CsNAC73 promotes cold-induced theanine biosynthesis by activating CsAlaDC expression in albino Camellia sinensis cv....

Camellia sinensis cv. Baiye 1 is a temperature-sensitive albino tea cultivar. During early spring, the albino tender shoots exhibited a distinct temperature-dependent fluctuation in theanine content and chlorophyll levels, but the underlying regulatory mechanism remains unclear. This study elucidates a regulatory mechanism involving the transcription factor CsNAC73, which mediates cold signal transduction to regulate theanine biosynthesis during the seasonal greening process of 'Baiye 1'. Leaf albino phenotypes and metabolite determinations in field-grown plants revealed close but distinct correlation of both theanine and chlorophyll levels with the accumulated effective temperature. Gene expression-metabolite correlation analysis identified CsNAC73 as a positive regulator of both theanine accumulation and the expression of its biosynthetic gene, alanine decarboxylase (CsAlaDC), in response to temperature fluctuations. Controlled cold treatments demonstrated a significant increase in CsNAC73 expression under cold stress, which was accompanied by concomitant increases in CsAlaDC expression and theanine content. Furthermore, transient suppression of CsNAC73 in albino tender shoots resulted in a reduction of CsAlaDC transcripts and a corresponding decrease in theanine levels. Yeast-one-hybrid, electrophoretic mobility shift assays, and luciferase assays demonstrated that CsNAC73 directly binds to the CsAlaDC promoter, thus enhancing its transcription and subsequently increasing theanine biosynthesis during the seasonal greening of spring albino tender shoots. These findings establish CsNAC73 as a critical transcriptional activator of cold-induced theanine biosynthesis by enhancing CsAlaDC expression during the seasonal greening of 'Baiye 1'.