CAF Proteins Help SOT1 Regulate the Stability of Chloroplast ndhA Transcripts

Protein-mediated RNA stabilization plays profound roles in chloroplast gene expression. Genetic studies have indicated that chloroplast ndhA transcripts, encoding a key subunit of the NADH dehydrogenase-like complex that mediates photosystem I cyclic electron transport and facilitates chlororespiration, are stabilized by PPR53 and its orthologs, but the underlying mechanisms are unclear. Here, we report that CHLOROPLAST RNA SPLICING 2 (CRS2)-ASSOCIATED FACTOR (CAF) proteins activate SUPPRESSOR OF THYLAKOID FORMATION 1 (SOT1), an ortholog of PPR53 in Arabidopsis thaliana, enhancing their affinity for the 5′ ends of ndhA transcripts to stabilize these molecules while inhibiting the RNA endonuclease activity of the SOT1 C-terminal SMR domain. In addition, we established that SOT1 improves the splicing efficiency of ndhA by facilitating the association of CAF2 with the ndhA intron, which may be due to the SOT1-mediated stability of the ndhA transcripts. Our findings shed light on the importance of PPR protein interaction partners in moderating RNA metabolism.

Pas de Trois: An Overview of Penta-, Tetra-, and Octo-Tricopeptide Repeat Proteins From Chlamydomonas reinhardtii and Their Role in Chloroplast Gene Expression

Penta-, Tetra-, and Octo-tricopeptide repeat (PPR, TPR, and OPR) proteins are nucleus-encoded proteins composed of tandem repeats of 35, 34, and 38–40 amino acids, respectively. They form helix-turn-helix structures that interact with mRNA or other proteins and participate in RNA stabilization, processing, maturation, and act as translation enhancers of chloroplast and mitochondrial mRNAs. These helical repeat proteins are unevenly present in plants and algae. While PPR proteins are more abundant in plants than in algae, OPR proteins are more abundant in algae. In Arabidopsis, maize, and rice there have been 450, 661, and 477 PPR proteins identified, respectively, which contrasts with only 14 PPR proteins identified in Chlamydomonas reinhardtii. Likewise, more than 120 OPR proteins members have been predicted from the nuclear genome of C. reinhardtii and only one has been identified in Arabidopsis thaliana. Due to their abundance in land plants, PPR proteins have been largely characterized making it possible to elucidate their RNA-binding code. This has even allowed researchers to generate engineered PPR proteins with defined affinity to a particular target, which has served as the basis to develop tools for gene expression in biotechnological applications. However, fine elucidation of the helical repeat proteins code in Chlamydomonas is a pending task. In this review, we summarize the current knowledge on the role PPR, TPR, and OPR proteins play in chloroplast gene expression in the green algae C. reinhardtii, pointing to relevant similarities and differences with their counterparts in plants. We also recapitulate on how these proteins have been engineered and shown to serve as mRNA regulatory factors for biotechnological applications in plants and how this could be used as a starting point for applications in algae.

The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism

Sustainable and economically viable support for an ever-increasing global population requires a paradigm shift in agricultural productivity, including the application of biotechnology to generate future crop plants. Current genetic engineering approaches aimed at enhancing the photosynthetic efficiency or composition of the harvested tissues involve relatively simple manipulations of endogenous metabolism. However, radical rewiring of central metabolism using new-to-nature pathways, so-called “synthetic metabolism”, may be needed to really bring about significant step changes. In many cases, this will require re-programming the metabolism of the chloroplast, or other plastids in non-green tissues, through a combination of chloroplast and nuclear engineering. However, current technologies for sophisticated chloroplast engineering (“transplastomics”) of plants are limited to just a handful of species. Moreover, the testing of metabolic rewiring in the chloroplast of plant models is often impractical given their obligate phototrophy, the extended time needed to create stable non-chimeric transplastomic lines, and the technical challenges associated with regeneration of whole plants. In contrast, the unicellular green alga, Chlamydomonas reinhardtii is a facultative heterotroph that allows for extensive modification of chloroplast function, including non-photosynthetic designs. Moreover, chloroplast engineering in C. reinhardtii is facile, with the ability to generate novel lines in a matter of weeks, and a well-defined molecular toolbox allows for rapid iterations of the “Design-Build-Test-Learn” (DBTL) cycle of modern synthetic biology approaches. The recent development of combinatorial DNA assembly pipelines for designing and building transgene clusters, simple methods for marker-free delivery of these clusters into the chloroplast genome, and the pre-existing wealth of knowledge regarding chloroplast gene expression and regulation in C. reinhardtii further adds to the versatility of transplastomics using this organism. Herein, we review the inherent advantages of the algal chloroplast as a simple and tractable testbed for metabolic engineering designs, which could then be implemented in higher plants.

