Table_3_CRISPR/Cas9-Induced Loss-of-Function Mutation in the Barley Mitogen-Activated Protein Kinase 6 Gene Causes Abnormal Embryo Development Leading to Severely Reduced Grain Germination and Seedling Shootless Phenotype.XLSX

The diverse roles of mitogen-activated protein kinases (MAPKs, MPKs) in plant development could be efficiently revealed by reverse genetic studies. In Arabidopsis, mpk6 knockout mutants complete the life cycle; however, ~40% of their embryos show defects in the development leading to abnormal phenotypes of seeds and seedlings' roots. Contrary to the Arabidopsis MPK6, the rice MPK6 (OsMPK6) is an essential gene as transfer DNA (T-DNA) insertion and CRISPR/Cas9 induced loss-of-function mutations in the OsMPK6 cause early embryo arrest. In this study, we successfully developed a viable transgenic barley line with the CRISPR/Cas9-induced heterozygous single base pair cytosine-guanine (CG) deletion [wild type (WT)/−1C] in the third exon of the HvMPK6 gene, a barley ortholog of the Arabidopsis and rice MPK6. There were no obvious macroscopic phenotype differences between the WT/−1C plants and WT plants. All the grains collected from the WT/−1C plants were of similar size and appearance. However, seedling emergence percentage (SEP) from these grains was substantially decreased in the soil in the T2 and T3 generation. The mutation analysis of the 248 emerged T2 and T3 generation plants showed that none of them was a biallelic mutant in the HvMPK6 gene, suggesting lethality of the −1C/−1C homozygous knockout mutation. In the soil, the majority of the −1C/−1C grains did not germinate and the minority of them developed into abnormal seedlings with a shootless phenotype and a reduced root system. Some of the −1C/−1C seedlings also developed one or more small chlorotic leaf blade-like structure/structures. The −1C/−1C grains contained the late-stage developed abnormal embryos with the morphologically obvious scutellum and root part of the embryonic axis but with the missing or substantially reduced shoot part of the embryonic axis. The observed embryonic abnormalities correlated well with the shootless phenotype of the seedlings and suggested that the later-stage defect is predetermined already during the embryo development. In conclusion, our results indicate that barley MPK6 is essential for the embryologically predetermined shoot formation, but not for the most aspects of the embryo and early seedling development.

丝裂原活化蛋白激酶（mitogen-activated protein kinases, MAPKs, 简称MPKs）在植物发育中的多样功能可通过反向遗传学研究得到高效揭示。在拟南芥中，mpk6敲除突变体可完成整个生命周期；然而其约40%的胚胎存在发育缺陷，进而导致种子及幼苗根系出现异常表型。与拟南芥MPK6不同，水稻MPK6（OsMPK6）是必需基因，转移DNA（T-DNA）插入及CRISPR/Cas9诱导的OsMPK6功能丧失突变会引发胚胎早期停滞。本研究成功培育了一株可存活的转基因大麦株系，其大麦MPK6（HvMPK6，拟南芥与水稻MPK6的同源基因）的第三个外显子中存在CRISPR/Cas9诱导的杂合单碱基胞嘧啶-鸟嘌呤（CG）缺失[野生型（WT）/−1C]。WT/−1C植株与野生型植株之间未观察到明显的宏观表型差异，其收获的所有籽粒大小与外观均一致，但在土壤中播种后，该株系T2及T3代籽粒的幼苗出苗率（SEP）显著降低。对248株出苗的T2、T3代植株进行突变分析后发现，未检测到任何HvMPK6基因的双等位突变体，这表明−1C/−1C纯合敲除突变具有致死性。在土壤中，绝大多数−1C/−1C籽粒无法萌发，少数萌发的籽粒会长成具有无苗表型且根系发育不全的异常幼苗；部分−1C/−1C幼苗还会形成一个或多个小型褪绿叶片状结构。−1C/−1C籽粒内含发育后期的异常胚胎，该胚胎具有形态学上清晰可见的盾片及胚轴根部，但胚轴的苗部缺失或显著缩短。本次观察到的胚胎异常与幼苗的无苗表型高度吻合，表明后期发育缺陷在胚胎发育阶段就已预先决定。综上，本研究结果表明，大麦MPK6对于胚胎预先决定的苗部形成是必需的，但并非胚胎及早期幼苗发育的大多数环节所必需。