Transcriptome analysis and phenotyping of walnut seedling roots under nitrogen stresses

Nitrogen is an essential core element in walnut seedling growth and development. However, nitrogen starvation and excessive nitrogen stress can cause stunted growth and development of walnut seedlings, and environmental pollution is also of concern. Therefore, it is necessary to study the mechanism of walnut seedling resistance to nitrogen stress. In this study, morphological and physiological observations and transcriptome sequencing of walnut seedlings under nitrogen starvation and excess nitrogen stress were performed. The results showed that walnut seedlings under nitrogen starvation and excess stress could adapt to the changes in the nitrogen environment by changing the coordination of their root morphology and physiological indexes. Based on an analysis of transcriptome data, 4911 differential genes (DEGs) were obtained (2180 were upregulated and 2731 were downregulated) in a comparison of nitrogen starvation and control groups. A total of 9497 DEGs (5091 upregulated and 4406 downregulated) were obtained in the comparison between the nitrogen overdose and control groups. When these DEGs were analysed, the differential genes in both groups were found to be significantly enriched in the plant’s circadian pathway. Therefore, we selected the circadian rhythm as the focus for further analysis. We made some discoveries by analysing the gene co-expression network of nitrogen metabolism, circadian rhythm, and hormone signal transduction. (a) Nitrite nitrogen (NO2−) or Glu may act as a nitrogen signal to the circadian clock. (b) Nitrogen signalling may be input into the circadian clock by regulating changes in the abundance of the CRY1 gene. (c) After the nitrogen signal enters the circadian clock, the expression of the LHY gene is upregulated, which causes a phase shift in the circadian clock. (d) The RVE protein may send information about the circadian clock’s response to nitrogen stress back to the nitrogen metabolic pathway via the hormone transduction pathway. In conclusion, various metabolic pathways in the roots of walnut seedlings coordinated with one another to resist the ill effects of nitrogen stress on the root cells, and these coordination relationships were regulated by the circadian clock. This study is expected to provide valuable information on the circadian clock regulation of plant resistance to nitrogen stress.