Plant microRNAs (miRNAs), as post-transcriptional regulators, widely participate in growth, development, and environmental adaptation processes, serving as the genetic foundation for many important agronomic traits. While the regulatory principles of miRNAs targeting transcription factors or other regulatory factors are relatively well understood, there is still a lack of in-depth understanding regarding miRNAs targeting non-transcription factors. miR408 is one of the most conserved miRNAs in land plants and targets genes encoding blue copper proteins, such as plantacyanin, and multi-copper oxidases like laccases in Arabidopsis. Although miR408's involvement in the regulation of copper homeostasis has long been known, its underlying molecular mechanisms have remained elusive.

 

Recently, the Lei Li Research Group from Peking University Institute of Advanced Agricultural Sciences (PKU-IAAS), School of Advanced Agricultural Sciences, and State Key Laboratory of Protein and Plant Gene Research published a research article titled "The microRNA408–plantacyanin module balances plant growth and drought resistance by regulating reactive oxygen species homeostasis in guard cells" in The Plant Cell, revealing the molecular mechanism by which miR408 balances plant growth and drought resistance through the regulation of reactive oxygen species (ROS) homeostasis in guard cells.

 

The research group conducted a systematic study on the miR408-plantacyanin module. Plantacyanin is a compact blue copper protein with only one type I copper-binding domain, aside from the N-terminal signal peptide. Previous studies have found a coordinated regulation of miR408 by light signals and core factors involved in copper homeostasis, promoting photosynthesis by regulating plastocyanin accumulation in the chloroplast (Zhang et al., 2014). Analysis of multiple plants revealed that overexpression of miR408 led to improved photosynthetic efficiency, increased seed size, and significantly enhanced yield (Pan et al., 2018). Subsequently, it was observed that plantacyanin accumulates in mature seeds but rapidly degrades during light-induced seed germination, uncovering the mechanism by which the miR408-plantacyanin module regulates hormone balance during seed germination (Jiang et al., 2021). In vegetative organs, plantacyanin accumulates in senescent leaves, and further investigation revealed the mechanism by which miR408 suppresses the SAG14-plantacyanin blue copper protein module, localized in the endoplasmic reticulum, to regulate leaf senescence (Hao et al., 2022). These studies have revealed the conserved mechanism of miR408 in regulating copper ion allocation to plastids. However, the evolutionary reasons for these regulatory mechanisms remain unclear.

 

The study initially conducted expression pattern analysis to determine plantacyanin as the main target of miR408 in Arabidopsis vegetative tissues. Fluorescence imaging revealed the expression of plantacyanin in guard cells, with subcellular localization associated with the inner membrane surrounding individual chloroplasts (Figure 1). Molecular biology techniques such as ChIP-seq, EMSA, dual-luciferase assays, and live imaging were employed. These experiments demonstrated that the promoter of miR408 is inhibited by multiple ABA-responsive transcription factors, leading to the accumulation of plantacyanin under stress conditions. Genetic analysis indicated that plantacyanin enhances the levels of reactive oxygen species (ROS) in guard cells (Figure), resulting in stomatal closure, reduced photosynthetic gas exchange, and enhanced drought resistance. Moreover, the miR408-plantacyanin module was capable of rescuing growth and drought-related phenotypes caused by overexpression or loss of ABA-responsive transcription factors. These findings suggest that the miR408-plantacyanin module regulates ROS homeostasis and stomatal movement by transducing ABA signals. Under favorable growth conditions, miR408 expression increases, inhibiting the accumulation of plantacyanin and ROS, thereby promoting stomatal opening, photosynthesis, and growth. Conversely, under stress conditions, ABA signaling suppresses miR408 expression, leading to the accumulation of plantacyanin and elevated ROS levels, promoting stomatal closure and enhancing drought resistance.

 

Figure Subcellular localization of plantacyanin and its role in promoting ROS accumulation

 

The inability of plants to move and evade environmental stress necessitates a delicate balance between growth and stress responses to adapt to constantly changing surroundings. Understanding how environmental changes affect plant growth is not only a fundamental scientific question but also crucial for agriculture and food security. Statistics indicate that adverse environmental conditions account for approximately 70% of overall yield losses in key crops, resulting in an average yield of only about 30% of their genetic potential. This study demonstrates that the highly conserved miR408 in terrestrial plants plays a fundamental role in balancing growth and drought resistance through the regulation of stomatal movement. This study not only provides mechanistic insights into the function and evolution of miR408 but also offers new avenues for the development of drought-tolerant and high-yielding crops.

 

Dr. Yanzhi Yang, a postdoctoral researcher at the School of Advanced Agricultural Sciences, Peking University, is the first author of this paper, while Dr. Lei Li, a research professor at Peking University, is the corresponding author. This work was supported by the National Natural Science Foundation of China and the State Key Laboratory of Protein and Plant Gene Research.