g -1 DW) (calculated from Table 1, P < 0.05). In addition, signifi cant differences were observed for Zn +2 allocation rates (i.e., sum of Zn +2 in all non-root tissues divided by the uptake period and total shoot dry weight) between the genotypes where the Zn +2 effi cient and ineffi cient genotypes translocated 6.0 and 3.9 pmol·h -1 ·g -1 DW, respectively (calculated from Table 1) EFFECTS OF CU +2 AND MG +2 ON ZN +2 UPTAKE AND SHOOT PAR-TITIONING. As compared to the ineffi cient genotype, the effi cient genotype absorbed signifi cantly greater amounts of Zn +2 regardless of the competing ion ( Fig. 2A). In general, Cu +2 reduced overall Zn +2 uptake while Mg +2 facilitated uptake, regardless of genotype or exposure time. After 6 h, Zn +2 uptake in the presence of Cu +2 was signifi cantly reduced in the effi cient genotype from 1129.2 to 745.3 pmol while in the ineffi cient genotype, Zn +2 uptake was signifi cantly reduced from 568.9 to 312.9 pmol ( Fig. 2A). On average, Cu +2 reduced root Zn +2 uptake compared to the control by 64% and 51% for the Zn +2 effi cient and ineffi cient genotypes, respectively (Fig. 2B).
Magnesium enhanced Zn +2 uptake in both genotypes at both time intervals. After 6 h, Zn +2 uptake in the presence of Mg +2 was   signifi cantly increased in the effi cient genotype from 1129.2 to 1309.9 pmol while in the ineffi cient genotype, Zn +2 uptake was signifi cantly increased from 568.9 to 676.9 pmol ( Fig. 2A). These increases were 116% and 119%, over the controls for the effi cient and ineffi cient genotypes, respectively (Fig. 2B). After 6 h, Zn +2 partitioning to shoot tissue categories significantly differed between the two genotypes with the addition of either Cu +2 or Mg +2 to the uptake solution. In the Zn +2 effi cient genotype, the lower stem tissue contained <60% of the shoot Zn +2 regardless of competing ion (Fig. 3). In addition, Mg +2 altered Zn +2 distribution within the plant by facilitating movement from the lower stem to the upper shoot category (i.e., reducing the percentage of allocated Zn +2 in lower stem from 50% to 20%) while both the mid shoot and lower shoot categories were unchanged. Copper appeared to have a less pronounced effect than Mg +2 in that more Zn +2 was present in the upper than the mid shoot categories while the percentage in the lower stem remained approximately equal. In the ineffi cient genotype, neither Mg +2 nor Cu +2 induced the same responses observed in the effi cient genotype. Overall, the lower stem category contained over 60% of the shoot Zn +2 while the upper shoot accounted for ≈4%, regardless of competing ion. In addition, Cu +2 appeared to signifi cantly reduce allocation out of the lower stem to any other tissue category while Mg +2 had no overall effect on distribution.

Discussion
Our results have identifi ed several signifi cant differences between Zn +2 effi cient and ineffi cient genotypes of exacum. The Zn +2 effi cient genotype possessed greater root CEC, greater Zn +2 uptake, greater Zn +2 partitioning to the upper shoot and greater ability to absorb and translocate Zn +2 in the presence of competing ions. We believe these differences are the basis for the Zn +2 effi cient phenotype previously observed in common glasshouse experiments (Riseman, 1997). The presence of multiple traits associated with a nutrient effi ciency is not surprising based on Rengelʼs (2001) generalizations for genotypic differences in nutrient effi ciency already mentioned. In addition to these generalizations, mechanisms associated with nutrient effi ciency may operate at various levels of plant organization (i.e., molecular, physiological, structural, or developmental). Our results indicate multiple traits, across different levels of plant organization are associated with Zn +2 effi ciency in exacum.
The Zn +2 effi cient genotype was found to have signifi cantly higher root CEC and greater overall Zn +2 uptake. As in soil, root CEC is a measure of the ability to electrostatically bind cations to negative surface charges. In roots, this is often called apoplastic binding and is comprised of charges on both the cell wall and exterior plasma membrane. These charges often arise from ligands containing negatively charged organic functional groups such as the carboxyl residues of pectins (Ostateck-Boczynski et al., 1995). Apoplastic CEC can infl uence cation selectivity and ion competition for membrane transporters. This is because diverse cations have different affi nities for apoplastic exchange sites, and because the distribution of one ionic species around a charged surface will effect the distribution of other cationic species through repulsion of like charges (Shomer et al., 2003). In relation to the observed differences in Zn +2 uptake, apoplastic CEC may selectively position greater amounts of Zn +2 near transporters thereby  Zn efficient Zn inefficient resulting in greater uptake by effi cient genotypes. However, other possibilities exist and include differences in transporter affi nities, kinetics, number, or transporter gene expression/regulation. To evaluate whether specifi c ions affected Zn +2 uptake, Mg +2 and Cu +2 were used in equimolar concentrations to Zn +2 as competitive inhibitors. In both genotypes, Mg +2 enhanced Zn +2 uptake while Cu +2 inhibited uptake. Two models, each with empirical support, have been proposed for Zn +2 uptake: 1) a general divalent cation channel and 2) a specifi c Zn +2 /Cu +2 transporter. In support of the general divalent cation model, Chaudhry and Loneragan (1972) demonstrated that all of the alkaline earth cations (Ca +2 , Mg +2 , Sr +2 and Ba +2 ) inhibited Zn +2 uptake in wheat (Triticum aestivum L.), indicating a common uptake mechanism. Kochian (1993), in summarizing zinc uptake research, proposed that Zn +2 , as well as Cu +2 , Mn +2 and Mg +2 , is probably absorbed through a common divalent cation channel and not by specifi c ion transporters. In opposition to this model, there is research that supports the existence of specifi c Zn +2 /Cu +2 transporters. Schmid et al. (1965) demonstrated using