Effect Of Nitrogen Forms And Abiotic Stress On Tea Plant Physiological Characteristics And Nitrogen Metabolism

Nitrogen,one of the most important elements that are vital to plant growth,is more urgently needed for tea,an economic crop for leaf use.The entire nitrogen metabolism of plants not only has an important influence on plant growth and development,but also can regulate the stress resistance of plants.In this study,effects of different nitrogen forms and nitrate stress on the physiological characteristics and nitrogen metabolism of tea plants was explored,which can provide useful information for the rational fertilization in tea plantation,the improvement of nitrogen use efficiency and the researches on stress resistance of tea plants.Results are as follows:1.Cutting seedlings from annual Shuchazao were applied as the experimental materials to measure the photosynthesis in tea leaves under the treatments of distinct nitrogen forms by the Li-6400 photosynthetic instrument.Results showed that the net photosynthesis rate?Pn?under the treatment at the NH₄⁺-N/NO₃^--N ratio of 3:1 didn't have significant differences when compared with that at the NH₄⁺-N/NO₃^--N ratio 2:2,both of which showed outstandingly higher level than those under treatments of only ammonium,only nitrite and at the NH₄⁺-N/NO₃^--N ratio of 1:3.The highest stomatal conductance?Cond?of tea leaves under the treatment of only ammonium didn't differ notably from those under the treatments at the NH₄⁺-N/NO₃^--N ratio of 3:1 and 2:2,but it showed distinct differences in comparison with those under the treatments of only nitrite and at the NH₄⁺-N/NO₃^--N ratio of 1:3.The transpiration rate?Tr?of tea leaves under the treatment at the NH₄⁺-N/NO₃^--N ratio of 3:1 didn't differ notably from those under the treatments of only ammonium,and at the NH₄⁺-N/NO₃^--N ratio of 2:2,but compared with those under the treatments of only nitrite and at the NH₄⁺-N/NO₃^--N ratio of 1:3,it showed evidently differences.In conclusion,the photosynthesis rates of tea leaves under the treatments at the NH₄⁺-N/NO₃^--N ratio of 3:1 and 2:2 were stronger.2.Under the treatments of different nitrogen forms,by applying the national standard methods,cutting seedlings from annual Shuchazao were applied as the experimental materials to detect the content of the water extract,amino acids,tea polyphenols and the caffeine.Under the treatment of only ammonium,the highest amino acids content in tea leaves was 3.97%,which differed not notably from those under the treatments at the NH₄⁺-N/NO₃^--N ratio of 3:1 and 2:2 but showed distinct differences in comparison with those under the treatments of only nitrite and at the NH₄⁺-N/NO₃^--N ratio of 1:3.Under the treatment of only ammonium,tea polyphenols content was evidently higher than those under other treatments of nitrogen forms.The second highest tea polyphenols content existed in the tea leaves under the treatment at the NH₄⁺-N/NO₃^--N ratio of 3:1,which was much higher than those under the treatments of only nitrite and at the NH₄⁺-N/NO₃^--N ratio of 1:3 and 2:2?that content of these three treatments showed no outstanding differences?.The content of the water extract and the caffeine didn't differ significantly among tea leaves under the treatments of different nitrogen forms.To summarize,tea leaves under the treatment of only ammonium were indicated to own the best tea quality,followed by those under the treatments with the NH₄⁺-N/NO₃^--N ratio of 3:1,2:2,only nitrite and at the NH₄⁺-N/NO₃^--N ratio of 1:3.3.Total RNA was extracted as the templates from the second leaves of Shuchazao under the treatments of diverse nitrogen forms to detect the gene expressions related to the regulation of the nitrogen metabolism with the Fluorescent Quantitative PCR.The expressions of AMT3;1 gene in tea leaves under the treatments at the NH₄⁺-N/NO₃^--N ratio of 2:2 and 3:1 didn't have notable difference?the former was slightly higher than the latter?,which were outstandingly higher than those under other treatments of nitrogen forms.The expressions of NRT1;2 gene showed negative correlation to the NH₄⁺-N/NO₃^--N ratio in the treatments of diverse nitrogen forms,so did the expressions of NR.Under the treatment with a NH₄⁺-N/NO₃^--N ratio of 2:2,GS1;1 gene and GS1;2 gene reached the highest level of expression?significantly higher than those under other treatments?,followed by those under the treatments with a NH₄⁺-N/NO₃^--N ratio of3:1?notable higher than those under the latter treatments?,only ammonium,only nitrite and the treatment with a NH₄⁺-N/NO₃^--N ratio of 1:3.The expressions of GOGAT gene and GDH gene increased significantly with the increase of NH₄⁺-N/NO₃^--N