A prion-like domain is required for phase separation and chloroplast RNA processing during cold acclimation in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) plants can produce photosynthetic tissue with active chloroplasts at temperatures as low as 4°C, and this process depends on the presence of the nuclear-encoded, chloroplast-localized RNA-binding protein CP29A. In this study, we demonstrate that CP29A undergoes phase separation in vitro and in vivo in a temperature-dependent manner, which is mediated by a prion-like domain (PLD) located between the two RNA recognition motif (RRM) domains of CP29A. The resulting droplets display liquid-like properties and are found near chloroplast nucleoids. The PLD is required to support chloroplast RNA splicing and translation in cold-treated tissue. Together, our findings suggest that plant chloroplast gene expression is compartmentalized by inducible condensation of CP29A at low temperatures, a mechanism that could play a crucial role in plant cold resistance.

A CRR2-Dependent sRNA Sequence Supports Papillomavirus Vaccine Expression in Tobacco Chloroplasts.

Human papillomavirus (HPV) infection is the leading cause of cervical cancer, and vaccination with HPV L1 capsid proteins has been successful in controlling it. However, vaccination coverage is not universal, particularly in developing countries, where 80% of all cervical cancer cases occur. Cost-effective vaccination could be achieved by expressing the L1 protein in plants. Various efforts have been made to produce the L1 protein in plants, including attempts to express it in chloroplasts for high-yield performance. However, manipulating chloroplast gene expression requires complex and difficult-to-control expression elements. In recent years, a family of nuclear-encoded, chloroplast-targeted RNA-binding proteins, the pentatricopeptide repeat (PPR) proteins, were described as key regulators of chloroplast gene expression. For example, PPR proteins are used by plants to stabilize and translate chloroplast mRNAs. The objective is to demonstrate that a PPR target site can be used to drive HPV L1 expression in chloroplasts. To test our hypothesis, we used biolistic chloroplast transformation to establish tobacco lines that express two variants of the HPV L1 protein under the control of the target site of the PPR protein CHLORORESPIRATORY REDUCTION2 (CRR2). The transgenes were inserted into a dicistronic operon driven by the plastid rRNA promoter. To determine the effectiveness of the PPR target site for the expression of the HPV L1 protein in the chloroplasts, we analyzed the accumulation of the transgenic mRNA and its processing, as well as the accumulation of the L1 protein in the transgenic lines. We established homoplastomic lines carrying either the HPV18 L1 protein or an HPV16B Enterotoxin::L1 fusion protein. The latter line showed severe growth retardation and pigment loss, suggesting that the fusion protein is toxic to the chloroplasts. Despite the presence of dicistronic mRNAs, we observed very little accumulation of monocistronic transgenic mRNA and no significant increase in CRR2-associated small RNAs. Although both lines expressed the L1 protein, quantification using an external standard suggested that the amounts were low. Our results suggest that PPR binding sites can be used to drive vaccine expression in plant chloroplasts; however, the factors that modulate the effectiveness of target gene expression remain unclear. The identification of dozens of PPR binding sites through small RNA sequencing expands the set of expression elements available for high-value protein production in chloroplasts.

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.

Ectopic Transplastomic Expression of a Synthetic MatK Gene Leads to Cotyledon-Specific Leaf Variegation.

Chloroplasts (and other plastids) harbor their own genetic material, with a bacterial-like gene-expression systems. Chloroplast RNA metabolism is complex and is predominantly mediated by nuclear-encoded RNA-binding proteins. In addition to these nuclear factors, the chloroplast-encoded intron maturase MatK has been suggested to perform as a splicing factor for a subset of chloroplast introns. MatK is essential for plant cell survival in tobacco, and thus null mutants have not yet been isolated. We therefore attempted to over-express MatK from a neutral site in the chloroplast, placing it under the control of a theophylline-inducible riboswitch. This ectopic insertion of MatK lead to a variegated cotyledons phenotype. The addition of the inducer theophylline exacerbated the phenotype in a concentration-dependent manner. The extent of variegation was further modulated by light, sucrose and spectinomycin, suggesting that the function of MatK is intertwined with photosynthesis and plastid translation. Inhibiting translation in the transplastomic lines has a profound effect on the accumulation of several chloroplast mRNAs, including the accumulation of an RNA antisense to rpl33, a gene coding for an essential chloroplast ribosomal protein. Our study further supports the idea that MatK expression needs to be tightly regulated to prevent detrimental effects and establishes another link between leaf variegation and chloroplast translation.

