Theanine transporters are involved in nitrogen deficiency response in tea plant (Camellia sinensis L.)

Abstract

Nitrogen in soil directly influences the production and quality of tea. However, high nitrogen application in tea plantation leads to soil acidification and environmental pollution. Studies in model plants showed that plasma membrane localized amino acid transporter can regulate the distribution of amino acids to enhance nitrogen use efficiency. Our recent study identified six CsAAPs as transporters for theanine, a unique and most abundant non-proteinaceous amino acid in tea plant. In this work, we found these theanine transporters can also transport Glutamine, Glutamate, aspartate, alanine and γ-aminobutyric acid. Tissue-specific expression analyses showed that CsAAP1, CsAAP5 and CsAAP6 mainly expressed in leaves, CsAAP8 in root, CsAAP4 and CsAAP2 in stem. Furthermore, the expression of these CsAAPs was induced by nitrogen deficiency in a tissue-specific manner. Subcellular localization analyses showed that CsAAP1, CsAAP2 and CsAAP6 location were in the plasma membrane and endoplasmic reticulum. Taken together, these results suggested theanine transporters are involved in nitrogen deficiency response probably by mediating amino acid transport from roots to new shoots and from source to sink tissues in tea plants.

Keywords: Camellia sinensis L; CsAAPs; Tea; amino acid transporter; nitrogen.

Figure 1.

Yeast 22Δ10α growth complementation assay with amino acid as the sole nitrogen source. Yeast mutant line 22Δ10α was used to analyze growth of yeast cells expressing CsAAPs on medium with Gln, Glu, Asp, Ala, or GABA with the sole nitrogen source, respectively. For control, yeast cells grow with the supply of 2-mM ammonium sulfate. The yeast strains were transformed with pYES2-CsAAPs. 4 yeast colonies in each row represent different dilutions.

Figure 2.

CsAAP expression in various tissues in response to nitrogen (n) deficiency. Results of RT-qPCR of CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6 and CsAAP8 expression in first and second leaves, third and fourth leaves, tender stem and root of the tea plants under normal N supply (Control) and N deficiency conditions. Mean of three independent biological replicates of 4 plants with three technical replicates. Error bars are standard error of the mean. Asterisks represent statistical significance determined by Student’s t-test (*p = 0.05, **p = 0.01, ***p = 0.01).

Figure 3.

Plasma membrane localization of CsAAP in Arabidopsis protoplasts and tobacco epidermal cells. GFP-CsAAP1, GFP-CsAAP2, or GFP-CsAAP6 were transiently expressed in Arabidopsis protoplasts or expressed in tobacco epidermal cells together with AtWAK2-mCherry. (a) GFP-CsAAP1, GFP-CsAAP2 or GFP-CsAAP6 in Arabidopsis protoplasts; (b) GFP-CsAAP1 and AtWAK2-mCherry in tobacco epidermal cells.