Plant materials.

Unless otherwise stated, vegetatively propagated Phalaenopsis Sogo Yukidian ‘V3’ plants grown in sphagnum moss in 10.5-cm pots (0.75 L) were purchased from a commercial grower (Clone International Biotech, Pingtung, Taiwan). During the experimental period, plants were grown in a Venlo-type greenhouse at National Taiwan University with a pad and fan system with maximum monthly temperature of 27 °C in summer, minimum monthly temperature of 18 °C in winter, and average daily minimum relative humidity of 70%. Light intensity was maintained below a maximum level of 400 μmol·m⁻²·s⁻¹ using an adjustable double layer of shadecloth.

Expt. 1: Differences in fertilizer N absorption and partitioning through Phalaenopsis roots and leaves.

The aim of this experiment was to compare the absorption of fertilizer N by Phalaenopsis roots and leaves, and how it is subsequently partitioned. Plants were removed from their pots and the sphagnum moss medium was removed. The roots were divided into two halves based on their total length: young roots and old roots. To prevent ¹⁵N contamination between these two halves, the young and old roots were fixed onto separate tree fern slabs, as shown in Fig. 1A. Johnson’s solution (Johnson et al., 1957) labeled with ¹⁵N (22.5 atom% ¹⁵N) and supplemented with 0.1% surfactant (Tween-20; Nacalai Tesque, Kyoto, Japan) was applied with a paintbrush to the upper or lower surfaces of all leaves excluding the top leaf, or to the young or old roots once per day for 3 d (consecutive). There were four treatments (i.e., four fertilizer application sites) with six single-plant replications in each treatment. The roots were then wrapped in sphagnum moss and nets after ¹⁵N treatment (Fig. 1B) to retain moisture. To prevent ¹⁵N contamination between leaves and roots, fertigation was done by spraying regular Johnson’s solution (224 mg·L⁻¹ N) to the sphagnum moss substrate when it was almost dry. Four weeks after ¹⁵N treatment, some organ parts were sampled from newly grown leaves (top leaf, without ¹⁵N application), mature leaves (lowest healthy leaf), young roots, and old roots. The leaves were sampled at 2.5-cm length measured from the leaf tip. The roots were sampled as 10-cm-long pieces. Eight weeks after ¹⁵N treatment, whole plants were harvested and separated into the following components: newly grown leaves (leaves younger than those fully expanded before ¹⁵N treatment), mature leaves (fully expanded leaves before ¹⁵N treatment), young roots, old roots, and newly grown roots (roots newly formed on the nodes of the shoot above the sphagnum moss wrapping after ¹⁵N treatment). The experimental period was from 24 July to 18 Sept.

Phalaenopsis Sogo Yukidian ‘V3’ plant fixed onto tree fern slabs. (A) The young roots and old roots were fixed onto the right and the left tree fern slabs, respectively. (B) The roots were wrapped in sphagnum moss and nets after ¹⁵N treatment.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.229

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Phalaenopsis Sogo Yukidian ‘V3’ plant fixed onto tree fern slabs. (A) The young roots and old roots were fixed onto the right and the left tree fern slabs, respectively. (B) The roots were wrapped in sphagnum moss and nets after ¹⁵N treatment.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.229

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Phalaenopsis Sogo Yukidian ‘V3’ plant fixed onto tree fern slabs. (A) The young roots and old roots were fixed onto the right and the left tree fern slabs, respectively. (B) The roots were wrapped in sphagnum moss and nets after ¹⁵N treatment.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.229

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Expt. 2: Short-term transport of fertilizer N to Phalaenopsis leaves after fertilizer application to the roots.

The aim of this experiment was to determine the fate of fertilizer N in Phalaenopsis leaves shortly after absorption by the roots. Plants with seven leaves were used, and they were chosen such that the youngest leaves were ≈90% to 95% expanded. The plants were removed from the substrate and then repotted into fresh sphagnum moss substrate (rinsed and wrung beforehand to remove soluble nutrients). The ¹⁵N-labeling was performed by subirrigating the plants from the bottom of the pots with Johnson’s solution enriched with 22.5 atom% ¹⁵N. Just before ¹⁵N-labeling was performed (0 d), and 0.5, 1, 2, 4, and 8 d after ¹⁵N labeling, the first leaf and fourth leaf of each sampled plant were harvested. There were six sampling time points with seven single-plant replications in each sampling time. The samples were then analyzed for total N concentration and ¹⁵N concentration. The experimental period was from 20 Nov. to 28 Nov.

Expt. 3: Sink strengths of Phalaenopsis leaves at various ages.

