Why old duplicated genes are not thrown away in (paleo)polyploids? Example from the petC gene in Brassica napus.

Duplication of genes at different time period, through recurrent and frequent polyploidization events, have played a major role in plant evolution, adaptation and diversification. Interestingly, some of the ancestral duplicated genes (referred as paleologs), have been maintained for millions of years, and there is still a poor knowledge of the reasons of their retention, especially when testing the phenotypic effect of individual copies by using functional genetic approaches. To fill this gap, we performed functional genetic (CRISPR-Cas9), physiological, transcriptomic and evolutionary studies to finely investigate this open question, taking the example of the petC gene (involved in cytochrome b6/f and thus impacting photosynthesis) that is present in four paleologous copies in the oilseed crop Brassica napus. RNA-Seq and selective pressure analyses suggested that all paleologous copies conserved the same function and that they were all highly transcribed. Thereafter, the Knock Out (K.O.) of one, several or all petC copies highlighted that all paleologous copies have to be K.O. to suppress the gene function. In addition, we could determine that phenotypic effects in single and double mutants could only be deciphered in high light conditions. Interestingly, we did not detect any significant differences between single mutants K.O. for either the A03 or A09 copy (despite being differentially transcribed), or even between mutants for a single or two petC copies. Altogether, this work revealed that petC paleologs have retained their ancestral function and that the retention of these copies is explained by their compensatory role, especially in optimal environmental conditions.

I-SceI and customized meganucleases-mediated genome editing in tomato and oilseed rape.

Meganucleases are rare cutting enzymes that can generate DNA modifications and are part of the plant genome editing toolkit although they lack versatility. Here, we evaluated the use of two meganucleases, I-SceI and a customized meganuclease, in tomato and oilseed rape. Different strategies were explored for the use of these meganucleases. The activity of a customized and a I-SceI meganucleases was first estimated by the use of a reporter construct GFFP with the target sequences and enabled to demonstrate that both meganucleases can generate double-strand break and HDR mediated recombination in a reporter gene. Interestingly, I-SceI seems to have a higher DSB efficiency than the customized meganuclease: up to 62.5% in tomato and 44.8% in oilseed rape. Secondly, the same exogenous landing pad was introduced in both species. Despite being less efficient compared to I-SceI, the customized meganuclease was able to generate the excision of an exogenous transgene (large deletion of up to 3316 bp) present in tomato. In this paper, we also present some pitfalls to be considered before using meganucleases (e.g., potential toxicity) for plant genome editing.

Molecular evolution and transcriptional regulation of the oilseed rape proline dehydrogenase genes suggest distinct roles of proline catabolism during development.

MAIN CONCLUSION: Six BnaProDH1 and two BnaProDH2 genes were identified in Brassica napus genome. The BnaProDH1 genes are mainly expressed in pollen and roots' organs while BnaProDH2 gene expression is associated with leaf vascular tissues at senescence. Proline dehydrogenase (ProDH) catalyzes the first step in the catabolism of proline. The ProDH gene family in oilseed rape (Brassica napus) was characterized and compared to other Brassicaceae ProDH sequences to establish the phylogenetic relationships between genes. Six BnaProDH1 genes and two BnaProDH2 genes were identified in the B. napus genome. Expression of the three paralogous pairs of BnaProDH1 genes and the two homoeologous BnaProDH2 genes was measured by real-time quantitative RT-PCR in plants at vegetative and reproductive stages. The BnaProDH2 genes are specifically expressed in vasculature in an age-dependent manner, while BnaProDH1 genes are strongly expressed in pollen grains and roots. Compared to the abundant expression of BnaProDH1, the overall expression of BnaProDH2 is low except in roots and senescent leaves. The BnaProDH1 paralogs showed different levels of expression with BnaA&C.ProDH1.a the most strongly expressed and BnaA&C.ProDH1.c the least. The promoters of two BnaProDH1 and two BnaProDH2 genes were fused with uidA reporter gene (GUS) to characterize organ and tissue expression profiles in transformed B. napus plants. The transformants with promoters from different genes showed contrasting patterns of GUS activity, which corresponded to the spatial expression of their respective transcripts. ProDHs probably have non-redundant functions in different organs and at different phenological stages. In terms of molecular evolution, all BnaProDH sequences appear to have undergone strong purifying selection and some copies are becoming subfunctionalized. This detailed description of oilseed rape ProDH genes provides new elements to investigate the function of proline metabolism in plant development.

Evaluation of genome and base editing tools in maize protoplasts.

Introduction: Despite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing remains a labor-intensive process requiring often several months of in vitro culture to obtain mutant plantlets. To avoid a waste in time and money and to test, in only a few days, the efficiency of molecular constructs or novel Cas9 variants (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9) prior to stable transformation, rapid analysis tools are helpful. Methods: To this end, a streamlined maize protoplast system for transient expression of CRISPR/Cas9 tools coupled to NGS (next generation sequencing) analysis and a novel bioinformatics pipeline was established. Results and discussion: Mutation types found with high frequency in maize leaf protoplasts had a trend to be the ones observed after stable transformation of immature maize embryos. The protoplast system also allowed to conclude that modifications of the sgRNA (single guide RNA) scaffold leave little room for improvement, that relaxed PAM (protospacer adjacent motif) sites increase the choice of target sites for genome editing, albeit with decreased frequency, and that efficient base editing in maize could be achieved for certain but not all target sites. Phenotypic analysis of base edited mutant maize plants demonstrated that the introduction of a stop codon but not the mutation of a serine predicted to be phosphorylated in the bHLH (basic helix loop helix) transcription factor ZmICEa (INDUCER OF CBF EXPRESSIONa) caused abnormal stomata, pale leaves and eventual plant death two months after sowing.

Identifying and Isolating Meiotic Mutants in a Polyploid Brassica Crop.

This chapter provides a detailed description of TILLING and CRISPR-Cas9 approaches for the purpose of studying genes/factors involved in meiotic recombination in the polyploid species B. napus. The TILLING approach involves the screening and identification of EMS-mutagenized M2 B. napus plants. The strategy for high-throughput plant pooling, the set up for microfluidic PCR and sequencing is provided and the parameters for the analysis of sequence results and the detection of mutants are explained. The CRISPR-Cas system relies on the optimal design of guide RNAs and their efficient expression. The procedure for the generation and detection of knockout mutants is described with the aims to simultaneously target homologous genes.