Applications of Synthetic Pentatricopeptide Repeat Proteins.

RNA binding proteins play integral roles in the regulation of essential processes in cells, and as such are attractive targets for engineering to manipulate gene expression at the RNA level. Expression of transcripts in chloroplasts and mitochondria is heavily regulated by pentatricopeptide repeat (PPR) proteins. The diverse roles of PPR proteins, and their naturally modular architecture, makes them ideal candidates for engineering. Synthetic PPR proteins are showing great potential to become valuable tools for controlling the expression of plastid and mitochondrial transcripts. In this review, by 'synthetic' we mean both rationally modified natural PPR proteins and completely novel proteins designed using the principles learnt from their natural counterparts. We focus on the many different applications of synthetic PPR proteins, covering both their use in basic research to learn more about protein-RNA interactions, and their use to achieve specific outcomes in RNA processing and the control of gene expression. We describe the challenges associated with the design, construction and deployment of synthetic PPR proteins and provide perspectives on how they might be assembled and used in future biotechnology applications.

The GENOMES UNCOUPLED1 protein has an ancient, highly conserved role but not in retrograde signalling.

The pentatricopeptide repeat protein GENOMES UNCOUPLED1 (GUN1) is required for chloroplast-to-nucleus signalling when plastid translation becomes inhibited during chloroplast development in Arabidopsis thaliana, but its exact molecular function remains unknown. We analysed GUN1 sequences in land plants and streptophyte algae. We tested functional conservation by complementation of the Arabidopsis gun1 mutant with GUN1 genes from the streptophyte alga Coleochate orbicularis or the liverwort Marchantia polymorpha. We also analysed the transcriptomes of M. polymorpha gun1 knockout mutant lines during chloroplast development. GUN1 evolved within the streptophyte algal ancestors of land plants and is highly conserved among land plants but missing from the Rafflesiaceae that lack chloroplast genomes. GUN1 genes from C. orbicularis and M. polymorpha suppress the cold-sensitive phenotype of the Arabidopsis gun1 mutant and restore typical retrograde responses to treatments with inhibitors of plastid translation, even though M. polymorpha responds very differently to such treatments. Our findings suggest that GUN1 is an ancient protein that evolved within the streptophyte algal ancestors of land plants before the first plants colonized land more than 470 million years ago. Its primary role is likely to be in chloroplast gene expression and its role in chloroplast retrograde signalling probably evolved more recently.

Microtubule associated protein WAVE DAMPENED2-LIKE (WDL) controls microtubule bundling and the stability of the site of tip-growth in Marchantia polymorpha rhizoids.

Tip-growth is a mode of polarized cell expansion where incorporation of new membrane and wall is stably restricted to a single, small domain of the cell surface resulting in the formation of a tubular projection that extends away from the body of the cell. The organization of the microtubule cytoskeleton is conserved among tip-growing cells of land plants: bundles of microtubules run longitudinally along the non-growing shank and a network of fine microtubules grow into the apical dome where growth occurs. Together, these microtubule networks control the stable positioning of the growth site at the cell surface. This conserved dynamic organization is required for the spatial stability of tip-growth, as demonstrated by the formation of sinuous tip-growing cells upon treatment with microtubule-stabilizing or microtubule-destabilizing drugs. Microtubule associated proteins (MAPs) that either stabilize or destabilize microtubule networks are required for the maintenance of stable tip-growth in root hairs of flowering plants. NIMA RELATED KINASE (NEK) is a MAP that destabilizes microtubule growing ends in the apical dome of tip-growing rhizoid cells in the liverwort Marchantia polymorpha. We hypothesized that both microtubule stabilizing and destabilizing MAPs are required for the maintenance of the stable tip-growth in liverworts. To identify genes encoding microtubule-stabilizing and microtubule-destabilizing activities we generated 120,000 UV-B mutagenized and 336,000 T-DNA transformed Marchantia polymorpha plants and screened for defective rhizoid phenotypes. We identified 119 mutants and retained 30 mutants in which the sinuous rhizoid phenotype was inherited. The 30 mutants were classified into at least 4 linkage groups. Characterisation of two of the linkage groups showed that MAP genes-WAVE DAMPENED2-LIKE (WDL) and NIMA-RELATED KINASE (NEK)-are required to stabilize the site of tip growth in elongating rhizoids. Furthermore, we show that MpWDL is required for the formation of a bundled array of parallel and longitudinally orientated microtubules in the non-growing shank of rhizoids where MpWDL-YFP localizes to microtubule bundles. We propose a model where the opposite functions of MpWDL and MpNEK on microtubule bundling are spatially separated and promote tip-growth spatial stability.

