Root cDNA Library Research Articles

Comparative Transcriptome Analysis Reveals the Genes and Pathways Related to Wheat Root Hair Length.

Tube-like outgrowths from root epidermal cells, known as root hairs, enhance water and nutrient absorption, facilitate microbial interactions, and contribute to plant anchorage by expanding the root surface area. Genetically regulated and strongly influenced by environmental conditions, longer root hairs generally enhance water and nutrient absorption, correlating with increased stress resistance. Wheat, a globally predominant crop pivotal for human nutrition, necessitates the identification of long root hair genotypes and their regulatory genes to enhance nutrient capture and yield potential. This study focused on 261 wheat samples of diverse genotypes during germination, revealing noticeable disparities in the length of the root hair among the genotypes. Notably, two long root hair genotypes (W106 and W136) and two short root hair genotypes (W90 and W100) were identified. Transcriptome sequencing resulted in the development of 12 root cDNA libraries, unveiling 1180 shared differentially expressed genes (DEGs). Further analyses, including GO function annotation, KEGG enrichment, MapMan metabolic pathway analysis, and protein-protein interaction (PPI) network prediction, underscored the upregulation of root hair length regulatory genes in the long root hair genotypes. These included genes are associated with GA and BA hormone signaling pathways, FRS/FRF and bHLH transcription factors, phenylpropanoid, lignin, lignan secondary metabolic pathways, the peroxidase gene for maintaining ROS steady state, and the ankyrin gene with diverse biological functions. This study contributes valuable insights into modulating the length of wheat root hair and identifies candidate genes for the genetic improvement of wheat root traits.

Role of a complex of two proteins in alleviating sodium ion stress in an economic crop.

An economically valuable woody plant species tree bean (Cajanus cajan (L.) Millsp.) is predominantly cultivated in tropical and subtropical areas and is regarded as an important food legume (or pulse) crop that is facing serious sodium ion stress. NAM (N-acetyl-5-methoxytryptamine) has been implicated in abiotic and biotic stress tolerance in plants. However, the role of NAM in sodium ion stress tolerance has not been determined. In this study, the effect of NAM was investigated in the economically valuable woody plant species, challenged with stress at 40 mM sodium ion for 3 days. NAM-treated plants (200 μM) had significantly higher fresh weight, average root length, significantly reduced cell size, increased cell number, and increased cytoskeleton filaments in single cells. The expression pattern of one of 10 Tree bean Dynamic Balance Movement Related Protein (TbDMP), TbDMP was consistent with the sodium ion-stress alleviation by NAM. Using TbDMP as bait, Dynamic Balance Movement Related Kinase Protein (TbDBK) was determined to interact with TbDMP by screening the tree bean root cDNA library in yeast. Biochemical experiments showed that NAM enhanced the interaction between the two proteins which promoted resist sodium ion stress resistance. This study provides evidence of a pathway through which the skeleton participates in NAM signaling.

Decreased expression of fructosyltransferase genes in asparagus roots may contribute to efficient fructan degradation during asparagus spear harvesting

Asparagus (Asparagus officinalis L.) accumulates inulin and inulin neoseries-type fructans in root, which are synthesized by three fructosyltransferases—sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), and fructan:fructan 6G-fructosyltransferase (6G-FFT, EC 2.4.1.243). Fructans in roots are considered as energy sources for emerging of spears, and it has been demonstrated that a gradual decrease in root fructan content occurs during the spear harvesting season (budding and shooting up period). However, the roles of certain three fructosyltransferases during the harvest season have not yet been elucidated. Here, we investigated the variation in enzymatic activities and gene expression levels of three fructosyltransferases and examined sugar contents in roots before and during the spear harvest period. Two cDNAs, aoft2 and aoft3, were isolated from the cDNA library of roots. The respective recombinant proteins (rAoFT2 and rAoFT3), produced by Pichia pastoris, were characterized: rAoFT2 showed 1-FFT activity (producing nystose from 1-kestose), whereas rAoFT3 showed 1-SST activity (producing 1-kestose from sucrose). These reaction profiles of recombinant proteins were similar to those of native enzymes purified previously. These results indicate that aoft2 and aoft3 encoding 1-FFT and 1-SST are involved in fructan synthesis in roots. A gradual downregulation of fructosyltransferase genes and activity of respective enzymes was observed in roots during the harvest period, which also coincided with the decrease in fructooligosaccharides and increase in fructose due to fructan exohydrolase activity. These findings suggest that downregulation of fructosyltransferases genes during harvest time may contribute to efficient degradation of fructan required for the emergence of spears.

