Transcriptome analysis reveals candidate genes involved in nitrogen deficiency stress in apples

Nitrogen is one of the macroelements required for plant growth and development and the identification of candidate genes involved in nitrogen deficiency stress is of great importance to the sustainable development of agriculture. Here, we found that the color of apple leaves changed from dark green to yellow-green, the malondialdehyde (MDA) content, soluble protein content, and proline content significantly increased, the chlorophyll content significantly decreased in response to nitrate deficiency stress. According to the physio-logical and biochemical changes of apple leaves during nitrate deficiency stress, nitrogen deficiency stress was divided into two stages: early nitrogen deficiency stage (ES) and late nitrogen deficiency stage (LS). Tran-scriptome sequencing was performed in these two stress stages. 5773 differential expression genes (DEGs) were identified in the early nitrogen deficiency stress stage and 6130 DEGs were identified in the late nitrogen deficiency stress stage. Functional analysis of these DEGs revealed that a large number of DEGs were enriched in 'porphyrin and chlorophyll metabolic' pathways, the 'photosynthesis' pathway, the 'photosynthesis-antenna protein' pathway, and the 'ABA', 'ETH', and 'JA' signal transduction pathways, and the metabolic networks of these pathways were constructed. In addition, overexpression of MdNAC4 weakened the tolerance of apple calli to nitrogen deficiency stress. Taken together, our results reveal possible pathways for apple adaptation to ni-trogen deficiency stress and identify the function of MdNAC4, a key transcription factor regulating nitrogen deficiency stress, which enriches the molecular mechanism of apple adapting to a nitrogen deficiency environment.