Stabilization and translation of synthetic operon-derived mRNAs in chloroplasts by sequences representing PPR protein-binding sites.

The chloroplast is a prime target for genetic engineering in plants, offering various advantages over nuclear transformation. For example, chloroplasts allow the expression of polycistronic transcripts and thus to engineer complex metabolic pathways. Each cistron within such a longer transcript needs its own expression elements. Within the 5'-UTR, such expression elements are needed for stabilizing mRNAs and for translation of the downstream reading frame. One of the few effective expression elements used so far in transplastomic approaches is the intercistronic expression element (IEE). The IEE is derived from the psbT-psbH intergenic region and includes a target sequence of the RNA binding protein HCF107. We here show that excessive expression of the IEE can lead to specific defects of endogenous chloroplast mRNA stabilization, likely via depletion of HCF107. Key players in chloroplast transcript stabilization and translation are pentatricopeptide repeat (PPR) proteins, which are structurally related to HCF107. PPR proteins that stabilize mRNAs leave behind short RNA footprints that are indicators of their activity. We identified such sRNAs in tobacco, and demonstrate that they are sufficient to stabilize and stimulate translation of mRNAs from synthetic dicistronic transgenes in chloroplasts. Thus, minimal sequence elements are generally adequate to support key steps in chloroplast gene expression, i.e. RNA stability and translation. Furthermore, our analysis expands the repertoire of available expression elements to facilitate the assembly and expression of multi-gene ensembles in the chloroplast.

Stable Membrane-Association of mRNAs in Etiolated, Greening and Mature Plastids.

Chloroplast genes are transcribed as polycistronic precursor RNAs that give rise to a multitude of processing products down to monocistronic forms. Translation of these mRNAs is realized by bacterial type 70S ribosomes. A larger fraction of these ribosomes is attached to chloroplast membranes. This study analyzed transcriptome-wide distribution of plastid mRNAs between soluble and membrane fractions of purified plastids using microarray analyses and validating RNA gel blot hybridizations. To determine the impact of light on mRNA localization, we used etioplasts, greening plastids and mature chloroplasts from Zea mays as a source for membrane and soluble extracts. The results show that the three plastid types display an almost identical distribution of RNAs between the two organellar fractions, which is confirmed by quantitative RNA gel blot analyses. Furthermore, they reveal that different RNAs processed from polycistronic precursors show transcript-autonomous distribution between stroma and membrane fractions. Disruption of ribosomes leads to release of mRNAs from membranes, demonstrating that attachment is likely a direct consequence of translation. We conclude that plastid mRNA distribution is a stable feature of different plastid types, setting up rapid chloroplast translation in any plastid type.

Function of calcium-dependent protein kinase CPK28 of Arabidopsis thaliana in plant stem elongation and vascular development.

After a period of vegetative growth, plants undergo a developmental switch to the reproductive phase, inducing the transition to bolting, elongation of the inflorescence and flowering. We have identified calcium-dependent protein kinase CPK28 from Arabidopsis thaliana as a regulatory component that controls stem elongation and vascular development. In two independent mutant alleles of cpk28, a reduction of stem elongation, accompanied by shorter leaf petioles and enhanced anthocyanin levels, is observed upon the transition to the generative phase. Anatomical analysis revealed an altered vascular pattern characterised by fewer xylem tracheary elements but at the same time increased lignification and secondary growth. Coincident with these morphological changes, cpk28 mutants showed altered expression of NAC transcriptional regulators NST1 and NST3 as well as of GA3ox1, a key regulator of gibberellic acid homeostasis. In vitro protein kinase activity of CPK28 is strictly calcium-dependent. Furthermore, CPK28 is phosphorylated in vivo at several sites. Site-specific amino acid substitutions at these phosphorylation sites resulted in reduced in vitro activity. However, when introduced into a cpk28 mutant background, wild-type and phosphorylation site variants, but not kinase-inactive variants of CPK28 complemented the morphological and developmental defects. Our data identify CPK28 as a developmentally controlled regulator for coordinated stem elongation and secondary growth.

Transcriptome analysis of the Euglena gracilis plastid chromosome.

