Single cell sequencing reveals phloem loading occurs via the abaxial bundle sheath cells in maize leaves [bulk RNA-seq]

Leaves are asymmetric, with differential functionalization of abaxial and adaxial tissues. The bundle sheath (BS) surrounding the vasculature of the C3 crop barley is dorsoventrally differentiated into three domains: adaxial structural, lateral S-type, and abaxial L-type. S-type cells seem to transfer assimilates towards the phloem. Here we used single-cell RNA sequencing to investigate BS differentiation in C4 maize. Abaxial BS (abBS) 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 were also identified in the phloem parenchyma (PP). Thus maize acquired a unique mechanism for phloem loading in which abBS cells provide the main pathway for apoplasmic sucrose transfer towards the phloem. This pathway predominates in veins responsible for phloem loading (rank-2 intermediate), while rank-1 intermediate and major veins export sucrose from the phloem parenchyma (PP) adjacent to the sieve element companion cell (SE/CC) complex, as in Arabidopsis. We surmise that abBS identity is subject to dorsoventral patterning and has components of PP identity. The observations provide first insights into the unique properties of abBS cells, cells previously considered to fulfill the same functions as other bundle sheath cells (BSCs), and a basis for understanding the C4 syndrome. Overall design: To determine which genes are affected by protoplasting in order to remove them from the analysis, we performed RNAseq on maize leaf tissue and protoplasts generated from the equilvant tissue

叶片呈不对称形态，其远轴（abaxial）与近轴（adaxial）组织的功能特化存在显著差异。环绕C3作物大麦维管束的维管束鞘（bundle sheath, BS）沿背腹轴分化为三个区域：近轴结构域、侧向S型区域以及远轴L型区域。S型细胞似乎负责将同化物转运至韧皮部。本研究借助单细胞RNA测序（single-cell RNA sequencing），探究C4玉米的维管束鞘分化过程。二级中间叶脉的远轴维管束鞘（abaxial BS, abBS）细胞特异性表达3种SWEET家族蔗糖单向转运蛋白（SWEET13a、SWEET13b及SWEET13c）与UmamiT氨基酸外排转运蛋白。SWEET13a、SWEET13b、SWEET13c同样在韧皮部薄壁组织（phloem parenchyma, PP）中被检测到。据此，玉米演化出一套独特的韧皮部装载机制：远轴维管束鞘细胞为质外体蔗糖向韧皮部转运的主要通路。该通路在负责韧皮部装载的二级中间叶脉中占主导地位；而一级中间叶脉与主叶脉则如拟南芥一般，通过紧邻筛分子伴胞（sieve element companion cell, SE/CC）复合体的韧皮部薄壁组织完成蔗糖外运。我们推测，远轴维管束鞘的细胞身份受背腹轴模式调控，并兼具韧皮部薄壁组织的细胞身份特征。本研究首次揭示了远轴维管束鞘细胞的独特属性——此前这类细胞被认为与其他维管束鞘细胞功能一致——同时为理解C4光合综合征提供了理论基础。实验设计：为明确哪些基因会因原生质体制备过程受到影响并需从分析中剔除，我们对玉米叶片组织以及从对应组织中制备的原生质体开展了RNA测序（RNAseq）。