Elicitor hydrophobin Hyd1 interacts with Ubiquilin1-like to induce maize systemic resistance.

Trichoderma harzianum is a plant-beneficial fungus that secretes small cysteine-rich proteins that induce plant defense responses; however, the molecular mechanism involved in this induction is largely unknown. Here, we report that the class II hydrophobin ThHyd1 acts as an elicitor of induced systemic resistance (ISR) in plants. Immunogold labeling and immunofluorescence revealed ThHyd1 localized on maize (Zea mays) root cell plasma membranes. To identify host plant protein interactors of Hyd1, we screened a maize B73 root cDNA library. ThHyd1 interacted directly with ubiquilin 1-like (UBL). Furthermore, the N-terminal fragment of UBL was primarily responsible for binding with Hyd1 and the eight-cysteine amino acid of Hyd1 participated in the protein-protein interactions. Hyd1 from T. harzianum (Thhyd1) and ubl from maize were co-expressed in Arabidopsis thaliana, they synergistically promoted plant resistance against Botrytis cinerea. RNA-sequencing analysis of global gene expression in maize leaves 24 h after spraying with Curvularia lunata spore suspension showed that Thhyd1-induced systemic resistance was primarily associated with brassinosteroid signaling, likely mediated through BAK1. Jasmonate/ethylene (JA/ET) signaling was also involved to some extent in this response. Our results suggest that the Hyd1-UBL axis might play a key role in inducing systemic resistance as a result of Trichoderma-plant interactions.

Exploring the adaptive mechanism of Passiflora edulis in karst areas via an integrative analysis of nutrient elements and transcriptional profiles

BackgroundPassiflora edulis, known as passion fruit and native to South America, is now widely cultivated throughout southern China for its edible value, medicinal efficacy and ornamental properties. We have developed a cold-tolerant variety of P. edulis (‘Pingtang 1’) that can survive subzero temperatures and is highly adaptable in Karst areas. In this study, cuttings of ‘Pingtang 1’ were cultivated in a limestone (L) rocky desertification area and a sandy dolomite (D) rock desertification area. Changes in nutrient elements in both the soils and plants were revealed in the two plots. Moreover, RNA sequencing (RNA-Seq) was performed to profile the root transcriptomes for further exploration of nutrient adaptative mechanism of Passiflora edulis in Karst regions.ResultsIn this study, a total of, 244,705,162 clean reads were generated from four cDNA libraries and assembled into 84,198 unigenes, of which 56,962 were annotated by publicly available databases. Transcriptome profiles were generated, and 1314 unigenes (531 upregulated and 801 downregulated) were significantly differentially expressed between the L and D root cDNA libraries (L_R and D_R, respectively); these profiles provide a global overview of the gene expression patterns associated with P. edulis adaptability to Karst soils. Most unigenes including a number of differentially expressed genes (DEGs) were involved in nutrient element uptake, utilization, signal regulation. And DEGs enriched in KEGG pathways of plant hormone signal transduction, phenylpropanoid biosynthesis, and biosynthesis of unsaturated fatty acids were significantly expressed.ConclusionThese results could contribute to better understanding the adaptation of this species to environmental stress and thus enhance the potential for successfully introducing and commercially deploying P. edulis.

MePHD1 as a PHD-Finger Protein Negatively Regulates ADP-Glucose Pyrophosphorylase Small Subunit1a Gene in Cassava.

