Analyses of Ca²⁺ dynamics using a ubiquitin‐10 promoter‐driven Yellow Cameleon 3.6 indicator reveal reliable transgene expression and differences in cytoplasmic Ca²⁺ responses in Arabidopsis and rice (Oryza sativa) roots

Abstract

both species were analyzed during exposure to hyperpolarization/depolarization cycles or in response to application of the amino acid glutamate. We found a superior performance of the UBQ10 promoter with regard to expression pattern, levels and expression stabilities in both species. We observed remarkable differences between the two species in the spatiotemporal parameters of the observed Ca 2+ signatures.Rice appeared in general to respond with a lower maximal signal amplitude but greatly increased signal duration when compared with Arabidopsis. Our results identify important advantages to using the UBQ10 promoter in Arabidopsis and rice and in T-DNA mutant backgrounds. Moreover, the observed differences in Ca 2+ signaling in the two species underscore the need for comparative studies to achieve a comprehensive understanding of Ca 2+ signaling in plants.

Chloroplast-Specific in Vivo Ca2+ Imaging Using Yellow Cameleon Fluorescent Protein Sensors Reveals Organelle-Autonomous Ca2+ Signatures in the Stroma

“…S2). This was indicative of strong transgene silencing, which we and others have already observed in other fluorescent protein lines (Deuschle et al, 2006;Jones et al, 2014;Behera et al, 2015;Schwarzländer et al, 2016). To overcome this limitation, we introduced the 35S:2Bam4-YC3.6 construct into the Arabidopsis rdr6-11 background (here referred to as rdr6 for simplicity), which is impaired in gene silencing (Peragine et al, 2004).…”

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase

“…Lack of AVP1 overexpression is most likely due to transgene silencing, as indicated by the complete absence or patchy kanamycin resistance in AVP1-1 and AVP1-2 in subsequent generations (Supplemental Figures 1M and 1N). We thus generated transgenic lines expressing AVP1 under the control of the UBQ10 promoter, which leads to constitutive and robust overexpression (Grefen et al, 2010;Behera et al, 2015) and resulted in 2-to 3-fold higher V-PPase activity ( Figures 1A and 1B). Notably, constitutive overexpression of AVP1 did not correlate with an increase in rosette size and fresh weight ( Figures 1C and 1D) or with altered auxin transport and content as reported previously Supplemental Tables 1 and 2).…”

A Rice Ca2+ Binding Protein Is Required for Tapetum Function and Pollen Formation

“…The image parameters for YFP channel were as follows: smart gain (531.0); offset (21.3%). For the statistical analysis of [Ca 2+ ] cyt , the ratio of fluorescence intensity in channel CFP and YFP in the whole region of the cell was measured and calculated as described (Behera et al, 2015). For the statistical analysis of [Ca 2+ ] PM , the ratios of fluorescence intensity in channels CFP and YFP around the plasma membrane were measured (n .…”