THE PROTECTION STRATEGIES REALIZED IN A RESURRECTION PLANT UNDER DROUGHT STRESS

The homoiochlorophyllous resurrection plants adapt unique capabilities against damage caused by reactive oxygen species (ROS) from photosynthesis during desiccation. Under severe drought stress, the plants can maintain most of the photosynthetic apparatus and chlorophylls while minimizing the ROS damage and recover efficiently after re-watering. This study aims to reveal the protection mechanisms under drought stress at different ages of the life cycle in the homoiochlorophyllous resurrection plant Craterostigma pumilum and provides comprehensive information on the regulation of photosynthesis. The investigations in younger ( six months) C. pumilum plants included high energy quenching analyses, photosynthetic protein composition and functional analyses, as well as ultrastructure imaging of chloroplast alteration in the cause of de- and rehydration. Both younger and older C. pumilum show similar general responses to drought, including dehydration rate, sucrose accumulation, partial chlorophyll breakdown, and having the capability of swift recovery of the relative water content. The chlorophyll fluorescence analysis shows that high-energy quenching (qE) increases in younger plants for protection, but it decreases in older plants during dehydration. The difference in qE protection depends on the change of a proton motive force (pmf) between both plant types. Prominent photosynthetic protein degradation was observed in older C. pumilum. The specific degradation of the D1 subunit of photosystem II and in particular of the cytochrome b6f complex leads to lower linear electron transport (LET) in older plants than that in younger plants. Furthermore, ultrastructure analysis provides evidence that dehydrated older plants possess senescence-like chloroplast degradation with a significant increase in plastoglobuli accumulation and appearance of degradation vesicles. This chloroplast degradation is suppressed in younger plants. The results conclude that younger plants employ a photoprotection-based strategy and maintain a high capability of D1 subunit repair against drought stress which results in a faster recovery. In contrast, older plants employ a degradation-based protection strategy through a breakdown of D1 proteins and of subunits of the cyt b6f complex when dehydrated. It follows that the protection strategies depend on the ontogenetic state of C. pumilum. Both strategies are capable of protecting C. pumilum from severe drought stress

H2O2-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies

Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H2O2 to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2O2 content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H2O2 and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H2O2 by 3,3′-diaminobenzidine (DAB) staining suggested that H2O2 could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H2O2-based method. Moreover, HyB induced overproduction of H2O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice