Net NO3- and NH4+ flux rate
In our first set of analysis, we found that LT (8℃) treatment significantly depressed the net NO3- flux rate in root hair zones and in the vascular bundle of other detection sites. The net NO3- influx rate in the root hair zone of LT (8℃) treatment was reduced to 19.3% of the NT (26 ℃) treatment. The net NO3- eflux rate in the vascular bundles of main roots, stems, petioles, midribs, lateral veins, and shoot tips were reduced to 36.2%, 11.7%, 11.0%, 21.5%, 7.6%, and 23.1% of the NT (26 ℃) treatment, respectively (Fig. 2). This indicated that low temperature inhibited the uptake and upward transport of nitrate.
However, compared with the net NO3- flux rate, the change of net NH4+ flux rate under LT treatment was different. The net NH4+ influx rate in the root hair zone of LT (8℃) treatment was reduced to 68.7% of the NT (26 ℃) treatment. The net NH4+ eflux rate in the vascular bundles of main roots, stems and petioles under LT (8℃) treatment were reduced to 37.6%, 9.4%, and 14.5% of NT (26 ℃) treatment, respectively (Fig. 3). While the net NH4+ flux rate in the vascular bundles of midribs, lateral veins and shoot tips increased to 160.9%, 303.0%, and 164.1% of NT (26 ℃) treatment, respectively.
Compared with the net NO3- flux rate, the net NH4+ flux rate of detection sites were much lower under NT treatment, but significantly higher in lateral vein and shoot tip under LT treatment. This indicated that the inhibition of net NO3- flux rate at low temperature was more serious than that of net NH4+ flux rate.
N uptake per plant, N concentration and N distribution in different tissues of the seedling
In order to further explore the above phenomenon, the effects of low temperature on the uptake and distribution of NO3- and NH4+ in plants was studied by isotope tracer method. Compared with NT treatment, NO3- -N, NH4+-N and total N uptake per plant under LT treatment decreased 78.1%, 58.8% and 72.6%, respectively (Table 1). After LT treatment, the ratio of NO3--N/total N decreased significantly, while the ratio of NH4+-N/total N increased significantly. This indicated that low temperature decreased NO3- and NH4+ uptake significantly, especially NO3-.
After 5 hours of LT treatment, the NO3--N contents in the root, stem, cotyledon, 1st petiole, 1st blade, 2nd petiole, 2nd blade, and shoot tip were reduced to 33.8%, 13.7%, 15.8%, 6.8%, 18.8%, 8.2%, 14.4%, and 10.6% of NT treatment, respectively. While the NH4+-N contents in the root, stem, cotyledon, 1st petiole, 1st blade, 2nd petiole, 2nd blade, and shoot tip were reduced to 78.3%, 43.0%, 29.7%, 23.4%, 30.4%, 24.6%, 23.7%, and 11.0% of NT treatment, respectively (Fig. 4). This result was consistent with Fig. 2-3.
Table 1 NO3--N, NH4+-N, and total N uptake of per cucumber seedlings exposed to 26℃ and 8℃for 5 h.
Treatment
|
NO3--N uptake
(µmol per plant)
|
NH4+-N uptake
(µmol per plant)
|
Total N uptake
(µmol per plant)
|
NO3--N/total N
(%)
|
NH4+-N/total N
(%)
|
NT(26℃)
|
229.60 ± 12.13 a
|
92.13 ± 6.60 a
|
321.73 ± 18.73 a
|
71.37 ± 3.78 a
|
28.63 ± 1.86 b
|
LT(8℃)
|
50.33 ± 3.00 b
|
37.93 ± 2.33 b
|
88.27 ± 5.33 b
|
57.02 ± 3.36 b
|
42.98 ± 2.53 a
|
Note: Total N refers to NO3--N plus NH4+-N. Values were means ± SE (n = 3). Different lowercase letters indicate significant differences (P < 0.05).
As shown in Fig. 5, exposure of cucumber seedlings to low temperature resulted in a significant increase in not only NO3--N (24.8%) but also NH4+-N (26.0%) distribution proportion in roots. In other words, LT treatment significantly reduced the distribution ratio of NO3--N and NH4+-N in the shoot. This result indicated that low temperature inhibited the transportation of NO3- and NH4+ from root to shoot, and resulted in the accumulation of nitrogen in root.
