Cationic lipid nanoparticle-mediated delivery of a Cas9/crRNA ribonucleoprotein complex for transgene-free editing of the citrus plant genome.

KEY MESSAGE: A lipofectamine-mediated transfection protocol for DNA-free genome editing of citrus protoplast cells using a Cas9/gRNA ribonucleoprotein (RNP) complex resulted in the production of transgene free genome edited citrus.

Shading-Dependent Greening Process of the Leaves in the Light-Sensitive Albino Tea Plant 'Huangjinya': Possible Involvement of the Light-Harvesting Complex II Subunit of Photosystem II in the Phenotypic Characteristic.

The light-sensitive albino tea plant can produce pale-yellow shoots with high levels of amino acids which are suitable to process high-quality tea. In order to understand the mechanism of the albino phenotype formation, the changes in the physio-chemical characteristics, chloroplast ultrastructure, chlorophyll-binding proteins, and the relevant gene expression were comprehensively investigated in the leaves of the light-sensitive albino cultivar 'Huangjinya' ('HJY') during short-term shading treatment. In the content of photosynthetic pigments, the ultrastructure of the chloroplast, and parameters of the photosynthesis in the leaves of 'HJY' could be gradually normalized along with the extension of the shading time, resulting in the leaf color transformed from pale yellow to green. BN-PAGE and SDS-PAGE revealed that function restoration of the photosynthetic apparatus was attributed to the proper formation of the pigment-protein complexes on the thylakoid membrane that benefited from the increased levels of the LHCII subunits in the shaded leaves of 'HJY', indicating the low level of LHCII subunits, especially the lack of the Lhcb1 might be responsible for the albino phenotype of the 'HJY' under natural light condition. The deficiency of the Lhcb1 was mainly subject to the strongly suppressed expression of the Lhcb1.x which might be modulated by the chloroplast retrograde signaling pathway GUN1 (GENOMES UNCOUPLED 1)-PTM (PHD type transcription factor with transmembrane domains)-ABI4 (ABSCISIC ACID INSENSITIVE 4).

Efficient knockout of the phytoene desaturase gene in a hybrid poplar (Populus alba × Populus glandulosa) using the CRISPR/Cas9 system with a single gRNA.

The CRISPR/Cas9 system has been used for genome editing in several plant species; however, there are few reports on its use in trees. Here, CRISPR/Cas9 was used to mutate a target gene in Populus alba × Populus glandulosa hybrid poplars. The hybrid poplar is routinely used in molecular biological studies due to the well-established Agrobacterium-mediated transformation method. A single guide RNA (sgRNA) with reported high mutation efficiency in other popular species was designed with a protospacer adjacent motif sequence for the phytoene desaturase 1 (PagPDS1) gene. The pHSE/Cas9-PagPDS1 sgRNA vector was delivered into hybrid poplar cells using Agrobacterium-mediated transformation. The transgenic plants were propagated and classified them into three groups according to their phenotypes. Among a total of 110 lines of transgenic hybrid poplars, 82 lines showed either an albino or a pale green phenotype, indicating around 74.5% phenotypic mutation efficiency of the PagPDS1 gene. The albino phenotypes were observed when the CRISPR/Cas9-mediated mutations in both PagPDS1 alleles in the transgenic plants. There was no off-target modification of the PagPDS2 gene, which has a potential sgRNA target sequence with two mismatches. The results confirmed that the sgRNA can specifically edit PagPDS1 rather than PagPDS2, indicating that CRISPR/Cas9-mediated genome editing can effectively induce target mutations in the hybrid poplar. This technique will be useful to improve tree quality in hybrid poplars (P. alba × P. glandulosa); for example, by enhancing biomass or stress tolerance.

Generation of Albino Cynops pyrrhogaster by Genomic Editing of the tyrosinase Gene.