ADP-glucose pyrophosphorylase (AGPase) is an important enzyme in the starch synthesis pathway. Its enzyme activity can determine the efficiency of starch biosynthesis. Cassava (Manihot esculenta Crantz) is the main staple crop worldwide and has a high starch content in its storage root. However, the inner regulatory mechanism of AGPase gene family is unclear. MePHD1; a plant homeodomain transcription factor; was isolated through a yeast one-hybrid screening using the promoter of ADP-glucose pyrophosphorylase small subunit1a (MeAGPS1a) as bait, and cassava storage root cDNA library as prey. This factor could bind to the MeAGPS1a promoter in vitro and in vivo, and its predicted binding region ranged from −400 bp to −201 bp, at the translation initiation site. The transcript level of MePHD1 could be induced by five plant hormones, and a temperature of 42 °C. This was down-regulated during the maturation process of the storage root. MePHD1 protein could repress the promoter activity of MeAGPS1a gene by a dual-luciferase assay; which indicated that MePHD1 is a negative regulator for the transcript level of MeAGPS1a gene.

Generation and characterization of expressed sequence tags (ESTs) from coralloid root cDNA library of Cycas debaoensis

A normalized full-length cDNA library was constructed from the coralloid roots of Cycas debaoensis by the DSN (duplex-specific nuclease) normalization method combined with the SMART (Switching Mechanism At 5′ end of the RNA Transcript) technique. The titer of the original cDNA library was about 1.5 × 106 cfu·mL−1 and the average insertion size was about 1 kb with a high recombination rate (97%). The 5011 high-quality expressed sequence tags (ESTs) were obtained from 5393 randomly picked cDNA clones. Clustering and assembly of ESTs resulted in 2984 unique sequences, consisting of 618 contigs and 2366 singlets. EST sequence annotation revealed that 2333 and 1901 unigenes were functionally annotated in the NCBI non-redundant database and Swiss-Prot protein database, respectively. Functional analysis demonstrated that 1495 (50.1%) unigenes were associated with 4082 Gene Ontology (GO) terms. A total of 847 unigenes were grouped into 22 Cluster of Orthologous Groups (COG) functional categories. Based on the EST dataset, 22 ESTs that encoded putative receptor-like protein kinase (RLK) genes were screened. Furthermore, a total of 94 simple sequence repeats (SSRs) were discovered, of which 20 loci were successfully amplified in C. debaoensis. This study is the first EST analysis for the coralloid roots of C. debaoensis and provides a valuable genomic resource for novel gene discovery, gene expression and comparative genomics, conservation and management studies as well as applications in C. debaoensis and related cycad species.

MeSAUR1, Encoded by a Small Auxin-Up RNA Gene, Acts as a Transcription Regulator to Positively Regulate ADP-Glucose Pyrophosphorylase Small Subunit1a Gene in Cassava.

Cassava, being one of the top three tuberous crops, features highly efficient starch accumulation in the storage root to adapt the tropical resources and environments. The molecular mechanism for the process, however, is still unclear. ADP-glucose pyrophosphorylase, the first and rate-limited enzyme in starch biosynthesis pathway, is a heterotetramer comprised of two small/catalytic and two large/modulatory subunits. To understand the regulation of MeAGPase, the promoter of a highly expressed small subunit, MeAGPs1a, was used as bait for a yeast one-hybrid assay to screen storage root cDNA library. One cDNA, coding for a small auxin-up RNA protein, named MeSAUR1, was isolated from cassava. MeSAUR1 could bind to the promoter of MeAGPS1a in yeast one-hybrid test and in vitro, and was located in cell nucleus. MeSAUR1 displayed a higher transcript level in cassava root cortex, and its expression was induced by indole-3-acetic acid, gibberellin and ethylene, but repressed by abscisic acid. A dual-luciferase interaction test further convinced that MeSAUR1 could bind to the promoter of MeAGPS1a, and positively regulate the transcription of MeAGPS1a in cassava.

