Gene Expression Studies In Plants Research Articles

Soil texture is a stronger driver of the maize rhizosphere microbiome and extracellular enzyme activities than soil depth or the presence of root hairs

AimsDifferent drivers are known to shape rhizosphere microbiome assembly. How soil texture (Texture) and presence or lack of root hairs (Root Hair) of plants affect the rhizosphere microbiome assembly and soil potential extracellular enzyme activities (EEA) at defined rooting depth (Depth) is still a knowledge gap. We investigated effects of these drivers on microbial assembly in rhizosphere and on potential EEA in root-affected soil of maize.MethodsSamples were taken from three depths of root hair defective mutant rth3 and wild-type WT maize planted on loam and sand in soil columns after 22 days. Rhizosphere bacterial, archaeal, fungal and cercozoan communities were analysed by sequencing of 16S rRNA gene, ITS and 18S rRNA gene fragments. Soil potential EEA of ß-glucosidase, acid phosphatase and chitinase were estimated using fluorogenic substrates.ResultsThe bacterial, archaeal and cercozoan alpha- and beta-diversities were significantly and strongly altered by Texture, followed by Depth and Root Hair. Texture and Depth had a small impact on fungal assembly, and only fungal beta-diversity was significantly affected. Significant impacts by Depth and Root Hair on beta-diversity and relative abundances at taxonomic levels of bacteria, archaea, fungi and cercozoa were dependent on Texture. Likewise, the patterns of potential EEA followed the trends of microbial communities, and the potential EEA correlated with the relative abundances of several taxa.ConclusionsTexture was the strongest driver of rhizosphere microbiome and of soil potential EEA, followed by Depth and Root Hair, similarly to findings in maize root architecture and plant gene expression studies.

Efficient expression and function of a receptor-like kinase in wheat powdery mildew defence require an intron-located MYB binding site.

SummaryThe LRK10‐like receptor kinases (LRK10L‐RLKs) are ubiquitously present in higher plants, but knowledge of their expression and function is still limited. Here, we report expression and functional analysis of TtdLRK10L‐1, a typical LRK10L‐RLK in durum wheat (Triticum turgidum L. ssp. durum). The introns of TtdLRK10L‐1 contained multiple kinds of predicted cis‐elements. To investigate the potential effect of these cis‐elements on TtdLRK10L‐1 expression and function, two types of transgenic wheat lines were prepared, which expressed a GFP‐tagged TtdLRK10L‐1 protein (TtdLRK10L‐1:GFP) from the cDNA or genomic DNA (gDNA) sequence of TtdLRK10L‐1 under the native promoter. TtdLRK10L‐1:GFP expression was up‐regulated by the powdery mildew pathogen Blumeria graminis f. sp. tritici (Bgt) in both types of transgenic plants, with the scale of the elevation being much stronger in the gDNA lines. Both types of transgenic plants exhibited enhanced resistance to Bgt infection relative to wild type control. Notably, the Bgt defence activated in the gDNA lines was significantly stronger than that in the cDNA lines. Further analysis revealed that a putative MYB transcription factor binding site (MYB‐BS, CAGTTA) located in TtdLRK10L‐1 intron I was critical for the efficient expression and function of TtdLRK10L‐1 in Bgt defence. This MYB‐BS could also increase the activity of a superpromoter widely used in ectopic gene expression studies in plants. Together, our results deepen the understanding of the expression and functional characteristics of LRK10L‐RLKs. TtdLRK10L‐1 is likely useful for further dissecting the molecular processes underlying wheat defence against Bgt and for developing Bgt resistant wheat crops.

Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization

BackgroundIn situ analysis of biomarkers such as DNA, RNA and proteins are important for research and diagnostic purposes. At the RNA level, plant gene expression studies rely on qPCR, RNAseq and probe-based in situ hybridization (ISH). However, for ISH experiments poor stability of RNA and RNA based probes commonly results in poor detection or poor reproducibility. Recently, the development and availability of the RNAscope RNA-ISH method addressed these problems by novel signal amplification and background suppression. This method is capable of simultaneous detection of multiple target RNAs down to the single molecule level in individual cells, allowing researchers to study spatio-temporal patterning of gene expression. However, this method has not been optimized thus poorly utilized for plant specific gene expression studies which would allow for fluorescent multiplex detection. Here we provide a step-by-step method for sample collection and pretreatment optimization to perform the RNAscope assay in the leaf tissues of model monocot plant barley. We have shown the spatial distribution pattern of HvGAPDH and the low expressed disease resistance gene Rpg1 in leaf tissue sections of barley and discuss precautions that should be followed during image analysis.ResultsWe have shown the ubiquitous HvGAPH and predominantly stomatal guard cell associated subsidiary cell expressed Rpg1 expression pattern in barley leaf sections and described the improve RNAscope methodology suitable for plant tissues using confocal laser microscope. By addressing the problems in the sample collection and incorporating additional sample backing steps we have significantly reduced the section detachment and experiment failure problems. Further, by reducing the time of protease treatment, we minimized the sample disintegration due to over digestion of barley tissues.ConclusionsRNAscope multiplex fluorescent RNA-ISH detection is well described and adapted for animal tissue samples, however due to morphological and structural differences in the plant tissues the standard protocol is deficient and required optimization. Utilizing barley specific HvGAPDH and Rpg1 RNA probes we report an optimized method which can be used for RNAscope detection to determine the spatial expression and semi-quantification of target RNAs. This optimized method will be immensely useful in other plant species such as the widely utilized Arabidopsis.

Identification and validation of reference genes for qRT-PCR analysis in mulberry (Morus alba L.).

Mulberry (Morus alba L.) is an important economic tree species in many countries. Quantitative real time PCR (qRT-PCR) has become a widely used method for gene expression studies in plants. A suitable reference gene is essential to ensure accurate and reliable results for qRT-PCR analyses. However, no reports describing the selection of reference genes have been published for mulberry. In this work, we evaluated the stability of twenty candidate reference genes in different plant tissues and under different stress conditions by qRT-PCR in mulberry using algorithms in two programs—geNorm and NormFinder. The results revealed that TUB2, UBI4, ACTIN3 and RPL4 were ranked as the most stable reference genes in the samples subsets, whereas EF1α4 and TUB3showed the least stability with both algorithms. To further validate the stability of the reference genes, the expression patterns of six genes of mulberry were analyzed by normalization with the selected reference genes. Our study will benefit future analyses of gene expression in mulberry.

Differential contribution of Clavibacter michiganensis ssp. michiganensis virulence factors to systemic and local infection in tomato.

Clavibacter michiganensis ssp. michiganensis (Cmm) causes substantial economic losses in tomato production worldwide. The disease symptoms observed in plants infected systemically by Cmm are wilting and canker on the stem, whereas blister-like spots develop in locally infected leaves. A wide repertoire of serine proteases and cell wall-degrading enzymes has been implicated in the development of wilt and canker symptoms. However, virulence factors involved in the formation of blister-like spots, which play an important role in Cmm secondary spread in tomato nurseries, are largely unknown. Here, we demonstrate that Cmm virulence factors play different roles during blister formation relative to wilting. Inoculation with a green fluorescent protein (GFP)-labelled Cmm382 indicates that penetration occurs mainly through trichomes. When spray inoculated on tomato leaves, the wild-type Cmm382 and Cmm100 (lacking plasmids pCM1 and pCM2) strains form blister-like spots on leaves, whereas Cmm27 (lacking the chp/tomA pathogenicity island) is non-pathogenic, indicating that plasmid-borne genes, which have a crucial role in wilting, are not required for blister formation. Conversely, mutations in chromosomal genes encoding serine proteases (chpC and sbtA), cell wall-degrading enzymes (pgaA and endX/Y), a transcriptional regulator (vatr2), a putative perforin (perF) and a putative sortase (srtA) significantly affect disease incidence and the severity of blister formation. The transcript levels of these genes, as measured by quantitative reverse transcription-polymerase chain reaction, showed that, during blister formation, they are expressed early at 8-16 h after inoculation, whereas, during wilting, they are expressed after 24-72 h or expressed at low levels. Plant gene expression studies suggest that chpC is involved in the suppression of host defence.

A rapid, simple, and inexpensive method for the preparation of strand-specific RNA-Seq libraries.

