Differential gene expression and transport functionality in the bundle sheath versus mesophyll - a potential role in leaf mineral homeostasis. Arabidopsis thaliana

The bundle sheath cells (BSCs) layer – a presumed control point for radial transport of water and solutes between the vasculature and the leaf mesophyll cells (MCs) – is still largely understudied. Using isolated protoplasts, we found that 45% of the 90 genes differentially expressed in BSCs vs. MCs are membrane related and 20% are transport related, suggesting unique functionality of membrane transport in the BSCs, supported also by functional assays (electrophysiology and fluorescence imaging). A tight control of long distance transport is required for the optimization of the hydro-mineral homeostasis of organs in plants under fluctuating ambient conditions. The mechanism of ion selectivity and absorption from the xylem to the leaf is still poorly understood. The bundle sheath (BS), tightly enwrapping the leaf vasculature, has been suggested as a part of this mechanism, acting as a selective barrier which regulates the radial transport of water and solutes from the xylem to leaf cells. This suggestion relies on the anatomy, as well as on the recent physiological transport assays of the BS and its cells (BSCs). We hypothesized that the unique transport functionality of the BSCs is manifested in its transcriptome. To test this, we compared the transcriptomes of individually hand-picked protoplasts of GFP-labeled BSCs and non-labeled mesophyll cells (MCs) of Arabidopsis thaliana leaves. Indeed, in conformation of our hypothesis, 45% of the 90 genes differentially expressed in BSCs vs. MCs are membrane related and 20% are transport related, with the proton-pump AHA2 as a prominent example. Moreover, fluorescence imaging using the potentiometric dye (di-8 ANEPPS) revealed a more negative membrane potential of the BSCs protoplasts compared to those of MCs, and electrophysiological assays (patch-clamp) showed that the major, AKT2-like, membrane K+ conductances of BSCs and MCs had different voltage dependency ranges. Combined, these differences are compatible with an expected possibility of simultaneous but oppositely directed transmembrane K+ fluxes in BSCs and MCs in otherwise similar conditions. Overall design: 6 Samples (arrays) were performed. We generated pairwise comparison between the 2 different cell types (triplicates) using Partek Genomics Suite.

束鞘细胞（bundle sheath cells, BSCs）层——被认为是维管束与叶肉细胞（mesophyll cells, MCs）之间水分和溶质径向运输的调控节点——目前仍未得到充分研究。本研究通过分离原生质体（protoplasts）发现，在束鞘细胞与叶肉细胞间差异表达的90个基因中，45%与膜相关，20%与转运相关，提示束鞘细胞的膜转运功能具有独特性，该结论亦得到了功能实验（电生理检测与荧光成像）的支持。在环境条件波动的情况下，植物要优化各器官的水矿质稳态，需对长距离转运进行严格调控。然而，从木质部（xylem）到叶片的离子选择性吸收机制仍不甚明晰。紧密包裹叶片维管束的束鞘（BS）被认为是该机制的组成部分，可作为选择性屏障调控水分与溶质从木质部向叶细胞的径向运输。这一假说基于束鞘及其细胞的解剖学特征与近期的生理转运实验结果。我们提出假说：束鞘细胞独特的转运功能可通过其转录组（transcriptome）体现。为验证该假说，我们对比了拟南芥（Arabidopsis thaliana）叶片中绿色荧光蛋白（GFP）标记的束鞘细胞与未标记叶肉细胞的原生质体转录组，所有原生质体均经手工单独挑取。结果证实了我们的假说：在束鞘细胞与叶肉细胞间差异表达的90个基因中，45%与膜相关，20%与转运相关，质子泵AHA2便是典型代表。此外，使用电位敏感染料di-8 ANEPPS开展的荧光成像实验显示，束鞘细胞原生质体的膜电位较叶肉细胞更为负向；而膜片钳（patch-clamp）电生理实验表明，束鞘细胞与叶肉细胞中主要的AKT2样膜钾离子电导具有不同的电压依赖范围。综上，这些差异符合在相似环境条件下，束鞘细胞与叶肉细胞可同时发生方向相反的跨膜钾离子流动的预期。整体实验设计：本研究共设置6个样本（阵列），通过Partek Genomics Suite软件对两种细胞类型开展三组重复的两两对比分析。