MuDR, the founder member of the Mutator superfamily and its MURA transcripts, has been identified as toxic sequences to Escherichia coli (E. coli), which heavily hindered the elucidation of the biochemical features of MURA transposase and confined the broader application of the Mutator system in other organisms. To harness less constrained systems as alternatives, we attempted to clone TED and Jittery, two recently isolated autonomous Mutator-like elements (MULEs) from maize, respectively. Their full-length transcripts and genomic copies are successfully cloned when the incubation time for bacteria to recover from heat shock is extended appropriately prior to plating. However, during their proliferation in E. coli, TED transformed plasmids are unstable, as evidenced by derivatives from which frameshift, deletion mutations, or IS transposon insertions are readily detected. Our results suggest that neither leaky expression of the transposase nor the presence of terminal inverse repeats (TIRs) are responsible for the cloning barriers, which were once ascribed to the presence of the Shine–Dalgarno-like sequence. Instead, the internal sequence of TED (from 1250 to 2845 bp), especially the exons in this region, was the most likely causer. The findings provide novel insights into the property and function of the Mutator superfamily and shed light on the dissection of toxic effects on cloning from MULEs.

Transcription factors GATAs were a member of zinc finger protein, which could bind DNA regulatory regions to control expression of target genes, thus influencing plant growth and development either in normal condition or environmental stresses. Recently, GATA genes have been found and functionally characterized in a number of plant species. However, little information of GATA genes were annotated in wheat. In the current study, 79 GATA genes were identified in wheat, which were unevenly located on 21 chromosomes. According to the analysis of phylogenetic tree and functional domain structures, TaGATAs were classified into four subfamilies (I, II, III, and IV), consist of 35, 21, 12, and 11 genes, respectively. Meanwhile, the amino acids of 79 TaGATAs exhibited apparent difference in four subfamilies according to GATA domains comparison, gene structures and conserved motif analysis. We then analyze the gene duplication and synteny between the genomes of wheat and Arabidopsis, rice and barley, which provided insights into evolutionary characteristics. In addition, expression patterns of TaGATAs were analyzed, and they showed obvious difference in diverse tissues and abiotic stresses. In general, these results provide useful information for future TaGATA gene function analysis, and it helps to better understand molecular breeding and stress response in wheat.

Helitrons, a novel type of mysterious DNA transposons discovered computationally prior to bench work confirmation, are components ubiquitous in most sequenced genomes of various eukaryotes, including plants, animals, and fungi. There is a paucity of empirical evidence to elucidate the mechanism of Helitrons transposition in plants. Here, by constructing several artificial defective Helitron (dHel) reporter systems, we aim to identify the autonomous Helitrons (aHel) in maize genetically and to demonstrate the transposition and repair mechanisms of Helitrons upon the dHel-GFP excision in maize. When crossing with various inbred lines, several transgenic lines produced progeny of segregated, purple-blotched kernels, resulting from a leaky expression of the C1 gene driven by the dHel-interrupted promoter. Transcription analysis indicated that the insertion of different dHels into the C1 promoter or exon would lead to multiple distinct mRNA transcripts corresponding to transgenes in the host genome. Simple excision products and circular intermediates of dHel-GFP transposition have been detected from the leaf tissue of the seedlings in F1 hybrids of transgenic lines with corresponding c1 tester, although they failed to be detected in all primary transgenic lines. These results revealed the transposition and repair mechanism of Helitrons in maize. It is strongly suggested that this reporter system can detect the genetic activity of autonomic Helitron at the molecular level. Sequence features of dHel itself, together with the flanking regions, impact the excision activity of dHel and the regulation of the dHel on the transcription level of the host gene.

Heterotrimeric GTP-binding proteins (G proteins) are a group of regulators essential for signal transmission into cells. Regulator of G-protein signaling 1 (AtRGS1) possesses intrinsic GTPase-accelerating protein (GAP) activity and could suppress G-protein and glucose signal transduction in Arabidopsis (Arabidopsis thaliana). However, how AtRGS1 activity is regulated is poorly understood. Here we identified a knockout mutant of OXYSTEROL BINDING PROTEIN-RELATED PROTEIN 2A, orp2a-1, which exhibits similar phenotypes to the arabidopsis g-protein beta 1-2 (agb1-2) mutant. Transgenic lines overexpressing ORP2A displayed short hypocotyls, a hypersensitive response to sugar and lower intracellular AtRGS1 levels than the control. Consistently, ORP2A interacted with AtRGS1 in vitro and in vivo. Tissue-specific expression of two ORP2A alternative splicing isoforms implied functions in controlling organ size and shape. Bioinformatic data and phenotypes of orp2a-1, agb1-2 and the orp2a-1 agb1-2 double mutant revealed the genetic interactions between ORP2A and Gβ in the regulation of G-protein signaling and sugar response. Both alternative protein isoforms of ORP2A localized in the ER, plasma membrane (PM) and ER-PM Contact Sites and interacted with vesicle-associated membrane protein-associated protein 27-1 (VAP27-1) in vivo and in vitro through their FFAT-like motif. ORP2A also displayed differential phosphatidyl phosphoinositide binding activity mediated by the PH domain in vitro. Taken together, the Arabidopsis membrane protein ORP2A interacts with AtRGS1 and VAP27-1 to positively regulate G-protein and sugar signaling by facilitating AtRGS1 degradation.

Heterotrimeric GTP-binding proteins are a group of regulators essential for signal transmission into cells. AtRGS1 with intrinsic GTPase-accelerating protein activity could suppress G protein and glucose signal transduction in Arabidopsis. However, how AtRGS1 activity is regulated is currently poor understood. Here we identified a knockout mutant orp2a-1 which shows phenotypes similar to agb1-2. With overexpression of ORP2A, transgenic lines display short hypocotyl, hypersensitivity to sugar and lower intracellular AtRGS1 level than control. Consistently, ORP2A shows interaction with AtRGS1 in vitro and vivo. Tissue specificity of ORP2A with two alternative protein forms imply its functions in organ size and shape controlling. Bioinformatic data and phenotypes of orp2a-1, agb1-2 and double mutant reveal genetic interactions in the regulation of G protein signaling and sugar response between ORP2A and Gβ. Both alternative splicing forms of ORP2A locate in the ER, PM and EPCS, and interact with VAP27-1 mediated by a FFAT-like motif in vivo and vitro. ORP2A also displays differential phosphatidyl phosphoinositide binding activity mediated by its PH domain in vitro. Taken together, it is suggested that Arabidopsis membrane protein ORP2A interacts with AtRGS1 and VAP27-1 to positively regulate G protein and sugar signaling by facilitating AtRGS1 degradation.