*3.1. Transcription and Environmental Stress Responses*

The transcriptional regulation of chloroplast gene expression is crucial not only for photosynthesis but also for plant development. Recent studies have revealed that the transcriptional control of chloroplast gene expression also plays important roles in plant responses to environmental changes. The chloroplast gene *psbA* encodes the D1 reaction center protein of photosystem II (PSII) [61–63]. Due to the nature of PSII photochemistry, D1 protein is continuously subjected to photodamage, which decreases photosynthetic activity (an effect known as photoinhibition). These damaged D1 proteins are replaced by *de novo* synthesized D1 proteins following the partial disassembly of the PSII complex [64,65]. Hence, the capacity to repair photodamaged PSII strongly depends on the ability to generate new D1 protein. Chloroplasts can adjust the transcriptional efficiency of *psbA* during photoinhibition under adverse environmental conditions such as high light and temperature [66–68]. During chloroplast evolution, several nucleus-encoded proteins have developed the ability to regulate *psbA* transcription in order to repair photodamaged PSII under adverse environmental conditions. Tomato (*Solanum lycopersicum*) WHIRLY1 (SlWHY1) was recently found to upregulate *psbA* transcription under chilling conditions. Under these conditions, the chloroplast-localized SlWHY1 promotes the transcription of *psbA* by directly binding to the upstream region of its promoter (the sequence "GTTACCCT"), resulting in increased D1 abundance to relieve photoinhibition [69,70]. Overexpression of *SlWHY1* leads to increased *de novo* synthesis of D1 protein and increased resistance to photoinhibition under chilling conditions [69]. These findings suggest that *psbA* transcription is an important target for regulating PSII activity to adjust plant resistance to environmental stresses.

**Table 1.** Chloroplast gene expression and stress response mutants.



**Table 1.** *Cont.*

The chloroplast gene *PsbD* encodes the reaction center protein D2 of PSII [86]. The expression of *psbD* is controlled by four PEP promoters. One of these is the blue-light-responsive promoter *psbD* BLRP [87–89]. The structure of *psbD* BLRP is distinct from that of common PEP promoters, which are characterized by conserved −35 and −10 elements. The *psbD* BLRP contains three *cis*-elements, including the AAG box, PGT box, and −10 element, but lacks the conserved −35 element [32,89,90]. This promoter has been well characterized. *psbD* BLRP transcription is specifically regulated by chloroplast-localized sigma factor 5 (SIG5) [71,91]. *psbD* BLRP transcription is also induced by environmental stresses, such as high salinity, low temperature, and osmotic stress [71]. In addition, *psbD* BLRP transcription is modulated in response to the relative proportions of red and far red light in a process mediated by signals from phytochromes [92]. Thus, *psbD* BLRP transcription is modulated during plant responses to environmental stress and sensing of light signals. Indeed, high *psbD* BLRP activity favors the synthesis of D2, thus relieving high-light-induced damage to PSII [93]. On the other hand, *psbD* BLRP transcription mediated by SIG5 shows obvious circadian oscillation, revealing how chloroplast gene expression is involved in the circadian oscillator [94].

*psbD* BLRP transcription may be also involved in biotic stress responses. Pathogens deliver various effectors into plant host cells when pathogens attack plants. These effectors assist pathogen proliferation and suppress plant defense responses [95–101]. Two *Pseudomonas* effectors, HopR1 and HopBB1, has been suggested to be involved in *psbD* transcription by targeting PTF1 (PLASTID TRANSCRIPTION FACTOR 1), a transcription factor for *psbD* BLRP transcription [102,103]. Moreover, the loss of PTF1 leads to more resistant to *Pseudomonas syringae* pv. *tomato* strain DC3000 in Arabidopsis [104]. Thus, *psbD* BLRP transcription may play a role in biotic stress responses.