The distribution proportion of NO3--N and NH4+-N in blade, stem, cotyledon, and other aerial tissues all decreased under LT treatment. Among the detection aerial tissues, the proportion of NO3--N distribution in stems, 1st petioles, 1st blades, 2nd petioles, 2nd blades and shoot tips of LT treatment was 61.8%, 32.7%, 85.4%, 38.2%, 66.9%, and 47.6% of that of NT treatment, respectively. The NH4+-N distribution ratios in stems, 1st petioles, 1st blades, 2nd petioles, 2nd blades, and shoot tips treated with 8℃ was 103.5%, 59.2%, 73.3%, 61.0% 58.7%, and 26.2% of those treated with 26℃, respectively. This indicated that low temperature decreased the NO3--N distribution in petioles, stems and shoot tips more seriously than that in blades. While the effects of low temperature on the NH4+-N distribution in stems and petioles were almost the same, except that the proportion of NH4+-N distribution in stems increased under low temperature.
Gene expression of CsNRTs, CsCLCs,CsSLAHs, and CsAMTs in petioles and midribs
The effects of low temperature on the transcription levels of CsNRTs, CsCLCs, and CsAMTs were shown in Fig.6. The results showed that exposure of cucumber seedlings to low temperature decreased the relative expression of CsNRT1.4a in petioles and midribs, and CsAMT3.3 in midribs, whereas significantly enhanced the expression levels of CsNRT1.1, CsNRT1.3, CsNRT1.7, CsNRT1.8, CsCLCa, CsCLCe, CsAMT1.2c in petioles and midribs, CsNRT1.2b, CsAMT1.2a, and CsAMT1.2b in midribs.
The expression of CsNRT1.2a, CsNRT1.5a, CsNRT1.10, CsCLCc, CsCLCd, CsAMT2, and CsAMT3.3 in petioles and midribs, CsNRT1.2b, CsNRT1.4a, CsNRT1.4b, CsCLCa, CsCLCb, CsAMT1.2a and CsAMT1.2b in petioles were not significantly affected by low temperature (Fig. 6). The relative expression of CsNRT1.2c, CsNRT1.5b, CsNRT1.5c, CsNRT1.9, CsSLAH1~4, CsCLCf, CsCLCg, CsAMT1.1a, and CsAMT1.1b in petioles and midribs of the seedlings were much lower than the genes shown in Fig. 6. So we didn't mention their relative expressions in this article.
NRAmax, NRAact, NiRA in stems and petioles, gene expressions of CsNRs and CsNiR
NR and NiR catalyze the nitrate-to-nitrite and nitrite-to-ammonium reduction process in plants, respectively [10, 39]. NRAmax can reflect the amount of enzyme protein indirectly, and NRAact indicates actual NR activity in situ [40]. After 5 hours LT treatment, NRAmax in roots were depressed significantly (61.5% of NT treatment), while NRAmax in stems, petioles and midribs increased significantly (Fig. 7A). There was no significant difference in NRAmax in blades between the two treatments.
Compared with NT treatment, NRAact in stems and petioles of LT treatment increased by 113.2% and 96.2%, respectively (Fig. 7B). While NRAact in midribs and blades of LT treatment decreased significantly. There was no significant difference in NRAact in roots between NT and LT treatment. Except for midribs, NiR activity in roots, stems, petioles and blades was not significantly decreased by LT treatment (Fig. 7C).
Cucumber sativus has 3 NR family genes (CsNR1, CsNR2, and CsNR3) according to Reda et al. [41]. Among them, the relative expression of CsNR1 in roots were much higher than that in other organs (Fig. 8A). Its expression in roots, stems, petioles, and midribs of cucumber were down-regulated by low temperature significantly. While it’s expression in blades was not affected significantly by LT treatment. The relative expression of CsNR2 in leaves (petioles, midribs and blades) were higher than that in roots and stems, and low temperature up-regulated the expression of CsNR2 in leaves (Fig. 8B). The relative expression of CsNR2 in roots was down-regulated by LT treatment, while the expression in stems was not affected significantly. High expression of CsNR3 was also found in leaves too, especially in blades. The relative expression of CsNR3 in blade of NT and LT treatment was 284 and 355 times higher than that in stems, respectively. LT treatment increased it’s expression in all the detected tissues significantly (Fig. 8C). These results suggested that CsNR1 may be the dominant gene of NR in cucumber roots, and CsNR3 may be the dominant gene in cucumber leaves. CsNR2 and CsNR3 may play a leading role together in stem and petiole.
Compared with the CsNRs, the difference of relative expression of CsNiR in different tissues was small (Fig. 8D). LT treatment enhanced the expression of CsNiR in petioles, midribs, and blades. The highest expression of CsNiR was observed in roots in both NT and LT treatment, but not affected by low temperature.