High-throughput sequencing of short cDNA tags, or RNA-Seq, has become a staple of genome-wide gene expression studies in plants. RNA-Seq libraries necessarily contain tags that correspond to the mRNA-poly(A) junction, or polyadenylation site, and thus may be mined for data that can help study alternative polyadenylation. This report presents a simple, rapid, and inexpensive method for preparing strand-specific RNA-Seq libraries from varying quantities of total RNA.

Transcriptional profiling of Zea mays roots reveals roles for jasmonic acid and terpenoids in resistance against Phytophthora cinnamomi

Phytophthora cinnamomi is a soil-borne plant pathogen that has caused widespread damage to vulnerable native ecosystems and agriculture systems across the world and shows no sign of abating. Management of the pathogen in the natural environment is difficult and the options are limited. In order to discover more about how resistant plants are able to defend themselves against this generalist pathogen, a microarray study of plant gene expression following root inoculation with P. cinnamomi was undertaken. Zea mays was used as a resistant model plant, and microarray analysis was conducted using the Affymetrix GeneChip Maize Genome Array on root samples collected at 6- and 24-h post-inoculation. Over 300 genes were differentially expressed in inoculated roots compared with controls across the two time points. Following Gene Ontology enrichment analysis and REVIGO visualisation of the up-regulated genes, many were implicated in plant defence responses to biotic stress. Genes that were up-regulated included those involved in phytoalexin biosynthesis and jasmonic acid/ethylene biosynthesis and other defence-related genes including those encoding glutathione S-transferases and serine-protease inhibitors. Of particular interest was the identification of the two most highly up-regulated genes, terpene synthase11 (Tps11) and kaurene synthase2 (An2), which are both involved in production of terpenoid phytoalexins. This is the first study that has investigated gene expression at a global level in roots in response to P. cinnamomi in a model plant species and provides valuable insights into the mechanisms involved in defence.

Gene Expression: A Review on Methods for the Study of Defense-Related Gene Differential Expression in Plants

The plant genes involved in cellular signaling and metabolism have not been fully identified, while the function(s) of many of those which have are as yet incompletely characterized. Gene expression analysis allows the identification of genes and the study of their relationship with cellular processes. There are several options available for studying gene expression, including the use of cDNA and microarray libraries and techniques such as suppression subtractive hybridization (SSH), differential display (DD), RNA fingerprinting by arbitrary primed PCR (RAP), expressed sequence tags (EST), serial analysis of gene expression (SAGE), representational difference analysis (RDA), cDNA-amplified fragment length polymorphism (cDNA-AFLP) and RNA sequencing (RNA-Seq). Focusing on defense-related processes in plants, we present a brief review and examples of each of these methodologies and their advantages and limitations regarding the study of plant gene expression.

PlantRGS: A Web Server for the Identification of Most Suitable Candidate Reference Genes for Quantitative Gene Expression Studies in Plants

Normalization of quantitative gene expression data with a suitable reference gene is essential for accurate and reliable results. However, the availability and choice of most suitable reference gene(s) showing uniform expression across all the experimental conditions remain a drawback. We have developed a web server, PlantRGS (http://www.nipgr.res.in/PlantRGS), for the identification of most suitable candidate reference gene(s) at the whole-genome level using microarray data for quantitative gene expression studies in plants. Microarray data from more than 11 000 tissue samples for nine plant species have been included in the PlantRGS for meta-analysis. The web server provides a user-friendly graphical user interface-based analysis tool for the identification of most suitable reference genes in the selected plant species under user-defined experimental conditions. Various parameter options and output formats will help users to investigate desired number of most suitable reference genes with wide range of expression levels. Validation of results revealed that novel reference genes identified by the PlantRGS outperforms the traditionally used reference genes in terms of expression stability. We anticipate that the PlantRGS will provide a platform for the identification of most suitable reference gene(s) under given experimental conditions and facilitate quantitative gene expression studies in plants.

