Analysis of cis-acting expression determinants of the tobacco psbA 5’UTR in vivo

Chloroplast gene expression is predominantly regulated at the posttranscriptional levels of mRNA stability and translation efficiency. The expression of psbA, an important photosynthesis-related chloroplast gene, has been revealed to be regulated via its 5’- untranslated region (UTR). Some cis-acting elements within this 5’UTR and the correlated trans-acting factors have been defined in Chlamydomonas. However, no in vivo evidence with respect to the cis-acting elements of the psbA 5’UTR has been so far achieved in higher plants such as tobacco. To attempt this, we generated a series of mutants of the tobacco psbA 5’UTR by base alterations and sequence deletions, with special regard to the stem-loop structure and the putative target sites for ribosome association and binding of nuclear regulatory factors. In addition, a versatile plastid transformation vector pKCZ with an insertion site in the inverted repeat region of the plastid genome was constructed. In all constructs, the psbA 5’UTR (Wt or modified) was used as the 5’ leader of the reporter gene uidA under control of the same promoter, Prrn, the promoter of the rRNA operon. Through biolistic DNA delivery to tobacco chloroplasts, transplastomic plants were obtained. DNA and RNA analyses of these transplastomic plants demonstrated that the transgenes aadA and uidA had been correctly integrated into the plastome at the insertion site, and transcribed in discrete sizes. Quantitative assays were also done to determine the proportion of intact transplastome, the uidA mRNA level, Gus activity, and uidA translation efficiency. The main results are the following: 1) The insertion site at the unique MunI between two tRNA genes (trnR-ACG and trnNGUU) is functional. Vector pKCZ has a large flexibility for further DNA manipulations and hence is useful for future applications. 2) The stem-loop of the psbA 5’UTR is required for mRNA stabilisation and translation. All mutants related to this region showed a 2~3 fold decrease in mRNA stability and a 1.5~6 fold reduction in translation efficiency. The function of this stem-loop depends on its correct sequence and secondary conformation. 3) the AU-box of the psbA 5’UTR is a crucial translation determinant. Mutations of this element almost abolished translation efficacy (up to 175-fold decrease), but did not significantly affect mRNA accumulation. The regulatory role of the AU-Box is sequencedependent and might be affected by its inner secondary structure. 4) The internal AUG codon of the psbA 5’UTR is unable to initiate translation. An introduction of mRNA translatability from this codon failed to direct the translation of reporter uidA gene, overriding the mutation of the AU-Box. 5) The 5’end poly(A) sequence does not confer a distinct regulatory signal. The deletion of this element only insignificantly affected mRNA abundance and translation. However, this mutation might slightly disturb the conformation of the stem-loop, resulting in a moderate decrease in translation efficiency (~1.5 fold). 6) The SD(Shine-Dalgarno)-like RBS (ribosome binding site) of the psbA 5’UTR appears to be an indispensable element for translation initiation. Mutation of this element led to a dramatically low expression of the uidA gene as seen by Gus staining. 7) The 5’end structural sequence of the rbcL 5’UTR does not convey a high mRNA stabilising effect to the psbA 5’UTR in a cycling condition of the light and the dark. Their distinct roles appear to be involved in darkness adaptation. Furthermore, with respect to the overall regulatory function of the psbA 5’UTR, two models are proposed, i.e. dual RBS-mediated translation initiation, and cpRBPs-mediated mRNA stability and translation. The mechanisms for mRNA stabilisation entailed by the rbcL 5’UTR are also discussed. Direct repeat-mediated transgene loss after chloroplast transformation and other aspects related to the choice of insertion site and plastid promoter are also analysed

A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in Chlamydomonas reinhardtii

In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont

A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in Chlamydomonas reinhardtii

In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont

5'UTR sequence requirements for stability of rbcL transcripts in the chloroplast of Chlamydomonas reinhardtii

A reporter gene was constructed of the Chlamydomonas reinhardtii rbcL 5 f region from position -70 to +47, relative to the transcription start point (+1), followed by the E. coli uidA (GUS) gene and the Chlamydomonas psaB 3 f region downstream of it. Two constructs were made in order to examine the importance of the sequence of a predicted stem-loop structure between positions +1 and +41 of the C. reinhardtii rbcL 5 e region. The first construct tested consisted of a reversed nucleotide sequence between positions +5 and +37, while the second construct examined consisted of a complete change of sequence between positions +6 and +36 of the region, in which the nucleotides were changed so that each purine is replaced by another purine (A ¨G; G ¨A), and each pyrimidine by another pyrimidine (C ¨T; T ¨C). The constructs were inserted into the chloroplast genome downstream of the atpB gene. Transcript accumulation of the reporter gene was determined by Northern blot. Both constructs did not exhibit a change in accumulation of GUS transcripts in comparison to the original reporter gene construct, proving that the altered nucleotides are not significant in stabilizing the rbcL transcript in the C. reinhardtii chloroplast

ISOLATION AND CHARACTERIZATION OF A SECOND PROTEIN L-ISOASPARTYL METHYLTRANSFERASE GENE IN ARABIDOPSIS THALIANA

Conversion of aspartate and asparagine residues to isoaspartate is a prevalent covalent protein modification in cells. The accumulation of these altered residues can lead to the loss of protein function and the consequent loss of cellular function. The L-ISOASPARTATE METHYLTRANSFERASE (EC 2.1.1.77) (PIMT) iteratively methylates abnormal isoaspartyl residues leading to conversion to L-aspartate, thereby mitigating the injurious effects of aging. Arabidopsis thaliana is unique among eukaryotes studied to date in that it possesses two genes (At3g48330 (PIMT1) and At5g50240 (PIMT2)) encoding PIMT. The PIMT2 gene exhibits a complex transcriptional control involving different transcriptional initiation sites and 5\u27- and 3\u27- alternative splice site selection in the first intron. Varying the transcriptional initiation site results in alternative targeting of the PIMT2 proteins thus produced to: 1) the nucleus, or 2) the cytoplasm, while PIMT1 is cytosolic. Inclusion of a 51 nucleotide 5 alternatively spliced sequence with or without a nine nucleotide 3 alternatively spliced sequence dramatically alteres the subcellular protein localization from the cytoplasm and around the chloroplast to inside the chloroplast. All recombinant PIMT2 isoform tested exhibit PIMT activity, although solubility varied among them. Multiplex RT-PCR was used to establish PIMT1 and PIMT2 transcript presence and abundance, relative to -TUBULIN, in various tissues and under a variety of stresses imposed on seeds and seedlings. PIMT1 transcript is constitutively present but can increase, along with PIMT2, in developing seeds presumably in response to increasing endogenous ABA. Transcript from PIMT2 also increases in establishing seedlings due to exogenous ABA application or applied stress presumably through an ABA-dependent pathway. Furthermore, Cleaved Amplified Polymorphic Sequence analysis of the PIMT2 amplicons has shown that the ratio among the splicing variants alters upon ABA application, implicating a role for the spliceosome or differential RNA stability in orchestrating the plant\u27s response to stress. T-DNA insertional mutants of both genes were isolated but no obvious phenotype has been identified. The double mutant has been generated and will be evaluated

Bioinformatic Analysis Of Solanaceae Chloroplast Genomes And Characterization Of An Arabidopsis Protein Disulfide Isomerase In Transgenic Tobacco Chloroplasts

Throughout history, traditional plant breeding has been used to provide resistance to pests, disease and other forms of environmental stress, as well as to increase yield and improve upon quality and processing attributes. Over the last decade, the advancement in sequencing technology and bioinformatic analysis has unleashed a wealth of knowledge about chloroplast genetic organization and evolution. The lack of complete plastid genome sequences is one of the major limitations in advancing plastid genetic engineering to other useful crops. This is due to the fact that plastid genome sequences are essential for the identification of endogenous regulatory sequences and optimal sites for homologous recombination. Analysis of four Solanaceae genomes revealed significant genetic modifications in both coding and non-coding regions. Repeat analysis with Reputer revealed 33 to 45 direct and inverted repeats ≥ 30bp with at least 90% homology. All but five of the 42 repeats shared among all four genomes were located in the exact same genes or intergenic regions, suggesting a functional role. Intergenic analysis found four regions that are 100 percent identical in all four Solanaceae genomes. Such highly conserved intergenic regions are ideal targets for multi-species transformation cassettes. Protein disulfide isomerases (PDI) are a family of proteins known to function as molecular chaperones and aid in the formation of disulfide bonds during protein folding. They contain at least one thioredoxin domain used for the formation, isomerization, and reduction/oxidation of disulfide bonds. Bioinformatic analysis identified 13 PDI-like (PDIL) proteins found in Arabidopsis that contain at least one thioredoxin domain. In addition to the above-mentioned characteristics, PDIs have been shown to be directly involved in the translational regulation of the psbA mRNA in response to light and could potentially increase the efficiency of chloroplast engineering in plants. Human serum albumin (HSA) requires 17 disulfide bonds to be properly folded and is an ideal candidate for assessing the disulfide bond formation, protein folding, and other chaperone-like characteristics of PDIL proteins. Therefore, I have coexpressed HSA in order to further characterize an Arabidopsis PDIL protein, atPDIL5-4, and in particular, the redox control of the psbA 5\u27UTR. Interestingly, the polyclonal antibody used for identifying the PDIL protein cross-reacted and identified other proteins, but not the transgenic atPDIL5-4. Results of these investigations will be presented