Albino animals are useful for in situ hybridization experiments that demonstrate gene expression in embryos and organs, for the immunological rejection of skin grafts transplanted to host animals, and to identify tissues with regenerative ability during limbs and retina regeneration processes. Cynops pyrrhogaster has extensive regenerating capacities. To facilitate regenerative research, in the present study, we produced albino C. pyrrhogaster using genomic editing. The DNA fragment containing part of the tyrosinase gene from C. pyrrhogaster was amplified using degenerate primers corresponding to evolutionarily conserved nucleotide sequences among several species, and the nucleotide sequence was determined. We designed a transcription activator-like effector nuclease (TALEN) that targets a candidate of the C. pyrrhogaster tyrosinase gene. Fertilized eggs were injected with TALEN mRNA, and albinos of C. pyrrhogaster were obtained. The results of the present study demonstrated that TALEN can be used effectively for genomic editing in C. pyrrhogaster and that the candidates of the tyrosinase gene that were cloned by us are essential for melanin synthesis. The albino newts created in the present study can be used as versatile experimental material.

Identification of the CsFtsH genes from Camellia sinensis reveals its potential role in leaf color phenotype.

The filamentation temperature-sensitive H (FtsH) metalloprotease participates in the chloroplast photosystem II (PSII) repair cycle, playing a crucial role in regulating leaf coloration. However, the evolutionary history and biological function of the FtsH family in albino tea plants are still unknown. In this study, 35 CsFtsH members, including 7 CsFtsH-like (CsFtsHi1-CsFtsHi7) proteins, mapping onto 11 chromosomes in 6 subgroups, were identified in the 'Shuchazao2' tea genome, and their exon/intron structure, domain characteristics, collinearity, protein interaction network, and secondary structure were comprehensively analyzed. Furthermore, real-time fluorescence quantitative PCR (RT-qPCR) analysis revealed that the expression levels of CsFtsH1/2/5/8 were significantly positively correlated with the leaf color of tea plants. The subcellular localization revealed that they were located in the chloroplast. The transgenic Arabidopsis has demonstrated that CsFtsH2 and CsFtsH5 could restore the chlorophyll content and chlorophyll fluorescence intensity in var1 and var2 mutants, respectively. Moreover, protein-protein interactions have confirmed that CsFtsH1 with CsFtsH5, and CsFtsH2 with CsFtsH8 could form a hetero-comples and function in chloroplasts. In summary, this study aims to not only increase the understanding of the underlying molecular mechanisms of CsFtsH but also to provide a solid and detailed theoretical foundation for the breeding of albino tea plant varieties.

Conversion obstacle from Mg-protoporphyrin IX to protochlorophyllide might be responsible for chlorophyll-deficient phenotype of the Huangjinya's albino offspring.

The albino tea cultivar is one of the most important germplasms for key gene mining and high-quality tea producing. In order to elucidate the chlorophyll-deficient mechanism of albino cultivar 'Huangjinya' and its offspring, color difference, photosynthetic pigments and the relevant genes' expression of the tender shoots were comprehensively investigated in this study. Among the tested 16 offspring, 5 exhibited albino phenotype in spring and autumn, 3 showed albino phenotype in spring but normal green in autumn, while the rests were all normal green. The shoot of albino offspring had significantly higher lightness and/or yellowness than that of green ones, and possessed dramatically lower photosynthetic pigments and chlorophyll precursor protochlorophyllide (Pchlide), as well as higher chlorophyll a/chlorophyll b but lower chlorophylls/carotenoids in comparison with green ones. Among the tested genes involved in chlorophyll and carotenoid metabolism pathways, expression of the magnesium protoporphyrin IX monomethyl ester cyclase (CRD), 3,8-divinyl chlorophyllide 8-vinyl reductase (DVR), 5-aminolevulinate dehydratase 1 (HEMB1), 1-deoxy-D-xylulose 5-phosphate synthase 1 (DXS1) and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ISPH) was remarkably down-regulated in shoots of the albino offspring. Color difference indices of the offspring were significantly correlated with the levels of photosynthetic pigments and Pchlide, and low level of chlorophylls in shoot of albino offspring was mainly due to conversion obstacle from magnesium protoporphyrin Ⅸ (Mg-Proto IX) to Pchlide which might be attributed to down-regulatory expression of CRD and DVR.

