Abstract
The hyperthermostable β-glucosidase BglB of Thermotoga maritima was modified by adding a short C-terminal tetrapeptide (AFVY, which transports phaseolin to the vacuole, to its C-terminal sequence). The modified β-glucosidase BglB was transformed into tobacco (Nicotiana tabacum L.) plants. We observed a range of significant phenotypic changes in the transgenic plants compared to the wild-type (WT) plants. The transgenic plants had faster stem growth, earlier flowering, enhanced root systems development, an increased biomass biosynthesis rate, and higher salt stress tolerance in young plants compared to WT. In addition, programed cell death was enhanced in mature plants. Furthermore, the C-terminal AFVY tetrapeptide efficiently sorted T. maritima BglB into the vacuole, which was maintained in an active form and could perform its glycoside hydrolysis function on hormone conjugates, leading to elevated hormone [abscisic acid (ABA), indole 3-acetic acid (IAA), and cytokinin] levels that likely contributed to the phenotypic changes in the transgenic plants. The elevation of cytokinin led to upregulation of the transcription factor WUSCHELL, a homeodomain factor that regulates the development, division, and reproduction of stem cells in the shoot apical meristems. Elevation of IAA led to enhanced root development, and the elevation of ABA contributed to enhanced tolerance to salt stress and programed cell death. These results suggest that overexpressing vacuole-targeted T. maritima BglB may have several advantages for molecular farming technology to improve multiple targets, including enhanced production of the β-glucosidase BglB, increased biomass, and shortened developmental stages, that could play pivotal roles in bioenergy and biofuel production.
Highlights
Vacuole-Targeted BglB Elevate Hormone heterologous β-glucosidase can release active forms of phytohormones from their inactive conjugates that consist of glycoside links (Spena et al, 1992; Brzobohaty et al, 1993)
10 and 12 lines of CB and VB transgenic plants, respectively, were confirmed, and three of the lines were used for further analysis after confirmation of the transgenes by genomic DNA polymerase chain reaction (PCR), reverse transcription (RT)-PCR, and Western blotting (Figures 1B–E)
The presence of the nucleotide sequences encoding the AFVY tetrapeptide in the VB construct was confirmed using PCR with a reverse primer specific to the VB construct (Figure 1C), and the transcript of the heterologous BglB in the transgenic plants were confirmed by RTPCR (Figure 1D)
Summary
Vacuole-Targeted BglB Elevate Hormone heterologous β-glucosidase can release active forms of phytohormones from their inactive conjugates that consist of glycoside links (Spena et al, 1992; Brzobohaty et al, 1993). Many studies have revealed that the inactive forms of phytohormone conjugates act as reversible deactivated storage molecules, and are important for the regulation of physiologically active hormone levels; their normal biological functions remain unknown (Staswick, 2009; Piotrowska and Bajguz, 2011). We targeted the vacuole by insertion of the AFVY tetrapeptide to examine whether BglB maintain its functions of hydrolyzing glycoside bonds to release free hormones from its conjugates, and to determine how such changes in hormones levels may affect the growth and development of transgenic plants. In the shoot apical meristem, the transcription factor WUSCHELL (WUS) can be upregulated via cytokinin, and a group of dividing cells called the quiescent center (QC) is upregulated by indole 3-acetic acid (IAA) in the root apical meristem (Kerk et al, 2000; Overvoorde et al, 2010; Yadav et al, 2010; Zhao et al, 2010)
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