Daniela Gargano

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

Transformation of potato plastids is limited by low transformation frequencies and low transgene expression in tubers. In order to improve the transformation efficiency, we modified the regeneration procedure and prepared novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome. Vector delivery was performed by the biolistic approach. By using the improved regeneration procedure and the potato flanking sequences, we regenerated about one shoot every bombardment. This efficiency corresponds to 15–18-fold improvement compared to previous results with potato and is comparable to that usually achieved with tobacco. Further, we tested five promoters and terminators, and four 5′-UTRs, to increase the expression of the gfp transgene in tubers. In leaves, accumulation of GFP to about 4% of total soluble protein (TSP) was obtained with the strong promoter of the rrn operon, a synthetic rbcL-derived 5′-UTR and the bacterial rrnB terminator. GFP protein was detected in tubers of plants transformed with only four constructs out of eleven. Best results (up to approximately 0.02% TSP) were achieved with the rrn promoter and rbcL 5′-UTR construct, described above, and another containing the same terminator, but with the promoter and 5′-UTR from the plastid clpP gene. The results obtained suggest the potential use of clpP as source of novel regulatory sequences in constructs aiming to express transgenes in amyloplasts and other non-green plastids. Furthermore, they represent a significant advancement of the plastid transformation technology in potato, of relevance to its implementation in potato breeding and biotechnology.

Direct gene transfer in potato: A comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts

Direct gene transfer methods in potato would facilitate the transfer of multiple genes and the manipulation of metabolic pathways in this species. In this study, up to 1.8 transformation events per shot (=0.5 per bombarded leaf) and 67.2 events per million protoplasts treated were obtained with particle bombardment and PEG-mediated direct DNA uptake, respectively. Limited disassociation of both HPT and GUS genes appeared to occur during the process of integration in only 19% of transformants. A large number of transformed potato plants with transgene expression at levels comparable to Agrobacterium-mediated transformation was obtained. High levels of GUS expression were only obtained in lines derived from PEG treatment. No correlation between the number of gene insertions and gene expression levels was found, suggesting that multiple insertions may have little or no effect on transgene expression.

Genome-wide analysis of plastome sequence variation and development of plastidial CAPS markers in common potato and related Solanum species

The plastome sequence of the European cultivated potato, Solanum tuberosum subsp. tuberosum (tbr, GenBank accession no. DQ386163), was compared with that of S. bulbocastanum, a wild potato relative (blb, GenBank accession no. DQ347958), in order to characterize the degree and type of variability in different genomic regions, and develop molecular markers relevant to genetics, breeding and biotechnology of potato. One hundred forty-two and 251 PICs (Potentially Informative Characters) were found in coding and non-coding sequences (NCSs), respectively. Further, while variation in coding regions was almost exclusively due to nucleotide substitutions, 25% of PICs in NCSs of tbr and blb were due to indels, most of them mononucleotide or longer tandem repeats (micro and minisatellites). Four intergenic regions were selected for further analyses in other 16 tuber-bearing Solanum species. The rps16-trnQUUGgene spacer was found to be the most variable, forty-six PICs in this region distinguishing 18 haplotypes. Analysis of haplotype relationships, based on variability in the four intergenic regions, confirmed that the most primitive species from Central America were the most distant to S. tuberosum. Finally, polymorphic sites in the same regions were used to develop a set of CAPS (Cleaved Amplified Polymorphic Sequences) markers for species/cytoplasm identification in Solanum spp.

Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley

The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.).

Lil3 dimerization and chlorophyll binding in Arabidopsis thaliana.

The two‐helix light harvesting like (Lil) protein Lil3 belongs to the family of chlorophyll binding light harvesting proteins of photosynthetic membranes. A function in tetrapyrrol synthesis and stabilization of geranylgeraniol reductase has been shown. Lil proteins contain the chlorophyll a/b‐binding motif; however, binding of chlorophyll has not been demonstrated. We find that Lil3.2 fromArabidopsis thaliana forms heterodimers with Lil3.1 and binds chlorophyll. Lil3.2 heterodimerization (25 ± 7.8 nM) is favored relative to homodimerization (431 ± 59 nM). Interaction of Lil3.2 with chlorophyll a (231 ± 49 nM) suggests that heterodimerization precedes binding of chlorophyll inArabidopsis thaliana.

