Nitrogen Form Preference And Related Mechanism Of Blackberry

Blackberry（Rubus spp.）is a third-generation small berry fruit tree with special taste and nutritional value,which is widely grown around the world.Nitrogen（N）is an important life element,also an important component of many living substances such as nucleic acids,enzymes,ATP,amino acids and proteins,which plays a vital role in the yield and quality of blackberry.The current research on blackberry fertilization mainly focuses on the level of N fertilizer application,while there are few reports on the effects of different N fertilizer types on the nutritional growth and fruit quality.In this study,we used annual and biennial‘Ningzhi 4’blackberry potted plants as the research materials,and set up different forms of N fertilizer treatments（no nitrogen（CK）,nitrate nitrogen（NO₃^—-N）,ammonium nitrogen（NH₄⁺-N）and amide nitrogen（urea））,and observed and measured the growth and development indexes,physiological characteristics of blackberry plants under different treatments.The key candidate genes and regulatory factors in the carbon and N metabolic network of blackberries were predicted by transcriptome and metabolome analysis.The main findings are as follows:1.Annual blackberry plants were used as test material to study the effects of different N forms on the growth and development of blackberries.The results showed that NH₄⁺or urea-fed plants had a better growth state,showed a greater plant height,biomass,chlorophyll and N,and enhanced antioxidant enzyme activities and photosynthesis.NH₄⁺-N was beneficial to the accumulation of sugars and amino acids in leaves and roots,and promoted the transport of auxin and cytokinin to leaves.NH₄⁺-N treatment stimulated stomatal number and stomatal conductance,which accelerated the exchange of CO₂and water.NO₃^—-N significantly inhibited root growth and increased the contents of active oxygen,malondialdehyde and antioxidants in roots.Correlation and principal component analysis showed that growth and dry matter accumulation were closely related to the antioxidant system,photosynthetic characteristics,amino acids and hormone content.It was concluded that blackberry plants prefer to absorb NH₄⁺-N and can adapt to NH₄⁺-N environment for a long time.2.Two-year-old blackberry plants were used to explore the effects of different N forms on fruit quality at different ripening stages.The results showed that NH₄⁺-N or Urea could significantly improve the appearance of blackberry fruit,including fruit size,firmness and color,and help to accumulate more soluble solids,fructose,sucrose,glucose,anthocyanin,ellagic acid and VC,while NO₃^—-N treatment could accumulate more flavonoids and organic acids and improve the scavenging ability of DPPH free radicals.The sugar accumulation in blackberry was mainly fructose(34.13～47.80 mg?g^-1FW)and glucose(19.01～35.50 mg?g^-1FW).The fruit size,weight,firmness and color brightness decreased with the harvest period,while the contents of sugar,anthocyanin,ellagic acid,flavonoids and VC increased at first and then decreased,the highest content was in the middle and later stage.With the passage of maturity,the total antioxidant capacity and DPPH free radical scavenging capacity increased.The total phenol content was maintained at about 8 mg?g^-1FW in each harvest period,while the acid content decreased at first and then increased.It was concluded that in the process of blackberry planting,it is recommended to apply NH₄⁺-N mainly,which is more beneficial to improve fruit appearance,fruit taste and nutritional quality.3.Transcriptome and metabolome analysis of one-year-old blackberry leaves under different N treatments were conducted to explore the relationship between N preference and key genes of C and N metabolism in blackberry.Transcriptome analysis showed that there were4163,2705,5415,379,330 and 551 DEGs in the six comparison groups,i.e.,NH₄?-N vs.CK,NO₃^—-N vs.CK,urea vs.CK,NH₄?-N vs.NO₃^—-N,NH₄?-N vs.urea and urea vs.NO₃^—-N,respectively.Different N forms significantly affected photosynthesis,flavonoid biosynthesis and the TCA cycle.Metabolome analysis indicated that the levels of lipids,carbohydrates,flavonoids and amino acids were markedly changed under different N treatments.Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis indicated that N metabolism and amino acid metabolism were significantly affected by the N form.Overall,blackberry plants prefer NH₄?-N and urea.Integrated transcriptomic and metabolomic data revealed that amino acids,including proline,arginine,L-isoleucine,L-aspartate,threonine,and L-glutamate,played important roles in maintaining normal plant growth by regulating N metabolism and amino acid metabolism.Overall,blackberry plants preferentially take up NH₄⁺-N.Under the NH₄⁺-N treatment,N assimilation was stronger,flavonoid biosynthesis was decreased,and the promoting influence of NH₄⁺-N on N metabolism was better than that of NO₃^—-N.However,the NO₃^—-N treatment enhanced the C/N ratio,accelerated the process of C metabolism and increased the synthesis of flavonoids,thereby accelerating the flow of N metabolism to C metabolism.