**1. Introduction**

Seedlings, growing in the darkness before emerging from the soil, undergo etiolation with long hypocotyls and closed cotyledons which contain undeveloped plastids called etioplasts that have no chlorophyll [1,2]. The greening process initiates when exposed to light as the seedlings come out of soil, and this de-etiolation process includes the reduction of hypocotyl elongation rate, cotyledon opening, chlorophyll synthesis, and chloroplast biogenesis, and subsequently, seedlings absorb light energy and transition to autotrophy [3,4].

Chlorophyll (Chl) has unique and essential roles in harvesting and transducing light energy in antenna systems, and charge separation and electron transport in reaction centers [5,6]. Chlorophyll level is an important index used to evaluate photosynthetic capacity. Chlorophyll content decreases under cold stress, which might be because low temperature suppresses chlorophyll biosynthesis, probably by inhibiting the activities of chlorophyll biosynthetic enzymes [7]. A previous study showed that the optimum temperature of divinyl reductase (DVR) activity was 30 ◦C, and low activity was observed at 10 ◦C and no activity was found at 50 ◦C [7].

Chls share a common biosynthetic pathway with other tetrapyrroles, including siroheme, heme, and phytochromobilin in plants, algae, and many bacteria [6,8,9]. Since tetrapyrrole intermediates are all photosensitizers that are easily activated by light, leading to highly toxic levels of reactive oxygen species (ROS) and photooxidative damage and growth retardation, Chl biosynthesis must be finely controlled to ensure healthy growth during the greening process [9,10]. Chl biosynthesis is a very complex process that is executed via a series of coordinated reactions catalyzed by numerous enzymes [11]. The process of Chl biosynthesis can be divided into three distinct phases. The first phase involves the synthesis of protoporphyrin IX (Proto IX) from glutamate [12]. The first committed precursor is δ-aminolevulinic acid (ALA), and its synthesis is a key control point in Chl biosynthesis. This step is catalyzed by glutamyl-tRNA reductase (GluTR), which is encoded by *HEMA* gene [8]. δ-Aminolevulinic acid dehydratase (ALAD) catalyzes aggregation of two ALA molecules into porphobilinogen (PBG). Four PBGs polymerize and further cyclize to yield uroporphyrinogen III (urogen III), when then decarboxylates to form coproporphyrinogen III (coprogen III). Coprogen III occurs via oxidative decarboxylation and oxygen-dependent aromatization reaction to form protoporphyrin IX (Proto IX) [9]. The second phase includes the synthesis of chlorophyll *a* (Chl*a*) from Proto IX. Proto IX is the branch point of the chlorophyll and heme biosynthetic pathways [6]. The insertion of Mg2<sup>+</sup> into Proto IX forms Mg-protoporphyrin IX (Mg-proto IX), which is catalyzed by Mg-chelatase, but the insertion of Fe2<sup>+</sup> into Proto IX catalyzed by ferrochelatase starts the heme branch [9]. Meanwhile, the photoreduction of Pchlide to chlorophyllide (Chlide), catalyzed by protochlorophyllide oxidoreductase (POR), is another important step. POR is the key enzyme in the light-dependent greening of higher plants [13]. Two POR proteins (PORA and PORB) and three POR proteins (PORA, PORB, and PORC) have been found in rice and *Arabidopsis*, respectively [14,15]. DVR catalyzes 3,8-divinyl-chlide *a* to yield chlorophyllide a (Chlide a), which further forms Chl *a* catalyzed by chlorophyll synthase (CHLG). The third phase is the interconversion of Chl *a* and chlorophyll *b* (Chl *b*) catalyzed by chlorophyllide a oxygenase (CAO) which is known as the chlorophyll cycle [16].

Chloroplasts are responsible for photosynthesis and the production of hormones and metabolites [17,18], which develop from proplastids that are present in the immature cells of plant meristems. Chloroplast biogenesis involves a coordinated function of plastid- and nuclear-encoded genes [19]. The process of chloroplast biogenesis accompanies the biosynthesis of chlorophylls, chloroplast proteins, carotenoids, and lipids, and the assembly of photosynthetic protein complexes including the light-harvesting complex (LHC), photosystem I (PSI), photosystem II (PSII), cytochrome *f*/*b6* (cyt*f*/*b6*), and adenosine triphosphate (ATP) synthase [1,17]. Many factors have direct impacts on chloroplast biogenesis, such as light, water, salt, leaf age, etc. [1,20].

Low temperature is one of the most severe weather events that destructively affect crop growth, quality, and yield [21,22]. Cold or chilling stress impairs the chloroplast microstructure, photosynthetic metabolism, and energy production [23], directly leading to inhibited photosynthesis, which is a severe threat to crop production. Additionally, the efficiency of photosynthetic electron transport in plants is significantly decreased under chilling stress, resulting in a burst of ROS that directly causes cellular oxidative damages and increases membrane rigidity [24–27]. Low temperature also disrupts the carbon reduction cycle and the control of stomatal conductance [28].

Rice (*Oryza sativa* L.) is a kind of thermophilic cereal crop that feeds almost half of the world's population. Originating from tropical and subtropical areas, rice is highly sensitive to low temperatures, particularly during the greening process of the early seedling growth. In China, cold spells in late

spring cause serious suppression of rice seed germination and young seedling growth, especially in the middle–lower Yangtze River region and southern China. In the past 30 years, 30%–50% rice seedlings suffered from chilling or cold stress, resulting in a reduction of 3–5 megatons every year [29].

Chlorophyll biosynthesis is seriously blocked by low temperatures during greening. However, the inhibitory mechanism by which this occurs is still unclear. Understanding how rice responds to low temperatures will provide valuable information and genetic resources for improving cold-stress tolerance. Therefore, the object of this study was to explore the response mechanism of chlorophyll biosynthesis and chloroplast biogenesis during the greening process under chilling stress and to provide valuable data for crop production.

#### **2. Results**
