Distribution And Utilization Characteristics Of Carbon And Nitrogen In Different Nutritional Stages Of Apple Annual Cycle

The study was carried out in guanzhuang village,Yantai city from March to July 2018,the 3th years old GaLa/M9T337 dwarf apple were used as the test material,The ¹⁵N and ¹³C isotope tracer technology was used to study the carbon and nitrogen nutrition distribution and utilization characteristics of apples at different nutrient stages.The characteristics of nitrogen transport and distribution from different sources of M9T337 dwarf rootstocks seedlings and effects of exogenous IAA on apple seedling growth,NO₃^-ion velocity,carbon and nitrogen distribution were carried out in the orchard of Shandong Agricultural University fruit research station from 2018 to 2019,the M9T337 dwarf rootstock seedling was used as a test material,using ¹⁵N and ¹³C isotope tracing technology and non-damaging micro-testing technology,Meanwhile,the expression of nitrogen metabolizing enzyme genes under different nitrogen supply levels was analyzed by using PCR fluorescence quantitative technology.The main results are as follows:1.Study on the characteristics of carbon and nitrogen distribution and utilization in different nutrient stages of apples.Using 3 year old Gala/M9T337 apple as a test material,¹⁵N and ¹³C isotope tracing techniques were used to study the distribution and utilization of carbon and nitrogen in different nutrient stages of apples.The results showed that:In different nutrient stages of the annual cycle,the organs’ability to regulate ¹⁵N differs greatly.During the period of nutrient storage（April 20）,the leaves have the strongest ability to regulate new ¹⁵N absorption,followed by the new organs such as fruits（flowers）and new shoots,the lowest or lower Ndff value in the root system;During the nutrition conversion period（May 20）,the Ndff value of storage organs such as perennial branches and central stems gradually increased;During the use of the new nutrient period（June 20）,the Ndff value of each organ reached a high level.After 30 days of fertilization,the highest Ndff value（0.487%）in fruits.There were significant differences in ¹⁵N distribution rates among different organs at different nutritional stages.The ¹⁵N distribution rate of vegetative organs was the largest during the nutritional period during storage;the ¹⁵N distribution rate of the storage organs was the largest at 20 and 30 days after fertilization during the nutrient conversion period;the ¹⁵N distribution rate of the reproductive organs was significantly increased during the nutritional period during the new period.From the storage nutrition period to the fresh nutrition period,the ¹⁵N utilization rate of the whole plant showed an increasing trend,and the highest was 2.73%after 30 days of fertilization during the fresh nutrition period.The ¹³C distribution rate of each organ at different nutritional stages in the annual cycle is quite different.During the storage nutrition period,the ¹³C distribution rate of leaves is 66.67%,which is significantly higher than other organs,followed by the new one,and the root system ¹³C distribution rate（1.64%）is the lowest;During the nutrient conversion period,the ¹³C distribution rate of the root system was 6.27 times that of the previous stage,and the ¹³C distribution rate of the fruit reached9.24%.During the utilization of the new nutrition period,the ¹³C distribution rate in the fruit increased significantly,reaching 38.79%.Therefore,fertilization during the storage nutrition period is beneficial to the construction of vegetative organs,fertilization during the nutrition conversion period is conducive to the nutritional supplementation of the storehouse,and fertilization during the new nutrition period is used to ensure sufficient supply of nitrogen during the critical period of fruit demand.2.Study on the Characteristics of Nitrogen Transport and Distribution from Different Sources of M9T337dwarf rootstocks seedlings.The M9T337 dwarf rootstocks seedlings were used as test materials for hydroponic experiments to investigate the dynamics of original（storage）nitrogen and newly absorbed nitrogen in apple Distribution law.The results showed that there were significant differences in the accumulation of ¹⁵N from different sources in different organs.The accumulated amount of absorbed ¹⁵N in leaves showed an increasing trend,while the accumulation of ¹⁵N in roots and new shoots showed a downward trend,and the ¹⁵N accumulation in new shoots changed little;The cumulative amount of newly absorbed ¹⁵N in each organ showed an upward trend,and the cumulative increase of ¹⁵N in leaves was the largest over time,and the smallest in