Truncated Cotton Subtilase Promoter Directs Guard Cell-Specific Expression of Foreign Genes in Tobacco and Arabidopsis

Abstract

A 993-bp regulatory region upstream of the translation start codon of subtilisin-like serine protease gene was isolated from Gossypium barbadense. This (T/A)AAAG-rich region, GbSLSP, and its 5′- and 3′-truncated versions were transferred into tobacco and Arabidopsis after fusing with GUS or GFP. Histochemical and quantitative GUS analysis and confocal GFP fluorescence scanning in the transgenic plants showed that the GbSLSP-driven GUS and GFP expressed preferentially in guard cells, whereas driven by GbSLSPF2 to GbSLSPF4, the 5′-truncated GbSLSP versions with progressively reduced Dof1 elements, both GUS and GFP expressed exclusively in guard cells, and the expression strength declined with (T/A)AAAG copy decrement. Deletion of 5′-untranslated region from GbSLSP markedly weakened the activity of GUS and GFP, while deletion from the strongest guard cell-specific promoter, GbSLSPF2, not only significantly decreased the expression strength, but also completely abolished the guard cell specificity. These results suggested both guard cell specificity and expression strength of the promoters be coordinately controlled by 5′-untranslated region and a cluster of at least 3 (T/A)AAAG elements within a region of about 100 bp relative to transcription start site. Our guard cell-specific promoters will enrich tools to manipulate gene expression in guard cells for scientific research and crop improvement.

Differential fine-tuning of gene expression regulation in coffee leaves by CcDREB1D promoter haplotypes under water deficit

“…The increase in leaf-specific GUS activities observed from the short to the medium- and full-length constructs also tallied with the fact that cluster arrangements of DOF-binding sites are essential in determining guard cell-specific gene expression ( Galbiati et al , 2008 ; Yang et al , 2008 ). However, this does not preclude the participation of other CREs functioning conjointly with DOF-binding sites to fine-tune the transcriptional regulation of gene expression in guard cells, as previously proposed in grape VvMYB60 , potato KST1 and cotton GbSLSP promoters, for example ( Plesch et al , 2001 ; Gardner et al , 2009 ; Cominelli et al , 2011 ; Galbiati et al , 2011 ; Han et al , 2013 ). Of the CREs that could play such roles, it is worth noting the presence of several GT-1 DNA binding sites in the CcDREB1D promoter haplotypes, with three of them (one in the medium (−608/−966) and two in the distal (−966/−1308) regions of the CcDREB1D promoters) closely located to DOF-binding elements.…”

Subcellular Localization of a Plant Catalase-Phenol Oxidase, AcCATPO, from Amaranthus and Identification of a Non-canonical Peroxisome Targeting Signal

“…The peroxisomal marker, pBin20-mCherry-PTS1, and all GFP constructs were transformed into Agrobacterium tumefaciens strain GV3101. For transient expression of single construct, the resultant bacterial suspension was directly infiltrated into young leaves of tobacco ( N. benthamiana ) as previously reported ( Han et al, 2013 ). For co-expression of the fused GFP and peroxisomal marker RFP, the bacterial suspension harboring GFP construct and that carrying peroxisomal RFP marker were mixed in a ratio of 1:1, and then the resulting mixture was co-infiltrated into young leaves as reported before ( Chen et al, 2017 ).…”

A PPO Promoter from Betalain-Producing Red Swiss Chard, Directs Petiole- and Root-Preferential Expression of Foreign Gene in Anthocyanins-Producing Plants

“…Histochemical GUS staining and quantitative analysis for GUS activity in the transgenic plants were performed according to the method described by Jefferson et al [ 43 ] and slightly modified by Han et al [ 44 ]. Briefly, the Arabidopsis whole-plantlets of different developmental stages (5-day-old, 10-day-old, 15-day-old, after anthesis) were incubated at 37 °C for 12 to 16 h in GUS staining solution (50 mM phosphate buffer, pH 6.7, 1 mM EDTA pH 8.0, 0.2% ( v / v ) Triton-100, 1 mM K 3 FeCN 6 , 1 mM K 4 FeCN 6 , 0.5 mg/mL 5-bromo-4-chloro-3-indoxyl- d -glucuronic acid (X-gluc)).…”

“…For quantitative GUS activity assay, the samples were prepared as previously described [ 45 ], and the enzymatic reaction was carried out according to Han et al [ 44 ].…”