Self-pollination of the orchid Cycnoches haagii from Brazilian Cerrado results in albino phenotype seedlings

Cycnoches haagii Barb. Rodr. is an epiphytic orchid very targeted by collectors, but no reference was found in the literature about its reproductive biology. Thus, the purpose of this study was to obtain initial information regarding pollination types and its influence on seed viability of this native orchid of the Brazilian Cerrado, in order to enable future propagation and preservation programs. Pollination among flowers of the same plant (geitonogamy) or different plants (xenogamy) were carried out. Seeds extracted from the capsules were sown in B&G medium, with full and half strength. Seeds from geitonogamic resulted in 25% of albino protocorms and consequently in albino seedlings. This phenomenon did not occur in seedlings derived from xenogamic pollination. Pigment analysis showed that even the albino seedlings presented chlorophylls and carotenoids, however, in significantly minor concentrations, 16% and 37% respectively, in relation to green seedlings. Geitonogamic and xenogamic pollinations resulted in C. haagii viable seeds with high germination percentage (90%) under in vitro conditions. The germination of seeds from xenogamic pollination resulted in chlorophyll or normal seedlings only, and can be recommended at conservation programs. On the other hand, although geitonogamic pollination should be avoided at conservation programs of this orchid species as it leads to albino seedlings, it showed a very interesting system to obtain seedlings with this phenotype, an interesting plant material to future investigation.

The Component of the TAC Complex, TCD7, Controls Rice Chloroplast Development at the Early Seedling Stage under Cold Stress

Transcriptionally active chromosome (TAC) is a component of protein-DNA complexes with RNA polymerase activity found in chloroplasts. Although TAC in Arabidopsis thaliana has been extensively investigated, how the rice (Oryza sativa L.) TAC complex functions remains largely unknown. We report the characterization of the mutant thermosensitive chlorophyll-deficient7 (tcd7) and the cloning of TCD7. tcd7 mutant seedlings displayed an albino phenotype specifically at low temperatures and before the four-leaf stage. We identified TCD7 by map-based cloning followed by transgenic rescue and genome editing tests, showing that TCD7 encodes the putative TAC component FRUCTOKINASE-LIKE 2 (OsFLN2). TCD7 transcripts were highly abundant in green tissues, and the protein localized to chloroplasts. In agreement with the albino phenotype, transcript levels of genes controlling chloroplast development and the establishment of photosynthetic capacity were severely reduced in tcd7 seedlings at low temperatures, but were expressed as in the wild type at high temperatures, implying that TCD7 regulates the PEP pathway and chloroplast development. Moreover, TCD7 interacted with the thioredoxin OsTRXz to form an OsTRXz-TCD7 regulatory module, which might regulate plastid transcription under cold stress. Our results demonstrate that the nucleus-encoded TAC protein TCD7 protects chloroplast development from cold stress via a TRXz-FLN regulatory module.

Albinism in F1 Hybrids Hinders Geneflow Between Cultivated Chickpea (Cicer arietinum L.) and the Tertiary Gene Pool Species, Cicer Pinnatifidum

Wild Cicer species, especially those in the tertiary gene pool, carry useful alleles for chickpea improvement. The aim of this study was to evaluate the crossability and geneflow between three chickpea cultivars (as female parents) and four cross-incompatible Cicer pinnatifidum accessions (as pollen parents) from the tertiary gene pool. Ten crosses were conducted. One fully developed healthy F1 seed was harvested in vivo from the ICC 4958 × ICC 17269 cross, but the seedling developed an albino phenotype at 4–5 days after germination. Unlike other crosses, those involving the cultivar ICCV 96030 generated a large number of pods with comparatively large ovules. One albino plantlet was obtained from the ICCV 96030 × ICC 17269 cross by embryo rescue. Crosses involving ICCV 10 resulted in flower drop and poor pod set. These variable genotype-specific responses of pod, ovule, and seed development indicate that genetic factors affect the formation of interspecific hybrids. Although pod and seed formation in these interspecific crosses can be improved, geneflow between these materials is hindered by a strong genetic factor conferring albinism in the F1 hybrids.

