*Article* **E**ff**ect of Low Temperature on Chlorophyll Biosynthesis and Chloroplast Biogenesis of Rice Seedlings during Greening**

**Yuqing Zhao 1,**†**, Qiaohong Han 1,**†**, Chunbang Ding 1, Yan Huang 1, Jinqiu Liao 1, Tao Chen 1, Shiling Feng 1, Lijun Zhou 1, Zhongwei Zhang 2, Yanger Chen 1, Shu Yuan <sup>2</sup> and Ming Yuan 1,\***


Received: 14 December 2019; Accepted: 17 February 2020; Published: 19 February 2020

**Abstract:** Rice (*Oryza sativa* L.) frequently suffers in late spring from severe damage due to cold spells, which causes the block of chlorophyll biosynthesis during early rice seedling greening. However, the inhibitory mechanism by which this occurs is still unclear. To explore the responsive mechanism of rice seedlings to low temperatures during greening, the effects of chilling stress on chlorophyll biosynthesis and plastid development were studied in rice seedlings. Chlorophyll biosynthesis was obviously inhibited and chlorophyll accumulation declined under low temperatures during greening. The decrease in chlorophyll synthesis was due to the inhibited synthesis of δ-aminolevulinic acid (ALA) and the suppression of conversion from protochlorophyllide (Pchlide) into chlorophylls (Chls). Meanwhile, the activities of glutamate-1-semialdehyde transaminase (GSA-AT), Mg-chelatase, and protochlorophyllide oxidoreductase (POR) were downregulated under low temperatures. Further investigations showed that chloroplasts at 18 ◦C had loose granum lamellae, while the thylakoid and lamellar structures of grana could hardly develop at 12 ◦C after 48 h of greening. Additionally, photosystem II (PSII) and photosystem I (PSI) proteins obviously declined in the stressed seedlings, to the point that the PSII and PSI proteins could hardly be detected after 48 h of greening at 12 ◦C. Furthermore, the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) and cell death were all induced by low temperature. Chilling stress had no effect on the development of epidermis cells, but the stomata were smaller under chilling stress than those at 28 ◦C. Taken together, our study promotes more comprehensive understanding in that chilling could inhibit chlorophyll biosynthesis and cause oxidative damages during greening.

**Keywords:** *Oryza sativa* L.; chilling stress; chlorophyll biosynthesis; chloroplast biogenesis; epidermal characteristics