MOLECULAR BASES OF SVP REGULATORY FUNCTIONS IN ARABIDOPSIS THALIANA

Flowering time regulation has a strong impact on plant life cycle, since it allows plants to flower and to reproduce under environmental permissive conditions. Several genes are involved in the regulatory pathways that determine the floral transition step, i.e. the switch from the plant vegetative phase to the reproductive phase and the consequent flower formation and fruit set. Among those genes, SHORT VEGETATIVE PHASE (SVP), a MADS box transcription factor, acts as strong repressor of the so called florigen promoting genes, FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1). Moreover, SVP has been also reported to act as a repressor of flower homeotic gene expression, thus ensuring the correct maintenance of floral meristem identity. Due to the relevance of SVP in both such important plant developmental stages, during my Ph.D. research program I tried to elucidate the molecular mechanisms at the basis of SVP activities. That has been done through different and complementary strategies that had the dual aim to identify SVP protein partners and to move the first steps towards the comprehension of the role of chloroplasts and chloroplast-nucleus signaling pathways in SVP functions. Co-immunoprecipitation assays followed by Mass Spectrometry analyses have allowed to draw up a list of Arabidopsis putative robust SVP interactors involved, at different levels, in chromatin organization and histone modification. Interestingly, the detailed characterization of the major Arabidopsis trimethyltransferase enzyme, SET DOMAIN GROUP 2 (SDG2), has revealed the existence of an SVP-SDG2 containing protein complex able to regulate the expression of SVP gene at the vegetative and reproductive meristems, by affecting the H3K4 methylation pattern within the first exon of SVP. Furthermore, our interests on the role of chloroplast-nucleus communication and its possible interactions with the flowering time regulation, have been met through the detailed characterization of two chloroplast-located PENTATRICO-PEPTIDE-REPEAT (PPR) containing proteins, which share three main features: i) they are part of the chloroplast gene expression machinery, ii) they are involved in chloroplast-nucleus communication, iii) they have been reported to be target genes of SVP by ChiP-seq assays. The detailed characterization of the Arabidopsis PPR proteins, GENOME UNCOUPLED 1 (GUN1) and CHLOROPLAST RNA PROCESSING 1 (AtCRP1), has provided the first preliminary insights into how chloroplast-nucleus signaling mechanisms may enable higher plants to more effectively adapt to the ever-changing internal and external conditions and mitigate detrimental effects to fitness

The molecular control of oil biosynthesis in Arabidopsis

The enzyme acetyl-CoA carboxylase exists in Arabidopsis as one plastidic heteromeric form and two homomeric isoforms, ACC1 and ACC2; with different subcellular localisations, ACC1 is cytosolic, while ACC2 is plastidial. Plants that are mutant for ACC1 show that this protein is essential for the synthesis of very long chain fatty acids in the developing seed of Arabidopsis, and is also required for correct patterning of cell division in the developing embryo. While the ACC1 gene is transcribed in both seed and leaves, the enzyme is only active in the seed at high levels, leading to the accumulation of storage triacyl glycerols (TAGs). In investigating the possible post-transcriptional control of ACC1 activity, we found that the ACC1 transcript is alternatively spliced. Analysis of ACC1 splice isoforms in both wildtype seedlings and in seedlings of the mdf1 loss-of-function mutant, which is defective in splicing control, shows that the ACC1 transcript has an alternative donor site in the 5' UTR, upstream of the translation start site. Leaf and seed show different proportions of spliced and non-spliced versions of the ACC1 transcript, with the non-spliced isoform being abundant in leaf, consistent with a role for splicing in regulating ACC1 activity. Mis-splicing of ACC1 in the mdf mutant or in transgenic MDF overexpressors is associated with aberrant cell division in the root meristem and ectopic lipid accumulation. Genetic complementation of the mdf mutant with a genomic sequence of the ACC1 gene under the control of the MDF gene promoter leads to a partial rescue of the meristematic activity in the root and shoot systems, enhancing the development of lateral roots and true leaves, respectively. These results show that correct splicing of ACC1 is required for both correct cell division and tissue development in Arabidopsis