A synthetic RNA editing factor edits its target site in chloroplasts and bacteria.

Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that our synthetic editing factor specifically recognises the target sequence in in vitro binding assays. The designed factor is equally specific for the target rpoA site when expressed in chloroplasts and in the bacterium E. coli. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.

Cofactor-independent RNA editing by a synthetic S-type PPR protein.

Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors and particularly prevalent in the lycophyte Selaginella. With the aid of a novel plate-based screening method, we show that synthetic S-type PPR proteins are easy to design and bind with high affinity and specificity and are functional in a wide range of pH, salt and temperature conditions. We find that they outperform a synthetic P-type PPR scaffold in many situations. We designed an S-type editing factor to edit an RNA target in E. coli and demonstrate that it edits effectively without requiring any additional cofactors to be added to the system. These qualities make S-type PPR scaffolds ideal for developing new RNA processing tools.

Negative regulation of conserved RSL class I bHLH transcription factors evolved independently among land plants.

Basic helix-loop-helix transcription factors encoded by RSL class I genes control a gene regulatory network that positively regulates the development of filamentous rooting cells - root hairs and rhizoids - in land plants. The GLABRA2 transcription factor negatively regulates these genes in the angiosperm Arabidopsis thaliana. To find negative regulators of RSL class I genes in early diverging land plants we conducted a mutant screen in the liverwort Marchantia polymorpha. This identified FEW RHIZOIDS1 (MpFRH1) microRNA (miRNA) that negatively regulates the RSL class I gene MpRSL1. The miRNA and its mRNA target constitute a feedback mechanism that controls epidermal cell differentiation. MpFRH1 miRNA target sites are conserved among liverwort RSL class I mRNAs but are not present in RSL class I mRNAs of other land plants. These findings indicate that while RSL class I genes are ancient and conserved, independent negative regulatory mechanisms evolved in different lineages during land plant evolution.

The Mechanism Forming the Cell Surface of Tip-Growing Rooting Cells Is Conserved among Land Plants.

To discover mechanisms that controlled the growth of the rooting system in the earliest land plants, we identified genes that control the development of rhizoids in the liverwort Marchantia polymorpha. 336,000 T-DNA transformed lines were screened for mutants with defects in rhizoid growth, and a de novo genome assembly was generated to identify the mutant genes. We report the identification of 33 genes required for rhizoid growth, of which 6 had not previously been functionally characterized in green plants. We demonstrate that members of the same orthogroup are active in cell wall synthesis, cell wall integrity sensing, and vesicle trafficking during M. polymorpha rhizoid and Arabidopsis thaliana root hair growth. This indicates that the mechanism for constructing the cell surface of tip-growing rooting cells is conserved among land plants and was active in the earliest land plants that existed sometime more than 470 million years ago [1, 2].

Growth regulation in tip-growing cells that develop on the epidermis.

Plants develop tip-growing extensions-root hairs and rhizoids-that initiate as swellings on the outer surface of individual epidermal cells. A conserved genetic mechanism controls the earliest stages in the initiation of these swellings. The same mechanism controls the formation of multicellular structures that develop from swellings on epidermal cells in early diverging land plants. Details of the molecular events that regulate the positioning of the swellings involve sterols and phosphatidylinositol phosphates. The final length of root hairs is determined by the intensity of a pulse of transcription factor synthesis. Genes encoding similar transcription factors control root hair development in cereals and are potential targets for crop improvement.

RSL Class I Genes Controlled the Development of Epidermal Structures in the Common Ancestor of Land Plants.

The colonization of the land by plants, sometime before 470 million years ago, was accompanied by the evolution tissue systems [1-3]. Specialized structures with diverse functions-from nutrient acquisition to reproduction-derived from single cells in the outermost layer (epidermis) were important sources of morphological innovation at this time [2, 4, 5]. In extant plants, these structures may be unicellular extensions, such as root hairs or rhizoids [6-9], or multicellular structures, such as asexual propagules or secretory hairs (papillae) [10-12]. Here, we show that a ROOTHAIR DEFECTIVE SIX-LIKE (RSL) class I basic helix-loop-helix transcription factor positively regulates the development of the unicellular and multicellular structures that develop from individual cells that expand out of the epidermal plane of the liverwort Marchantia polymorpha; mutants that lack MpRSL1 function do not develop rhizoids, slime papillae, mucilage papillae, or gemmae. Furthermore, we discovered that RSL class I genes are also required for the development of multicellular axillary hairs on the gametophyte of the moss Physcomitrella patens. Because class I RSL proteins also control the development of rhizoids in mosses and root hairs in angiosperms [13, 14], these data demonstrate that the function of RSL class I genes was to control the development of structures derived from single epidermal cells in the common ancestor of the land plants. Class I RSL genes therefore controlled the generation of adaptive morphological diversity as plants colonized the land from the water.

Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum.

This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and ß polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila.

Investigation of triterpene synthesis and regulation in oats reveals a role for ß-amyrin in determining root epidermal cell patterning.

Sterols have important functions in membranes and signaling. Plant sterols are synthesized via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to cycloartenol. Plants also convert 2,3-oxidosqualene to other sterol-like cyclization products, including the simple triterpene ß-amyrin. The function of ß-amyrin per se is unknown, but this molecule can serve as an intermediate in the synthesis of more complex triterpene glycosides associated with plant defense. ß-Amyrin is present at low levels in the roots of diploid oat (Avena strigosa). Oat roots also synthesize the ß-amyrin-derived triterpene glycoside avenacin A-1, which provides protection against soil-borne diseases. The genes for the early steps in avenacin A-1 synthesis [saponin-deficient 1 and 2 (Sad1 and Sad2)] have been recruited from the sterol pathway by gene duplication and neofunctionalization. Here we show that Sad1 and Sad2 are regulated by an ancient root developmental process that is conserved across diverse species. Sad1 promoter activity is dependent on an L1 box motif, implicating sterol/lipid-binding class IV homeodomain leucine zipper transcription factors as potential regulators. The metabolism of ß-amyrin is blocked in sad2 mutants, which therefore accumulate abnormally high levels of this triterpene. The accumulation of elevated levels of ß-amyrin in these mutants triggers a "superhairy" root phenotype. Importantly, this effect is manifested very early in the establishment of the root epidermis, causing a greater proportion of epidermal cells to be specified as root hair cells rather than nonhair cells. Together these findings suggest that simple triterpenes may have widespread and as yet largely unrecognized functions in plant growth and development.

Modularity of plant metabolic gene clusters: a trio of linked genes that are collectively required for acylation of triterpenes in oat.

Operon-like gene clusters are an emerging phenomenon in the field of plant natural products. The genes encoding some of the best-characterized plant secondary metabolite biosynthetic pathways are scattered across plant genomes. However, an increasing number of gene clusters encoding the synthesis of diverse natural products have recently been reported in plant genomes. These clusters have arisen through the neo-functionalization and relocation of existing genes within the genome, and not by horizontal gene transfer from microbes. The reasons for clustering are not yet clear, although this form of gene organization is likely to facilitate co-inheritance and co-regulation. Oats (Avena spp) synthesize antimicrobial triterpenoids (avenacins) that provide protection against disease. The synthesis of these compounds is encoded by a gene cluster. Here we show that a module of three adjacent genes within the wider biosynthetic gene cluster is required for avenacin acylation. Through the characterization of these genes and their encoded proteins we present a model of the subcellular organization of triterpenoid biosynthesis.

Spectroscopic studies of two spectral variants of light-harvesting complex 2 (LH2) from the photosynthetic purple sulfur bacterium Allochromatium vinosum.

Two spectral forms of the peripheral light-harvesting complex (LH2) from the purple sulfur photosynthetic bacterium Allochromatium vinosum were purified and their photophysical properties characterized. The complexes contain bacteriochlorophyll a (BChl a) and multiple species of carotenoids. The composition of carotenoids depends on the light conditions applied during growth of the cultures. In addition, LH2 grown under high light has a noticeable split of the B800 absorption band. The influence of the change of carotenoid distribution as well as the spectral change of the excitonic absorption of the bacteriochlorophylls on the light-harvesting ability was studied using steady-state absorption, fluorescence and femtosecond time-resolved absorption at 77K. The results demonstrate that the change of the distribution of the carotenoids when cells were grown at low light adapts the absorptive properties of the complex to the light conditions and maintains maximum photon-capture performance. In addition, an explanation for the origin of the enigmatic split of the B800 absorption band is provided. This spectral splitting is also observed in LH2 complexes from other photosynthetic sulfur purple bacterial species. According to results obtained from transient absorption spectroscopy, the B800 band split originates from two spectral forms of the associated BChl a monomeric molecules bound within the same complex.