The objective of this experiment was to compare Phalaenopsis leaves at various ages for their relative sink strengths for N. Plants were replanted into 10.5-cm pots after purchase. After the plants were transplanted, ¹⁵N-labeled Johnson’s solution (11.25 atom% ¹⁵N) was applied by subirrigation. There were six single-plant replications and leaves were sampled from each replication 8 weeks after ¹⁵N treatment and numbered from the first (top) leaf to the sixth (lowest) leaf. Plants were fertigated with regular Johnson’s solution (224 mg·L⁻¹ N) every 2 to 3 weeks after the first fertigation with labeled fertilizer solution. To prevent ¹⁵N contamination between leaves resulting from overhead fertigation, the fertilizer solution was applied by subirrigation. The experimental period was from 12 July to 6 Sept.

Expt. 4: Absorption and partitioning of N by Phalaenopsis plants during vegetative and reproductive phases.

The aim of this experiment was to trace the fate of a single application of fertilizer N over a long cultivation period involving vegetative and reproductive stages. Vegetatively propagated Phalaenopsis Sogo Yukidian ‘V3’ plants, grown in sphagnum moss in 8.5-cm pots, were purchased and transplanted into 10.5-cm pots. After plants were transplanted, ¹⁵N-labeled Johnson’s solution (11.25 atom% ¹⁵N) was applied by subirrigation. Plants were sampled 1, 2, 4, 8, 16, and 38 weeks after ¹⁵N treatment. There were six sampling time points with three to four single-plant replications at each sampling time: n = 4 at 1, 2, 4, 8, and 16 weeks and n = 3 at 38 weeks after ¹⁵N treatment. The sampled plants were dissected into newly grown leaves, mature leaves, and roots. Plants began to spike at Week 16 because of natural low temperature, which marked the shift from vegetative growth stage to the reproductive growth stage (when growth was mainly that of reproductive organs). The flowers (including unopened floral buds) and stalks were sampled at Weeks 16 and 38. Fertigation and maintenance of plants were the same as in Expt. 3. The experimental period was from 6 July to 10 May.

Isotopic labeling.

Modified Johnson’s solution was used as the fertilizer in all experiments. It contained 16 mm N, 2 mm phosphorus, 6 mm potassium, 4 mm calcium, 1 mm sulfur, 1 mm magnesium, 50 μm chlorine, 25 μm boron, 5 μm magnesium, 4 μm iron, 2 μm zinc, 0.5 μm copper, 0.1 μm molybdenum, and 0.1 μm nickel with ammonium-to-nitrate ratio of 1:7. In Expts. 1 and 2, the potassium nitrate in the Johnson’s solution was completely substituted with 60 atom% ¹⁵N-labeled potassium nitrate (ISOTEC, Miamisburg, OH). The ¹⁵N-labeled solution thus had a ¹⁵N concentration of 22.5 atom%. In Expts. 3 and 4, half of the potassium nitrate in the Johnson’s solution was substituted with 60 atom% ¹⁵N-labeled potassium nitrate. The ¹⁵N-labeled solution had a ¹⁵N concentration of 11.25 atom%.

Sample analysis and data calculation.

All samples were rinsed with deionized water, dried in a 70-°C, forced-air oven for 2 weeks, and ground to fine powders with an electric crusher before analysis. The N concentration (%) and ¹⁵N concentration (atom%) were determined by the Automatic Nitrogen and Carbon Analyzer–Mass spectrometry (MS) system (Europa Scientific, Crewe, UK) comprising a sample preparation unit (autosampler to gas chromatography), a capillary interface, and a MS system. The atom percent ¹⁵N excess (atom% excess) was obtained by subtracting natural abundance of ¹⁵N (0.366 atom%) from the results of the ¹⁵N analyses. The N content = N concentration × dry weight. The ¹⁵N content excess = weight % of excess ¹⁵N out of total N × N content. For each site of fertilizer application in Expt. 1, translocated ¹⁵N content excess was calculated by subtracting ¹⁵N content excess at the site of application from the whole-plant ¹⁵N content excess.

Statistical analysis.

All experiments were single-factor experiments carried out in a completely randomized design. Statistical tests were done using CoStat software (Version 6.101; CoHort Software, Monterey, CA). Data were subjected to one-way analysis of variance to compare differences among treatments. The least significant difference test at P ≤ 0.05 was used for multiple mean comparisons. The t test was used for comparing difference between two sample means. Graphs were plotted with SigmaPlot software (Version 10.0; Systat Software, San Jose, CA).

Expt. 1: Differences in fertilizer N absorption and partitioning through Phalaenopsis roots and leaves.