Rice TCD8 Encoding a Multi-Domain GTPase Is Crucial for Chloroplast Development of Early Leaf Stage at Low Temperatures.

The multi-domain GTPase (MnmE) is conservative from bacteria to human and participates in tRNA modified synthesis. However, our understanding of how the MnmE is involved in plant chloroplast development is scarce, let alone in rice. A novel rice mutant, thermo-sensitive chlorophyll-deficient mutant 8 (tcd8) was identified in this study, which apparently presented an albino phenotype at 20 °C but a normal green over 24 °C, coincided with chloroplast development and chlorophyll content. Map-based cloning and complementary test revealed the TCD8 encoded a multi-domain GTPase localized in chloroplasts. In addition, the disturbance of TCD8 suppressed the transcripts of certain chloroplast-related genes at low temperature, although the genes were recoverable to nearly normal levels at high temperature (32 °C), indicating that TCD8 governs chloroplast development at low temperature. The multi-domain GTPase gene in rice is first reported in this study, which endorses the importance in exploring chloroplast development in rice.

Improvement of Gene Delivery and Mutation Efficiency in the CRISPR-Cas9 Wheat (Triticum aestivum L.) Genomics System via Biolistics.

Discovery of the CRISPR-Cas9 gene editing system revolutionized the field of plant genomics. Despite advantages in the ease of designing gRNA and the low cost of the CRISPR-Cas9 system, there are still hurdles to overcome in low mutation efficiencies, specifically in hexaploid wheat. In conjunction with gene delivery and transformation frequency, the mutation efficiency bottleneck has the potential to slow down advancements in genomic editing of wheat. In this study, nine bombardment parameter combinations using three gold particle sizes and three rupture disk pressures were tested to establish optimal stable transformation frequencies in wheat. Utilizing the best transformation protocol and a knockout cassette of the phytoene desaturase gene, we subjected transformed embryos to four temperature treatments and compared mutation efficiencies. The use of 0.6 µm gold particles for bombardment increased transformation frequencies across all delivery pressures. A heat treatment of 34 °C for 24 h resulted in the highest mutation efficiency with no or minimal reduction in transformation frequency. The 34 °C treatment produced two M0 mutant events with albino phenotypes, requiring biallelic mutations in all three genomes of hexaploid wheat. Utilizing optimal transformation and heat treatment parameters greatly increases mutation efficiency and can help advance research efforts in wheat genomics.

Rice TSV2 encoding threonyl-tRNA synthetase is needed for early chloroplast development and seedling growth under cold stress.

Threonyl-tRNA synthetase (ThrRS), one of the aminoacyl-tRNA synthetases (AARSs), plays a crucial role in protein synthesis. However, the AARS functions on rice chloroplast development and growth were not fully appraised. In this study, a thermo-sensitive virescent mutant tsv2, which showed albino phenotype and lethal after the 4-leaf stage at 20°C but recovered to normal when the temperatures rose, was identified and characterized. Map-based cloning and complementation tests showed that TSV2 encoded a chloroplast-located ThrRS protein in rice. The Lys-to-Arg mutation in the anticodon-binding domain hampered chloroplast development under cold stress, while the loss of function of the ThrRS core domain in TSV2 fatally led to seedling death regardless of growing temperatures. In addition, TSV2 had a specific expression in early leaves. Its disruption obviously resulted in the downregulation of certain genes associated with chlorophyll biosynthesis, photosynthesis, and chloroplast development at cold conditions. Our observations revealed that rice nuclear-encoded TSV2 plays an important role in chloroplast development at the early leaf stage under cold stress.

The Effect of Daminozide, Dark/Light Schedule and Copper Sulphate in Tissue Culture of Triticum timopheevii.

