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.

HighlightBased on the aequorin system, we describe the establishment of a calcium reporter line in rice and test the responses of the reporter towards salt and oxidative stress.

Summary 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.

BnSIP1-1 is a trihelix transcription factor family gene which functions in abiotic stress response and abscisic acid (ABA) signaling during seed germination and seedling growth of Brassica napus. In the present study, further sequence analysis and phenotype identification indicated that this gene had roles in light regulation and flowering of reproductive growth stage. Many phytohormones responsive cis-acting elements, including TC-rich repeats, GARE-motif, and TCA and TGA elements, were identified in the promoter sequence of BnSIP1-1. The expression of BnSIP1-1 was regulated by light period and remarkable higher expression level of BnSIP1-1 was detected in roots than in leaves. Overexpression of BnSIP1-1 in Arabidopsis delayed flowering time for 3–5 days in transgenic plants. In addition, we also found BnSIP1-1 can respond to abiotic and ABA stress (treated with 200 mM NaCl, 300 mM mannitol or 50 μM ABA for 0, 1, 6, and 24 h) in B. napus through adjusting not only ABA but also other endogenous hormones, including indole-3-acetic acid and salicylic acid. Moreover, jasmonates (JA) signaling pathway was found not involving in the pathway of BnSIP1-1 responding to abiotic stresses.

Fruit ripening is a developmentally and genetically programmed process. In tomato (Solanum lycopersicum), ripening determines fruit quality, commodity value, shelf life and many important attributes. To understand this intricate process and its underpinning mechanism, an efficient and effective approach for screening and functional analysis of ripening-associated genes (RAGs) is required. Virus-induced gene silencing (VIGS) is a powerful reverse genetics tool for uncovering gene functions in plants. VIGS has been exploited to investigate roles of RAGs in tomato ripening. However in most cases, virus-induced RAG silencing is only assessed and correlated with the chromatic change of fruits. Here we report that silencing of LeSPL-CNR through a Potato virus X-based VIGS inhibited fruit ripening and led to development of non-ripening sectors in Ailsa Craig (AC) tomatoes. Non-ripening sectors remained firmer and possessed greater relative electric conductivity and acidity as well as a higher amount of chlorophyll, but a lower quantity of anthocyanin. VIGS of LeSPL-CNR also affects expression of other key RAGs and genes associated with biogenesis of ripening hormone ethylene. These findings indicate that AC fruits undergoing VIGS of LeSPL-CNR phenocopied physical, physiological, agrochemical, biochemical and molecular characteristics of the Colourless non-ripening epimutant. Thus, the overall phenotypical changes from visual appearance to RAG expression caused by LeSPL-CNR silencing reaffirm the great usefulness of VIGS to reveal biological functions of genes crucial in tomato ripening and fruit quality.

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.

RNAs can be imaged in living cells using molecular beacons, RNA-binding labeled proteins and RNA aptamer-based approaches. However, Spinach RNA-mimicking GFP (RMG) has not been successfully used to monitor cellular RNAs in plants. In this study, we re-evaluated Spinach-based RNA visualization in different plants via transient, transgenic, and virus-based expression strategies. We found that like bacterial, yeast and human cellular tRNAs, plant tRNAs such as tRNALys (K) 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 (KSK), once delivered into “chloroplast-free” onion epidermal cells can emit strong green fluorescence in the presence of DFHBI. Transgenic or virus-based expression of monomer KSK, in either stably transformed or virus-infected Nicotinana benthamiana plants, failed to show RMG fluorescence. However, incorporating tandem repeats of KSK into recombinant viral RNAs, enabled qualitative and quantitative detection, both in vitro and ex vivo (ex planta), of KSK-specific green fluorescence, though RMG was less obvious in vivo (in planta). These findings demonstrate Spinach-based RNA visualization has the potential for ex vivo and in vivo monitoring RNAs in plant cells.One sentence summarySpinach-based RMG technology was reevaluated to have potential for ex vivo and in vivo monitoring RNAs in plant cells.