Spinach-based RNA mimicking GFP in plant cells.

Spinach RNA-mimicking GFP (S-RMG) has been successfully used to monitor cellular RNAs including microRNAs in bacterium, yeast, and human cells. However, S-RMG has not been established in plants. In this study, we found that like bacterial, yeast, and human cellular tRNAs, plant tRNAs such as tRNALys can protect and/or stabilize the Spinach RNA aptamer interaction with the fluorophore DFHBI enabling detectable levels of green fluorescence to be emitted. The tRNALys-Spinach-tRNALys, once delivered into "chloroplast-free" onion epidermal cells can emit strong green fluorescence in the presence of DFHBI. Our results demonstrate for the first time that Spinach-based RNA visualization has the potential for in vivo monitoring of RNAs in plant cells.

Multigene editing via CRISPR/Cas9 guided by a single-sgRNA seed in Arabidopsis.

We report that a solo single-guide RNA (sgRNA) seed is capable of guiding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR -associated 9 (CRISRP/Cas9) to simultaneously edit multiple genes AtRPL10A, AtRPL10B and AtRPL10C in Arabidopsis. Our results also demonstrate that it is possible to use CRISPR/Cas9 technology to create AtRPL10 triple mutants which otherwise cannot be generated by conventional genetic crossing. Compared to other conventional multiplex CRISPR/Cas systems, a single sgRNA seed has the advantage of reducing off-target gene-editing. Such a gene editing system might be also applicable to modify other homologous genes, or even less-homologous sequences for multiple gene-editing in plants and other organisms.

Aequorin-based luminescence imaging reveals differential calcium signalling responses to salt and reactive oxygen species in rice roots.

It is well established that both salt and reactive oxygen species (ROS) stresses are able to increase the concentration of cytosolic free Ca(2+) ([Ca(2+)]i), which is caused by the flux of calcium (Ca(2+)). However, the differences between these two processes are largely unknown. Here, we introduced recombinant aequorin into rice (Oryza sativa) and examined the change in [Ca(2+)]i in response to salt and ROS stresses. The transgenic rice harbouring aequorin showed strong luminescence in roots when treated with exogenous Ca(2+). Considering the histological differences in roots between rice and Arabidopsis, we reappraised the discharging solution, and suggested that the percentage of ethanol should be 25%. Different concentrations of NaCl induced immediate [Ca(2+)]i spikes with the same durations and phases. In contrast, H2O2 induced delayed [Ca(2+)]i spikes with different peaks according to the concentrations of H2O2. According to the Ca(2+) inhibitor research, we also showed that the sources of Ca(2+) induced by NaCl and H2O2 are different. Furthermore, we evaluated the contribution of [Ca(2+)]i responses in the NaCl- and H2O2-induced gene expressions respectively, and present a Ca(2+)- and H2O2-mediated molecular signalling model for the initial response to NaCl in rice.

BnSIP1-1, a Trihelix Family Gene, Mediates Abiotic Stress Tolerance and ABA Signaling in Brassica napus.

The trihelix family genes have important functions in light-relevant and other developmental processes, but their roles in response to adverse environment are largely unclear. In this study, we identified a new gene, BnSIP1-1, which fell in the SIP1 (6b INTERACTING PROTEIN1) clade of the trihelix family with two trihelix DNA binding domains and a fourth amphipathic α-helix. BnSIP1-1 protein specifically targeted to the nucleus, and its expression can be induced by abscisic acid (ABA) and different stresses. Overexpression of BnSIP1-1 improved seed germination under osmotic pressure, salt, and ABA treatments. Moreover, BnSIP1-1 decreased the susceptibility of transgenic seedlings to osmotic pressure and ABA treatments, whereas there was no difference under salt stress between the transgenic and wild-type seedlings. ABA level in the transgenic seedlings leaves was higher than those in the control plants under normal condition. Under exogenous ABA treatment and mannitol stress, the accumulation of ABA in the transgenic plants was higher than that in the control plants; while under salt stress, the difference of ABA content before treatment was gradually smaller with the prolongation of salt treatment time, then after 24 h of treatment the ABA level was similar in transgenic and wild-type plants. The transcription levels of several general stress marker genes (BnRD29A, BnERD15, and BnLEA1) were higher in the transgenic plants than the wild-type plants, whereas salt-responsive genes (BnSOS1, BnNHX1, and BnHKT) were not significantly different or even reduced compared with the wild-type plants, which indicated that BnSIP1-1 specifically exerted different regulatory mechanisms on the osmotic- and salt-response pathways in seedling period. Overall, these findings suggested that BnSIP1-1 played roles in ABA synthesis and signaling, salt and osmotic stress response. To date, information about the involvement of the Brassica napus trihelix gene in abiotic response is scarce. Here, we firstly reported abiotic stress response and possible function mechanisms of a new trihelix gene in B. napus.