Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)

Field cress (Lepidium campestre) is a potential oilseed crop that has been under domestication in recent decades. CRISPR/Cas9 is a powerful tool for rapid trait improvement and gene characterization and for generating transgene-free mutants using protoplast transfection system. However, protoplast regeneration remains challenging for many plant species. Here we report an efficient protoplast regeneration and transfection protocol for field cress. Important factors such as type of basal media, type/combination of plant growth regulators, and culture duration on different media were optimized. Among the basal media tested, Nitsch was the best for protoplast growth in MI and MII media. For cell wall formation during the early stage of protoplast growth, relatively high auxin concentrations (0.5 mg L−1 NAA and 2,4-D), without addition of cytokinin was preferred for maintaining protoplast viability. After cell wall formation, 1.1 mg L−1 TDZ combined with either 0.05 mg L−1 NAA or 2,4-D was found to efficiently promote protoplast growth. On solid shoot induction medium, 1.1 mg L−1 TDZ without any auxin resulted in over 80% shoot generation frequency. A longer culture duration in MI medium would inhibit protoplast growth, while a longer culture duration in MII medium significantly delayed shoot formation. Using this optimized protoplast regeneration protocol, we have established an efficient PEG-mediated transfection protocol using a vector harboring the GFP gene, with transfection efficiencies of 50–80%. This efficient protoplast protocol would facilitate further genetic improvement of field cress via genome editing, and be beneficial to development of protoplast regeneration protocols for related plant species.

Integrated multi-omics analysis uncovers roles of mdm-miR164b-MdORE1 in strigolactone mediated inhibition of adventitious root formation in apple

Adventitious root (AR) formation is important for the vegetative propagation. The effects of strigolactones (SLs) on AR formation have been rarely reported, especially in woody plants. In this study, we first verified the inhibitory effects of SLs on AR formation in apple materials. Transcriptome analysis identified 12,051 differentially expressed genes over the course of AR formation, with functions related to organogenesis, cell wall biogenesis or plant senescence. WGCNA suggests SLs might inhibit AR formation through repressing the expression of two core hub genes, MdLAC3 and MdORE1. We further verified that enhanced cell wall formation and accelerated senescence were involved in the AR inhibition caused by SLs. Combining small RNA and degradome sequencing, as well as a dual-luciferase sensor system, we identified and validated three negatively correlated miRNA–mRNA pairs, including mdm-miR397–MdLAC3 involved in secondary cell wall formation, and mdm-miR164a/b–MdORE1 involved in senescence. Finally, we have experimentally demonstrated the role of mdm-miR164b–MdORE1 in SLs-mediated inhibition of AR formation. Overall, our findings not only propose a comprehensive regulatory network for the function of SLs on AR formation, but also provide novel candidate genes for the potential genetic improvement of AR formation in woody plants using transgenic or CRISPR technology.

Exocytosis of the silicified cell wall of diatoms involves extensive membrane disintegration

Diatoms are unicellular algae that are characterized by their silica cell walls. The silica elements form intracellularly in a membrane-bound organelle, and are exocytosed after completion. How diatoms maintain membrane homeostasis during the exocytosis of these large and rigid silica elements is a long-standing enigma. We studied membrane dynamics during cell wall formation and exocytosis in the diatom Stephanopyxis turris, using live-cell confocal microscopy and advanced electron microscopy. Our results provide detailed information on the ultrastructure and dynamics of the silicification process, showing that during cell wall formation, the organelle membranes tightly enclose the mineral phase, creating a precise mold of the delicate geometrical patterns. Surprisingly, during exocytosis of the mature silica elements, the proximal organelle membrane becomes the new plasma membrane, and the distal membranes gradually disintegrate into the extracellular space without any noticeable endocytic retrieval or extracellular repurposing. These observations suggest that diatoms evolved an extraordinary exocytosis mechanism in order to secrete their cell wall elements.

Transcriptome analysis of Pará rubber tree (H. brasiliensis) seedlings under ethylene stimulation

Background Natural rubber (cis-1,4-polyioprene, NR) is an indispensable industrial raw material obtained from the Pará rubber tree (H. brasiliensis). Natural rubber cannot be replaced by synthetic rubber compounds because of the superior resilience, elasticity, abrasion resistance, efficient heat dispersion, and impact resistance of NR. In NR production, latex is harvested by periodical tapping of the trunk bark. Ethylene enhances and prolongs latex flow and latex regeneration. Ethephon, which is an ethylene-releasing compound, applied to the trunk before tapping usually results in a 1.5- to 2-fold increase in latex yield. However, intense mechanical damage to bark tissues by excessive tapping and/or over-stimulation with ethephon induces severe oxidative stress in laticifer cells, which often causes tapping panel dryness (TPD) syndrome. To enhance NR production without causing TPD, an improved understanding of the molecular mechanism of the ethylene response in the Pará rubber tree is required. Therefore, we investigated gene expression in response to ethephon treatment using Pará rubber tree seedlings as a model system. Results After ethephon treatment, 3270 genes showed significant differences in expression compared with the mock treatment. Genes associated with carotenoids, flavonoids, and abscisic acid biosynthesis were significantly upregulated by ethephon treatment, which might contribute to an increase in latex flow. Genes associated with secondary cell wall formation were downregulated, which might be because of the reduced sugar supply. Given that sucrose is an important molecule for NR production, a trade-off may arise between NR production and cell wall formation for plant growth and for wound healing at the tapping panel. Conclusions Dynamic changes in gene expression occur specifically in response to ethephon treatment. Certain genes identified may potentially contribute to latex production or TPD suppression. These data provide valuable information to understand the mechanism of ethylene stimulation, and will contribute to improved management practices and/or molecular breeding to attain higher yields of latex from Pará rubber trees.

Investigation of cell wall proteins of C. sinensis leaves by combining cell wall proteomics and N-glycoproteomics

Background C. sinensis is an important economic crop with fluoride over-accumulation in its leaves, which poses a serious threat to human health due to its leaf consumption as tea. Recently, our study has indicated that cell wall proteins (CWPs) probably play a vital role in fluoride accumulation/detoxification in C. sinensis. However, there has been a lack in CWP identification and characterization up to now. This study is aimed to characterize cell wall proteome of C. sinensis leaves and to develop more CWPs related to stress response. A strategy of combined cell wall proteomics and N-glycoproteomics was employed to investigate CWPs. CWPs were extracted by sequential salt buffers, while N-glycoproteins were enriched by hydrophilic interaction chromatography method using C. sinensis leaves as a material. Afterwards all the proteins were subjected to UPLC-MS/MS analysis. Results A total of 501 CWPs and 195 CWPs were identified respectively by cell wall proteomics and N-glycoproteomics profiling with 118 CWPs in common. Notably, N-glycoproteomics is a feasible method for CWP identification, and it can enhance CWP coverage. Among identified CWPs, proteins acting on cell wall polysaccharides constitute the largest functional class, most of which might be involved in cell wall structure remodeling. The second largest functional class mainly encompass various proteases related to CWP turnover and maturation. Oxidoreductases represent the third largest functional class, most of which (especially Class III peroxidases) participate in defense response. As expected, identified CWPs are mainly related to plant cell wall formation and defense response. Conclusion This was the first large-scale investigation of CWPs in C. sinensis through cell wall proteomics and N-glycoproteomics. Our results not only provide a database for further research on CWPs, but also an insight into cell wall formation and defense response in C. sinensis.