HalALMT1 mediates malate efflux in the cortex of mature cluster rootlets of Hakea laurina, occurring naturally in phosphorus-poor soil

Hakea laurina, a woody plant in Proteaceae family, naturally occurs in severely phosphorus (P)-impoverished environments in southwest Australia. It develops distinctive “cluster roots” that exhibit a high capacity for carboxylate exudation and acid phosphatase activity, contributing to P acquisition. However, the molecular mechanisms underlying these physiological functions remain poorly understood. We explored the cluster root transcriptome using de novo RNA-Seq, which detected homologous transcripts of an aluminum (Al)-activated malate transporter (ALMT), designated HalALMT1, and two multidrug and toxic compound extrusion (MATE) proteins specifically expressed in mature cluster roots. Deferentially-expressed genes highlighted multiple carboxylate-related processes at maturity. Electrophysiological assays and overexpressed transgenic experiments confirmed HalALMT1’s ability to release malate and that its activity is enhanced by exposure to Al3+. Notably, HalALMT1 was specifically expressed in the mature cortex cells of cluster rootlets, which do not develop a suberized exodermis. Furthermore, acid phosphatase activity was pronounced throughout the rootlets, whereas it was only present in the epidermis and stele of non-cluster roots, revealing its extensive production in the rootlets. These results suggest that malate release and acid phosphatase production in the dominant cortex cells in cluster rootlets increase the amount exuded. This study sheds light on an outstanding P-acquisition strategy of Proteaceae. After germinating the Hakea laurina seedlings, six seedlings were transferred to a 4 L hydroponic solution with low P, specifically in a basic 0 µM P hydroponic setup supplemented with 6.4 µM NaH2PO4 every two to three months, and cultivated in the glasshouse at Hiroshima University. Mature cluster roots (MCR) and tips of cluster-bearing lateral roots (TCR) produced by approximate-three-year-old seedlings were harvested (n = 3). Roots were promptly frozen in liquid nitrogen and stored at -80˚C for RNA extraction, which was subjected to de novo RNA-Seq analsis. We investigated the genes related in phosphorus absorption and up-regulated in MCR.