Identifying and validating housekeeping hybrid Prunus spp. genes for root gene-expression studies

Prunus rootstock belonging to subgenera Amygdalus (peach), Prunus (plum) and Cerasus (cherry) are either from the same species as the scion or another one. The number of inter-species (including inter-subgenera) hybrids has increased as a result of broadening the genetic basis for stress (biotic and abiotic) resistance/tolerance. Identifying genes associated with important traits and responses requires expression analysis. Relative quantification is the simplest and most popular alternative, which requires reference genes (housekeeping) to normalize RT-qPCR data. However, there is a scarcity of validated housekeeping genes for hybrid Prunus rootstock species. This research aims to increase the number of housekeeping genes suitable for Prunus rootstock expression analysis. Twenty-one candidate housekeeping genes were pre-selected from previous RNAseq data that compared the response of root transcriptomes of two rootstocks subgenera to hypoxia treatment, ‘Mariana 2624’ (P. cerasifera Ehrh.× P. munsoniana W. Wight & Hedrick), and ‘Mazzard F12/1’ (P. avium L.). Representing groups of low, intermediate or high levels of expression, the genes were assayed by RT-qPCR at 72 hours of hypoxia treatment and analyzed with NormFinder software. A sub-set of seven housekeeping genes that presented the highest level of stability were selected, two with low levels of expression (Unknown 3, Unknown 7) and five with medium levels (GTB 1, TUA 3, ATPase P, PRT 6, RP II). The stability of these genes was evaluated under different stress conditions, cold and heat with the hybrid ‘Mariana 2624’ and N nutrition with the hybrids ‘Colt’ (P. avium × P. pseudocerasus Lindl.) and ‘Garnem’ [P. dulcis Mill.× (P. persica L.× P. davidiana Carr.)]. The algorithms of geNorm and BestKeeper software also were used to analyze the performance of these genes as housekeepers. Stability rankings varied according to treatments, genotypes and the software for evaluation, but the gene GBT 1 often had the highest ranking. However, most of the genes are suitable depending on the stressor and/or genotype to be evaluated. No optimal number of reference genes could be determined with geNorm software when all conditions and genotypes were considered. These results strongly suggest that relative RT-qPCR should be analyzed separately with their respective best housekeeper according to the treatment and/or genotypes in Prunus spp. rootstocks.

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title: Arabidopsis Nitrate Transporter NRT1.9 Is Important in Phloem Nitrate Transport[W][OA]
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title: Defining reference genes in Oryza sativa using organ, development, biotic and abiotic transcriptome datasets
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title: Genome-Wide Identification of New Reference Genes for qRT-PCR Normalization under High Temperature Stress in Rice Endosperm
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title: Selection and Validation of Reference Genes for Real-Time Quantitative PCR in Hyperaccumulating Ecotype of Sedum alfredii under Different Heavy Metals Stresses
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abstract: Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization.

title: Evaluation of stability and validation of reference genes for real time PCR expression studies in leaves and roots of Prunus spp. rootstocks under flooding
abstract: Molecular oxygen (O2) is involved in several biochemical reactions of plants. The low availability of oxygen to cells can lead to an energy crisis, since O2 is the main substrate of respiratory energy metabolism in aerobic organisms, acting as the final acceptor of electrons in the mitochondrial transport chain. When oxygen becomes insufficient for respiration, the plants begin a hypoxia condition. This situation can occur naturally due to excessive rainfall and the existence of poorly drained soils, causing soil flooding. This condition occurs in the South region of Rio Grande do Sul State, in Brazil, which is the largest Brazilian producer of stone fruits, mainly peaches, therefore, its production is adversely affected by flooding. Since the production of stone fruits plants (Prunus spp.) in the region occurs using rootstocks, the aim of this study was to identify potential reference genes for normalization of RT-qPCR data in leaves and roots of four rootstock of Prunus spp. (Capdeboscq, NR0170401, Marianna 2624 and Myrabolan 29-C) in the soil flooding condition. Eight candidate genes were evaluated: PLA2 (phospholipase A2), CYP2 (cyclophilin 2), Ef1α (elongantion factor 1-alpha), RPL13 (ribosomal protein L13), TUA (tubulin alpha), TUB (tubulin beta), RPII (RNA polymerase II), and 18S rRNA (18S ribosomal RNA). Some algorithms for the stability analysis were used, such as the ΔCt comparative method, BestKeeper, NormFinder, geNorm, and the RefFinder tool. As result, the CYP2 and RPII genes presented greater stability in the peach roostocks, whereas RPII and RPL13 were more stable in the plum rootstocks. The Ef1α gene showed no expression stability in the rootstocks ‘NR0170401’ (both tissues) and ‘Myrabolan 29-C’ (roots). The 18S rRNA gene was shown to be unstable for ‘Capdeboscq’ and ‘Marianna 2624’ leaves, while the TUB and PLA2 genes were not stable for roots of these cultivars, respectively. Analysis of the expression of LDH1 (Lactate Dehydrogenase) and ADH1 (alcohol Dehydrogenase) proved the importance of validation of the reference genes to obtain accurate results in RT-qPCR. Taken together, this study selected the reference genes suitable for the reliable normalization of gene expression data in Prunus spp. under flooding.

