*Article* **OsCAF1, a CRM Domain Containing Protein, Influences Chloroplast Development**

**Qiang Zhang 1,**†**, Lan Shen 1,**†**, Zhongwei Wang 2, Guanglian Hu 1, Deyong Ren 1, Jiang Hu 1, Li Zhu 1, Zhenyu Gao 1, Guangheng Zhang 1, Longbiao Guo 1, Dali Zeng <sup>1</sup> and Qian Qian 1,\***


Received: 6 July 2019; Accepted: 3 September 2019; Published: 6 September 2019

**Abstract:** The chloroplast RNA splicing and ribosome maturation (CRM) domain proteins are involved in the splicing of chloroplast gene introns. Numerous CRM domain proteins have been reported to play key roles in chloroplast development in several plant species. However, the functions of CRM domain proteins in chloroplast development in rice remain poorly understood. In the study, we generated *oscaf1* albino mutants, which eventually died at the seedling stage, through the editing of *OsCAF1* with two CRM domains using CRISPR/Cas9 technology. The mesophyll cells in *oscaf1* mutant had decreased chloroplast numbers and damaged chloroplast structures. OsCAF1 was located in the chloroplast, and transcripts revealed high levels in green tissues. In addition, the OsCAF1 promoted the splicing of group IIA and group IIB introns, unlike orthologous proteins of AtCAF1 and ZmCAF1, which only affected the splicing of subgroup IIB introns. We also observed that the C-terminal of OsCAF1 interacts with OsCRS2, and OsCAF1–OsCRS2 complex may participate in the splicing of group IIA and group IIB introns in rice chloroplasts. OsCAF1 regulates chloroplast development by influencing the splicing of group II introns.

**Keywords:** chloroplast RNA splicing and ribosome maturation (CRM) domain; intron splicing; chloroplast development; rice

### **1. Introduction**

Chloroplasts are important organelles in plants. A series of metabolic processes occur in chloroplasts, including photosynthesis and anabolism of compounds such as tetrapyrroles, terpenoids, lipids, amino acids, and hormones [1]. Chloroplasts are considered semi-autonomous organelles because their development is not only influenced by their own genetic material but also by fine regulation by nuclear-encoded genes [2,3]. Studies have shown that by plastid-encoded polymerases (PEPs) and nucleus-encoded polymerases (NEPs) influence the development of chloroplasts [4–6]. Chloroplast genes encode approximately 100 proteins, some of which have one or more introns that cannot be self-spliced, and the primary RNA transcription of such chloroplast genes require splicing by ribozymes potentially via chemical steps similar to spliceosome-mediated splicing in the nucleus [7–9]. In plants, based on the primary sequences, predicted structures, and splicing mechanisms, the introns of the chloroplast are mainly classified into two categories, including group I and group II [10]. Group II introns are mainly divided further into two subgroups, including subgroup IIA introns and subgroup IIB introns [11]. *Arabidopsis thaliana*, maize, and rice chloroplast genomes all have only 1 group I intron, and 20, 17, and 17 group II introns, respectively [8]. In plant chloroplasts, such introns have lost the capacity to self-splice in vivo and require nuclear gene-encoded proteins as co-factors to participate in splicing [8,12].

Numerous studies have shown that nuclear-encoded pentatricopeptide repeat (PPR) proteins participate in chloroplast RNA editing and splicing, which are critical for chloroplast development and function [8,13]. Currently, the large PPR protein families, including AtOTP51, ZmPPR5, OsPPR6, and OsPPR1, are considered to participate in different chloroplast group II intron splicing activities [14–18]. Previous studies have shown that the disruption of the normal functions of such PPR proteins could lead to abnormal chloroplast development, which would lead to albino seedlings or death of plants [14–18]. In addition to the PPR proteins mentioned above, chloroplast RNA splicing and ribosome maturation (CRM) domain proteins participate in the splicing of chloroplast gene introns [8].

In plants, the splicing of chloroplast introns requires splicing factors encoded by nuclear genes and the abnormal splicing of chloroplast intron will affect the development of chloroplast [14–16]. Several proteins with CRM domains have been identified as splicing co-factors, including CFM2, CFM3, CRS1, CAF1, and CAF2 [8]. Both ZmCFM2 and AtCFM2 have four CRM domains, and they participate in the splicing of *ndhA* and *ycf3-1* subgroup IIB and group I introns. In *A. thaliana,* AtCFM2 potentially promotes the splicing of *clpP* introns [19]. CFM3 and CRS1 contain three CRM domains, and AtCRS1 and ZmCRS1 have been associated with the splicing of the *atpF* intron, which belongs to subgroup IIA introns [20,21]. In addition, CFM3 has been reported to be dual-localized in chloroplasts and mitochondria, and CFM3a is required for the splicing of group II introns, including *ndhB*, *rpl16*, *rps16*, *petD*, and *petB* introns in chloroplasts [19]. The CAF1 and CAF2 contain two CRM domains, which are required for the splicing of group IIB introns, including *ndhA*, *ndhB*, *petB*, *petD*, *rpl16*, *rps16*, *trnG*, and *ycf3-1* in maize and *A. thaliana* [11,21]. In *A. thaliana,* AtCAF2 potentially promotes the splicing of *rpoC1* and *ClpP* introns that are absent in the maize chloroplast genome [21]. In addition, CAF1 and CAF2 could interact with CRS2, forming the CRS2–CAF1 and CRS2–CAF2 complexes, which participate in the splicing of chloroplast group II introns and the regulation of chloroplast development in maize [22].

Previous studies have shown that there are 14 proteins that contain one or more CRM domains in rice [8]. To date, researchers have only studied the functions of OsCRS1 and OsCFM3, which contain three CRM domains. The studies revealed that *oscrs1* mutants exhibited albino leaf phenotypes in rice [23]. In addition, OsCRS1 has been reported to participate in the splicing of *ndhA*, *ndhB*, *petD*, *ycf3-1*, and *trnL* introns and it could influence the expression of PEP-dependent genes such as *psaA* and *psbA* [23]. Furthermore, *oscfm3* T-DNA insertion mutants exhibited albino seedling phenotypes; however, the OsCFM3 participates in the intron splicing of *ndhB*, *petD*, *rps16,* and *rpl16* in rice [19]. The functions of other CRM domain proteins have hardly been reported in rice. Previous studies have shown that homologous *CAF1* plays important roles in chloroplast development in *A. thaliana* and *Zea mays* L. In rice, the function of *OsCAF1* remains unknown and investigating its function would enhance our understanding of mechanisms of chloroplast development.

In this study, we investigated the function of OsCAF1 in rice chloroplast development, and *oscaf1* mutants were obtained using the CRISPR-Cas9 system. The results of our study revealed that *oscaf1* mutants exhibited albino seedlings phenotype along with a damaged chloroplast structure. In rice, chloroplast development is regulated by the expression of PEP-dependent genes, which could be affected by *OsCAF1*. In addition, we observed that the leaves of *oscaf1* mutants accumulated higher hydrogen peroxide (H2O2) contents. In addition, OsCAF1 could interact with the OsCRS2 via C-terminal, and then form an OsCAF1–OsCRS2 complex that regulates the splicing of chloroplast gene introns. Chloroplast RNA splicing analysis showed that OsCAF1 influenced the splicing of both chloroplast subgroup IIA and subgroup IIB introns, which is different from the orthologous proteins of AtCAF1 and ZmCAF1, which both influence the splicing of chloroplast subgroup IIB introns. The results of the present study reveal the functions of OsCAF1 in the regulation of rice chloroplast development.
