Optimization of an Efficient Protoplast Transformation System for Transient Expression Analysis Using Leaves of Torenia fournieri.

Torenia fournieri (T. fournieri) is one of the most widely used horticultural flowers and is considered a potential model plant for the genetic investigation of ornamental traits. In this study, we optimized an efficient protocol for high efficiency preparation and transformation of T. fournieri protoplast. The transformation rate reached ~75% when a 35S:GFP construct was used for the transformation. Using this system, we characterized the subcellular localization of several TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors (TFs), and found a distinct localization pattern between the CIN and CYC classes of TCP TFs. Furthermore, we also demonstrated the feasibility of the expression of dual luciferase assay system in T. fournieri protoplasts for the measurement of the activity of cis-regulatory elements. Taken together, a well-optimized transient expression system in T. fournieri protoplasts would be crucial for rapid exploration of the gene function or cis-regulatory elements.

A reference-grade wild soybean genome.

Efficient crop improvement depends on the application of accurate genetic information contained in diverse germplasm resources. Here we report a reference-grade genome of wild soybean accession W05, with a final assembled genome size of 1013.2 Mb and a contig N50 of 3.3 Mb. The analytical power of the W05 genome is demonstrated by several examples. First, we identify an inversion at the locus determining seed coat color during domestication. Second, a translocation event between chromosomes 11 and 13 of some genotypes is shown to interfere with the assignment of QTLs. Third, we find a region containing copy number variations of the Kunitz trypsin inhibitor (KTI) genes. Such findings illustrate the power of this assembly in the analysis of large structural variations in soybean germplasm collections. The wild soybean genome assembly has wide applications in comparative genomic and evolutionary studies, as well as in crop breeding and improvement programs.

Paraformaldehyde Fixation May Lead to Misinterpretation of the Subcellular Localization of Plant High Mobility Group Box Proteins.

Arabidopsis High Mobility Group Box (HMBG) proteins were previously found associated with the interphase chromatin but not the metaphase chromosome. However, these studies are usually based on immunolocalization analysis involving paraformaldehyde fixation. Paraformaldehyde fixation has been widely adapted to preserved cell morphology before immunofluorescence staining. On one hand, the processed cells are no longer living. On the other hand, the processing may lead to misinterpretation of localization. HMGBs from Arabidopsis were fused with enhanced green fluorescence protein (EGFP) and transformed into tobacco BY-2 cells. Basically, the localization of these HMGB proteins detected with EGFP fluorescence in interphase agreed with previous publications. Upon 4% paraformaldehyde fixation, AtHMGB1 was found associated with interphase but not the metaphase chromosomes as previously reported. However, when EGFP fluorescence signal was directly observed under confocal microscope without fixation, association of AtHMGB1 with metaphase chromosomes can be detected. Paraformaldehyde fixation led to dissociation of EGFP tagged AtHMBG1 protein from metaphase chromosomes. This kind of pre-processing of live specimen may lead to dissociation of protein-protein or protein-nucleic acid interaction. Therefore, using of EGFP fusion proteins in live specimen is a better way to determine the correct localization and interaction of proteins.

GmSAL1 hydrolyzes inositol-1,4,5-trisphosphate and regulates stomatal closure in detached leaves and ion compartmentalization in plant cells.

Inositol polyphosphatases are important regulators since they control the catabolism of phosphoinositol derivatives, which are often signaling molecules for cellular processes. Here we report on the characterization of one of their members in soybean, GmSAL1. In contrast to the substrate specificity of its Arabidopsis homologues (AtSAL1 and AtSAL2), GmSAL1 only hydrolyzes inositol-1,4,5-trisphosphate (IP3) but not inositol-1,3,4-trisphosphate or inositol-1,4-bisphosphate.The ectopic expression of GmSAL1 in transgenic Arabidopsis thaliana led to a reduction in IP3 signals, which was inferred from the reduction in the cytoplasmic signals of the in vivo biomarker pleckstrin homology domain-green florescent protein fusion protein and the suppression of abscisic acid-induced stomatal closure. At the cellular level, the ectopic expression of GmSAL1 in transgenic BY-2 cells enhanced vacuolar Na(+) compartmentalization and therefore could partially alleviate salinity stress.

The unconventional P-loop NTPase OsYchF1 and its regulator OsGAP1 play opposite roles in salinity stress tolerance.

YchF proteins are a group of mysterious but ubiquitous unconventional G-proteins found in all kingdoms of life except Archaea. Their functions have been documented in microorganisms, protozoa and human, but those of plant YchF homologues are largely unknown. Our group has previously shown that OsYchF1 and its interacting protein, OsGAP1, play opposite roles in plant defense responses. OsGAP1 was found to stimulate the GTPase/ATPase activities of OsYchF1 and regulate its subcellular localization. In this report, we demonstrate that both OsYchF1 and OsGAP1 are localized mainly in the cytosol under NaCl treatment. The ectopic expression of OsYchF1 in transgenic Arabidopsis thaliana leads to reduced tolerance towards salinity stress, while the ectopic expression of OsGAP1 has the opposite effect. Similar results were also obtained with the Arabidopsis homologues, AtYchF1 and AtGAP1, by using AtGAP1 overexpressors and underexpressors, as well as an AtYchF1-knockdown mutant. OsYchF1 and OsGAP1 also exhibit highly significant effects on salinity-induced oxidative stress tolerance. The expression of OsYchF1 suppresses the anti-oxidation enzymatic activities and increases lipid peroxidation in transgenic Arabidopsis, and leads to the accumulation of reactive oxygen species (ROS) in tobacco BY-2 cells, while the ectopic expression of OsGAP1 has the opposite effects in these two model systems.

The GmCLC1 protein from soybean functions as a chloride ion transporter.

Soil salinization is a global issue that hampers agricultural production. Chloride is one of the prominent anions on saline land that cause toxicity to the plant. We previously identified the GmCLC1 gene from soybean (Glycine max) that encodes a putative tonoplast-localized chloride transporter. In this study, using electrophysiological analysis, we demonstrated the chloride transport function of GmCLC1. Interestingly, this chloride transport activity is pH dependent, suggesting that GmCLC1 is probably a chloride/proton antiporter. When the cDNA of GmCLC1 was expressed in tobacco BY-2 cells under the control of a constitutive promoter, the protective effect against salinity stress in transgenic tobacco BY-2 cells was also found to be pH sensitive. In the native host soybean, the expression of GmCLC1 gene is regulated by pH. All these findings support the notion that the function of GmCLC1 is regulated by pH.