PPR647 Protein Is Required for Chloroplast RNA Editing, Splicing and Chloroplast Development in Maize

Chloroplasts play an essential role in plant growth and development. Any factors affecting chloroplast development will lead to abnormal plant growth. Here, we characterized a new maize mutant, albino seedling mutant 81647 (as-81647), which exhibits an entirely albino phenotype in leaves and eventually died before the three-leaf stage. Transmission electron microscopy (TEM) demonstrated that the chloroplast thylakoid membrane was impaired and the granum lamellae significantly decreased in as-81647. Map-based cloning and transgenic analysis confirmed that PPR647 encodes a new chloroplast protein consisting of 11 pentratricopeptide repeat domains. Quantitative real-time PCR (qRT-PCR) assays and transcriptome analysis (RNA-seq) showed that the PPR647 mutation significantly disrupted the expression of PEP-dependent plastid genes. In addition, RNA splicing and RNA editing of multiple chloroplast genes showed severe defects in as-81647. These results indicated that PPR647 is crucial for RNA editing, RNA splicing of chloroplast genes, and plays an essential role in chloroplast development.

Mutation of the ALBOSTRIANS Ohnologous Gene HvCMF3 Impairs Chloroplast Development and Thylakoid Architecture in Barley

Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained if at least one member of the pair undergoes neo- or sub-functionalization. Phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed them as members of a subfamily of genes coding for CCT motif (CONSTANS, CONSTANS-LIKE and TIMING OF CAB1) proteins characterized by a single CCT domain and a putative N-terminal chloroplast transit peptide. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a yellowish/light green – xantha – phenotype and successively develop pale green leaves. Compared to wild type, plastids of mutant seedlings show a decreased PSII efficiency, impaired processing and reduced amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional domain, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs were localized to plastids and nucleus. Hvcmf3Hvcmf7 double mutants exhibited a xantha-albino or albino phenotype depending on the strength of molecular lesion of the HvCMF7 allele. The chloroplast ribosome deficiency is discussed as the primary observed defect of the Hvcmf3 mutants. Based on our observations, the genes HvCMF3 and HvCMF7 have similar but not identical functions in chloroplast development of barley supporting our hypothesis of neo-/sub-functionalization between both ohnologous genes.

Rice TSV2 Encoding Threonyl-tRNA Synthetase is Needed for Early Chloroplast Development and Seedling Growth under Cold Stress

Threonyl-tRNA synthetase (ThrRS), one of 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 down-regulation 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.

Application of CRISPR/Cas9 technology in wild apple (Malus sieverii) for paired sites gene editing

Background Xinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities. It is necessary to use new technologies to research its genomic function and molecular improvement. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability varies depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Results In this study, we used 2 systems of vectors with paired sgRNAs targeting to MsPDS. As expected, we successfully induced the albino phenotype of calli and buds in both systems. Conclusions We conclude that CRISPR/Cas9 is a powerful system for editing the wild apple genome and expands the range of plants available for gene editing.

Correction of Frameshift Mutations in the atpB Gene by Translational Recoding in Chloroplasts of Oenothera and Tobacco

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding a β-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, non-functional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of mutation that otherwise would be lethal.

New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for “regenerated T0” lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase—ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.

First record of albinism in spiny rats of genus Proechimys (Rodentia: Echimyidae) from Western Amazon

Albinism is a rare condition in natural populations. One of the factors that would lead to higher allele frequency for the albino phenotype is bottleneck events, causing small population sizes and loss of genetic diversity. Here, we report the first record of albinism in one specimen of Proechimys gardneri from Porto Velho, Rondônia, Brazil. This is also the first record of albinism for the genus and for the family Echimyidae. We identified the specimen through morphology and confirmed the identification by 801 base pairs (bp) of mitochondrial marker cytochrome b (Cyt b). We also discuss the selection against albino phenotype in natural populations.