Plant–rhizobacteria interactions alleviate abiotic stress conditions

Root-colonizing non-pathogenic bacteria can increase plant resistance to biotic and abiotic stress factors. Bacterial inoculates have been applied as biofertilizers and can increase the effectiveness of phytoremediation. Inoculating plants with non-pathogenic bacteria can provide 'bioprotection' against biotic stresses, and some root-colonizing bacteria increase tolerance against abiotic stresses such as drought, salinity and metal toxicity. Systematic identification of bacterial strains providing cross-protection against multiple stressors would be highly valuable for agricultural production in changing environmental conditions. For bacterial cross-protection to be an effective tool, a better understanding of the underlying morphological, physiological and molecular mechanisms of bacterially mediated stress tolerance, and the phenomenon of cross-protection is critical. Beneficial bacteria-mediated plant gene expression studies under non-stress conditions or during pathogenic rhizobacteria-plant interactions are plentiful, but only few molecular studies on beneficial interactions under abiotic stress situations have been reported. Thus, here we attempt an overview of current knowledge on physiological impacts and modes of action of bacterial mitigation of abiotic stress symptoms in plants. Where available, molecular data will be provided to support physiological or morphological observations. We indicate further research avenues to enable better use of cross-protection capacities of root-colonizing non-pathogenic bacteria in agricultural production systems affected by a changing climate.

Purification, Cloning and Functional Characterization of an Endogenous beta-Glucuronidase in Arabidopsis thaliana

Beta-glucuronidase (GUS) activities have been extensively characterized in bacteria, fungi, and animals, and the bacterial enzyme GUSA from Escherichia coli is commonly used as a reporter for gene expression studies in plants. Although endogenous GUS activity has been observed in plants, the nature and function of the enzymes involved remain elusive. Here we report on tissue-specific localization, partial purification and identification of AtGUS2, a GUS active under acidic conditions from Arabidopsis thaliana. This enzyme belongs to the GH79 family in the Carbohydrate-Active Enzymes database, which also includes mammalian heparanases that degrade the carbohydrate moieties of cell surface proteoglycans, and fungal enzymes active on arabinogalactan proteins (AGPs). We characterized a knockout insertion line (atgus2-1) and transgenic lines overexpressing AtGUS2 (Pro(35S):AtGUS2). Endogenous GUS activity assayed histochemically and biochemically was absent in atgus2-1 tissues and four times higher in Pro(35S):AtGUS2 lines. AGPs purified from atgus2-1 and Pro(35S):AtGUS2 seedlings showed higher and markedly lower glucuronic acid content, respectively. Our results suggest that endogenous GUS activity influences the sugar composition of the complex polysaccharide chains of AGPs. We also show that transgenics display hypocotyl and root growth defects compared to wild-type plants. Hypocotyl and root lengths are increased in Pro(35S):AtGUS2 seedlings, whereas hypocoyl length is reduced in atgus2-1 seedlings. These data are consistent with a role for the carbohydrate moieties of AGPs in cell growth.

Evaluation and Comparison of the GUS, LUC and GFP Reporter System for Gene Expression Studies in Plants

Abstract: The detailed analysis of the expression pattern of a plant gene can give important clues about its function in plant development, cell differentiation and defence reactions. Gene expression studies have been greatly facilitated by the employment of proteins like β‐glucuronidase (GUS), green fluorescent protein (GFP), and firefly luciferase (LUC) as reporters of gene activity. The application of reporter genes in plants, specifically in the field of gene expression studies, has expanded over the years from a mere tool to quantify (trans) gene expression in tissue samples, to real‐time imaging of in planta promoter dynamics. To correctly interpret the activity that is given by each reporter, it is important to have a good understanding of the intrinsic properties of the different reporter proteins. Here we discuss those properties of GUS, LUC and GFP that are of interest in gene expression studies.

Structure, expression pattern and chromosomal localization of the rice Osgrp-2 gene

Glycine-rich proteins (GRPs) belong to a kind of important structural proteins of plant cell walls. The expression of GRP genes is regulated spatially and developmentally as well as by various environmental stresses, thus providing a good model for the study of plant gene expression. We obtained the genomic sequence of a new GRP gene (Osgrp- 2) from a rice genomic library. The transcription start site of Osgrp- 2 was determined by 5'-rapid amplification of cDNA ends (RACE) and a 2.4-kb promoter sequence was thus delimited. The spatial and developmental expression pattern as well as the wound-inducible character of Osgrp- 2 in rice plants was analyzed in detail. Furthermore, the gene was mapped onto rice chromosome 10 by analysis of restriction fragment length polymorphism (RFLP).