Nitrogen concentration at 4 weeks after ¹⁵N-labeling treatments was highest in newly grown leaves (2.26% to 2.49%) and lowest in old roots (1.30% to 1.40%), and within the sampled organs, no difference in N concentrations was found among treatments (data not shown). Four weeks after ¹⁵N application to the lower or upper surface of mature leaves, the bulk of the absorbed ¹⁵N remained on the mature leaves (i.e., the site of application) with much less translocation to the other organs (Table 1). Atom% excess of 0.040% and 0.056% were detected in the mature leaves 4 weeks after ¹⁵N was applied to upper and lower leaf surfaces, respectively, whereas much lower atom% excess (0.004% to 0.011%) was found in other organs (Table 1). After application of ¹⁵N to old roots and young roots, these sites of application also had the greatest ¹⁵N concentrations 4 weeks later; application to old roots resulted in 0.356 atom% excess in old roots, whereas application to young roots resulted in 0.220 atom% excess in young roots (Table 1). The ¹⁵N concentrations in other organs were significantly lower (ranging from 0.014% to 0.028% in atom% excess) than in the old or young roots where labeled fertilizer was applied. Four weeks after ¹⁵N application, ¹⁵N was detectable at low levels in other organs where ¹⁵N had not been applied (Table 1).

Table 1.

The ¹⁵N concentrations of newly grown leaves, mature leaves, young roots, old roots, and newly grown roots of Phalaenopsis Sogo Yukidian ‘V3’ 4 and 8 weeks after application of isotopic nitrogen (¹⁵N) to different organs.

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Whole plants were harvested 8 weeks after treatment. The various treatments produced no difference in the dry weight, N concentration, and N content of various organs (data not shown). Nitrogen concentration was highest in newly grown leaves (2.32% to 2.49%) followed by young roots [1.72% to 1.94% (data not shown)]. Similar to Week 4, application of ¹⁵N to either the upper or lower surface of mature leaves or to either old roots or young roots resulted in the highest ¹⁵N concentration being found in the respective sites of application (Table 1). Irrespective of the site of application of the labeled fertilizer, newly grown leaves and newly grown roots had the highest ¹⁵N concentrations among the organs that were not sites of fertilizer application (Table 1). Between Weeks 4 and 8, the ¹⁵N concentrations in sites of fertilizer application generally decreased, whereas the ¹⁵N concentrations in the organs that were not sites of fertilizer application generally increased (Table 1). This shows that greater extent of ¹⁵N translocation from the sites of application occurred between Weeks 4 and 8.

Eight weeks after application of ¹⁵N-labeled fertilizer, plant ¹⁵N content excess varied with the site of application, where it was greatest after application to young roots [82.8 μg (Table 2)]. The second highest plant ¹⁵N content excess was found after application to old roots (60.3 μg), whereas plant ¹⁵N content excess was lowest after application to either the upper (34.1 μg) or lower surfaces of leaves [25.2 μg (Table 2)].

Table 2.

The ¹⁵N content excess of newly grown leaves, mature leaves, young roots, old roots, and newly grown roots of Phalaenopsis Sogo Yukidian ‘V3’ 8 weeks after application of isotopic nitrogen (¹⁵N) to different organs.

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Expt. 2: Short-term transport of fertilizer N to Phalaenopsis leaves after fertilizer application to the roots.

Dry weight of the first leaf ranged from 0.91 to 1.23 g, and increased significantly as days after fertilizer application increased (Table 3). Dry weight of the fourth leaf ranged from 1.01 to 1.18 g, but no obvious trend was found as days after fertilizer application increased (Table 3). Nitrogen concentration in the first leaf ranged from 1.84% to 1.96%, which was significantly higher than 1.09% to 1.23% found in the fourth leaf (Table 3). In both the first leaf and the fourth leaf, N concentration did not change with any obvious pattern over the experimental period (Table 3).

Table 3.

Changes in dry weight, nitrogen (N) concentration, and N content at different leaf positions of Phalaenopsis Sogo Yukidian ‘V3’ after application of ¹⁵N-labeled fertilizer solution at the roots.^z

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Nitrogen content in the first leaf ranged from 17.5 to 22.5 mg, which was significantly higher than the range found in the fourth leaf (Table 3). In the first leaf, N content increased as the number of days after fertilization increased, whereas in the fourth leaf, N content fluctuated between 12.4 and 14.5 mg, but did not seem to be related to the number of days after fertilization (Table 3).

In both the first leaf and the fourth leaf, a significant increase in ¹⁵N concentration could be detected as early as 0.5 d after application of ¹⁵N-labeled fertilizer at the roots. Between Day 0 and Day 0.5, ¹⁵N concentration increased from 0.0006 atom% excess to 0.0016 atom% excess in the first leaf and from 0.0005 atom% excess to 0.0014 atom% excess in the fourth leaf (Fig. 2A). Translocation of fertilizer N to both young and mature leaves 0.5 d after fertilizer application at the roots was also evident as shown by ¹⁵N content excess. Between Day 0 and Day 0.5, ¹⁵N content excess increased from 0.1 to 0.3 μg in the first leaf, and from 0.1 to 0.2 μg in the fourth leaf (Fig. 2B). Nitrogen-15 concentration and content continued to increase in both the first and fourth leaves over the experimental period, indicating that fertilizer N continued to accumulate in both young and mature leaves by Day 8 (Fig. 2).

