Two aquaporins, LcPIP1;4 and LcPIP1;4a, cooperatively regulate the onset of dormancy of the terminal buds in evergreen perennial litchi (<i>Litchi chinensis</i> Sonn<i>.</i>)

Although extensively studied in various plants, the roles of aquaporin proteins in litchi remain unclear. In this study, low moisture content was observed in the dormant terminal buds of litchi. Transcriptome analysis revealed that two aquaporin genes, <i>PLASMA MEMBRANE INTRINSIC PROTEIN 1;4</i> (<i>LcPIP1;4</i>) and <i>LcPIP1;5</i>, could be remarkably inhibited by exogenous ethylene (ETH), which also reduced the moisture content of litchi buds. Quantitative real-time polymerase chain reaction assays indicated that <i>LcPIP1;4</i> expression was relatively elevated in the dormancy stage of litchi terminal buds. Inhibition of <i>LcPIP1;4</i> in the buds of litchi during the growth stage delayed the onset of dormancy, resulting in a significantly reduced dormancy rate and increased moisture content. Further study indicated that LcPIP1;4 interacts with LcPIP1;4a, and they are capable of self-interaction<i>.</i> Silencing of <i>LcPIP1;4a</i> in litchi buds resulted in a phenotype consistent with silencing of <i>LcPIP1;4.</i> Additionally, simultaneous silencing of both <i>LcPIP1;4</i> and <i>LcPIP1;4a</i> resulted in a more severe bud dormancy phenotype. Moreover, <i>LcPIP1;4</i> was directly upregulated by LcRAP2.4. Silencing of <i>LcRAP2.4</i> also delayed the onset of dormancy in litchi terminal buds, which is regulated by LcSVP2. ETH treatment at 1000 mg/l significantly downregulated the expression of <i>LcPIP1;4</i> and <i>LcRAP2.4,</i> but had no significant effect on <i>LcPIP1;4a</i>. In contrast, abscisic acid (ABA) treatment at 200 mg/l significantly upregulated the expression of <i>LcPIP1;4</i>, <i>LcPIP1;4a</i>, and <i>LcRAP2.4</i>. Combined treatment with ETH and ABA exerted a stronger inhibitory effect on the bud break and upregulated <i>LcPIP1;4</i> and <i>LcRAP2.4</i> to lower degrees than ABA alone, suggesting that ABA reversed the inhibitory effect of ETH on the expression of <i>LcPIP1;4</i> and <i>LcRAP2.4</i>. ABA treatment and combined treatment with ETH and ABA effectively reduced the moisture content of the terminal buds. These results demonstrate that LcRAP2.4, LcPIP1;4, and LcPIP1;4a play a vital role in dormancy onset of litchi terminal buds by regulating moisture levels.