C2H2 zinc finger proteins (ZFPs) play important roles in plant development and response to abiotic stresses, and have been studied extensively. However, there are few studies on ZFPs in mangroves and mangrove associates, which represent a unique plant community with robust stress tolerance. MpZFP1, which is highly induced by salt stress in the mangrove associate Millettia pinnata, was cloned and functionally characterized in this study. MpZFP1 protein contains two zinc finger domains with conserved QALGGH motifs and targets to the nucleus. The heterologous expression of MpZFP1 in Arabidopsis increased the seeds’ germination rate, seedling survival rate, and biomass accumulation under salt stress. The transgenic plants also increased the expression of stress-responsive genes, including RD22 and RD29A, and reduced the accumulation of reactive oxygen species (ROS). These results indicate that MpZFP1 is a positive regulator of plant responses to salt stress due to its activation of gene expression and efficient scavenging of ROS.

Salt stress is a major increasing threat to global agriculture. Pongamia (Millettia pinnata), a semi-mangrove, is a good model to study the molecular mechanism of plant adaptation to the saline environment. Calcium signaling pathways play critical roles in the model plants such as Arabidopsis in responding to salt stress, but little is known about their function in Pongamia. Here, we have isolated and characterized a salt-responsive MpCML40, a calmodulin-like (CML) gene from Pongamia. MpCML40 protein has 140 amino acids and is homologous with Arabidopsis AtCML40. MpCML40 contains four EF-hand motifs and a bipartite NLS (Nuclear Localization Signal) and localizes both at the plasma membrane and in the nucleus. MpCML40 was highly induced after salt treatment, especially in Pongamia roots. Heterologous expression of MpCML40 in yeast cells improved their salt tolerance. The 35S::MpCML40 transgenic Arabidopsis highly enhanced seed germination rate and root length under salt and osmotic stresses. The transgenic plants had a higher level of proline and a lower level of MDA (malondialdehyde) under normal and stress conditions, which suggested that heterologous expression of MpCML40 contributed to proline accumulation to improve salt tolerance and protect plants from the ROS (reactive oxygen species) destructive effects. Furthermore, we did not observe any measurable discrepancies in the development and growth between the transgenic plants and wild-type plants under normal growth conditions. Our results suggest that MpCML40 is an important positive regulator in response to salt stress and of potential application in producing salt-tolerant crops.

Plant growth and development are precisely controlled by light and temperature during their life span. However, the mechanism by which photoperiod and seasonal changes influence the physiological response of day-neutral plants, such as tomato (Solanum lycopersicum), remains unclear. Here, we found that the tomato CONSTANS (CO) close homolog, CONSTANS-Like1 (SlCOL1), does not affect the flowering of tomato under either long-day or short-day conditions. However, CRISPR/Cas9-mediated editing of SlCOL1 showed a much lower anthocyanin accumulation in mutant than in wild-type plants, especially under SD at suboptimal low-temperature conditions. SlCOL1 directly activated the Hoffman's Anthocyanin 1 (SlAN1) promoter and interacted with SlAN1 to promote anthocyanin biosynthesis under SD. The cold-induced upregulation of SlCOL1 further promoted anthocyanin accumulation and enhanced reactive oxygen species scavenging under SD at low-temperature conditions. These results reveal that the SlCOL1-SlAN1 module collaboratively regulates anthocyanin accumulation under SD and cold conditions, which could help tomato counteract the cold autumn/winter season in nature.

Summary: Exploring the new elements to re‐design the expression cassette is crucial in synthetic biology. Viruses are one of the most important sources for exploring gene expression elements. In this study, we found that the DNA sequence of the SBG51 deltasatellite from the Sweet potato leaf curl virus (SPLCV) greatly enhanced the gene expression when flanked downstream of the terminator. The SBG51 sequence increased transient GFP gene expression in Nicotiana benthamiana leaves by up to ~6 times and ~10 times compared to the gene expression controlled by the UBQ10 promoter and 35S promoter alone, respectively. The increased GFP gene expression level contributed to the continuous accumulation of GFP protein and GFP fluorescence until 8 days post‐inoculation (dpi). The SBG51 sequence also enhanced the gene expression in the transgenic Arabidopsis plants and maintained the spatio‐temporal pattern of the FLOWERING LOCUS T (FT) and TOO MANY MOUTHS (TMM) promoters. We identified a 123 bp of AT‐rich sequence containing seven “ATAAA” or “TTAAA” elements from the SBG51 DNA, which had the gene expression enhancement effect. Furthermore, the artificial synthetic sequences containing tandem repeated “ATAAA” or “TTAAA” elements were sufficient to increase the gene expression but did not alter the polyadenylation of mRNA, similar to the function of matrix attachment regions (MAR). Additionally, the compact artificial synthetic sequence also had an effect on yeast when the expression cassette was integrated into the genome. We conclude that the geminivirus deltasatellite‐derived sequence and the “ATAAA”/“TTAAA” elements are powerful tools for enhancing gene expression.