Presentation_1_Genome-wide identification of cystathionine beta synthase genes in wheat and its relationship with anther male sterility under heat stress.pptx

Cystathionine beta synthase (CBS) domains containing proteins (CDCPs) plays an important role in plant development through regulation of the thioredoxin system, as well as its ability to respond to biotic and abiotic stress conditions. Despite this, no systematic study has examined the wheat CBS gene family and its relation to high temperature-induced male sterility. In this study, 66 CBS family members were identified in the wheat genome, and their gene or protein sequences were used for subsequent analysis. The TaCBS gene family was found to be unevenly distributed on 21 chromosomes, and they were classified into four subgroups according to their gene structure and phylogeny. The results of collinearity analysis showed that there were 25 shared orthologous genes between wheat, rice and Brachypodium distachyon, and one shared orthologous gene between wheat, millet and barley. The cis-regulatory elements of the TaCBS were related to JA, IAA, MYB, etc. GO and KEGG pathway analysis identified these TaCBS genes to be associated with pollination, reproduction, and signaling and cellular processes, respectively. A heatmap of wheat plants based on transcriptome data showed that TaCBS genes were expressed to a higher extent in spikelets relative to other tissues. In addition, 29 putative tae-miRNAs were identified, targeting 41 TaCBS genes. Moreover, qRT-PCR validation of six TaCBS genes indicated their critical role in anther development, as five of them were expressed at lower levels in heat-stressed male sterile anthers than in Normal anthers. Together with anther phenotypes, paraffin sections, starch potassium iodide staining, and qRT-PCR data, we hypothesized that the TaCBS gene has a very important connection with the heat-stressed sterility process in wheat, and these data provide a basis for further insight into their relationship.</p

Table_1_Genome-wide identification of cystathionine beta synthase genes in wheat and its relationship with anther male sterility under heat stress.xlsx

Cystathionine beta synthase (CBS) domains containing proteins (CDCPs) plays an important role in plant development through regulation of the thioredoxin system, as well as its ability to respond to biotic and abiotic stress conditions. Despite this, no systematic study has examined the wheat CBS gene family and its relation to high temperature-induced male sterility. In this study, 66 CBS family members were identified in the wheat genome, and their gene or protein sequences were used for subsequent analysis. The TaCBS gene family was found to be unevenly distributed on 21 chromosomes, and they were classified into four subgroups according to their gene structure and phylogeny. The results of collinearity analysis showed that there were 25 shared orthologous genes between wheat, rice and Brachypodium distachyon, and one shared orthologous gene between wheat, millet and barley. The cis-regulatory elements of the TaCBS were related to JA, IAA, MYB, etc. GO and KEGG pathway analysis identified these TaCBS genes to be associated with pollination, reproduction, and signaling and cellular processes, respectively. A heatmap of wheat plants based on transcriptome data showed that TaCBS genes were expressed to a higher extent in spikelets relative to other tissues. In addition, 29 putative tae-miRNAs were identified, targeting 41 TaCBS genes. Moreover, qRT-PCR validation of six TaCBS genes indicated their critical role in anther development, as five of them were expressed at lower levels in heat-stressed male sterile anthers than in Normal anthers. Together with anther phenotypes, paraffin sections, starch potassium iodide staining, and qRT-PCR data, we hypothesized that the TaCBS gene has a very important connection with the heat-stressed sterility process in wheat, and these data provide a basis for further insight into their relationship.</p

A Rapid, Highly Efficient and Economical Method of <i>Agrobacterium</i>-Mediated <i>In planta</i> Transient Transformation in Living Onion Epidermis

<div><p>Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the <i>in vivo</i> behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and <i>Agrobacterium</i>-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient <i>in planta</i> transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and <i>Agrobacterium</i>-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.</p></div

Schematic diagram of forming agroinfiltration bubble by injecting agroinfiltration liquid into the interface between adaxial epidermis and mesophyll of onion bulb scale.

<p>(A) Syringe. (B) Agroinfiltration liquid. (C) Mesophyll of onion bulb scale. (D) Adaxial epidermis of onion bulb scale.</p

Effects of bacterial concentrations and durations of agroinfiltration.

<p>Note: three samples per combination of bacterial concentration and infiltration duration were investigated for ten 2 mm² epidermal areas. Each efficiency value represents the mean of transformation efficiencies of thirty replicates of 2 mm² epidermal areas from three samples, and the standard errors were calculated by using Excel. Different capital and lowercase letters within the same column exhibit significant difference at the 1% and 5% probability level according to the Duncan test of SPSS 10.0 statistic analysis.</p

Comparison of agroinfiltration and particle bombardment methods on transformation in onion epidermis.

<p>Note: three samples per unit of infiltration components were investigated for ten 2 mm² epidermal areas. The efficiency value represents the mean of transformation efficiencies of thirty replicates of 2 mm² epidermal areas from three samples (the percentage of positive cells in total cells per unit area) in the part of transformation efficiency, and the standard errors were calculated by using Excel.</p

<i>Agrobacterium</i> mediated <i>in planta</i> transient transoformationin living onion epidermal cells.

<p>(A–F) Operational process of the modified agroinfiltration, (A) Onion bulb without outer scales, (B, C) The cut onion bulb prepared for subsequent injection, (D) The injection of <i>Agrobacteria</i>, (E) Bind injected cut scales together with elastic for further incubation, scale bar = 2.5 cm. (F) The magnification of injection location, scale bar = 4 mm. (G–V) Onion epidermal cells were transformed with constructs of pCM1205-RFP (G, H, I and J), pLPGM202 (K, L, M and N), pLPGM413 (O, P, Q and R) and pLPGM113 (S, T, U and V). Bright field images (G, K, O and S), UV excited fluorescence images (L, P, T and H), UV excited DAPI staining images (I, M, Q and U) and the merged images of fluorescence and DAPI (J, N, R and V), scale bar = 10 µm.</p

Comparison of transient transformation efficiencies in different plant materials by using agroinfiltration.

<p>Note: three samples per material were investigated for ten 2 mm² areas of agroinfiltrated epidermal cells. Each efficiency value represents the mean of transformation efficiencies of thirty replicates of 2 mm² epidermal areas from three samples (the percentage of positive cells in total cells per unit area), and the standard errors were calculated by using Excel. Different capital and lowercase letters within the same column exhibit significant difference at the 1% and 5% probability level according to the Duncan test of SPSS 10.0 statistic analysis. For onion two-day pretreatment was conducted before agroinfiltration.</p

Effects of different pretreatment time of onion before <i>Agrobacterium</i> infection.

<p>Note: three repeat samples for each treatment were investigated by observing ten 2 mm² epidermal areas of each sample. Each efficiency value represents the mean of transformation efficiencies of thirty replicates of 2 mm² epidermal areas from three samples (the percentage of positive cells in total cells per unit area), and the standard errors were calculated by using Excel. Different capital and lowercase letters within the same column exhibit significant difference at the 1% and 5% probability level according to the Duncan test of SPSS 10.0 statistic analysis.</p

Effects of different components of agroinfiltration liquid (OD₆₀₀ = 0.10).

<p>Note: three samples per unit of infiltration components were investigated for ten 2 mm² epidermal areas. Each efficiency value represents the mean of transformation efficiencies of thirty replicates of 2 mm² epidermal areas from three samples (the percentage of positive cells in total cells per unit area), and the standard errors were calculated by using Excel. Different capital and lowercase letters within the same column exhibit significant difference at the 1% and 5% probability level according to the Duncan test of SPSS 10.0 statistic analysis. The symbol “-”, represents that the agroinfiltration liquid included all the components except the referred component.</p