Bulk RNA-sequencing of hypocotyl explant-derived calli of Arabidopsis WT and rap2.12-2

Plants are aerobic organisms that rely on molecular oxygen for respiratory energy production. Hypoxic conditions, with oxygen levels ranging between 1% and 5%, usually limit aerobic respiration and affect plant growth and development. Here, we demonstrate that hypoxic microenvironment induced by active cell proliferation during the two-step plant regeneration process intrinsically represses the regeneration competence of callus in Arabidopsis thaliana. Hypoxia-repressed plant regeneration was mediated by the RELATED TO APETALA 2.12 (RAP2.12) protein, a member of the Ethylene Response Factor VII (ERF-VII) family. The hypoxia-activated RAP2.12 protein promoted salicylic acid (SA) biosynthesis and defense responses, inhibiting pluripotency acquisition and de novo shoot regeneration in calli. RAP2.12 could bind directly to the SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) gene promoter and activate SA biosynthesis, repressing plant regeneration via a PLETHORA (PLT)-dependent pathway. The rap2.12 mutant calli exhibited enhanced shoot regeneration, which was impaired by SA treatment. Taken together, our findings demonstrate that cell proliferation-dependent hypoxic microenvironment reduces cellular pluripotency and plant regeneration through the RAP2.12–SID2 module. Overall design: Hypocotyls of 1-week-old Col-0 (WT) and rap2.12-2 Arabidopsis thaliana seedlings were excised and incubated on callus-inducing medium (CIM). Calli were collected 7 days after incubation on CIM. Three biological replicates for each sample were generated.

植物为需氧生物，依赖分子氧进行呼吸产能。氧含量介于1%~5%的低氧条件通常会抑制有氧呼吸，进而影响植物的生长发育。本研究证实，在拟南芥（Arabidopsis thaliana）的两步法植株再生过程中，活跃细胞增殖所诱导的低氧微环境，会内在抑制愈伤组织的再生能力。
低氧抑制植物再生的过程由乙烯响应因子VII（ERF-VII）家族成员APETALA2相关蛋白2.12（RAP2.12）介导。经低氧激活的RAP2.12蛋白可促进水杨酸（SA）的生物合成与防御响应，进而抑制愈伤组织的多能性获得与不定芽再生。
RAP2.12能够直接结合水杨酸诱导缺陷2（SID2）基因的启动子区域并激活SA的生物合成，最终通过依赖于PLETHORA（PLT）的信号通路抑制植物再生。rap2.12突变体的愈伤组织具有更强的芽再生能力，而该能力可被水杨酸处理削弱。
综上，本研究结果表明，依赖细胞增殖的低氧微环境可通过RAP2.12–SID2模块降低细胞多能性并抑制植物再生。
实验设计概况：将生长1周的Col-0（野生型，WT）与rap2.12-2拟南芥幼苗的下胚轴切下，接种于愈伤组织诱导培养基（CIM）中培养。在接种于CIM后的第7天收集愈伤组织，每个样本设置3次生物学重复。