Alternative Splicing Studies of the Reactive Oxygen Species Gene Network inPopulusReveal Two Isoforms of High-Isoelectric-Point Superoxide Dismutase    

Abstract

Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipISODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipISODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays.

Superoxide dismutase‐1 and other proteins in inclusions from transgenic amyotrophic lateral sclerosis model mice

“…The resulting peptides were collected by centrifugation, lyophilized, and resuspended in 0.1% formic acid. Peptide identification was performed as previously described (Srivastava et al. 2009).…”

OnPLS integration of transcriptomic, proteomic and metabolomic data shows multi-level oxidative stress responses in the cambium of transgenic hipI- superoxide dismutase Populus plants

“…HipI-SOD is a Cu/Zn-SOD with a suggested role in ROS regulation and plant development [18-20]. The transgenic hipI-SOD Populus plants have higher levels of O 2 - than WT counterparts and impaired growth rates, accompanied by histological and morphological perturbations, including compressed and disorganized cell structures in the cambial region of the stem Srivastava et al [17] (Additional file 1: Figure S1).…”

Redox-dependent functional switching of plant proteins accompanying with their structural changes

“…Among them, the redox-dependent transcriptional factor, Rap2.4a, was isolated and studied as an efficient redox-sensor and transducer of redox status of cells to nucleus to control transcriptional activity of chloroplast antioxidant proteins (Shaikhali et al, 2008). The protein was cloned from a yeast-one-hybrid screening with a cis -regulatory element of 2-Cys peroxiredoxin (Prx)-A gene (2CPA) is grouped into activator protein 2 (AP2)-type transcription factor (Srivastava et al, 2009). Rap2.4a transcriptional activity is regulated by dithiol/disulfide transition of regulatory cysteinyl residues (Shaikhali et al, 2008).…”