A ppGpp-mediated brake on photosynthesis is required for acclimation to nitrogen limitation in Arabidopsis.

Guanosine pentaphosphate and tetraphosphate (together referred to as ppGpp) are hyperphosphorylated nucleotides found in bacteria and the chloroplasts of plants and algae. In plants and algae artificial ppGpp accumulation can inhibit chloroplast gene expression, and influence photosynthesis, nutrient remobilisation, growth, and immunity. However, it is so far unknown whether ppGpp is required for abiotic stress acclimation in plants. Here, we demonstrate that ppGpp biosynthesis is necessary for acclimation to nitrogen starvation in Arabidopsis. We show that ppGpp is required for remodeling the photosynthetic electron transport chain to downregulate photosynthetic activity and for protection against oxidative stress. Furthermore, we demonstrate that ppGpp is required for coupling chloroplastic and nuclear gene expression during nitrogen starvation. Altogether, our work indicates that ppGpp is a pivotal regulator of chloroplast activity for stress acclimation in plants.

Overexpression of 7-hydroxymethyl Chlorophyll a Reductase from Cucumber in Tobacco Accelerates Dark-Induced Chlorophyll Degradation

7-hydroxymethyl chlorophyll (Chl) a reductase (HCAR) plays critical roles in the Chl cycle and degradation during leaf senescence, however, its function in horticultural crops remains unknown. Here, we identified an HCAR gene (CsHCAR) from cucumber (Cucumis sativus L.) and investigated its roles in response to dark-induced Chl degradation. CsHCAR encoded 459 amino acids, which were orthologous to Arabidopsis HCAR, had the conserved domains, and localized in the chloroplast. Gene expression analysis showed that CsHCAR expression was the highest in senescent leaves and was responsive to different stresses and phytohormone treatments. Overexpression of CsHCAR in tobacco accelerated dark-induced Chl degradation through enhancing the expression of Chl catabolic genes. After 10 d of darkness treatment, the biomass of CsHCAR overexpression plants was reduced. Furthermore, the value of net photosynthetic rate, maximum quantum yield of photosystem II, and effective quantum yield of photosystem II in CsHCAR overexpression plants was significantly reduced in comparison to that in wild-type (WT) plants. The photosynthetic protein content, including Lhcb1, Lhcb2, Lhcb4, RbcS, and RbcL in CsHCAR overexpression plants exhibited a lower level as compared to that observed in WT plants. In addition, the expression of genes encoding these proteins in CsHCAR overexpression plants was significantly lower than that in WT plants. Moreover, CsHCAR overexpression plants inhibited the dark-induced accumulation of reactive oxygen species (ROS). These results indicate that CsHCAR affects the stability of photosynthetic proteins in chloroplasts, positively regulates Chl degradation, and plays an important role in maintaining ROS homeostasis in leaves.

The Chloroplast Epitranscriptome: Factors, Sites, Regulation, and Detection Methods

Modifications in nucleic acids are present in all three domains of life. More than 170 distinct chemical modifications have been reported in cellular RNAs to date. Collectively termed as epitranscriptome, these RNA modifications are often dynamic and involve distinct regulatory proteins that install, remove, and interpret these marks in a site-specific manner. Covalent nucleotide modifications-such as methylations at diverse positions in the bases, polyuridylation, and pseudouridylation and many others impact various events in the lifecycle of an RNA such as folding, localization, processing, stability, ribosome assembly, and translational processes and are thus crucial regulators of the RNA metabolism. In plants, the nuclear/cytoplasmic epitranscriptome plays important roles in a wide range of biological processes, such as organ development, viral infection, and physiological means. Notably, recent transcriptome-wide analyses have also revealed novel dynamic modifications not only in plant nuclear/cytoplasmic RNAs related to photosynthesis but especially in chloroplast mRNAs, suggesting important and hitherto undefined regulatory steps in plastid functions and gene expression. Here we report on the latest findings of known plastid RNA modifications and highlight their relevance for the post-transcriptional regulation of chloroplast gene expression and their role in controlling plant development, stress reactions, and acclimation processes.