Zea mays Fe deficiency-related 4 (ZmFDR4) functions as an iron transporter in the plastids of monocots.

Iron (Fe)-homeostasis in the plastids is closely associated with Fe transport proteins that prevent Fe from occurring in its toxic free ionic forms. However, the number of known protein families related to Fe transport in the plastids (about five) and the function of iron in non-green plastids is limited. In the present study, we report the functional characterization of Zea mays Fe deficiency-related 4 (ZmFDR4), which was isolated from a differentially expressed clone of a cDNA library of Fe deficiency-induced maize roots. ZmFDR4 is homologous to the bacterial FliP superfamily, coexisted in both algae and terrestrial plants, and capable of restoring the normal growth of the yeast mutant fet3fet4, which possesses defective Fe uptake systems. ZmFDR4 mRNA is ubiquitous in maize and is inducible by iron deficiency in wheat. Transient expression of the 35S:ZmFDR4-eGFP fusion protein in rice protoplasts indicated that ZmFDR4 maybe localizes to the plastids envelope and thylakoid. In 35S:c-Myc-ZmFDR4 transgenic tobacco, immunohistochemistry and immunoblotting confirmed that ZmFDR4 is targeted to both the chloroplast envelope and thylakoid. Meanwhile, ultrastructure analysis indicates that ZmFDR4 promotes the density of plastids and accumulation of starch grains. Moreover, Bathophenanthroline disulfonate (BPDS) colorimetry and inductively coupled plasma mass spectrometry (ICP-MS) indicate that ZmFDR4 is related to Fe uptake by plastids and increases seed Fe content. Finally, 35S:c-Myc-ZmFDR4 transgenic tobacco show enhanced photosynthetic efficiency. Therefore, the results of the present study demonstrate that ZmFDR4 functions as an iron transporter in monocot plastids and provide insight into the process of Fe uptake by plastids.

Molecular and Bioinformatic Characterization of the Rice ROOT UV-B SENSITIVE Gene Family.

Background ROOT UV-B SENSITIVE (RUS) genes exist in most eukaryotic organisms, and encode proteins that contain a DUF647 (domain of unknown function 647). Although the RUS genes are known to play essential roles in Arabidopsis seedling development, their precise functions are not well understood in other plants, including rice.FindingsIn this study, six OsRUS genes were cloned from rice root and leaf cDNA libraries. Our analysis showed that the sequence and open reading frame of cloned OsRUS3 cDNA differs from the predictions reported in the RAP-DB and RGAP databases. Public microarray, MPSS, and EST databases were used to analyze the expression profiles of the six OsRUS genes. Expression profiles for all OsRUS genes at different rice developmental stages were also analyzed by qRT-PCR. The signal peptide, GPI-anchor, transmembrane domain and subcellular localization of OsRUS proteins were predicted by various bioinformatics tools. Furthermore OsRUS1 was determined to be localized to the chloroplast by a protoplast experiment.ConclusionsAll the characterization of the OsRUS family generated from this study will provide a crucial foundation from which to further dissect how OsRUS genes function in rice development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-016-0127-0) contains supplementary material, which is available to authorized users.

Reference Transcriptomes and Detection of Duplicated Copies in Hexaploid and Allododecaploid Spartina Species (Poaceae).

In this study, we report the assembly and annotation of five reference transcriptomes for the European hexaploid Spartina species (S. maritima, S. alterniflora and their homoploid hybrids S. x townsendii and S. x neyrautii) and the allododecaploid invasive species S. anglica. These transcriptomes were constructed from various leaf and root cDNA libraries that were sequenced using both Roche-454 and Illumina technologies. Considering the high ploidy levels of the Spartina genomes under study, and considering the absence of diploid reference genome and the need of an appropriate analytical strategy, we developed generic bioinformatics tools to (1) detect different haplotypes of each gene within each species and (2) assign a parental origin to haplotypes detected in the hexaploid hybrids and the neo-allopolyploid. The approach described here allows the detection of putative homeologs from sets of short reads. Synonymous substitution rate (KS) comparisons between haplotypes from the hexaploid species revealed the presence of one KS peak (likely resulting from the tetraploid duplication event). The procedure developed in this study can be applied for future differential gene expression or genomics experiments to study the fate of duplicated genes in the invasive allododecaploid S. anglica.