¹⁵N concentration (atom% excess) and ¹⁵N content excess in Phalaenopsis Sogo Yukidian ‘V3’ leaves after application of ¹⁵N-labeled fertilizer to the roots. **, *** Significant difference in the first leaf between sampling point and Day 0 by unpaired t test at P ≤ 0.01 and P ≤ 0.001, respectively. ##, ### Significant difference in the fourth leaf between sampling point and Day 0 by unpaired t test at P ≤ 0.01 and P ≤ 0.001, respectively. Bars indicate sem; n = 7.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.229

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¹⁵N concentration (atom% excess) and ¹⁵N content excess in Phalaenopsis Sogo Yukidian ‘V3’ leaves after application of ¹⁵N-labeled fertilizer to the roots. **, *** Significant difference in the first leaf between sampling point and Day 0 by unpaired t test at P ≤ 0.01 and P ≤ 0.001, respectively. ##, ### Significant difference in the fourth leaf between sampling point and Day 0 by unpaired t test at P ≤ 0.01 and P ≤ 0.001, respectively. Bars indicate sem; n = 7.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.229

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¹⁵N concentration (atom% excess) and ¹⁵N content excess in Phalaenopsis Sogo Yukidian ‘V3’ leaves after application of ¹⁵N-labeled fertilizer to the roots. **, *** Significant difference in the first leaf between sampling point and Day 0 by unpaired t test at P ≤ 0.01 and P ≤ 0.001, respectively. ##, ### Significant difference in the fourth leaf between sampling point and Day 0 by unpaired t test at P ≤ 0.01 and P ≤ 0.001, respectively. Bars indicate sem; n = 7.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.229

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Expt. 3: Sink strengths of Phalaenopsis leaves at various ages.

Dry weight was greatest for second leaves (1.54 g) and decreased successively with lower leaf position with only 0.35 g dry weight present in sixth leaves (Fig. 3A). The first leaves were not fully expanded when sampled and had a lower dry weight than the second and third leaves (Fig. 3A). Nitrogen concentration was highest in the first leaves (2.31%) and decreased successively with lower leaf position, down to 1.16% (Fig. 3B). Nitrogen content was highest in second leaves at 26.7 mg and the first leaves had the second highest at 22.2 mg (Fig. 3C) although first leaves had the highest N concentration. The lower N content was the result of the smaller dry weight of the first leaf compared with the second leaf. Nitrogen content decreased from the third leaf (19.7 mg) to the sixth leaf [4.0 mg (Fig. 3C)].

Dry weight, nitrogen (N) concentration, N content, ¹⁵N concentration, and ¹⁵N content excess of leaves in Phalaenopsis Sogo Yukidian ‘V3’ 8 weeks after application of ¹⁵N-labeled fertilizer to the roots. Leaf position 1 (first leaf) is the youngest leaf, and leaf position 6 (sixth leaf) is the oldest leaf of the plant. Columns marked with the same letter are not statistically different at P ≤ 0.05 by the least significant difference test. Bars indicate sem; n = 6.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.229

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Dry weight, nitrogen (N) concentration, N content, ¹⁵N concentration, and ¹⁵N content excess of leaves in Phalaenopsis Sogo Yukidian ‘V3’ 8 weeks after application of ¹⁵N-labeled fertilizer to the roots. Leaf position 1 (first leaf) is the youngest leaf, and leaf position 6 (sixth leaf) is the oldest leaf of the plant. Columns marked with the same letter are not statistically different at P ≤ 0.05 by the least significant difference test. Bars indicate sem; n = 6.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.229

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Dry weight, nitrogen (N) concentration, N content, ¹⁵N concentration, and ¹⁵N content excess of leaves in Phalaenopsis Sogo Yukidian ‘V3’ 8 weeks after application of ¹⁵N-labeled fertilizer to the roots. Leaf position 1 (first leaf) is the youngest leaf, and leaf position 6 (sixth leaf) is the oldest leaf of the plant. Columns marked with the same letter are not statistically different at P ≤ 0.05 by the least significant difference test. Bars indicate sem; n = 6.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.229

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The concentration of ¹⁵N was highest in first leaves (1.082 atom% excess) followed by second leaves [0.803 atom% excess (Fig. 3D)]. Compared with first and second leaves, the third to sixth leaves had lower ¹⁵N concentration (0.359 to 0.246 atom% excess), and the differences from third to sixth leaves were small (Fig. 3D). First leaves and second leaves had the highest ¹⁵N content excess [257 and 230 μg, respectively (Fig. 3E)]. Third leaves had higher ¹⁵N content excess (75 μg) than fourth to sixth leaves [15 to 36 μg (Fig. 3E)].