The *psbEFLJ* operon contains four chloroplast genes: *psbE*, *psbF*, *psbL*, and *psbJ*. These genes encode the α and β subunits of cytochrome b559, PsbL, and PsbJ, respectively, which are crucial for the proper functioning of PSII [105,106]. The transcriptional regulation of *psbEFLJ* was recently investigated. *psbEFLJ* transcription is positively regulated by the nucleus-encoded protein mTERF5 (mitochondrial Transcription Termination Factor 5), which acts as a pausing factor [72,107]. mTERF5 causes transcriptional pausing on *psbEFLJ* by binding to the nucleotides+30 to+51 from the transcription start site and recruits additional pTAC6 into the PEP complex at the pausing region to form an enhanced PEP complex, thus positively regulating *psbEFLJ* transcription. In addition, *mterf5* mutants are less sensitive to NaCl and abscisic acid (ABA) than wild-type plants, indicating that mTERF5 functions as a negative regulator of salt tolerance, perhaps via ABA signaling [73]. These findings point to functional links between *psbEFLJ* transcription and salt tolerance as well as ABA signaling.

#### *3.2. RNA Metabolism and Environmental Stress Responses*

RNA metabolism in chloroplasts is remarkably complex, involving a series of steps such as 5 and 3 trimming, RNA editing, splicing, and intergenic cleavage [31]. Analyses of mutants with defective RNA editing suggested that RNA editing, splicing, and stability help regulate environmental stress responses in plants [28,29].

An overall deficiency in chloroplast RNA editing (C-to-U base conversion) in Arabidopsis could be caused by the mutation of *ORRM1* (*Organelle RRM Protein 1*), encoding an essential plastid RNA editing factor. *orrm1* mutants exhibited greatly reduced RNA editing efficiency compared to wild-type Arabidopsis at 62% (21 of 34) of the chloroplast editing sites. Among these, the editing efficiency at 12 sites decreased by at least 90%, whereas that of the nine other sites decreased by 10% to 90% in *orrm1* vs. wild-type plants [74]. The reduced RNA editing deficiency at multiple sites in *orrm1* plants did not result in distinctive phenotypes at normal temperatures (22 ◦C), but the mutants were sensitive to chilling, displaying yellow emerging leaves under chilling conditions (4 ◦C) [75]. These findings suggest that chloroplast RNA editing confers low-temperature tolerance in Arabidopsis. However, the RNA editing site that confers this improved low-temperature tolerance is unknown.

The *indica* (*Oryza sativa* ssp. *indica*) rice cultivar *Dular*, referred to as *dua1*, is planted in tropical regions of Southeast Asia, including India and the Philippines. *dua1* plants are less tolerant of low temperatures than Nipponbare (*O. sativa* ssp. *japonica*) plants, which are grown in northern areas of Asia, as *dua1* plants display pale leaves under low-temperature conditions (19 ◦C). A recent study revealed that that the low-temperature sensitivity of *dua1* is caused by defective RNA editing of the plastid ribosome gene *rps8*, which is located 182 nt downstream of the translational start site (*rps8*-182). The edited *rps8* transcripts generate RPS8 protein with altered amino acid hydrophobicity, suggesting that RNA editing at *rps8*-182 improves low-temperature tolerance in rice by moderating the stability of RPS8 protein under low-temperature conditions [76]. Chloroplast genomes have very slow rates of sequence evolution, averaging ~5-fold slower than nuclear genomes [108,109], suggesting that chloroplast RNA editing evolved to improve low-temperature tolerance by increasing protein stability.

*ndhB* encodes the B subunit of the chloroplast NADH dehydrogenase-like complex that is required for cyclic electron flow around photosystem I [110,111]. The defective RNA editing of *ndhB*-2, *ndhB*-3, *ndhB*-4, and *ndhB*-6 sites enhances the disease resistance against fungal pathogens in Arabidopsis [112]. This finding suggests that chloroplast RNA editing is interlinked with plant immunity.