barley (Hordeum vulgare L.), that Cu +2 strongly inhibited Zn +2 uptake while Mn +2 had no effect. Bowen (1987), using rice (Oryza sativa L.) and tomato (Lycopersicon esculentum Mill.), showed that Cu +2 and Zn +2 mutually and competitively inhibited uptake of each other and suggested both micronutrients were absorbed through the same uptake mechanism or carrier site. These apparently confl icting models may, in reality, both be correct. Often, inadequate experimental methods, that do not account for uptake/release rates, uptake of ions from complex solutions (e.g., solutions with multiple ions), or electrostatic affi nities of ions present in low concentrations, inhibit the accurate characterization of ion specifi c transporters. Therefore, much of the published micronutrient uptake research must be viewed with care. However, research that attempted to account for these issues has identifi ed both high and low affi nity transporters within a single plant species (Reid et al., 1996). They state that the low affi nity transporter had broad substrate specifi city while they made no comment on the substrate specifi city for the high affi nity transporter. These two Zn +2 uptake mechanisms may individually represent the two proposed models where the low affi nity transporter is analogous to the general divalent cation channel and the high affi nity transporter is analogous to the Zn +2 /Cu +2 specifi c transporter. Data from this research, using the relatively low concentration of 2 μM Zn, support the presence of a high-affi nity transporter specifi c to Zn +2 and Cu +2 . However, future research should evaluate the presence of multiple uptake mechanisms and ionic affi nities of transporters in this germplasm.
The cause for increased Zn +2 uptake and shoot transport in the presence of Mg +2 is unclear. It may be due to apoplastic binding affi nities that may have otherwise been available to bind Zn +2 . With these binding sites occupied, Zn +2 transport through the apoplast was facilitated. Another possibility is that the experimental plants were in the initial stages of Mg +2 defi ciency due to exposure to a simple CaCl 2 + ZnCl 2 solution for 24 h prior to the uptake experiments. With the addition of Mg +2 to the uptake solution, the defi ciency was alleviated. In evaluating the effects of Mg +2 on Zn +2 toxicity, micromolar concentrations of Mg +2 were found to alleviate Zn +2 toxicity while simultaneously facilitating higher tissue Zn +2 concentrations (Pedler et al. 2004). The authors concluded that the protective effect of Mg +2 was not due to diminished Zn +2 uptake or translocation but rather, Mg +2 was involved in some type of internal detoxifi cation or sequestration mechanism. However, working with isolated vesicles from tonoplasts, Verkleij et al. (1998) found that a Zn +2 -tolerant genotype of Silene vulgaris (Moench.) Garcke displayed higher Zn +2 transport rates than the sensitive genotype and that transport required Mg-ATP. If our experimental plants did have an induced Mg +2 defi ciency, the addition of Mg +2 to the uptake solution may have replenished the biologically available Mg +2 pool required for normal Zn +2 uptake. Although no clear explanation or mechanism has been determined to explain this Zn +2 × Mg +2 interaction, we accept the possibility that a tissue-specifi c Mg +2 requiring Zn +2 transporter may be present in Exacum.
Zn effi ciency in exacum is a composite trait involving both apoplastic (i.e., CEC) and symplastic (i.e., Zn +2 transporters, Mg +2 requirement) traits that are most likely under independent regulation. When we consider the polyploid nature of the original taxa combined with distinct edaphic conditions of their native habitats, the opportunity for gene diversifi cation is great. Gene diversifi cation for micronutrient uptake can be a relatively simple process in which a single amino acid replacement can signifi cantly alter substrate specifi city (Rogers et al., 2000). In their native habitats, no Exacum taxon was observed with Zn +2 defi ciency symptoms despite the large range in divalent cation concentrations and ratios (Riseman, 1997). Therefore, we propose that Sri Lankan exacum have experienced edaphic selection where taxa have evolved unique genes, regulators, or linkage groups that allow for adequate Zn +2 uptake and transport under disparate nutrient conditions. With subsequent interspecifi c hybridization, these genes, regulators, or linkage groups have been rearranged and mismatched to the point where Zn +2 uptake mechanisms are less effective. These conclusions are consistent with Rengelʼs generalizations (2001) concerning genotypic differences in nutrient effi ciency in that 1) we identifi ed more than one mechanism associated with the effi ciency phenotype (i.e., root CEC, Zn +2 uptake, and Zn +2 × Mg +2 interaction), 2) our genotypic differences were based on mechanisms not present (or expressed) in the less effi cient genotype (i.e., lower root CEC, reduced Zn +2 uptake in ineffi cient genotype), and 3) mechanisms identifi ed in exacum operate at various levels of plant organization (i.e., genetic and physiological differences between genotypes).
This research has presented information on various physiological traits associated with Zn +2 effi ciency and has proposed a mechanism for their development and evolution. With this information, we are better able to breed and select genotypes suitable for commercial production because we now have specifi c evaluation and selection criteria linked to the zinc effi ciency phenotype. However, several important questions were raised that require additional research. Are multiple Zn +2 uptake mechanisms present in exacum that are analogous to the low and high affi nity transporters identifi ed in other organisms? What is the physiological basis for enhanced Zn +2 uptake and translocation in the presence of micromolar concentrations of Mg +2 ? And fi nally, is Zn +2 effi ciency in exacum the result of several interacting mechanisms that only operate effectively when present in their original arrangement?