Systematic Isolation and Characterization of Cadmium Tolerant Genes in Tobacco: A cDNA Library Construction and Screening Approach.

Heavy metal pollution is a major limiting factor that severely affects plant growth worldwide, and the accumulation of heavy metal in the plant may be hazardous to human health. To identify the processes involved in cadmium detoxification, we constructed a cDNA library of tobacco roots acclimated to cadmium (Cd) stress. According to the results of functional screening cDNA library with a yeast Cd-sensitive mutant, ycf1Δ, we obtained a series of candidate genes that were involved in Cd response. Sequence analysis and yeast functional complementation of 24 positive cDNA clones revealed that, in addition to antioxidant genes, genes implicated in abiotic and biotic stress defenses, cellular metabolism, and signal transduction showed Cd detoxification effects in yeast. The real time RT-PCR analyses revealed that some Cd tolerance/ detoxification genes may be able to anticipate in other stresses such as biotic defense and water balance in tobacco. Taken together, our data suggest that plants’ acclimation to Cd stress is a highly complex process associated with broad gene functions. Moreover, our results provide insights into the Cd detoxification mechanisms along with the antioxidant system, defense gene induction, and calcium signal pathway.

Gene Expression and Yeast Two-Hybrid Studies of 1R-MYB Transcription Factor Mediating Drought Stress Response in Chickpea (Cicer arietinum L.).

Drought stress has been one of the serious constraints affecting chickpea productivity to a great extent. Genomics-assisted breeding has a potential to accelerate breeding precisely and efficiently. In order to do so, understanding the molecular mechanisms for drought tolerance and identification of candidate genes are crucial. Transcription factors (TFs) have important roles in the regulation of plant stress related genes. In this context, quantitative real time-PCR (qRT-PCR) was used to study the differential gene expression of selected TFs, identified from large-scale expressed sequence tags (ESTs) analysis, in contrasting drought responsive genotypes. Root tissues of ICC 4958 (tolerant), ICC 1882 (sensitive), JG 11 (elite), and JG 11+ (introgression line) were used for the study. Subsequently, a candidate single repeat MYB (1R-MYB) transcript that was remarkably induced in the drought tolerant genotypes under drought stress was cloned (coding sequence region for the 1R-MYB protein) and subjected to yeast two-hybrid (Y2H) analysis. The screening of a root cDNA library with Y2H using the 1R-MYB bait protein, identified three CDS encoding peptides namely, galactinol-sucrose galactosyltransferase 2, CBL (Calcineurin B-like)-interacting serine/threonine-protein kinase 25, and ABA responsive 17-like, which were confirmed by co-transformation in yeast. These findings provide preliminary insights into the ability of this 1R-MYB transcription factor to co-regulate drought tolerance mechanism in chickpea.

TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea).

