*3.3. Translation and Environmental Stress Responses*

Translation is the final step in chloroplast gene expression. Chloroplast gene translation regulates protein accumulation to optimize photosynthetic performance and to attenuate photooxidative damage. Thus, the regulation of chloroplast gene translation represents a unique component of plant responses to internal and external stimuli.

Most plants growing in direct sunlight routinely encounter high-light stress; the resulting high photon flux exceeds the photosynthetic capacity, thereby damaging the chloroplast. To explore the regulation of chloroplast gene translation during the rapid adaptation of plants to high light, a systematic ribosome profiling study was performed to detect changes in chloroplast gene translation efficiency in tobacco seedlings following transfer from moderate light to high light. The ribosome

occupancy on *psbA* transcripts (encoding PSII reaction center protein D1) increased in response to high-light treatment [115]. Given that D1 protein is the main site prone to photodamage by high light, the upregulated *psbA* translation should substantially facilitate the repair of PSII under high-light stress. However, the molecular mechanisms underlying the translational activation of *psbA* under these conditions remain to be further explored.

Studies on the functions of chloroplast ribosome proteins have revealed that maintaining sufficiently high chloroplast gene translation efficiency is important for proper chloroplast development at low temperature. Maize (*Zea mays*) mutants with a loss of ribosomal protein RPS17 were pale green when grown at moderate temperature (27 ◦C) but appeared albino under cool conditions (17 ◦C) [83]. Tobacco mutants with a loss of the ribosomal protein Rpl33 showed no visible phenotypes at any stage of development under standard conditions, with similar development, growth rates, and onset of flowering to wild-type plants. However, the *Rpl33* knockout mutants were sensitive to cold stress, although not to heat or to low or high light levels. When *Rpl33* knockout mutants were transferred to cold-stress conditions (4 ◦C), they exhibited strong photooxidative damage symptoms and recovered much more slowly from low-temperature stress than wild-type plants [85]. As with Rpl33, the loss of the ribosomal protein Rps15 in tobacco resulted in a growth phenotype almost identical growth to that of wild-type plants, although young plants grew slightly more slowly and the onset of flowering was slightly delayed. However, the *Rps15* knockout mutants were cold sensitive, with more severe pigment loss and worse photosynthetic performance than wild-type plants [84]. Together, these findings suggest that the maintenance of plastid translational capacity is important in enabling plant tolerance to chilling stress.

In Arabidopsis, the expression of the chloroplast ribosome protein gene *RPS1* was considerably induced by heat stress (2 h at 38 ◦C). *RPS1* knockdown mutants (*rps1*) displayed retarded growth and slightly pale-green leaves. When *rps1* seedlings were exposed to transient high-temperature conditions (3 h at 45 ◦C), they were much more heat sensitive than wild-type seedlings, as almost no mutants survived after a 7-d recovery, whereas more than 90% of wild-type seedlings did. However, there were no significant differences between *rps1* and wild-type plants under osmotic and salinity stress. These results suggest that decreased *RPS1* expression alters cellular heat stress responses by disrupting chloroplast gene translation rather than through general physiological defects. RPS1 is required to activate the expression of *HsfA2* (*HEAT STRESS TRANSCRIPTION FACTOR A-2*), a highly heat-shock-inducible gene encoding a transcription factor that is crucial for triggering cellular responses to heat stress. The constitutive expression of *HsfA2* was sufficient to rescue the heat-sensitive phenotype of *rps1* mutants, suggesting that the defective expression of *HsfA2* is responsible for the heat-sensitive phenotype of *rps1* mutants. Like the *rps1* mutant phenotype, treatment with lincomycin, an inhibitor of chloroplast gene translation, also led to an obvious reduction in the expression of *HsfA2* in response to heat stress [82]. These findings reveal a plastid-to-nucleus retrograde signaling pathway that regulates chloroplast gene translational capacity to transcriptionally activate cellular heat stress responses, especially the HsfA2-dependent heat tolerance pathway.

#### **4. Conclusions and Future Perspectives**

Photosynthesis, one of the most important physiological processes in plants, is highly sensitive to environmental stresses. These stresses often inhibit photosynthesis considerably [116–124]. Many studies have reported that chloroplasts can act as sensors of the external environment [7,19]. Thus, in addition to hosting photosynthesis, chloroplasts play important roles in plant responses to various environmental stresses. Likewise, the proper expression of chloroplast genes is crucial for chloroplast development, photosynthesis, and plant development. However, as summarized in this review, many studies indicate that chloroplast gene expression is also important for plant stress responses.

Further elucidating the roles of chloroplast gene expression in plant responses to various environmental stresses would lay the foundation for genetically improving plant tolerance to the environment. However, the underlying molecular mechanisms remains largely unknown. Most studies have focused on the roles of nucleus-encoded proteins in regulating chloroplast gene expression and plant responses to environmental stress, while the direct connection between chloroplast gene expression and environmental stress responses has been largely ignored. Future studies should therefore address two major issues: How environmental stress triggers chloroplast gene expression, and what roles chloroplast gene expression plays in plant responses to environmental stress. Several specific issues need to be investigated. For example, D1 protein synthesis is important for PSII repair under high-light conditions [71,115,125–128]. Thus, enhanced *psbA* transcription and translation would help increase the tolerance of PSII to high light; however, it is still unclear to what extent high light triggers the transcription and translation of *psbA*. In addition, RNA editing, particularly C-to-U base conversion, is a widespread phenomenon in chloroplasts across nearly all plant species [48]. As discussed above, it is clear that RNA editing is important for plant tolerance of low temperature [74–76], yet the exact editing sites, and how RNA editing at these sites improves plant tolerance to low temperatures, remain to be explored. Finally, translation and splicing of several chloroplast genes trigger plastid-to-nucleus retrograde signaling and ABA signaling [73,77,79,80,114], but it remains unclear how the signals generated from these processes are transferred out of chloroplasts and integrated into these signaling pathways.

New technology is needed to better investigate the molecular mechanism of chloroplast gene expression in response to the environment. Chloroplast transformation is an extremely time-consuming and difficult process that has only been achieved in a few plant species, such as lettuce, poplar, and Arabidopsis. This make it extremely difficult to control chloroplast gene transcription using traditional genetic engineering approaches. To further explore the connection between chloroplast gene transcription and environmental stress responses, novel tools must be designed that are similar to CRISPR/Cas9 and RNA interference to knock out and knock down chloroplast genes. It is also critical to design an artificial RNA editing system to carry out RNA editing of specific sites in chloroplasts. This system would be useful for investigating the specific roles of different RNA editing sites in plant responses to environmental stresses and even creating new RNA editing sites that could improve stress tolerance in plants.

**Author Contributions:** Y.Z. and C.L. conceived and planned this review paper designed the project. Y.Z., C.L., A.Z., and X.L. prepared and drafted the manuscript. Y.Z. and C.L. revised the manuscript. All the authors read and approved the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Natural Science Foundation of China (reference numbers 31730102 and 31670238).

**Acknowledgments:** This work was supported by the National Natural Science Foundation of China (reference numbers 31730102 and 31670238). The authors are grateful to the authors of the excellent papers discussed. The authors also apologize to the authors of the other excellent papers in this area that could not be discussed because of lack of space.

**Conflicts of Interest:** The authors declare no conflict of interest.
