| Tomato (Lycopersicon esculentum Mill.) is one of the most popular vegetables with the largest planting areas in China and its growth and development are intimately depending on the supply of nitrogen (N). Since the mineralization and nitrification are occurring in soil, nitrate (NO3-) and ammonium (NH4+) are always mixed existed and unevenly distributed in soils. Previous reports showed that an optimal ratio of NO3" to NH4+ always enhanced the growth and N uptake of tomato, and N use efficiency was improved. However, the mechanism of enhancement of tomato growth by mixed supply of NO3- and NH4+ is still not clear. Thus, in order to deeply investigate the effects of NO3- and NH4+ nutrition on the growth and N metabolism of tomato (Variety'Xinpinbaoguan') seedlings, in the present work, a whole-root system and a split-root system were adopted simultaneously, which simulated homogeneous and uneven supply of NO3- and NH4+, respectively. In addition, the methods of extraction and purification of 6 endogenous cytokinin (CTK) components and indole-3-acetic acid (IAA) in plant tissue, and the determination by high performance liquid chromatography (HPLC) were established. The main results are shown as in follows.1. The effects of different N forms on the growth and root morphology of tomato seedlings were studied both under a homogeneous and a localized supply of NO3" and NH4+ nutrition. The results showed that:(1) In comparison to the sole NO3- nutrition (100NA), homogeneous supply of 75%NO3" plus 25%NH4+(75NA) to the root zone led to the maximum shoot and root dry matter (DM), while 50%NO3- plus 50%NH4+(50NA) supply significantly depressed the growth of shoot and root, and the sole NH4+nutrition led to the least shoot and root DM. Spatially separated supply of NO3- and NH4+ (N|A) resulted in an insignificant difference in shoot DM while a significant higher total root DM compared with sole NO3" supply (N|N). However, when the sole NO3- was replaced by the mixture of NO3- and NH4+ nutrition at the ratio of 75:25 (N|NA), the shoot and total root DM was not significantly affected. (2) In a homogenous supply, with the decrease of ratio of NO3- to NH4+, the root total length (RL) and root surface area (RS) declined significantly. In localized supply, however, compared with the control (N|N), spatially separated supply of sole NH4+ in N|A treatment led to a significant increase in the average of root diameter (AD) of the root exposed to the single NH4+ nutrition and also a significant increase in RV and RS of the other half root which was exposed to the sole NO3". However, a localized supply of mixed NO3- and NH4+ nutrition at the ratio of 75:25 in N|NA treatment did not bring about any significant increase in the root morphology parameters when compared with the control.2. Different N forms and culture mode brought about different effects on the concentration and accumulation of nutrient elements in different parts of tomato seedlings. Under both homogeneous and localized supply, the concentration of total N in flower was higher than those in leaf and root and they are all higher than that in stem. In a whole-root system, with the increase of NH4+/NO3- ratio in nutrient solution, the concentration of total N increased both in leaf and stem. The mixed supply of NO3- and NH4+ nutrition at the ratio of 75:25 led to a significant increase in the concentration of phosphorus (P) and potassium (K) in root. In a split-root system, however, when compared to the controle (N|N), localized supply of sole NH4+(N|A) significantly decreased the concentration of total N both in flower and stem, but increased the concentration of total N in the root which was exposed to sole NH4+, also it decreased the concentration of P and K in both half roots in N|A treatment. Localized supply of either sole NH4+ or the mixture of NO3- and NH4+at the ratio of 75:25 both decreased the concentration of calcium (Ca) and magnesium (Mg) in all parts of tomato plants but increased the concentration of sulphur (S) in leaves. With the increase of NH4+/NO3- ratio in nutrient solution in a homogeneous supply, the concentration of boron (B) and manganese (Mn) in leaves and Zinc (Zn) and molybdenum (Mo) in leaves and root all increased. Local supply of sole NH4+ in solution significantly increased the concentration of Fe, B, Zn, Cu and Mo in root. In addition, the mixed supply of NH4+/NO3-led to a significant increase in N, P and K accumulation in the plants no matter what culture mode was.3. Under two culture modes, the effects of N forms on the uptake of NO3- and NH4+ and distribution in tomato seedlings were studied by the use of 15N tracer method. With the increase of NH4+ ratio in nutrient solution, the amount of net 15NH4+ uptake was not affected but the amount of net 15NO3- uptake was significantly decreased. Compared with sole NO3- supply in whole-root system as 100NA and also in a split-root system as N|N, mixed nutrition to 22% increase in the amount of net 15N uptake no matter what culture mode was and the absorbed N was mainly distributed in leaves. Compared with 100NA, When NO3" was replaced by 50% NH4+ (50NA), it resulted in a significant decrease in the accumulation of 15N in root and no significant difference in leaf and stem. However, localized supply of NH4+ in N|A resulted in a significant decrease in the accumulation of 15N in NH4+-fed root but a significant increase in that of 15N in NO3--fed root. Therefore, no significant difference was found in the total accumulation of net 15N in leaf, stem and the root.4. Under a homogeneous supply, with the increase of NH4+ in nutrient solution, nitrate reductase (NR) activity was significantly decreased in both leaves and root but glutamine synthetase (GS) activity was increased in leaves.75NA treatment led to the highest GS activity in root. When spatially separately supplied by NO3- and NH4+, the activity of NR in leaves and the root which was exposed to the sole NH4+ were significantly decreased, and GS activity was significantly increased both in shoot and the root exposed to sole NH4+. In a whole-root system, comparing with the treatment of 100NA,75NA treatment resulted in an increase of soluble carbohydrate in shoot and also an increase in soluble protein both in shoot and root, whereas 50NA treatment showed no significant difference either in the shoot or in the root. However, with the same ratio of NO3- to NH4+ at 50:50 in a split-root system, the concentration of soluble carbohydrate and protein were found to be increased in the leaves and roots, no matter what N form was supplied.5. The expression patterns in transcriptional levels of genes which were involved in N uptake (LeNRT1.2, LeAMT1.1 and LeAMT1.2) and those related to N reduction and assimilation (LeNR,LeGS and LeGSH) in tomato seedlings were analyzed in tomato roots by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). LeNRT1.2 (accession number:X92852) is a putative low affinity nitrate transporter gene and can be inducible. Comparing with the expression of LeNRT1.2 in the root which was exposed to sole NO3- (100NA), homogeneous supply of 75% NO3- plus 25% NH4+ led to no effects in the expression of LeNRT1.2 whereas 50% NO3" plus 50% NH4+ resulted in a significant decrease in the expression of this gene. The tendency of LeNRT1.2 expression patterns was in accordance with that of net uptake of NO3- in tomato plants. However, with the same ratio of NO3- to NH4+ at 50:50 in the split-root system (N|A), comparing with the treatment of N|N, the expression of root LeNRT1.2 and LeAMTl.1 (accession number: X92854) was significantly depressed by sole NH4+ supply. It was found that there was no effect on the expression of LeAMTl.1 by N forms in homogeneous supply. LeAMT1.2 (accession number:X95098) gene was also found to be hardly affected by N forms and culture mode, which was in agreement with no difference in net NH4+ uptake no matter what N form was supplied.The expression of LeNR (accession number:X14060) was depresssed with the increase of NH4+ in nutrient solution in a homogeneous supply.In a split-root system, however, in comparison to N|N treatment, the expression of LeNR was significantly depressed in the root which was supplied by sole NH4+ but was significantly increased in the opposite root which was supplied with sole NO3- (N|A). Spatially separately supplied by sole NO3'and the mixture of 75% NO3- plus 25% NH4+(N|NA) led to a significant decrease in the expression of LeNR in both half roots. Both in a homogeneous and a localized supply, it was found that the expression of LeNRT1.2 and LeNR showed the same changing tendency and there was no significant difference in the expression of LeGS (accession number:LEU 15059) and LeGSH (accession number:AF017984) in tomato no matter what N form was supplied.6. The concentrations of 6 endogenous CTKs components and IAA in xylem sap were determined by HPLC. The results showed that, regardless of the culture mode, a close negative correlation was found between the concentration of NH4+ and total CTKs and zeatin riboside (ZR) in xylem sap. Dihydrozeatin and dihydrozeatin riboside (ZR+(diH)ZR) were the dominant CTK fraction in xylem exudates and accounted for up to 60-80% of total CTKs.75NA and N|A treatments both showed a higher level of ZR+(diH)ZR concentration compared to the other treatments in the respective culture mode. Regardless of the culture mode, a close negative correlation was found between the concentration of NH4+ and total CTKs in xylem sap (r=-0.951*, n= 6, ro.oi,4= 0.917), and ZR (r=-0.963*, n= 6, ro.05,4= 0.811). However, a significantly positive correlation was found between the xylem-NO3-and zeatin (ZT), in which the coefficient was 0.879* (n= 6, ro.05,4= 0.811) irrespective of culture mode. In the homogeneous supply, with the increase of NH4+ ratio in nutrient solutuion, the total concentrations of CTKs and IAA in xylem sap were significantly decreased during 24 h. In the localized supply, however, either the sole NH4+ supply or the mixed NO3- and NH4+ nutrition both led to a significant decrease of the total concentration of CTKs and IAA during 24 h, but there was no significant difference in the total accumulation of CTKs and IAA during 24 h between the sole NH4+ supply and the mixed NO3- and NH4+ nutrition.In summary, the results showed above indicated that whether a combined supply of NO3- and NH4+ was superior to either the sole NO3- or NH4+ nutrition or not was depending on the existence of NO3- and the ratio of NH4+ to NO3-. Generally speaking, NO3- was the main N form for tomato plant to uptake from the combined supply of NO3- and NH4+ but when the ratio of NH4+ to NO3- exceeded 25%, the activity of ZR+(diH)ZR in root and leaves and the expression of LeNRT1.2 and LeNR all significantly decreased, and then the efficiency of N absorption and utilization was decreased. An optimal ratio of NO3- to NH4+ (NO3-:NH4+=75:25) benefited the growth in that it could maintain a higher level of ZR+(diH)ZR in xylem exudation, thereby it could regulate the allocation of carbohydrate between the root and shoot, and then improve the root morphology and enhance the root growth, which was beneficial to the absorption and utility of N and other elements. |
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