Gene Expression In Plant Tissues Research Articles

PfbZIP85 Transcription Factor Mediates ω-3 Fatty Acid-Enriched Oil Biosynthesis by Down-Regulating PfLPAT1B Gene Expression in Plant Tissues.

The basic leucine zipper (bZIP) transcription factor (TF) family is one of the biggest TF families identified so far in the plant kingdom, functioning in diverse biological processes including plant growth and development, signal transduction, and stress responses. For Perilla frutescens, a novel oilseed crop abundant in polyunsaturated fatty acids (PUFAs) (especially α-linolenic acid, ALA), the identification and biological functions of bZIP members remain limited. In this study, 101 PfbZIPs were identified in the perilla genome and classified into eleven distinct groups (Groups A, B, C, D, E, F, G, H, I, S, and UC) based on their phylogenetic relationships and gene structures. These PfbZIP genes were distributed unevenly across 18 chromosomes, with 83 pairs of them being segmental duplication genes. Moreover, 78 and 148 pairs of orthologous bZIP genes were detected between perilla and Arabidopsis or sesame, respectively. PfbZIP members belonging to the same subgroup exhibited highly conserved gene structures and functional domains, although significant differences were detected between groups. RNA-seq and RT-qPCR analysis revealed differential expressions of 101 PfbZIP genes during perilla seed development, with several PfbZIPs exhibiting significant correlations with the key oil-related genes. Y1H and GUS activity assays evidenced that PfbZIP85 downregulated the expression of the PfLPAT1B gene by physical interaction with the promoter. PfLPAT1B encodes a lysophosphatidate acyltransferase (LPAT), one of the key enzymes for triacylglycerol (TAG) assembly. Heterogeneous expression of PfbZIP85 significantly reduced the levels of TAG and UFAs (mainly C18:1 and C18:2) but enhanced C18:3 accumulation in both seeds and non-seed tissues in the transgenic tobacco lines. Furthermore, these transgenic tobacco plants showed no significantly adverse phenotype for other agronomic traits such as plant growth, thousand seed weight, and seed germination rate. Collectively, these findings offer valuable perspectives for understanding the functions of PfbZIPs in perilla, particularly in lipid metabolism, showing PfbZIP85 as a suitable target in plant genetic improvement for high-value vegetable oil production.

Destruction of transgenic potato plants (Solanum tuberosum L.) remnants under the influence of soil microbiome

Scientific community has been considering various hypotheses regarding the possible impact of GM plants on non-target soil microorganisms for decades, distinguishing direct and indirect effects. Direct effects are usually easier to detect, as transgenic proteins, characterized by a range of elimination against certain types of insects, pathogenic fungi and bacteria, can affect non-target beneficial symbionts and soil microbial groups involved in the transformation of organic substances. Indirect effects are difficult to assess, as many different factors can affect the composition of root exudates and the metabolic activity of plants. Changes in metabolic pathways can affect the composition of soil exudates and gene expression in plant tissues. At the same time, these changes can affect the decomposition of organic matter and the course of microbiological processes in the soil. Based on experimental studies, an assessment of the rate of destruction of plant residues and DNA degradation in transgenic potato plants in soddy-podzolic soil of potato crop rotation was carried out. The results of the studies have established a significant role of the microbiome of soddy-podzolic soil of potato crop rotation in the destruction of residues of transgenic potato plants and DNA degradation. Destruction of residues of transgenic plants under the influence of microflora of soddy-podzolic soil of potato crop rotation was 30 days with a loss of raw mass 87.14±1.11%. DNA degradation over this period is 88% (186 ng/mg) from the initial amount of total DNA, and degradation of nptII and 35S genes — about 90% from their initial amount in PCR products. It was found that about 90% of nptII and 35S genes in DNA residues of transgenic potato plants under the influence of microbiome soddy-podzolic soil occurs on day 30.

