Evidence for phloem loading via the abaxial bundle sheath cells in maize leaves

Leaves are asymmetric, with different functions for adaxial and abaxial tissue. The bundle sheath (BS) of C-3 barley (Hordeum vulgare) is dorsoventrally differentiated into three types of cells: adaxial structural, lateral S-type, and abaxial L-type BS cells. Based on plasmodesmatal connections between S-type cells and mestome sheath (parenchymatous cell layer below bundle sheath), S-type cells likely transfer assimilates toward the phloem. Here, we used single-cell RNA sequencing to investigate BS differentiation in C-4 maize (Zea mays L.) plants. Abaxial BS ((BS)-B-ab) cells of rank-2 intermediate veins specifically expressed three SWEET sucrose uniporters (SWEET13a, b, and c) and UmamiT amino acid efflux transporters. SWEET13a, b, c mRNAs were also detected in the phloem parenchyma (PP). We show that maize has acquired a mechanism for phloem loading in which (BS)-B-ab cells provide the main route for apoplasmic sucrose transfer toward the phloem. This putative route predominates in veins responsible for phloem loading (rank-2 intermediate), whereas rank-1 intermediate and major veins export sucrose from the PP adjacent to the sieve element companion cell complex, as in Arabidopsis thaliana. We surmise that (BS)-B-ab identity is subject to dorsoventral patterning and has components of PP identity. These observations provide insights into the unique transport-specific properties of (BS)-B-ab cells and support a modification to the canonical phloem loading pathway in maize.