Expt. 4: Absorption and partitioning of N by Phalaenopsis plants during vegetative and reproductive phases.

Whole-plant dry weight increased from 10.5 g in Week 1 to 22.4 g in Week 38 (Fig. 4A). The increase in dry weight was mainly the result of the increase in the dry weight of newly grown leaves and the newly grown inflorescence; root dry weight increased only slightly, whereas a slight decrease was observed in the dry weight of mature leaves (Fig. 4A). The plants began to spike in Week 16 with inflorescence dry weight of 0.09 g. By Week 38, the inflorescences were in a stage where half of the blooms were open, and the dry weight of the stalk had increased to 3.9 g while the dry weight of the flower was 1.7 g (Fig. 4A).

Changes in dry weight, nitrogen (N) concentration, N content, ¹⁵N concentration, and ¹⁵N content excess of newly grown leaves, mature leaves, roots, stalk, and flowers in Phalaenopsis Sogo Yukidian ‘V3’ from the vegetative growth stage through to the reproductive growth stage after a single application of ¹⁵N-labeled fertilizer at Week 0. The plants shifted from the vegetative growth stage to the reproductive stage at approximately Week 16 when they began to spike. Bars indicate sem; n = 4 for samples collected at Weeks 1 to 16, n = 3 for samples collected at Week 38, and n = 1 for stalk sample at Week 16.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.229

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Changes in dry weight, nitrogen (N) concentration, N content, ¹⁵N concentration, and ¹⁵N content excess of newly grown leaves, mature leaves, roots, stalk, and flowers in Phalaenopsis Sogo Yukidian ‘V3’ from the vegetative growth stage through to the reproductive growth stage after a single application of ¹⁵N-labeled fertilizer at Week 0. The plants shifted from the vegetative growth stage to the reproductive stage at approximately Week 16 when they began to spike. Bars indicate sem; n = 4 for samples collected at Weeks 1 to 16, n = 3 for samples collected at Week 38, and n = 1 for stalk sample at Week 16.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.229

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Changes in dry weight, nitrogen (N) concentration, N content, ¹⁵N concentration, and ¹⁵N content excess of newly grown leaves, mature leaves, roots, stalk, and flowers in Phalaenopsis Sogo Yukidian ‘V3’ from the vegetative growth stage through to the reproductive growth stage after a single application of ¹⁵N-labeled fertilizer at Week 0. The plants shifted from the vegetative growth stage to the reproductive stage at approximately Week 16 when they began to spike. Bars indicate sem; n = 4 for samples collected at Weeks 1 to 16, n = 3 for samples collected at Week 38, and n = 1 for stalk sample at Week 16.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.229

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Among the various organs, newly grown leaves had the highest N concentration, which increased from 2.37% in Week 1 to 2.71% in Week 16 but decreased after flowering to 2.41% in Week 38 (Fig. 4B). Nitrogen concentration in roots increased from 1.67% in Week 1 to 2.47% in Week 38 (Fig. 4B). In mature leaves, N concentration was lowest, increasing from 1.70% in Week 1 to 2.09% in Week 16. As is the case with newly grown leaves, N concentration in mature leaves decreased after flowering, from 2.09% in Week 16 to 1.84% in Week 38 (Fig. 4B). Nitrogen concentration in newly emerging flower stalk in Week 16 was high at 3.43% but decreased after flowering so that by Week 38, the stalk contained 2.19% N, whereas N concentration was lower in flowers in comparison at only 1.43% (Fig. 4B).

Nitrogen content of mature leaves remained relatively stable from Week 1 to Week 38 [44.2 to 62.2 mg (Fig. 4C)]. As is the case with the dry weight of newly grown leaves, the N content of newly grown leaves increased as the experiment progressed, from 43.3 mg in Week 1 to 161.0 mg in Week 38 (Fig. 4C).

After application of ¹⁵N-labeled fertilizer at Week 0, the ¹⁵N concentration in roots, mature leaves, and newly grown leaves of the plants increased gradually. By Week 1, the ¹⁵N concentration in mature leaves was only 0.08 atom% excess, which was lowest among the organs. The increase in ¹⁵N concentration in mature leaves was comparatively small, reaching only 0.65 atom% excess in Week 16 with no further difference by Week 38 at 0.62 atom% excess (Fig. 4D). Changes in ¹⁵N concentration in roots and newly grown leaves were similar in the first 8 weeks. In roots, it increased substantially from 0.42 atom% excess in Week 1 to 1.48 atom% excess in Week 8, but decreased afterward to only 1.24 atom% excess in Week 16 (Fig. 4D). In newly grown leaves, it increased from 0.28 atom% excess to 1.61 atom% excess between Week 1 and Week 16, but decreased to 1.27 atom% excess during flowering at Week 38 (Fig. 4D). The concentration of ¹⁵N in newly grown flower stalk at Week 16 was 1.53 atom% excess, which was similar to that of newly grown leaves at 1.61 atom% excess (Fig. 4D). At Week 38, ¹⁵N concentration of the flower stalk decreased to 0.92 atom% excess, and a similar ¹⁵N concentration was found in the flowers at 0.87 atom% excess (Fig. 4D).