Triticum timopheevii Zhuk. is a tetraploid wheat that is utilized worldwide as a valuable breeding source for wheat improvement. Gene-based biotechnologies can contribute to this field; however, T. timopheevii exhibits recalcitrance and albinism in tissue cultures, making this species of little use for manipulation through genetic engineering and genome editing. This study tested various approaches to increasing in vitro somatic embryogenesis and plant regeneration, while reducing the portion of albinos in cultures derived from immature embryos (IEs) of T. timopheevii. They included (i) adjusting the balance between 2,4-D and daminozide in callus induction medium; (ii) cultivation using various darkness/illumination schedules; and (iii) inclusion of additional concentrations of copper ions in the tissue culture medium. We achieved a 2.5-fold increase in somatic embryogenesis (up to 80%) when 50 mg L-1 daminozide was included in the callus induction medium together with 3 mg L-1 2,4-D. It was found that the dark cultivation for 20-30 days was superior in terms of achieving maximum culture efficiency; moreover, switching to light in under 2 weeks from culture initiation significantly increased the number of albino plants, suppressed somatic embryogenesis, and decreased the regeneration of green plants. Media containing higher levels of copper ions did not have a positive effect on the regeneration of green plants; contrarily, the elevated concentrations caused albinism in plantlets. The results and relevant conclusions of the present study might be valuable for establishing an improved protocol for the regeneration of green plants in tissue cultures of T. timopheevii.

The rice white green leaf 2 gene causes defects in chloroplast development and affects the plastid ribosomal protein S9.

BACKGROUND: Plastid ribosomal proteins (PRPs) play important roles in the translation of key proteins involved in chloroplast development and photosynthesis. PRPs have been widely studied in many plant species; however, few studies have investigated their roles in rice. RESULT: In the present study, we used ethyl methane sulfonate mutagenesis and obtained a novel rice mutant called white green leaf 2 (wgl2). The wgl2 mutants exhibited an albino phenotype from germination through the three-leaf stage, and then gradually transitioned to green through the later developmental stages. Consistent with this albino phenotype, wgl2 mutants had abnormal chloroplasts and lower levels of photosynthetic pigments. Map-based cloning and DNA sequencing analyses of wgl2 revealed a single-nucleotide substitution (G to T) in the first exon of LOC_Os03g55930, which resulted in a substitution of glycine 92 to valine (G92 V). WGL2 encodes a conserved ribosomal protein, which localizes to the chloroplast. Complementation and targeted deletion experiments confirmed that the point mutation in WGL2 is responsible for the wgl2 mutant phenotype. WGL2 is preferentially expressed in the leaf, and mutating WGL2 led to obvious changes in the expression of genes related to chlorophyll biosynthesis, photosynthesis, chloroplast development, and ribosome development compared with wild-type. CONCLUSIONS: WGL2 encodes a conserved ribosomal protein, which localizes to the chloroplast. WGL2 is essential for early chloroplast development in rice. These results facilitate research that will further uncover the molecular mechanism of chloroplast development.

The Rice Pentatricopeptide Repeat Gene TCD10 is Needed for Chloroplast Development under Cold Stress.

BACKGROUND: Chloroplast plays a vital role in plant development and growth. The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in plants. In addition, cold stress affects a broad spectrum of cellular components, e.g. chloroplast, and metabolism in plants. However, the regulatory mechanism for rice PPR genes on chloroplast development still remains elusive under cold stress. RESULT: In this paper, we characterized a new rice PPR gene mutant tcd10 (thermo-sensitive chlorophyll-deficient mutant 10) that exhibits the albino phenotype, malformed chloroplast and could not survive after the 5-leaf stage when grown at 20 °C, but does the normal phenotype at 32 °C. Map-based cloning, followed by RNA interference and CRISPR/Cas9 genome editing techniques, revealed that TCD10 encoding a novel PPR protein, mainly localized to the chloroplasts, with 27 PPR motifs, is responsible for the mutant phenotype. In addition, TCD10 is specific expression in tissues. The disruption of TCD10 resulted in an evidently reduced expression of chloroplast-associated genes under cold stress (20 °C), whereas they did recovered to normal levels at high temperature (32 °C). These results showed an important role of TCD10 for chloroplast development under cold stress. CONCLUSIONS: The TCD10 encodes a novel rice PPR protein, mainly located in chloroplasts, which is important for chloroplast development, growth and the maintenance of photosynthetic electron transport and its disorder would lead to an aberrant chloroplast and abnormal expressions in these genes for chloroplast development and photosynthesis in rice under cold stress.