The characterisation of transcript levels of chloroplast genes and their changes under different conditions is an initial step towards understanding chloroplast gene expression and the functional integration of the plastid chromosome into the entire integrated compartmentalised genome of the plant cell. Using RNA from cells of 12 different developmental stages and stress treatments, we have studied the transcript patterns of all 96 genes of the circular plastid chromosome of Euglena gracilis, Pringsheim strain Z, by a macroarray-based approach and Northern analysis of selected genes representing approximately half a dozen operons. The unicellular alga possesses complex, triple-envelope chloroplasts that were acquired by secondary endosymbiosis. (1) Transcripts were detected from all genes, although stationary concentrations varied substantially between individual loci. No obvious economy in the expression pattern with respect to transcription units and genes for complex structures was noted. (2) The chromosome appears to be constitutively expressed under all chosen conditions including stresses such as UV light, temperature, antiplastidial agents, herbicide and heavy metal exposure. (3) The euglenoid organelle transcriptome is qualitatively relatively insensitive to the environment, but exhibited marked overall quantitative changes. The more or less global changes demonstrate that primarily RNA turnover, translational, proteolytic and/or metabolic control regulate organelle gene expression in the alga.

Role of the low-molecular-weight subunits PetL, PetG, and PetN in assembly, stability, and dimerization of the cytochrome b6f complex in tobacco.

The cytochrome b(6)f (Cyt b(6)f) complex in flowering plants contains nine conserved subunits, of which three, PetG, PetL, and PetN, are bitopic plastid-encoded low-molecular-weight proteins of largely unknown function. Homoplastomic knockout lines of the three genes have been generated in tobacco (Nicotiana tabacum 'Petit Havana') to analyze and compare their roles in assembly and stability of the complex. Deletion of petG or petN caused a bleached phenotype and loss of photosynthetic electron transport and photoautotrophy. Levels of all subunits that constitute the Cyt b(6)f complex were faintly detectable, indicating that both proteins are essential for the stability of the membrane complex. In contrast, DeltapetL plants accumulate about 50% of other Cyt b(6)f subunits, appear green, and grow photoautotrophically. However, DeltapetL plants show increased light sensitivity as compared to wild type. Assembly studies revealed that PetL is primarily required for proper conformation of the Rieske protein, leading to stability and formation of dimeric Cyt b(6)f complexes. Unlike wild type, phosphorylation levels of the outer antenna of photosystem II (PSII) are significantly decreased under state II conditions, although the plastoquinone pool is largely reduced in DeltapetL, as revealed by measurements of PSI and PSII redox states. This confirms the sensory role of the Cyt b(6)f complex in activation of the corresponding kinase. The reduced light-harvesting complex II phosphorylation did not affect state transition and association of light-harvesting complex II to PSI under state II conditions. Ferredoxin-dependent plastoquinone reduction, which functions in cyclic electron transport around PSI in vivo, was not impaired in DeltapetL.

Plastid tRNA genes trnC-GCA and trnN-GUU are essential for plant cell development.

Higher plant chloroplast genomes code for a conserved set of 30 tRNAs. This set is believed to be sufficient to support translation, although import of cytosolic tRNA has been proposed to provide additional tRNA species to the chloroplast. Previous knock-outs of tRNA genes, or the pronounced reduction of the level of selected tRNAs, has not led to severe phenotypes. We deleted the two tRNA genes trnN-GUU and trnC-GCA independently from the plastid chromosome of tobacco. No homoplastomic tissue of either DeltatrnN or DeltatrnC plants could be isolated. Both mutants exhibit occasional loss of leaf sectors, and mutant plastid chromosomes are rapidly lost upon relief of selective pressure. This suggests that the knock-out of both trn genes is lethal, and that both tRNA species are required for cell survival. Surprisingly, the impact on chloroplast and cell development differs pronouncedly between the two mutants. Heteroplastomic DeltatrnC and DeltatrnN tissue exhibit different aberrations of the internal membrane systems and, more importantly, heteroplastomic DeltatrnN plants are variegated. Accumulation of tRNA-N and plastid-encoded proteins is reduced in white sectors of DeltatrnN plants, and differentiation of palisade cells is abolished. Our data demonstrate that plastid tRNAs are essential, i.e. not complemented by cytosolic tRNA, and have a differential impact on chloroplast and plant cell development.

A Rab-E GTPase mutant acts downstream of the Rab-D subclass in biosynthetic membrane traffic to the plasma membrane in tobacco leaf epidermis.