BackgroundTargeting Induced Local Lesions in Genomes (TILLING) is a powerful reverse genetics approach for functional genomics studies. We used high-throughput sequencing, combined with a two-dimensional pooling strategy, with either minimum read percentage with non-reference nucleotide or minimum variance multiplier as mutation prediction parameters, to detect genes related to abiotic and biotic stress resistances. In peanut, lipoxygenase genes were reported to be highly induced in mature seeds infected with Aspergillus spp., indicating their importance in plant-fungus interactions. Recent studies showed that phospholipase D (PLD) expression was elevated more quickly in drought sensitive lines than in drought tolerant lines of peanut. A newly discovered lipoxygenase (LOX) gene in peanut, along with two peanut PLD genes from previous publications were selected for TILLING. Additionally, two major allergen genes Ara h 1 and Ara h 2, and fatty acid desaturase AhFAD2, a gene which controls the ratio of oleic to linoleic acid in the seed, were also used in our study. The objectives of this research were to develop a suitable TILLING by sequencing method for this allotetraploid, and use this method to identify mutations induced in stress related genes.ResultsWe screened a peanut root cDNA library and identified three candidate LOX genes. The gene AhLOX7 was selected for TILLING due to its high expression in seeds and roots. By screening 768 M2 lines from the TILLING population, four missense mutations were identified for AhLOX7, three missense mutations were identified for AhPLD, one missense and two silent mutations were identified for Ara h 1.01, three silent and five missense mutations were identified for Ara h 1.02, one missense mutation was identified for AhFAD2B, and one silent mutation was identified for Ara h 2.02. The overall mutation frequency was 1 SNP/1,066 kb. The SNP detection frequency for single copy genes was 1 SNP/344 kb and 1 SNP/3,028 kb for multiple copy genes.ConclusionsOur TILLING by sequencing approach is efficient to identify mutations in single and multi-copy genes. The mutations identified in our study can be used to further study gene function and have potential usefulness in breeding programs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1348-0) contains supplementary material, which is available to authorized users.

Comparison and development of EST–SSRs from two 454 sequencing libraries of Gossypium barbadense

EST–SSRs of Gossypium barbadense are mainly developed using traditional Sanger sequencing. However, due to the high cost and low throughput of Sanger sequencing, it is necessary to use high throughput sequencing technology for the development of more ESTs to more effectively analyze the structure and function of this species. In this study, a G. barbadense acc. 3–79 unnormalized fiber cDNA library (219.63 Mb) and a G. barbadense cv. Hai7124 normalized root cDNA library (204.61 Mb) were obtained by 454 sequencing. EST–SSRs were identified from the two libraries, and only 7,255 SSRs were obtained from the unnormalized library, with an average frequency of 1/31.00 kb. In contrast, 16,087 SSRs were obtained from the normalized library, with an average frequency of 1/13.02 kb. The frequencies of dinucleotides and tetranucleotides in the two libraries were very different. Comparing the two libraries, we found that a normalized cDNA library is more efficient for mining SSRs. Integrating the two libraries allowed the development of 1,129 EST–SSR markers, and 311 polymorphic loci were integrated into our interspecific BC1 genetic linkage map. The mapping results showed that the distribution of EST–SSRs on sub-genomes and chromosomes was uneven; however, the distribution of the mapped G. barbadense EST–SSRs on homologous chromosomes was similar, with the exception of Chr05 versus Chr19 and Chr12 versus Chr26. This study provided new EST–SSR markers that will facilitate studies on cotton genetics and breeding.

Generation of an expressed sequence tag (EST) library from salt‐stressed roots of Jatropha curcas for identification of abiotic stress‐responsive genes

Improving salinity and drought tolerance of crop plants has been an important aim of modern agricultural development, which depends on understanding the functions of genes expressed during the process of stress adaptation. EST resources are an efficient and cost-effective solution to gene discovery. Jatropha curcas is emerging as the most promising tree oil seed as a source of biodiesel. To identify genes that respond to abiotic stress, in the present study, we report 1240 ESTs generated from root cDNA libraries of J.curcas. ESTs were clustered and assembled into a collection of 865 unigenes, with 107 contigs and 758 singleton sequences. The putative functions of several ESTs could be assigned by similarity to plant gene sequence comparisons. It was found that 23 full-length CDS (34%) and the majority of transcription factors had sequence similarity to genes known to be involved in abiotic and biotic stress tolerance. The expression pattern of nine selected genes revealed that these genes are differentially expressed in various tissues during adaptation to stress. The data could serve as a critical resource to enable plant improvement programmes towards enhancing the adaptability of J.curcas to growth on marginal lands.

Identification of tissue-specific, abiotic stress-responsive gene expression patterns in wine grape (Vitis vinifera L.) based on curation and mining of large-scale EST data sets.