The ¹⁵N content excess of the whole plant had an increasing trend throughout the experimental period, but the rate of increase decreased gradually over time (Fig. 4E). The ¹⁵N content excess in roots and newly grown leaves increased to a larger extent compared with mature leaves (Fig. 4E).

Plants of Phalaenopsis Sogo Yukidian ‘V3’ can absorb nutrients through both their leaves and roots.

Our finding from Expt. 1 shows that foliar absorption of nutrients by Phalaenopsis plants is not directly related to stomatal density. Numerous studies have been conducted to elucidate the mechanism of foliar absorption of nutrients in various plant species, which is now known to occur primarily through the cuticular region (Fernandez and Eichert, 2009) facilitated by porous regions called ectodesmata (Haynes and Goh, 1977; Kannan, 1986). Phalaenopsis have more stomata on the lower leaf surface (Lee and Lee, 1991). Our observation that lower and upper leaf surfaces of Phalaenopsis had similar absorption efficiency of fertilizer N (Tables 1 and 2) despite the difference in stomatal density is in agreement with the hypothesis that foliar absorption is mostly through the cuticular region.

The absorption efficiency of Phalaenopsis leaves was less than that of the roots and fertilizer N absorption of the leaves was only approximately half that of the roots (Table 2). We also found that fertilizer N was more efficiently translocated when it was applied to roots than when it was applied to leaves. The ratio of ¹⁵N remaining at the site of application to the total ¹⁵N absorbed by the plant was much higher when fertilizer N was applied to the leaves {56% [i.e., (19.1/34.1) × 100%] and 56% [i.e., (14.1/25.2) × 100%]} than when it was applied to the roots {between 22% [i.e., (13.1/60.3) × 100%] and 35% [i.e., (29.3/82.8) × 100%]; calculated from Table 2}. The higher proportion of retained fertilizer N in leaves to the total absorbed N indicates that compared with roots, leaves are less efficient in releasing the absorbed N for translocation to distant organs.

Temporary storage of N likely occurs at the site of fertilizer application. From Expt. 1 we found that 4 and 8 weeks after ¹⁵N-labeled fertilizer was applied to the roots or leaves, the sites of fertilizer application consistently had the highest ¹⁵N concentrations. One possible explanation to this observation is that the bulk of the absorbed N was stored temporarily in the leaves or roots to which the fertilizer was applied. Another possibility is that labeled fertilizer residue, which could not be removed despite washing during the sampling procedure but had not been absorbed into the living tissue either, accounted for the higher ¹⁵N concentration at the sites of application. The first explanation is more probable because the ¹⁵N concentration at the sites of fertilizer application decreased from Week 4 to Week 8 (Table 1).

During cultivation, it is not feasible to maintain a film of moisture on leaf surfaces, whereas the roots are by necessity in contact for extended periods with moisture in the substrate. Thus, it is more feasible and logically sound to apply the fertilizer solution to the substrate rather than to the leaves. This is in agreement with the concept that foliar fertilization of crops is generally not sufficient and only supplemental in nature (Fritz, 1978). Wang (2010) reported that foliar fertilization alone was insufficient for obtaining optimal vegetative growth and flowering of Phalaenopsis. Based on this, we conclude that fertilizer N should mainly be applied to the roots of potted Phalaenopsis plants.

One very useful application of foliar fertilization is when irrigation or fertigation is withheld from potted Phalaenopsis plants. Drier conditions at the root system are to be maintained after repotting or before shipping to avoid diseases and are achieved by withholding irrigation or fertigation for a few weeks when using sphagnum moss as the substrate. It is during this period that foliar fertilization should prove useful for supplying the nutrient requirements of the plants.

Significance of stored N pool in Phalaenopsis.

By tracing the fate of fertilizer N with ¹⁵N-labeling in Expt. 2, we determined the contribution of fertilizer N to the N accumulated in actively growing Phalaenopsis leaves. The level of enrichment in the fertilizer solution was 22.5 atom% ¹⁵N, corresponding to 23.7% ¹⁵N excess by weight out of a total N in the labeled fertilizer, whereas ¹⁵N content excess in the first leaf was 117.9 μg on Day 8 (Fig. 2B); therefore, the amount of fertilizer N accumulated in the first leaf by Day 8 was 0.50 mg (i.e., 0.1179 mg/23.7%). Nitrogen content in the first leaf increased from 18.2 mg to 22.5 mg from Day 0 to Day 8 (Table 3), which was an increase of 4.3 mg. Comparing fertilizer N accumulation with the total N accumulated in the first leaf, we found that the contribution of fertilizer N to the increase of N content in the first leaves over the 8-d experimental period was only 12% (i.e., 0.50 mg/4.3 mg). Therefore, despite availability and evident absorption of N at the root zone, a significant amount of N is sourced from elsewhere to meet the requirements of actively growing Phalaenopsis leaves, presumably from storage in older tissues.

