Search

Background: Optimization of a highly efficient transient expression system is critical for the study of gene function, particularly in those plants in which stable transformation methods are not widely available. Agrobacterium tumefaciens‑mediated transient transformation is a simple and low-cost method that has been developed and applied to a wide variety of plant species. However, the transient expression in spinach (Spinacia oleracea L.) is still not reported. Results: We developed a transient expression system in spinach leaves of the Sp75 and Sp73 varieties. Several factors influencing the transformation efficiency were optimized such as Agrobacterium strain, spinach seedling stage, leaf position, and the expression time after injection. Agrobacterium strain GV3101 (pSoup-p19) was more efficient than AGL1 in expressing recombinant protein in spinach leaves. In general, Sp75 leaves were more suitable than Sp73 leaves, regardless of grow stage. At four-leaf stage, higher intensity and efficiency of transient expression were observed in group 1 (G1) of Sp75 at 53 h after injection (HAI) and in G1 of Sp73 at 64 HAI. At six-leaf stage of Sp75, group 3 (G3) at 72 HAI were the most effective condition for transient expression. Using the optimized expression system, we detected the subcellular localization of a transcriptional co-activator SoMBF1c and a NADPH oxidase SoRbohF. We also detected the interaction of the protein kinase SoCRK10 and the NADPH oxidase SoRbohB. Conclusion: This study established a method of highly efficient transient expression mediated by Agrobacterium in spinach leaves. The transient expression system will facilitate the analysis of gene function and lay a solid foundation for molecular design breeding of spinach. [ABSTRACT FROM AUTHOR]

Background: Salt stress seriously impacts the plantation and productivity of global alfalfa (Medicago sativa). Previously, we genetically defined the M. sativa DROUGHT-INDUCED UNKNOWN PROTEIN 1 (MsDIUP1) as a drought tolerance gene of alfalfa. However, whether it is associated with salt tolerance needs to be explored. Results: In this study, we re-classified MsDIUP1 as a putative novel late embryogenesis abundant protein, and found its promoter activity was strongly induced by salt and ABA treatment. Heterologous overexpression of MsDIUP1 in Arabidopsis led to enhanced salt tolerance and ABA insensitivity, with a higher fresh weight, lateral root number, primary root length, and survival rate compared to the wild-type (WT) plants. Similarly, homologous overexpression of MsDIUP1 also enhanced the salt tolerance of alfalfa over-expression (OE) plants, accompanied by reduced oxidative damage, higher K+/Na+ ratio, increased osmoprotectant accumulation, and improved photosynthetic capacity relative to the WT plants. In reverse, disruption of MsDIUP1 expression caused a salt-hypersensitive phenotype of alfalfa RNA interference (RNAi) plants, with higher oxidative damage, less osmoprotectant accumulation, and lower photosynthetic pigment content than that of the WT. These physiological discoveries were in accordance with differential transcript profiles of the relevant genes associated with stress signaling, antioxidant defense, and osmotic adjustment in the salt-treated alfalfa WT, OE, and RNAi lines. Furthermore, we found that alfalfa OE plants showed no apparent alteration in their forage quality compared with the WT plants. Conclusions: Overall, these results reveal that MsDIUP1 improves plant salt tolerance via regulating stress signaling, antioxidant defense, ion homeostasis, osmotic adjustment, and photosynthesis. [ABSTRACT FROM AUTHOR]

Summary: Strigolactones (SLs) play a critical role in regulating plant tiller number. LATERAL BRANCHING OXIDOREDUCTASE (LBO) encodes an important late‐acting enzyme for SL biosynthesis and regulates shoot branching in Arabidopsis. However, little is known about the function of LBO in monocots including switchgrass (Panicum virgatum L.), a dual‐purpose fodder and biofuel crop.We studied the function of PvLBO via the genetic manipulation of its expression levels in both the wild‐type and miR156 overexpressing (miR156OE) switchgrass. Co‐expression analysis, quantitative real‐time polymerase chain reaction (qRT‐PCR), transient dual luciferase assay, and chromatin immunoprecipitation‐qPCR were all used to determine the activation of PvLBO by miR156‐targeted Squamosa Promoter Binding Protein‐like 2 (PvSPL2) in regulating tillering of switchgrass.PvLBOtranscripts dramatically declined in miR156OE transgenic switchgrass, and the overexpression of PvLBO in the miR156OE transgenic line produce fewer tillers than the control. Furthermore, we found that PvSPL2 can directly bind to the promoter of PvLBO and activate its transcription, suggesting that PvLBO is a novel downstream gene of PvSPL2.We propose that PvLBO functions as an SL biosynthetic gene to mediate tillering and acts as an important downstream factor in the crosstalk between the SL biosynthetic pathway and the miR156‐SPL module in switchgrass. [ABSTRACT FROM AUTHOR]

Excess copper (Cu) in soil due to industrial and agricultural practices can result in reduced plant growth. Excess Cu resulted in severely retarded root growth with severe discoloration of Alfalfa (Medicago sativa) and Medicago truncatula. Growth in the presence of hydrogen peroxide resulted in similar symptoms that could be partially recovered by the addition of the reductant ascorbic acid revealing damage was likely due to oxidative stress. The addition of proanthocyanidins (PAs) in the presence of Cu prevented much of the damage, including plant growth and restoration of lignin synthesis which was inhibited in the presence of excess Cu. Transcriptome analyses of the impact of excess Cu and the amelioration after PAs treatment revealed that changes were enriched in functions associated with the cell wall and extracellular processes, indicating that inhibition of cell wall synthesis was likely the reason for retarded growth. Excess Cu appeared to induce a strong defense response, along with alterations in the expression of a number of genes encoding transcription factors, notably related to ethylene signaling. The addition of PAs greatly reduced this response, and also induced novel genes that likely help ameliorate the effects of excess Cu. These included induction of genes involved in the last step of ascorbic acid biosynthesis and of enzymes involved in cell wall synthesis. Combined, these results show that excess Cu causes severe oxidative stress damage and inhibition of cell wall synthesis, which can be relieved by the addition of PAs. [ABSTRACT FROM AUTHOR]