BackgroundAbiotic stresses, such as water deficit and soil salinity, result in changes in physiology, nutrient use, and vegetative growth in vines, and ultimately, yield and flavor in berries of wine grape, Vitis vinifera L. Large-scale expressed sequence tags (ESTs) were generated, curated, and analyzed to identify major genetic determinants responsible for stress-adaptive responses. Although roots serve as the first site of perception and/or injury for many types of abiotic stress, EST sequencing in root tissues of wine grape exposed to abiotic stresses has been extremely limited to date. To overcome this limitation, large-scale EST sequencing was conducted from root tissues exposed to multiple abiotic stresses.ResultsA total of 62,236 expressed sequence tags (ESTs) were generated from leaf, berry, and root tissues from vines subjected to abiotic stresses and compared with 32,286 ESTs sequenced from 20 public cDNA libraries. Curation to correct annotation errors, clustering and assembly of the berry and leaf ESTs with currently available V. vinifera full-length transcripts and ESTs yielded a total of 13,278 unique sequences, with 2302 singletons and 10,976 mapped to V. vinifera gene models. Of these, 739 transcripts were found to have significant differential expression in stressed leaves and berries including 250 genes not described previously as being abiotic stress responsive. In a second analysis of 16,452 ESTs from a normalized root cDNA library derived from roots exposed to multiple, short-term, abiotic stresses, 135 genes with root-enriched expression patterns were identified on the basis of their relative EST abundance in roots relative to other tissues.ConclusionsThe large-scale analysis of relative EST frequency counts among a diverse collection of 23 different cDNA libraries from leaf, berry, and root tissues of wine grape exposed to a variety of abiotic stress conditions revealed distinct, tissue-specific expression patterns, previously unrecognized stress-induced genes, and many novel genes with root-enriched mRNA expression for improving our understanding of root biology and manipulation of rootstock traits in wine grape. mRNA abundance estimates based on EST library-enriched expression patterns showed only modest correlations between microarray and quantitative, real-time reverse transcription-polymerase chain reaction (qRT-PCR) methods highlighting the need for deep-sequencing expression profiling methods.

Gene-based SSR markers for common bean (Phaseolus vulgaris L.) derived from root and leaf tissue ESTs: an integration of the BMc series

BackgroundSequencing of cDNA libraries for the development of expressed sequence tags (ESTs) as well as for the discovery of simple sequence repeats (SSRs) has been a common method of developing microsatellites or SSR-based markers. In this research, our objective was to further sequence and develop common bean microsatellites from leaf and root cDNA libraries derived from the Andean gene pool accession G19833 and the Mesoamerican gene pool accession DOR364, mapping parents of a commonly used reference map. The root libraries were made from high and low phosphorus treated plants.ResultsA total of 3,123 EST sequences from leaf and root cDNA libraries were screened and used for direct simple sequence repeat discovery. From these EST sequences we found 184 microsatellites; the majority containing tri-nucleotide motifs, many of which were GC rich (ACC, AGC and AGG in particular). Di-nucleotide motif microsatellites were about half as common as the tri-nucleotide motif microsatellites but most of these were AGn microsatellites with a moderate number of ATn microsatellites in root ESTs followed by few ACn and no GCn microsatellites. Out of the 184 new SSR loci, 120 new microsatellite markers were developed in the BMc (Bean Microsatellites from cDNAs) series and these were evaluated for their capacity to distinguish bean diversity in a germplasm panel of 18 genotypes. We developed a database with images of the microsatellites and their polymorphism information content (PIC), which averaged 0.310 for polymorphic markers.ConclusionsThe present study produced information about microsatellite frequency in root and leaf tissues of two important genotypes for common bean genomics: namely G19833, the Andean genotype selected for whole genome shotgun sequencing from race Peru, and DOR364 a race Mesoamerica subgroup 2 genotype that is a small-red seeded, released variety in Central America. Both race Peru and Mesoamerica subgroup 2 (small red beans) have been understudied in comparison to race Nueva Granada and Mesoamerica subgroup 1 (black beans) both with regards to gene expression and as sources of markers. However, we found few differences between SSR type and frequency between the G19833 leaf and DOR364 root tissue-derived ESTs. Overall, our work adds to the analysis of microsatellite frequency evaluation for common bean and provides a new set of 120 BMc markers which combined with the 248 previously developed BMc markers brings the total in this series to 368 markers. Once we include BMd markers, which are derived from GenBank sequences, the current total of gene-based markers from our laboratory surpasses 500 markers. These markers are basic for studies of the transcriptome of common bean and can form anchor points for genetic mapping studies in the future.