Leaf N concentration of the plants used in Expt. 2 ranged from 1.09% to 1.96% (Table 3). This range is within a normal range of fertility, because Wang and Konow (2002) reported a N concentration range of 1.10% to 1.60% in the leaves of Phalaenopsis plants fertilized with 200 mg·L⁻¹ N. Therefore, translocation of N still occurs even when Phalaenopsis plants are not in a N-deficient stage. In Phalaenopsis, not only is N readily transported from roots to the shoot (Expts. 1 and 2), but N is also transported from the leaves to roots (Expt. 1). This highly fluid movement of N agrees with the high mobilization rate of nutrients in epiphytic orchids suggested by Hew and Yong (2004).

The leaves of Phalaenopsis have N storage function.

Nitrogen-15 concentration data in Expts. 2 and 3 show a significant difference. In Expt. 2, first leaves and fourth leaves had very similar ¹⁵N atom% excess during the 8-d period after fertilizer application (Fig. 2A). By contrast, in Expt. 3 the fourth leaves had a much lower ¹⁵N atom% excess compared with first and second leaves 8 weeks after fertilizer application (Fig. 3D). Hence, accumulation of fertilizer N occurred in the fourth leaves shortly after fertilizer application, but after a longer-term period, the stored N was translocated elsewhere. This observation suggests that older leaves of Phalaenopsis can act as temporary storage for fertilizer N shortly after fertilizer application.

Based on the results of Expt. 4, the concentrations of both N and ¹⁵N decreased in Phalaenopsis leaves as the plant shifted from vegetative to reproductive stages (Figs. 4B and 4D). In newly grown leaves, this decrease could be the result of a dilution effect as more dry weight was accumulated. However, in mature leaves, the likely cause was the translocation of N out of the mature leaves, because there was no significant change in the dry weight of mature leaves (Fig. 4A).

Our finding thus indicated that the mature leaves of Phalaenopsis can store N and translocate it for future use, thus serving the function of N source. The cactus pear (Opuntia ficus-indica), a CAM crop that has strong buffering capacity to fertilizer applications, has a high water content and stores a large nutrient pool in the plant (Felker and Bunch, 2009). Nitrate can be stored in the cladode (photosynthetic organ that constitutes the aboveground portion of the cactus pear plant) during periods when excess nitrate is available or during periods of slow growth, to be used later (Nerd and Nobel, 1995). Our results obtained from different experiments in this study strongly indicate the capability of mature leaves to store N for later use in Phalaenopsis, which is also a CAM plant (Hung, 2012).

Phalaenopsis roots likely have the function of N storage.

In Expt. 1, the ¹⁵N concentration in roots receiving ¹⁵N treatment decreased significantly (P ≤ 0.01 by paired t test) between Week 4 and Week 8 after treatment (Table 1), showing that translocation of ¹⁵N out of the roots took place several weeks after a single application of labeled fertilizer. In Expt. 4, out of the whole-plant ¹⁵N content excess at 1, 2, 4, 8, 16, and 38 weeks after the application of ¹⁵N-labeled fertilizer, the root ¹⁵N content excess constituted 69%, 63%, 59%, 51%, 45%, and 35%, respectively (calculated from Fig. 4E); in other words, the proportion of ¹⁵N in the roots out of the total ¹⁵N absorbed was continually decreasing, demonstrating the likelihood of translocation of previously stored ¹⁵N from the roots.

Between Week 8 and Week 16 in Expt. 4, the dry weights of roots, mature leaves, and newly grown leaves changed little, but significant amounts of N were accumulated in these organs, and the rates of accumulation were similar (Fig. 4A–C). The bulk of the N accumulated in these organs (i.e., roots, mature leaves, and newly grown leaves) probably came from recently absorbed fertilizer N, because regular Johnson’s solution was applied regularly throughout the experimental period. At the same time, our data suggest that ¹⁵N from the labeled fertilizer was still present in the substrate and was still absorbed and accumulated, because whole-plant ¹⁵N content excess was still increasing between Week 16 and Week 38 (Fig. 4E). Considering the roots in particular, we can infer that accumulation of newly absorbed ¹⁵N in the root tissue still took place between Week 8 and Week 16; this is because the roots still accumulated fertilizer N as shown by the increase in N content despite a lack of change in dry weight (Figs. 4A and 4C), whereas part of this recently absorbed N should be in the form of ¹⁵N.