Analysis of the chloroplast proteome in arc mutants and identification of novel protein components associated with FtsZ2

Chloroplasts are descendants of cyanobacteria and divide by binary fission. The number of chloroplasts is regulated in a cell type-specific manner to ensure that specialized cell types can perform their functions optimally. Several protein components of the chloroplast division apparatus have been identified in the past several years, but how this process is regulated in response to developmental status, environmental signals and stress is still unknown. To begin to address this we undertook a proteomic analysis of three accumulation and replication of chloroplasts mutants that show a spectrum of plastid division perturbations. We show that defects in the chloroplast division process results in changes in the abundance of proteins when compared to wild type, but that the profile of the native stromal and membrane complexes remains unchanged. Furthermore, by combining BN-PAGE with protein interaction assays we show that AtFtsZ2-1 and AtFtsZ2-2 assemble together with rpl12A and EF-Tu into a novel chloroplast membrane complex.

In vivo phosphorylation of FtsZ2 in Arabidopsis thaliana.

The tubulin-like FtsZ protein initiates assembly of the bacterial and plastid division machineries. In bacteria, phosphorylation of FtsZ impairs GTPase activity, polymerization and interactions with other division proteins. Using a proteomics approach, we have shown that AtFtsZ2 is phosphorylated in vivo in Arabidopsis and that PGK1 (phosphoglycerate kinase 1) interacts with AtFtsZ2 in planta, suggesting a possible role in FtsZ phosphorylation.

DJ-1 is a redox sensitive adapter protein for high molecular weight complexes involved in regulation of catecholamine homeostasis

&NA; DJ‐1 is an oxidation sensitive protein encoded by the PARK7 gene. Mutations in PARK7 are a rare cause of familial recessive Parkinsons disease (PD), but growing evidence suggests involvement of DJ‐1 in idiopathic PD. The key clinical features of PD, rigidity and bradykinesia, result from neurotransmitter imbalance, particularly the catecholamines dopamine (DA) and noradrenaline. We report in human brain and human SH‐SY5Y neuroblastoma cell lines that DJ‐1 predominantly forms high molecular weight (HMW) complexes that included RNA metabolism proteins hnRNPA1 and PABP1 and the glycolysis enzyme GAPDH. In cell culture models the oxidation status of DJ‐1 determined the specific complex composition. RNA sequencing indicated that oxidative changes to DJ‐1 were concomitant with changes in mRNA transcripts mainly involved in catecholamine metabolism. Importantly, loss of DJ‐1 function upon knock down (KD) or expression of the PD associated form L166P resulted in the absence of HMW DJ‐1 complexes. In the KD model, the absence of DJ‐1 complexes was accompanied by impairment in catecholamine homeostasis, with significant increases in intracellular DA and noraderenaline levels. These changes in catecholamines could be rescued by re‐expression of DJ‐1. This catecholamine imbalance may contribute to the particular vulnerability of dopaminergic and noradrenergic neurons to neurodegeneration in PARK7‐related PD. Notably, oxidised DJ‐1 was significantly decreased in idiopathic PD brain, suggesting altered complex function may also play a role in the more common sporadic form of the disease.

Genome-wide gene expression profiles in response to plastid division perturbations

Plastids are vital organelles involved in important metabolic functions that directly affect plant growth and development. Plastids divide by binary fission involving the coordination of numerous protein components. A tight control of the plastid division process ensures that: there is a full plastid complement during and after cell division, specialized cell types have optimal plastid numbers; the division rate is modulated in response to stress, metabolic fluxes and developmental status. However, how this control is exerted by the host nucleus is unclear. Here, we report a genome-wide microarray analysis of three accumulation and replication of chloroplasts (arc) mutants that show a spectrum of altered plastid division characteristics. To ensure a comprehensive data set, we selected arc3, arc5 and arc11 because they harbour mutations in protein components of both the stromal and cytosolic division machinery, are of different evolutionary origin and display different phenotypic severities in terms of chloroplast number, size and volume. We show that a surprisingly low number of genes are affected by altered plastid division status, but that the affected genes encode proteins important for a variety of fundamental plant processes.

Plastid Transformation in Potato: Solanum tuberosum

Although plastid transformation has attractive advantages and potential applications in plant biotechnology, for long time it has been highly efficient only in tobacco. The lack of efficient selection and regeneration protocols and, for some species, the inefficient recombination using heterologous flanking regions in transformation vectors prevented the extension of the technology to major crops. However, the availability of this technology for species other than tobacco could offer new possibilities in plant breeding, such as resistance management or improvement of nutritional value, with no or limited environmental concerns. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum). By optimizing the tissue culture system and using transformation vectors carrying homologous potato flanking sequences, we obtained up to one transplastomic shoot per bombardment. Such efficiency is comparable to that usually achieved in tobacco. The method described in this chapter can be used to regenerate potato transplastomic plants expressing recombinant proteins in chloroplasts as well as in amyloplasts.