A Transcription Factor Regulates Gene Expression in Chloroplasts

The chloroplast is a semi-autonomous organelle with its own genome. The expression of chloroplast genes depends on both chloroplasts and the nucleus. Although many nucleus-encoded proteins have been shown to localize in chloroplasts and are essential for chloroplast gene expression, it is not clear whether transcription factors can regulate gene expression in chloroplasts. Here we report that the transcription factor NAC102 localizes in both chloroplasts and nucleus in Arabidopsis. Specifically, NAC102 localizes in chloroplast nucleoids. Yeast two-hybrid assay and co-immunoprecipitation assay suggested that NAC102 interacts with chloroplast RNA polymerases. Furthermore, overexpression of NAC102 in chloroplasts leads to reduced chloroplast gene expression and chlorophyll content, indicating that NAC102 functions as a repressor in chloroplasts. Our study not only revealed that transcription factors are new regulators of chloroplast gene expression, but also discovered that transcription factors can function in chloroplasts in addition to the canonical organelle nucleus.

Cellular and transcriptomic analyses reveal two-staged chloroplast biogenesis underpinning photosynthesis build-up in the wheat leaf

Background The developmental gradient in monocot leaves has been exploited to uncover leaf developmental gene expression programs and chloroplast biogenesis processes. However, the relationship between the two is barely understood, which limits the value of transcriptome data to understand the process of chloroplast development. Results Taking advantage of the developmental gradient in the bread wheat leaf, we provide a simultaneous quantitative analysis for the development of mesophyll cells and of chloroplasts as a cellular compartment. This allows us to generate the first biologically-informed gene expression map of this leaf, with the entire developmental gradient from meristematic to fully differentiated cells captured. We show that the first phase of plastid development begins with organelle proliferation, which extends well beyond cell proliferation, and continues with the establishment and then the build-up of the plastid genetic machinery. The second phase is marked by the development of photosynthetic chloroplasts which occupy the available cellular space. Using a network reconstruction algorithm, we predict that known chloroplast gene expression regulators are differentially involved across those developmental stages. Conclusions Our analysis generates both the first wheat leaf transcriptional map and one of the most comprehensive descriptions to date of the developmental history of chloroplasts in higher plants. It reveals functionally distinct plastid and chloroplast development stages, identifies processes occurring in each of them, and highlights our very limited knowledge of the earliest drivers of plastid biogenesis, while providing a basis for their future identification.

Modeling indicates degradation of mRNA and protein as a potential regulation mechanisms during cold acclimation

Plants are constantly exposed to temperature fluctuations, which have direct effects on all cellular reactions because temperature influences reaction likelihood and speed. Chloroplasts are crucial to temperature acclimation responses of plants, due to their photosynthetic reactions whose products play a central role in plant metabolism. Consequently, chloroplasts serve as sensors of temperature changes and are simultaneously major targets of temperature acclimation. The core subunits of the complexes involved in the light reactions of photosynthesis are encoded in the chloroplast. As a result, it is assumed that temperature acclimation in plants requires regulatory responses in chloroplast gene expression and protein turnover. We conducted western blot experiments to assess changes in the accumulation of two photosynthetic complexes (PSII, and Cytb6f complex) and the ATP synthase in tobacco plants over two days of acclimation to low temperature. Surprisingly, the concentration of proteins within the chloroplast varied negligibly compared to controls. To explain this observation, we used a simplified Ordinary Differential Equation (ODE) model of transcription, translation, mRNA degradation and protein degradation to explain how the protein concentration can be kept constant. This model takes into account temperature effects on these processes. Through simulations of the ODE model, we show that mRNA and protein degradation are possible targets for control during temperature acclimation. Our model provides a basis for future directions in research and the analysis of future results.