Comparative withanolide profiles, gene isolation, and differential gene expression in the leaves and roots of Withania somnifera

SummaryWithania somnifera plants derive their therapeutic properties by synthesising biologically-active, steroidal lactones known as withanolides. However, little is known about the genes that encode the enzymes involved in this biosynthetic pathway. Significant differences in the spectra of withanolides detected in the aerial and underground organs of plants suggest differential expression of distinct organ-specific pathways and genes. Since expressed sequence tags (ESTs) provide a robust method for gene prospecting, and few ESTs are available for W. somnifera in the public databases, this study used an EST-based approach to analyse gene expression profiles in W. somnifera plants. Separate leaf and root cDNA libraries of W. somnifera were generated and yielded 495 leaf and 76 root ESTs. Two of the leaf ESTs were then converted into full-length gene sequences using RACE procedures. Genes for sterol methyl transferase (SMT1) and obtusifoliol-14 -demethylase (ODM), whose products are claimed to be involved in the biosynthetic pathway that leads to withanolides, were thus cloned. Withaferin-A contents, as well as the abundance of ODM and SMT1 gene transcripts, were found to be higher in leaves than in roots, and increased in both organs as the plants matured. Genes encoding 1-deoxy-D-xylulose-5-phosphate reducto-isomerase (DXR) and hydroxymethylglutaryl-CoA reductase (HMGR) were also isolated via W. somnifera leaf mRNA using RT-PCR. Subtractive hybridisation between leaf and root cDNA populations yielded 36 leaf-abundant and 47 root-abundant ESTs, some of which showed sequence (80%) homology to glucosyltransferases, which may be significant in view of the highly glucosylated nature of withanolides.

Competition between uptake of ammonium and potassium in barley and Arabidopsis roots: molecular mechanisms and physiological consequences

Plants can use ammonium (NH4+) as the sole nitrogen source, but at high NH4+ concentrations in the root medium, particularly in combination with a low availability of K+, plants suffer from NH4+ toxicity. To understand the role of K+ transporters and non-selective cation channels in K+/NH4+ interactions better, growth, NH4+ and K+ accumulation and the specific fluxes of NH4+, K+, and H+ were examined in roots of barley (Hordeum vulgare L.) and Arabidopsis seedlings. Net fluxes of K+ and NH4+ were negatively correlated, as were their tissue concentrations, suggesting that there is direct competition during uptake. Pharmacological treatments with the K+ transport inhibitors tetraethyl ammonium (TEA+) and gadolinium (Gd3+) reduced NH4+ influx, and the addition of TEA+ alleviated the NH4+-induced depression of root growth in germinating Arabidopsis plants. Screening of a barley root cDNA library in a yeast mutant lacking all NH4+ and K+ uptake proteins through the deletion of MEP1–3 and TRK1 and TRK2 resulted in the cloning of the barley K+ transporter HvHKT2;1. Further analysis in yeast suggested that HvHKT2;1, AtAKT1, and AtHAK5 transported NH4+, and that K+ supplied at increasing concentrations competed with this NH4+ transport. On the other hand, uptake of K+ by AtHAK5, and to a lesser extent via HvHKT2;1 and AtAKT1, was inhibited by increasing concentrations of NH4+. Together, the results of this study show that plant K+ transporters and channels are able to transport NH4+. Unregulated NH4+ uptake via these transporters may contribute to NH4+ toxicity at low K+ levels, and may explain the alleviation of NH4+ toxicity by K+.