The complete 1st leaf (1L) and 3rd leaf (3L) blades were picked and surface sterilized with 75% alcohol for the following experiments. About 0.6 g of fresh leaf blades were cut and immediately put in a digestion solution for protoplast and tissue isolation. The digestion solution for leaf tissue and protoplast single cell preparation contained 0.4 M D-mannitol (Sigma, USA), 1.5% cellulase R10 (Yakult, Japan), 0.5% macerozyme R10 (Yakult, Tokyo, Japan), 0.7% snailase (YEASEN, Shanghai, China), 20 mM 4-morpholineethanesulfonic acid (pH = 5.5), 20 mM KCl (Sigma, USA), 10 mM CaCl2 (Sigma, USA), 10 mM β-mercaptoethanol and 0.1% BSA (Sigma, USA). Entire intact leaf blades were cut into strips and immersed into 30 mL of digest solution in a glass dish. The strips were evacuated for 5 min and then incubated in the enzyme solution in an incubator at 25 °C for 4 hrs. The digestion mixture was filtered through a 40 μm nylon mesh sieve. Protoplasts were collected after gentle washing twice (centrifugation at 100 g for 2 min with a swinging bucket rotor) and were re-suspended in 8% D-mannitol solution. The number of isolated protoplasts was measured with a hemocytometer counting chamber, and the viability of the protoplasts was assessed by 2 μg mL-1 fluorescein diacetate. The concentration of protoplasts was adjusted to 1500-2000 cells μL-1 by 0.4 M D-mannitol for loading onto the chromium controller of the 10xGenomics platform. The scRNA-seq libraries were constructed using the 10x Genomics 3' Reagent Kits v3.