Genome-wide analysis and metabolic profiling unveil the role of peroxidase CsGPX3 in theaflavin production in black tea processing

Plucked tea leaves can be processed into black tea (Camellia sinensis), which is rich in health-promoting molecules, including flavonoid antioxidants. During black tea processing, theaflavins (TFs) and thearubigins (TRs) are generated via the successive oxidation of catechins by endogenous polyphenol oxidase (PPO)- or peroxidase (POD)-mediated reactions. This process must be well controlled to achieve the proper TF/TR ratio, which is an important quality parameter of the tea beverage. However, little is known about the POD/PPO catalyzed TF formation process at the molecular genetic level. Here, we identified and characterized the POD genes responsible for TF production in tea. Genome-wide analysis of POD/PPO family genes, metabolite profiling, and expression analysis of PPO/POD genes in tea leaves enabled us to select several PPO/POD genes potentially involved in TF production. Differential gene expression in plant tissues and enzyme activity in several tea varieties traditionally used for processing of various beverage types indicate that black tea processing primarily depends on PPO/POD activity. Among these POD/PPO genes, the POD CsGPX3 is involved in the generation of TFs during black tea processing. The capacity of PPO/POD-catalysed TF production is potentially used for controlling catechin oxidation during black tea processing and could be used to create molecular markers for breeding of tea plant varieties suitable for the production of high-quality black tea beverages.

Performance of 3‑[4‑(bromomethyl)phenyl]‑7‑(diethylamino) coumarin as a derivatization reagent for the analysis of medium and long chain fatty acids using HPLC with LIF detection

Knowledge of the profile of fatty acids, including the profile of long chain fatty acids, is an important parameter for many areas like human health, food sciences, and the study of gene expression in plant tissues, among others. Moreover, very long chain fatty acids occur at low concentrations in many biological samples. The 3‑[4‑(bromomethyl)phenyl]‑7‑(diethylamino)coumarin (MPAC‑Br) reagent is an improved version of the coumarin bromomethyl-type reagents, used for the fluorescent labeling of carboxylic acids in a variety of sample types. Derivatization reactions are always a matter of concern in routine analyses, as they are seen as a time demanding step and a source of errors in analytical chemistry. MPAC‑Br is studied in the present work, including the robustness of its derivatization reaction, the stability of its derivatives, the limits of its detection and the repeatability of its results. An optimized version of its protocol was applied to determine the content and profiles of both medium and long chain fatty acids in eight samples of oils and fats. It was shown that the proposed derivatization reaction is a reliable, robust, fast and convenient method for the analysis of these compounds.

Divergent Expression Patterns in Two Vernicia Species Revealed the Potential Role of the Hub Gene VmAP2/ERF036 in Resistance to Fusarium oxysporum in Vernicia montana.

Tung oil tree (Vernicia fordii) is a promising industrial oil crop; however, this tree is highly susceptible to Fusarium wilt disease. Conversely, Vernicia montana is resistant to the pathogen. The APETALA2/ethylene-responsive element binding factor (AP2/ERF) transcription factor superfamily has been reported to play a significant role in resistance to Fusarium oxysporum. In this study, comprehensive analysis identified 75 and 81 putative Vf/VmAP2/ERF transcription factor-encoding genes in V. fordii and V. montana, respectively, which were divided into AP2, ERF, related to ABI3 and VP1 (RAV) and Soloist families. After F. oxysporum infection, a majority of AP2/ERF superfamily genes showed strong patterns of repression in both V. fordii and V. montana. We then identified 53 pairs of one-to-one orthologs in V. fordii and V. montana, with most pairs of orthologous genes exhibiting similar expression in response to the pathogen. Further investigation of Vf/VmAP2/ERF gene expression in plant tissues indicated that the pairs of genes with different expression patterns in response to F. oxysporum tended to exhibit different tissue profiles in the two species. In addition, VmAP2/ERF036, showing the strongest interactions with 666 genes, was identified as a core hub gene mediating resistance. Moreover, qRT-PCR results indicated VmAP2/ERF036 showed repressed expression while its orthologous gene VfAP2/ERF036 had the opposite expression pattern during pathogen infection. Overall, comparative analysis of the Vf/VmAP2/ERF superfamily and indication of a potential hub resistance gene in resistant and susceptible Vernicia species provides valuable information for understanding the molecular basis and selection of essential functional genes for V. fordii genetic engineering to control Fusarium wilt disease.

ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging.

Imaging techniques for visualizing and analyzing precise morphology and gene expression patterns are essential for understanding biological processes during development in all organisms. With the aid of chemical screening, we developed a clearing method using chemical solutions, termed ClearSee, for deep imaging of morphology and gene expression in plant tissues. ClearSee rapidly diminishes chlorophyll autofluorescence while maintaining fluorescent protein stability. By adjusting the refractive index mismatch, whole-organ and whole-plant imaging can be performed by both confocal and two-photon excitation microscopy in ClearSee-treated samples. Moreover, ClearSee is applicable to multicolor imaging of fluorescent proteins to allow structural analysis of multiple gene expression. Given that ClearSee is compatible with staining by chemical dyes, the technique is useful for deep imaging in conjunction with genetic markers and for plant species not amenable to transgenic approaches. This method is useful for whole imaging for intact morphology and will help to accelerate the discovery of new phenomena in plant biological research.

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

In Situ Hybridization in the Plant Kalanchoë daigremontiana

Here we describe in detail the detection of gene expression in plant tissues of Kalanchoë daigremontiana by in situ hybridization analyses. Included are methods for making RNA transcript probes, probe-tissue hybridization, and detection of antisense RNA probes. The in situ hybridization technique is used to determine which cells or group of cells in particular tissue(s) express a gene of interest.

New insights into plant transaldolase

The oxidative pentose phosphate pathway (OPPP) provides plants with important substrates for both primary and secondary metabolism via the oxidation of glucose-6-phosphate. The OPPP is also thought to generate large amounts of reducing power to drive various anabolic processes. In animals this major pathway is located within the cytoplasm of cells, but in plants its subcellular compartmentation is far from clear. Although several enzymes of the OPPP were demonstrated to have both cytosolic and plastidic counterparts, there is yet no evidence for a full set of functional enzymes in each compartment. We report here the isolation of two coding sequences from tomato (Lycopersicon esculentum L.) which encode phylogenetically distant sequences (ToTal1 and ToTal2) that putatively encode distinct plastidic TA isoforms. The kinetic characterization of ToTal1 revealed that, unlike other enzymes of the non-oxidative branch of the OPPP, ToTal1 does not follow a Michaelis-Menten mode of catalysis which has implications for its role in regulating carbon flux between primary and secondary metabolism. TA genes appear to be differentially regulated at the level of gene expression in plant tissues and in response to environmental factors which suggests that TA isoforms have a non-overlapping role for plant metabolism.

Assessment of Transient Gene Expression in Plant Tissues Using the Green Fluorescent Protein as a Reference

Four different promoters (35S and enhanced 35S of the cauliflower mosaic virus, polyubiquitin of maize and actin1 of rice) were compared in a transient assay using maize leaves and particle bombardment. A gene encoding the jellyfish green fluorescent protein (GFP) driven by the 358 promoter was used as an internal standard to monitor the effectiveness of each bombardment. Normalisation of the transient expression assay using the GFP reference significantly reduced the variability between separate bombardments and allowed for a rapid and accurate evaluation of different promoters in microprojectile-bombarded leaves.

Fusicoccin-Induced Changes in the Translatable RNAs of Etiolated Pea Stem Tissue

The fungal toxin fusicoccin (FC) was found to induce substantial increases in the abundance of specific translatable RNAs over several hours, concomitantly with tissue expansion, in segments of etiolated pea internodes. The FC-induced RNAs encoded some relatively basic polypeptides of MWs 27-5-29-2 kDa, and some relatively acidic polypeptides of MWs 12-5-15-5 kDa. The earliest detectable increases in the FC-induced translation products occurred after 1-5-2-5 h. Auxin treatment caused a different overall pattern of changes in translation product spots, though some induced spots appeared to be common to both treatments. The possible significance of these observations is discussed in relation to other factors known to alter gene expression in plant tissues.