A laser capture microdissection-based transcriptome analysis on salt glands highlights salt secretion mechanism in...

Salt glands, developed in the epidermis of salt-secreting mangrove plant Avicennia marina leaves and specialized in salt secretion, are critical for improving the survival under high saline habitats. However, the salt secretion mechanism in A. marina remains poorly understood. In this study, laser capture microdissection was employed to isolate salt glands from the upper epidermis of A. marina, followed by transcriptome sequencing (RNA-Seq) to comparatively analyze gene expression profiles of salt glands under 0 mM NaCl and 400 mM NaCl treatments. Additionally, non-invasive micro-test technology (NMT) was used to measure real-time Na⁺ and Cl^- efflux rates from individual salt glands; transmission electron microscopy (TEM) was applied to observe subcellular structural changes; and heterologous expression assays were performed for functional validation of candidate genes. Our results showed that 400 mM NaCl treatment led to an increase in Na⁺ and Cl^- fluxes from salt glands, with the Na⁺ efflux reaching 2389 pmol cm^-2 s^-1 and the Cl^- efflux reaching 1652 pmol cm^-2 s^-1. In addition, a significant increase in mitochondrial density and vesicle number in secretory cells was found in 400 mM NaCl treatment salt glands of A. marina leaves. LCM-RNAseq identified a total of 1741 differentially expressed genes, which were significantly enriched in pathways related to vesicle trafficking, ion transport, compatible solute transport, aquaporins-based water transport, hormone signaling, cell wall and cuticle wax biosynthesis. Functional validation demonstrated that AmNHX2 (Na⁺/H⁺ antiporter), AmCLC-a (chloride channel), and AmSCAMP1 (secretory carrier membrane protein) significantly enhanced salt tolerance when heterologously expressed in yeast, tobacco, or Arabidopsis, and were involved in Na⁺/Cl^- transport or vesicle formation and trafficking. Collectively, this study reveals the molecular mechanisms by which salt glands of A. marina coordinately respond to salt stress through multiple pathways, including ion transport, vesicle trafficking, osmotic regulation, and cell wall plasticity, providing crucial insights for understanding salt tolerance in salt-secreting mangrove plants.