Comparing the utility of β-glucuronidase and green fluorescent protein for detection of weak promoter activity inArabidopsis thaliana

Two important marker proteins used in plant gene expression studies are green fluorescent protein (GFP) and β-glucuronidase (GUS). We have compared the utility of each in the analysis of a relatively weakArabidopsis thaliana promoter. The background green fluorescence of arabidopsis tissues and organs has been catalogued. This background fluorescence makes it difficult to detect weak promoter activity driving GFP, a problem compounded by the lack of amplification of the GFP signal. In the case of β-glucuronidase, due to diffusion of the enzymatic product, GUS may over-report promoter activity. However, because of the enzymatic amplification of the signal and the low β-glucuronidase activity of untransformed arabidopsis tissues, weak promoter activity is more easily and more accurately detected using GUS.

Large-scale statistical analyses of rice ESTs reveal correlated patterns of gene expression.

Large, publicly available collections of expressed sequence tags (ESTs) have been generated from Arabidopsis thaliana and rice (Oryza sativa). A potential, but relatively unexplored application of this data is in the study of plant gene expression. Other EST data, mainly from human and mouse, have been successfully used to point out genes exhibiting tissue- or disease-specific expression, as well as for identification of alternative transcripts. In this report, we go a step further in showing that computer analyses of plant EST data can be used to generate evidence of correlated expression patterns of genes across various tissues. Furthermore, tissue types and organs can be classified with respect to one another on the basis of their global gene expression patterns. As in previous studies, expression profiles are first estimated from EST counts. By clustering gene expression profiles or whole cDNA library profiles, we show that genes with similar functions, or cDNA libraries expected to share patterns of gene expression, are grouped together. Promising uses of this technique include functional genomics, in which evidence of correlated expression might complement (or substitute for) those of sequence similarity in the annotation of anonymous genes and identification of surrogate markers. The analysis presented here combines the application of a correlation-based clustering method with a graphical color map allowing intuitive visualization of patterns within a large table of expression measurements.

Expression of recombinant Renilla luciferase in transgenic plants results in high levels of light emission

A 970 bp DNA fragment which encodes the luciferase enzyme of the marine soft coral Renilla reniformis was fused to the cauliflower mosaic virus (CaMV) promoter. The construct pPCV702‐ruc was transferred into alfalfa protoplasts by Ca‐PEG‐mediated transformation and into tobacco, tomato and potato plants by Agrobacterium‐mediated transformation. The light emission from homogenates of alfalfa protoplasts transformed with pPCV702‐ruc was 16‐fold higher than that of protoplasts transformed with the same vector carrying the bacterial luxF gene. Application of a 3 µM aequous solution of 2‐benzyl luciferin (luciferin) on to calli, leaves, roots and slices of tomato fruits and potato tubers of transformed plants resulted in strong light emission within seconds which could be easily visualized by a photon counting camera. Light emissions obtained from tissue homogenates of tobacco plants containing a single copy of the pPCV702‐ruc construct were around 20‐fold higher than those from plants carrying multiple copies of the firefly luciferase gene and around 360‐fold higher than those from plants transformed with the bacterial luciferase gene. Owing to its high efficiency the Renilla luciferase may become a useful and novel tool for gene expression studies in plants and other systems.

Transformation of carrot tissues derived from proembryogenic suspension cells: A useful model system for gene expression studies in plants

A method is described for the high frequency transformation of carrot proembryogenic suspension culture cells by a non-oncogenic Ti-plasmid vector (pGV3850::1103) which carried a chimaeric kanamycin resistance gene (nos-NPT-II). Plants were regenerated efficiently from transformed material by somatic embryogenesis in the presence of kanamycin. Transformed tissues expressed readily detectable levels of both NPT-II and nopaline. NPT-II could be detected in total protein extracts by Western blotting. This analysis indicated that NPT-II was produced as a single, full length polypeptide. The T-DNA copy number in individually selected transformants was analysed by Southern blotting and ranged from 1-8 per diploid genome. The copy number and organization of the T-DNA was retained in plants regenerated from these transformants by somatic embryogenesis. These data suggested a clonal origin for the selected kanamycin resistant colonies. NPT-II expression levels appeared to be directly related to gene dosage.

Luciferase reporter gene cassettes for plant gene expression studies.

Luciferase reporter gene cassettes for plant gene expression studies.