Ding Xinxin, Zhang Xiaoguo, Paez-Valencia Julio, McLoughlin Fionn, Reyes Francisca C, Morohashi Kengo, Grotewold Erich, Vierstra Richard D, Otegui Marisa S
Department of Botany, University of Wisconsin-Madison, Madison, WI, United States.
Center for Quantitative Cell Imaging, University of Wisconsin-Madison, Madison, WI, United States.
Front Plant Sci. 2022 Feb 18;13:833612. doi: 10.3389/fpls.2022.833612. eCollection 2022.
The molecular machinery orchestrating microautophagy, whereby eukaryotic cells sequester autophagic cargo by direct invagination of the vacuolar/lysosomal membrane, is still largely unknown, especially in plants. Here, we demonstrate microautophagy of storage proteins in the maize aleurone cells of the endosperm and analyzed proteins with potential regulatory roles in this process. Within the cereal endosperm, starchy endosperm cells accumulate storage proteins (mostly prolamins) and starch whereas the peripheral aleurone cells store oils, storage proteins, and specialized metabolites. Although both cell types synthesize prolamins, they employ different pathways for their subcellular trafficking. Starchy endosperm cells accumulate prolamins in protein bodies within the endoplasmic reticulum (ER), whereas aleurone cells deliver prolamins to vacuoles an autophagic mechanism, which we show is by direct association of ER prolamin bodies with the tonoplast followed by engulfment microautophagy. To identify candidate proteins regulating this process, we performed RNA-seq transcriptomic comparisons of aleurone and starchy endosperm tissues during seed development and proteomic analysis on tonoplast-enriched fractions of aleurone cells. From these datasets, we identified 10 candidate proteins with potential roles in membrane modification and/or microautophagy, including phospholipase-Dα5 and a possible EUL-like lectin. We found that both proteins increased the frequency of tonoplast invaginations when overexpressed in leaf protoplasts and are highly enriched at the tonoplast surface surrounding ER protein bodies in maize aleurone cells, thus supporting their potential connections to microautophagy. Collectively, this candidate list now provides useful tools to study microautophagy in plants.
协调微自噬的分子机制，即真核细胞通过液泡/溶酶体膜的直接内陷来隔离自噬货物，目前仍 largely unknown，尤其是在植物中。在这里，我们展示了胚乳玉米糊粉层细胞中储存蛋白的微自噬，并分析了在此过程中具有潜在调节作用的蛋白质。在谷物胚乳中，淀粉胚乳细胞积累储存蛋白（主要是醇溶蛋白）和淀粉，而外围糊粉层细胞储存油脂、储存蛋白和特殊代谢产物。尽管这两种细胞类型都合成醇溶蛋白，但它们采用不同的途径进行亚细胞运输。淀粉胚乳细胞在内质网（ER）中的蛋白体中积累醇溶蛋白，而糊粉层细胞将醇溶蛋白输送到液泡——这是一种自噬机制，我们发现这是通过内质网醇溶蛋白体与液泡膜的直接结合，随后被吞噬——微自噬。为了鉴定调节这一过程的候选蛋白，我们在种子发育过程中对糊粉层和淀粉胚乳组织进行了RNA测序转录组比较，并对糊粉层细胞富含液泡膜的部分进行了蛋白质组分析。从这些数据集中，我们鉴定出10种在膜修饰和/或微自噬中具有潜在作用的候选蛋白，包括磷脂酶-Dα5和一种可能的类EUL凝集素。我们发现，当这两种蛋白在叶原生质体中过表达时，都会增加液泡膜内陷的频率，并且在玉米糊粉层细胞内质网蛋白体周围的液泡膜表面高度富集，从而支持它们与微自噬的潜在联系。总的来说，这个候选蛋白列表现在为研究植物中的微自噬提供了有用的工具。
