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. 2020 Jun 5;11:663. doi: 10.3389/fpls.2020.00663

FIGURE 4.

FIGURE 4

Biological/molecular phenotype and Raman spectral analysis of early nitrogen deficiency in leafy vegetables, Pak Choi and Choy Sum. Two-week-old seedlings of Pak Choi (Brassica rapa chinensis) and Choy Sum (Brassica rapa var. parachinensis) were transferred into +N or –N hydroponic medium and grown for 5 days. (A) Morphological phenotype, scale bar, 1 cm. n = 8 (biologically independent experiments). (B) Total chlorophyll content of leaf No. 4 samples was analyzed in +N and –N plants. Data are mean values, n = 8 (biologically independent experiments) and individual data points are shown as overlays. Asterisks indicate statistically significant difference compared with + N. *P < 0.05, **P < 0.01, and ***P < 0.001; two-tailed t-test. FW; fresh weight. P values are shown in Supplementary Table S2. (C) Nitrate content of leaf No. 4 samples was analyzed in +N and –N plants. Data are mean values, n = 12 (biologically independent experiments) and individual data points are shown as overlays. Asterisks indicate statistically significant difference compared with + N. *P < 0.05, **P < 0.01, and ***P < 0.001; two-tailed t-test. FW; fresh weight. P values are shown in Supplementary Table S2. (D) ORE1 orthologous gene transcript levels were analyzed by qRT-PCR in leaf No. 4 samples of Pak Choi and Choy Sum grown under +N or –N condition for 5 days. Data are mean values, n = 12 (biologically independent experiments) and individual data points are shown as overlays. Asterisks indicate statistically significant difference compared with +N. *P < 0.05, **P < 0.01, and ***P < 0.001; two-tailed t-test. Supplementary Tables S1, S2 show primer sets and P values, respectively. (E,F) Leaf No. 4 samples from +N and -N plants were measured by Raman spectroscopy. Only the 1046 cm–1 of Raman shift (cm–1) is shown. Data are mean values, n = 12 (biologically independent experiments) and individual data points are shown as overlays. Asterisks indicate statistically significant difference compared with +N. *P < 0.05, **P < 0.01, and ***P < 0.001; two-tailed t-test. P values were shown in Supplementary Table S5. a.u; arbitrary unit.