*rpl2* encodes a component of the 50S subunit in the chloroplast ribosome. This gene contains only a group II intron. In rice, the splicing of this intron is specifically regulated by WHITE STRIPE LEAF (WSL), a pentatricopeptide repeat (PPR) protein. Compared to the wild type, *wsl* mutants exhibit a decreased germination rate and reduced shoot and root growth upon treatment with ABA but not with α-naphthaleneacetic acid (NAA, an auxin), gibberellic acid (GA), epi-brassinosteroid (BL), or 6-benzylaminopurine (6-BA, a cytokinin). This finding suggests that the ABA signaling process is

specifically affected in *wsl*. These mutants also display decreased germination rates when grown on medium supplemented with sugar and NaCl [77]. Sugar and salinity responses are closely connected with ABA signaling, and several ABA-related genes (e.g., *ABI3* and *WRKY24*) are induced by ABA treatment in *wsl* mutants, suggesting that *rpl2* splicing plays an important role in plant responses to ABA.

In Arabidopsis, the splicing of chloroplast *trnA*, *trnI*, *rpl2*, *rps12* intron 1, and *rps12* intron 2 is regulated by DEAD-BOX RNA HELICASE 3 (RH3) [78]. Null mutants of *RH3* are embryo lethal, whereas the weak allele *rh3-4* displays retarded plant growth and pale-green leaves, along with considerable decreases in the splicing efficiency of *trnA*, *trnI*, *rpl2*, *rps12* intron 1, and *rps12* intron 2. Moreover, the endogenous ABA contents of 1-week-old *rh3-4* seedlings are ~50% lower than those of wild-type plants, suggesting that RH3 plays a role in ABA biosynthesis. The mutation of *RH3* results in the reduced expression of nucleus-encoded gene *ABA1* and *NCDE4*, encoding two crucial enzymes of the ABA biosynthetic pathway, perhaps explaining the decreased ABA contents of *rh3-4* seedlings. Consistent with their decreased ABA contents, *rh3-4* mutants exhibit more severely inhibited plant growth and greening than the wild type under abiotic stress conditions including salinity, cold, and dehydration stress [78–80]. These findings suggest that chloroplast RNA splicing of these genes is required for environmental stress responses in plants, especially responses related to ABA signaling. Yet how chloroplast RNA splicing regulates environmental stress responses is currently unknown. A defect in chloroplast RNA splicing would be likely to result in defective photosynthetic performance, thus leading to enhanced sensitivity to environmental stresses. Alternatively, chloroplast RNA splicing might trigger plastid-to-nucleus retrograde signaling to regulate plant stress responses.

Chloroplast RNA stability is also crucial for the proper expression of chloroplast genes. Increasing evidence indicates that chloroplast RNA stability is involved in plant responses to environmental stresses. Chloroplast ribonucleoproteins CP31A and CP29A are RNA chaperone proteins that associate with large sets of chloroplast transcripts [81,113]. Arabidopsis mutants with deletions of *CP31A* and *CP29A* do not have unusual phenotypes under normal conditions but show bleaching of newly emerging leaves at the bases of the youngest leaves under cold stress (8 ◦C). Kupsch et al. demonstrated that CP31A and CP29A are required for the accumulation of transcripts of many chloroplast genes under cold stress (8 ◦C), such as *psaA*, *psbD*, *psbF*, *psbB*, *petB*, *ndhF*, and *rbcL*. This cold-sensitive phenotype could be explained by a decreased stability of chloroplast transcripts in the *cp31a* and *cp29a* mutants [81,113]. DEAD-box RNA helicase 22 (RH22) is another chloroplast RNA chaperone. In cabbage (*Brassica rapa*), *RH22* expression was significantly upregulated by drought, heat, salt, and cold stress but markedly downregulated by UV stress. The overexpression of cabbage *RH22* enhanced the stability of chloroplast transcripts and improved growth and survival in Arabidopsis under drought and salt stress [114]. Moreover, Arabidopsis plants overexpressing cabbage *RH22* displayed better growth and more green leaves upon ABA treatment than the wild type, along with decreased expression of *ABI3*, *ABI4*, and *ABI5*, suggesting that chloroplast RNA stability plays a part in ABA signaling pathways [114]. Chloroplast RNA stability might have a positive role in plant responses to environmental stress by enhancing the translation of chloroplast genes.