The function of the Rab-E subclass of plant Rab GTPases in membrane traffic was investigated using a dominant-inhibitory mutant (RAB-E1(d)[NI]) of Arabidopsis thaliana RAB-E1(d) and in vivo imaging approaches that have been used to characterize similar mutants in the plant Rab-D2 and Rab-F2 subclasses. RAB-E1(d)[NI] inhibited the transport of a secreted green fluorescent protein marker, secGFP, but in contrast with dominant-inhibitory RAB-D2 or RAB-F2 mutants, it did not affect the transport of Golgi or vacuolar markers. Quantitative imaging revealed that RAB-E1(d)[NI] caused less intracellular secGFP accumulation than RAB-D2(a)[NI], a dominant-inhibitory mutant of a member of the Arabidopsis Rab-D2 subclass. Furthermore, whereas RAB-D2(a)[NI] caused secGFP to accumulate exclusively in the endoplasmic reticulum, RAB-E1(d)[NI] caused secGFP to accumulate additionally in the Golgi apparatus and a prevacuolar compartment that could be labeled by FM4-64 and yellow fluorescent protein (YFP)-tagged Arabidopsis RAB-F2(b). Using the vacuolar protease inhibitor E64-d, it was shown that some secGFP was transported to the vacuole in control cells and in the presence of RAB-E1(d)[NI]. Consistent with the hypothesis that secGFP carries a weak vacuolar-sorting determinant, it was shown that a secreted form of DsRed reaches the apoplast without appearing in the prevacuolar compartment. When fused to RAB-E1(d), YFP was targeted specifically to the Golgi via a saturable nucleotide- and prenylation-dependent mechanism but was never observed on the prevacuolar compartment. We propose that RAB-E1(d)[NI] inhibits the secretory pathway at or after the Golgi, causing an accumulation of secGFP in the upstream compartments and an increase in the quantity of secGFP that enters the vacuolar pathway.

Comparative analysis of plastid transcription profiles of entire plastid chromosomes from tobacco attributed to wild-type and PEP-deficient transcription machineries.

Transcription of plastid chromosomes in vascular plants is accomplished by at least two RNA polymerases of different phylogenetic origin: the ancestral (endosymbiotic) cyanobacterial-type RNA polymerase (PEP), of which the core is encoded in the organelle chromosome, and an additional phage-type RNA polymerase (NEP) of nuclear origin. Disruption of PEP genes in tobacco leads to off-white phenotypes. A macroarray-based approach of transcription rates and of transcript patterns of the entire plastid chromosome from leaves of wild-type as well as from transplastomic tobacco lacking PEP shows that the plastid chromosome is completely transcribed in both wild-type and PEP-deficient plastids, though into polymerase-specific profiles. Different probe types, run-on transcripts, 5' or 3' labelled RNAs, as well as cDNAs, have been used to evaluate the array approach. The findings combined with Northern and Western analyses of a selected number of loci demonstrate further that frequently no correlation exists between transcription rates, transcript levels, transcript patterns, and amounts of corresponding polypeptides. Run-on transcription as well as stationary RNA concentrations may increase, decrease or remain similar between the two experimental materials, independent of the nature of the encoded gene product or of the multisubunit assembly (thylakoid membrane or ribosome). Our findings show (i) that the absence of photosynthesis-related, plastome-encoded polypeptides in PEP-deficient plants is not directly caused by a lack of transcription by PEP, and demonstrate (ii) that the functional integration of PEP and NEP into the genetic system of the plant cell during evolution is substantially more complex than presently supposed.

Six active phage-type RNA polymerase genes in Nicotiana tabacum.

In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA polymerases (RNAP) homologous to the bacteriophage T7-enzyme. The Arabidopsis genome contains three such RpoT genes, while in monocotyledonous plants only two copies have been found. Analysis of Nicotiana tabacum, a natural allotetraploid, identified six different RpoT sequences. The study of the progenitor species of tobacco, N. sylvestris and N. tomentosiformis, uncovered that the sequences represent two orthologous sets each of three RpoT genes (RpoT1, RpoT2 and RpoT3). Interestingly, while the organelles are inherited exclusively from the N. sylvestris maternal parent, all six RpoT genes are expressed in N. tabacum. GFP-fusions of Nicotiana RpoT1 revealed mitochondrial targeting properties. Constructs containing the amino-terminus of RpoT2 were imported into mitochondria as well as into plastids. Thus, the dual-targeting feature, first described for Arabidopsis RpoT;2, appears to be conserved among eudicotyledonous plants. Tobacco RpoT3 is targeted to chloroplasts and the RNA is differentially expressed in plants lacking the plastid-encoded RNAP. Remarkably, translation of RpoT3 mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis, Nicotiana RpoT3 provides a second example for a non-viral plant mRNA that is exclusively translated from a non-AUG codon.