the new shoots.The ¹⁵N distribution rate of the organs treated with¹⁵N accumulation in the plant was within 7 days of cultivation,and the distribution rate was root system>leaf>shoot,and after 14 days of cultivation,the distribution rate was leaf>roots>shoot.On the first day of the new ¹⁵N treatment,the ¹⁵N distribution rate of the root system was the largest（57.18%）.By the 7th day of the treatment,the new ¹⁵N distribution rate increased by 35.46%,and the ¹⁵N distribution rate of the leaves increased by 55.39%.After 7 days of treatment,the ¹⁵N distribution rate of the leaves reached the maximum.Therefore,the newly absorbed nitrogen in apple seedlings is more easily transported to the leaves of new organs than the nitrogen accumulated.3.Effect of nitrogen supply levels on growth and expression of glutamine、glutamate and asparagine synthase genes of M9T337 dwarf rootstocks seedlings.The gene expression characteristics of glutamine synthetase（GS）,glutamate synthetase（GOGAT）,asparagine synthetase（AS）of M9T337 apple seedlings at different nitrogen levels were analyzed in hydroponic conditions.The results showed that,under low nitrogen treatment for 1 day,GOGAT expression was lowest in the root system.Within 7 days of low nitrogen stress treatment,the expression of GS genes in the underground part was significantly higher than that of AS and GOGAT,and the expression of 3 genes in the underground part was down-regulated after 7days of treatment.With the increase of nitrogen level,the gene expression level of GS in overground part increased,reaching the highest value on the 7th day of treatment,and there was no significant difference in the change of AS.Under high nitrogen treatment,the gene expression of GS and GOGAT was inhibited,and the expression of GOGAT was low in the root system.So,the AS and GS are involved in the response of apple root system to low nitrogen stress,and appropriate nitrogen level can induce the nitrogen metabolism key enzymes play a role in nitrogen metabolism circulation;however,high nitrogen level can inhibit the gene expression of nitrogen metabolism key enzymes in nitrogen metabolism,which had a negative effect on nitrogen metabolism in plants.4.Effects of Exogenous IAA on the Regulation of Carbon and Nitrogen Metabolism and Distribution of M9T337 dwarf rootstocks Seedlings.Hydroponic experiments were performed using apple dwarfing rootstock M9T337 seedlings as test materials.¹⁵N and ¹³C dual-labeling technology and non-damaging micro-testing technology were used to study the effect of exogenous IAA and its inhibition TIBA on carbon and nitrogen distribution.The results showed that the root length and total surface area of the root application IAA treatment were the largest,followed by the leaf application IAA treatment and the root application TIBA treatment was the smallest;and the number of root tips was root application IAA>leaf application IAA>root application TIBA>CK.There was a significant difference in NO₃^-ion flow rate between the four treatments.Root application IAA treatment and leaf application IAA treatment increased the root NO₃^-ion flow rate by 99.35%and 67.77%compared with CK treatment.Under root application TIBA treatment,the NO₃^-ion flow rate showed a significant efflux,and the NO₃^-ion flow rate reached232.97 pmol/（cm²·s）.Within 14 days of treatment,the ¹⁵N distribution rate of the root system was CK>root application IAA>leaf application IAA>root application TIBA.At the 21st day treatment,the ¹⁵N allocation ratio of the root system was not significantly different among the treatments.After 21 days of treatment,the ¹⁵N distribution rate of the root application TIBA treatment was significantly higher than that of other treatments.During the treatment period,the maximum increase in the ¹⁵N distribution rate of the root system was 49.8%in the root application of TIBA treatment,and the largest increase in the ¹⁵N distribution rate was 131.7%in the leaf application IAA treatment.There were significant differences in ¹³C distribution rates between different exogenous IAA treatments.The ¹³C distribution rate of apple seedling roots was root application TIBA>root application IAA>CK>leaf application IAA,while the leaf ¹³C distribution ratio was leaf application IAA>root application IAA>CK>root application TIBA.This indicated that root application TIBA accumulated more carbon and nitrogen nutrients in the root system and inhibited its transfer to leaves.Root application IAA treatment significantly promoted ¹³C distribution in the root system,promoted root growth,and increased nitrogen uptake and utilization.IAA application of leaves could promote the transfer of carbon and nitrogen nutrients to leaves.