Transformation of Long-Lived Albino Epipremnum aureum ‘Golden Pothos’ and Restoring Chloroplast Development

Chloroplasts are organelles responsible for chlorophyll biosynthesis, photosynthesis, and biosynthesis of many metabolites, which are one of key targets for crop improvement. Elucidating and engineering genes involved in chloroplast development are important approaches for studying chloroplast functions as well as developing new crops. In this study, we report a long-lived albino mutant derived from a popular ornamental plant Epipremnum aureum ‘Golden Pothos’ which could be used as a model for analyzing the function of genes involved in chloroplast development and generating colorful plants. Albino mutant plants were isolated from regenerated populations of variegated ‘Golden Pothos’ whose albino phenotype was previously found to be due to impaired expression of EaZIP, encoding Mg-protoporphyrin IX monomethyl ester cyclase. Using petioles of the mutant plants as explants with a traceable sGFP gene, an efficient transformation system was developed. Expressing Arabidopsis CHL27 (a homolog of EaZIP) but not EaZIP in albino plants restored green color and chloroplast development. Interestingly, in addition to the occurrence of plants with solid green color, plants with variegated leaves and pale-yellow leaves were also obtained in the regenerated populations. Nevertheless, our study shows that these long-lived albino plants along with the established efficient transformation system could be used for creating colorful ornamental plants. This system could also potentially be used for investigating physiological processes associated with chlorophyll levels and chloroplast development as well as certain biological activities, which are difficult to achieve using green plants.

Genetic analysis of the barley variegation mutant, grandpa1.a

Background Providing the photosynthesis factory for plants, chloroplasts are critical for crop biomass and economic yield. However, chloroplast development is a complicated process, coordinated by the cross-communication between the nucleus and plastids, and the underlying biogenesis mechanism has not been fully revealed. Variegation mutants have provided ideal models to identify genes or factors involved in chloroplast development. Well-developed chloroplasts are present in the green tissue areas, while the white areas contain undifferentiated plastids that are deficient in chlorophyll. Unlike albino plants, variegation mutants survive to maturity and enable investigation into the signaling pathways underlying chloroplast biogenesis. The allelic variegated mutants in barley, grandpa 1 (gpa1), have long been identified but have not been genetically characterized. Results We characterized and genetically analyzed the grandpa1.a (gpa1.a) mutant. The chloroplast ultrastructure was evaluated using transmission electron microscopy (TEM), and it was confirmed that chloroplast biogenesis was disrupted in the white sections of gpa1.a. To determine the precise position of Gpa1, a high-resolution genetic map was constructed. Segregating individuals were genotyped with the barley 50 k iSelect SNP Array, and the linked SNPs were converted to PCR-based markers for genetic mapping. The Gpa1 gene was mapped to chromosome 2H within a gene cluster functionally related to photosynthesis or chloroplast differentiation. In the variegated gpa1.a mutant, we identified a large deletion in this gene cluster that eliminates a putative plastid terminal oxidase (PTOX). Conclusions Here we characterized and genetically mapped the gpa1.a mutation causing a variegation phenotype in barley. The PTOX-encoding gene in the delimited region is a promising candidate for Gpa1. Therefore, the present study provides a foundation for the cloning of Gpa1, which will elevate our understanding of the molecular mechanisms underlying chloroplast biogenesis, particularly in monocot plants.

Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

Background Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley. Results We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoints of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanisms underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

Genetic Analysis of the Barley Variegation Mutant, Grandpa1.α

Background Providing the photosynthesis factory for plants, chloroplasts are critical for crop biomass and economic yield. However, chloroplast development is a complicated process, coordinated by the cross-communication between the nucleus and plastids, and the underlying biogenesis mechanism has not been fully revealed. Variegation mutants have provided ideal models to identify genes or factors involved in chloroplast development. Well-developed chloroplasts are present in the green tissue areas, while the white areas contain undifferentiated plastids that are deficient in chlorophyll. Unlike albino plants, variegation mutants survive to maturity and enable investigation into the signaling pathways underlying chloroplast biogenesis. The allelic variegated mutants in barley, grandpa 1 (gpa1), have long been identified but have not been genetically characterized. Results We characterized and genetically analyzed the grandpa1.a (gpa1.a) mutant. The chloroplast ultrastructure was evaluated using transmission electron microscopy (TEM), and it was confirmed that chloroplast biogenesis was disrupted in the white sections of gpa1.a. To determine the precise position of Gpa1, a high-resolution genetic map was constructed. Segregating individuals were genotyped with the barley 50k iSelect SNP Array, and the linked SNPs were converted to PCR-based markers for genetic mapping. The Gpa1 gene was mapped to chromosome 2H within a gene cluster functionally related to photosynthesis or chloroplast differentiation. In the variegated gpa1.a mutant, we identified a large deletion in this gene cluster that eliminates a putative plastid terminal oxidase (PTOX).Conclusions Here we characterized and genetically mapped the gpa1.a mutation causing a variegation phenotype in barley. The PTOX-encoding gene in the delimited region is a promising candidate for Gpa1. Therefore, the present study provides a foundation for the cloning of Gpa1, which will elevate our understanding of the molecular mechanisms underlying chloroplast biogenesis, particularly in monocot plants.

Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

Background: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley.Results: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoint of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanism underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

Background: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley.Results: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoint of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanism underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

Expression analysis of photosynthesis-related genes in albino Artocarpus heterophyllus seedlings leaves

Albino Artocarpus heterophyllus Seedlings (AAS) were found in the preliminary investigation by our group and were used as materials for researching. The phenotype of AAS leaves were observed and measured. In parallel, the photosynthetic physiological parameters were determined under different photosynthetically active radiations (PAR). The results suggested that the length, width, area and thickness of AAS leaves were less than normal seedings. Likewise, the net photosynthetic rate, intercellular CO2 concentration, stomatal conductance and transpiration rate of AAS leaves were not susceptible to PAR in contrast to normal individuals. Furthermore, the transcriptome sequencing technology was performed to clarify the expression of genes related to photosynthesis. It is as expected that numerous down-regulated genes were found in the synthesis of photosynthetic pigments, as well as the pathways of photosynthesis - antenna proteins, photoreaction and carbon fixation reaction of AAS leaves. Compared to other albino plants, AAS have a longer life span and more stable phenotypic traits with larger leaves, which could provide ideal materials for investigating photosynthesis of woody plants.

Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

Background: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH), which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley.Results: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. The impaired PEP activity caused a very low level of 16S and 23S rRNA transcripts, lack of plastid translation machinery and inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP, the highly increased expression of Sig2, plastid rRNA and tRNAGlu transcripts, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoint of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was the main cause of the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanism underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

SUMOylation contributes to proteostasis of the chloroplast protein import receptor TOC159 during early development

Chloroplast biogenesis describes the transition of non-photosynthetic proplastids to photosynthetically active chloroplasts in the cells of germinating seeds. Chloroplast biogenesis requires the import of thousands of nuclear-encoded preproteins and depends on the essential import receptor TOC159, mutation of which results in non-photosynthetic albino plants. We previously showed that ubiquitin-proteasome system (UPS)-dependent regulation of TOC159 levels contributes to the regulation of chloroplast biogenesis during early plant development. Here, we demonstrate that the SUMO (Small Ubiquitin-related Modifier) pathway crosstalks with the ubiquitin-proteasome pathway to affect TOC159 stability during early plant development. We identified a SUMO3-interacting motif (SIM) in the TOC159 GTPase (G-) domain and a SUMO3 covalent SUMOylation site in the membrane (M-) domain. A single K to R substitution (K1370R) in the M-domain disables SUMOylation. Expression of the TOC159K1370R mutant in the toc159 mutant (ppi2) complemented the albino phenotype. Compared to wild type TOC159, TOC159K1370R was destabilized under UPS-inducing stress conditions. However, TOC159K1370R recovered to same protein level as wild type TOC159 in the presence of a proteasome inhibitor. Thus, SUMOylation partially stabilizes TOC159 against UPS-dependent degradation under stress conditions. Our data contribute to the evolving model of tightly controlled proteostasis of the TOC159 import receptor during proplastid to chloroplast transition.

Efficient knockout of phytoene desaturase gene using CRISPR/Cas9 in melon

CRISPR/Cas9 system has been widely applied in many plant species to induce mutations in the genome for studying gene function and improving crops. However, to our knowledge, there is no report of CRISPR/Cas9-mediated genome editing in melon (Cucumis melo). In our study, phytoene desaturase gene of melon (CmPDS) was selected as target for the CRISPR/Cas9 system with two designed gRNAs, targeting exons 1 and 2. A construct (pHSE-CmPDS) carrying both gRNAs and the Cas9 protein was delivered by PEG-mediated transformation in protoplasts. Mutations were detected in protoplasts for both gRNAs. Subsequently, Agrobacterium-mediated transformation of cotyledonary explants was carried out, and fully albino and chimeric albino plants were successfully regenerated. A regeneration efficiency of 71% of transformed plants was achieved from cotyledonary explants, a 39% of genetic transformed plants were successful gene edited, and finally, a 42–45% of mutation rate was detected by Sanger analysis. In melon protoplasts and plants most mutations were substitutions (91%), followed by insertions (7%) and deletions (2%). We set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and feasible in melon, generating multi-allelic mutations in both genomic target sites of the CmPDS gene showing an albino phenotype easily detectable after only few weeks after Agrobacterium-mediated transformation.