ratio except for the treatments of only ammonium,of which these expressions were lower than others.The content of nitrogen in tea leaves under the treatment with a NH₄⁺-N/NO₃^--N ratio of 3:1was 4.42%,which reached the highest level outstandingly higher than those under other 4treatments?mutually had no significant difference?.Though the treatment with a NH₄⁺-N/NO₃^--N ratio of 3:1 didn't contribute more than the treatment with a NH₄⁺-N/NO₃^--N ratio of 2:2 in the GS/GOGAT ammonium assimilation pathway,it played a more important role in the promotion of GDH ammonium assimilation pathway when compared with the latter.Thus,among the treatments of diverse nitrogen forms,the treatments with a NH₄⁺-N/NO₃^--N ratio of 3:1 and 2:2 leaded to the better nitrogen metabolism.4.The photochemical efficiency?Fv/Fm?and the photosynthesis rate of tea leaves?from Shuchazao?were mensurated under acid,alkaline,salt and saline-alkali stress respectively with the help of the chlorophyll fluorescence imaging instrument and photosynthesis instrument.Fv/Fm didn't change significantly under acid stress but decreased notably in the 11^th day under alkaline stress.For those under salt or saline-alkali stress,Fv/Fm value went down obviously in the 9^th day.The net photosynthesis rates of tea leaves declined evidently under stresses of acid,alkaline and salt,which was much higher than the rate under saline-alkali stress.Under the saline-alkali stress,Cond and Tr reduced notably under saline-alkali stress while these values didn't changed obviously under the stresses of acid,alkaline or salt.5.Applying tea leaves of Shuchazao under acid,alkaline,salt and saline-alkali stress as the experimental materials,the malondialdehyde?MDA?and soluble sugar?WSS?content was determined through the TCA method and the anthrone-colorimetric method respectively.MDA content reached its highest level?6.41nmol/g?with saline-alkali stress and the lowest level with acid stress,which was indicated to have significant difference mutually under these stresses.With each stress,WSS content in tea leaves raised notably and among these stress,the saline-alkali stress led to the highest value of WSS content?the value was slightly higher than that under salt stress but outstandingly higher than that under alkaline stress?while the acid stress caused the lowest.6.With the help of the Fluorescent Quantitative PCR,the tendency of gene expressions related to the regulation of the nitrogen metabolism was determined by applying the RNA in second leaves of Shuchazao under the stresses of acid,alkaline,salt and saline-alkali as the templates.The expression of AMT gene family reduced with each stress while the expression of NRT1;2 gene showed the decreasing tendency under the stress of salt or saline-alkali stress.Besides,the expression of NRT1;2 gene didn't changed obviously under the stress of acid or alkaline.Under the acid stress,the expression of NR gene showed the raising tendency and under the acid of alkaline,salt or saline-alkali stress it raised for response and then fell down to the control level.Under acid stress,the expressions of GS1;1 and GOGAT didn't change notably while those of GS1;2 and GS1;3showed reducing tendencies.Under the stress of alkaline,salt or saline-alkali stress,the expressions of GS1;1,GS1;3 and GOGAT gene decreased while the expression of GS1;2gene increased.Under each stress above,the expression of GDH showed the tendency of decrease.Under these stress,the activities of GS decreased obviously?with acid stress the activity was 17.49U/g,significantly higher than that with other stresses?and the activity with the stress of alkaline,salt or saline-alkali stress mutually had no outstanding difference.The activity value of GOGAT under acid stress was 98.98 nmol/min/g?not obviously changed?,which was notably higher than that under any other stress above.Besides,the activity value of GOGAT under alkaline stress was slightly higher than that under salt stress but obviously higher than that under saline-alkali stress.The least difference of nitrogen content between the tea leaves under acid stress and the control was1.82%?not significant?,followed by those under alkaline?outstanding higher than the following two stresses?,salt and saline-alkali stress.Although acid stress reduces the GDH ammonium assimilation,it may increase the GS/GOGAT ammonium assimilation by raising the NR activity,thus maintaining the nitrogen metabolism of tea leaves.Alkaline,salt and saline-alkali stress treatments all inhibited the GS/GOGAT ammonium assimilation pathway and GDH ammonium assimilation pathway,but the GS/GOGAT ammonium assimilation pathway was less inhibited under alkaline stress.