Although the accumulation of newly absorbed ¹⁵N in roots between Week 8 and Week 16 can be inferred from this, ¹⁵N content excess in roots remained at the same level during this period, indicating that some ¹⁵N previously stored in the root must have been translocated elsewhere. The decrease in ¹⁵N concentration in roots between Weeks 8 and 16 while ¹⁵N concentration increased in the other organs (Fig. 4D) is consistent with outward translocation of previously stored ¹⁵N from the roots. The aforementioned observations from Expts. 1 and 4 indicate that Phalaenopsis roots are likely to have the function of N storage.

Young actively growing tissues in Phalaenopsis show strong sink activity for N.

In Expt. 1, we found that regardless of site of application of the labeled fertilizer, newly grown leaves, which were not application sites of fertilizer for all treatments, consistently received the highest allocation of the fertilizer translocated from the application site; between 40% and 46% of the translocated ¹⁵N was allocated to the newly grown leaves [(4.5/11.2) × 100%, (6.9/15.0) × 100%; calculated from Table 2]. This result shows that among all organs during the vegetative growth stage, newly grown leaves had the highest sink strength for N. Furthermore, results from Expt. 3 show that sink strength of Phalaenopsis leaves decreases with increasing leaf age, which is in agreement with what has been stated above.

We also found that fertilizer N applied to leaves was still translocated to roots (Table 1). Hence, from Expt. 1 we can see that if we isolate the roots out of their role as a conduit of nutrients from substrate to shoot, the roots by themselves show sink activity for N; and just as is observed in leaves where the actively growing young leaves show higher sink activity than mature leaves (Tables 1 and 2; Fig. 3), actively growing young roots show higher sink activity than older roots (Tables 1 and 2). This is not surprising, because roots as living organs have basic metabolism, which requires N; this requirement is especially high in young roots, which have high metabolic activity.

Results of Expts. 1, 3, and 4 indicate that during the vegetative stage, newly grown roots and newly grown leaves are strong sink organs for N in Phalaenopsis (Table 2; Figs. 3 and 4), whereas stalks and flowers are strong sinks during the reproductive growth period (Fig. 4). Newly grown roots, newly grown leaves, and reproductive organs are all organs with high metabolic activity, whereas metabolism requires N. Therefore, actively growing tissues with high metabolic activities are to be expected to have high N requirements. Similar results were found in blueberry, where the current season’s stems and leaves receive the highest allocation of fertilizer N (Retamales and Hanson, 1989).

Fertilizer application thus has a long-term effect on Phalaenopsis growth.

Plants received a single application of labeled fertilizer N in Expt. 4. Throughout the 38-week period, the plant still accumulated fertilizer N that had been applied at the beginning of the period (Fig. 4E). This result provides evidence of the high nutrient-holding capacity of the sphagnum moss substrate, which has also been demonstrated by the work of Yao et al. (2008).

Storage and partitioning of N have been shown with ¹⁵N-labeling in other crops. In fruit trees, the woody trunk and roots are major storage organs for N. This storage function of woody tissues can perhaps be intuitively reasoned in the case of deciduous fruits, but it has been definitively shown with ¹⁵N-labeling in peach trees, the old woody tissues of which provide the bulk of N during fruit set in spring, whereas before leaf fall they receive translocation of N from the foliage (Munoz et al., 1993). In greenhouse roses, a perennial crop with shorter cycles of growth and harvest, the N stored in old stems and foliage significantly contributes to the N requirement of new flowering shoots (Cabrera et al., 1995). In some orchids that have pseudobulbs, the pseudobulbs have been shown to serve the function as nutrient and water storage (Ng and Hew, 2000).

We have here shown with ¹⁵N-labeling, the capability of the leaves of Phalaenopsis to store N. This ability may be coupled with ability to act as a reservoir of water as indicated by the water storage capacity of lower leaves of Phalaenopsis (Hung, 2012) and perhaps as a reservoir of other types of minerals and nutrients as well. Owing to the nutrient storage function in tissues and high nutrient-holding capacity of sphagnum moss substrate, fertilizer application thus has a long-term effect on Phalaenopsis growth.

In conclusion, although Phalaenopsis can absorb fertilizer N through both its roots and leaves, the roots have better absorption efficiency and translocate the absorbed N more readily compared with leaves. Nitrogen is a mobile nutrient. In many plants, N deficiency will result in transport of N from older tissues to young tissues. So the translocation of N is a common phenomenon. What is particular about Phalaenopsis is the fluidity with which N is translocated, even when N supply is not limiting. We have provided from these experiments numerical data indicating the presence of a significant N pool in Phalaenopsis tissues. A significant storage pool of various nutrients probably accounts for the great resilience of Phalaenopsis against nutrient deficiency stress in general.