title: Structure-function relationship in P-type ATPases--a biophysical approach.
abstract: P-type ATPases are a large family of membrane proteins that perform active ion transport across biological membranes. In these proteins the energy-providing ATP hydrolysis is coupled to ion-transport that builds up or maintains the electrochemical potential gradients of one or two ion species across the membrane. P-type ATPases are found in virtually all eukaryotic cells and also in bacteria, and they are transporters of a broad variety of ions. So far, a crystal structure with atomic resolution is available only for one species, the SR Ca-ATPase. However, biochemical and biophysical studies provide an abundance of details on the function of this class of ion pumps. The aim of this review is to summarize the results of preferentially biophysical investigations of the three best-studied ion pumps, the Na,K-ATPase, the gastric H,K-ATPase, and the SR Ca-ATPase, and to compare functional properties to recent structural insights with the aim of contributing to the understanding of their structure-function relationship.

title: PRT6/At5g02310 encodes an Arabidopsis ubiquitin ligase of the N‐end rule pathway with arginine specificity and is not the CER3 locus
abstract: The eukaryotic N‐end rule pathway mediates ubiquitin‐ and proteasome‐dependent turnover of proteins with a bulky amino‐terminal residue. Arabidopsis locus At5g02310 shows significant similarity to the yeast N‐end rule ligase Ubr1. We demonstrate that At5g02310 is a ubiquitin ligase and mediates degradation of proteins with amino‐terminal Arg residue. Unlike Ubr1, the Arabidopsis protein does not participate in degradation of proteins with amino‐terminal Phe or Leu. This modified target specificity coincides with characteristic differences in domain structure. In contrast to previous publications, our data indicate that At5g02310 is not identical to CER3, a gene involved in establishment of a protective surface wax layer. At5g02310 has therefore been re‐designated PROTEOLYSIS 6 (PRT6), in accordance with its ubiquitin ligase function.

title: SPT6L encoding a putative WG/GW-repeat protein regulates apical-basal polarity of embryo in Arabidopsis.
abstract: In eukaryotes, a protein motif consisting of WG/GW repeats, also called the Argonaute (AGO) hook, is thought to be essential for binding AGO proteins to fulfill their functions in RNA-mediated gene silencing. Although a number of WG/GW-containing proteins have been computationally identified in Arabidopsis, their roles in plant growth and development are unknown. Here, we show that the Arabidopsis Suppressor of Ty insertion 6-like (SPT6L) gene, which encodes a protein with C-terminal WG/GW repeats, plays critical roles in embryonic development. SPT6L is evolutionarily conserved only in vascular plants, with varying numbers of C-terminal WG/GW repeats, which are plant-species specific. spt6l mutants formed embryos with an aberrant apical-basal axis, showing insufficient development of the basal domain and embryonic lethality. Expression domains of the class-III homeodomain-leucine zipper (HD-ZIP III) genes PHABULOSA (PHB) and PHAVOLUTA (PHV) were expanded in the spt6l embryo. In contrast, the PLETHORA1 (PLT1) gene, which acts antagonistically to the HD-ZIP III genes in specification of basal fate, was severely down-regulated in the spt6l mutant. Furthermore, the phb phv double mutations partially rescued aberrant basal development in the spt6l background and restored PLT1 expression. Collectively, our results indicate that SPT6L is essential for specification of the apical-basal axis, partly by controlling the HD-ZIP III genes in embryos.

title: Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs.
abstract: Plant basic helix-loop-helix (bHLH) transcription factors play essential roles in abiotic stress tolerance. However, most bHLHs have not been functionally characterized. Here, we characterized the functional role of a bHLH transcription factor from Arabidopsis, AtbHLH112, in response to abiotic stress. AtbHLH112 is a nuclear-localized protein, and its nuclear localization is induced by salt, drought and abscisic acid (ABA). In addition, AtbHLH112 serves as a transcriptional activator, with the activation domain located at its N-terminus. In addition to binding to the E-box motifs of stress-responsive genes, AtbHLH112 binds to a novel motif with the sequence 'GG[GT]CC[GT][GA][TA]C' (GCG-box). Gain- and loss-of-function analyses showed that the transcript level of AtbHLH112 is positively correlated with salt and drought tolerance. AtbHLH112 mediates stress tolerance by increasing the expression of P5CS genes and reducing the expression of P5CDH and ProDH genes to increase proline levels. AtbHLH112 also increases the expression of POD and SOD genes to improve reactive oxygen species (ROS) scavenging ability. We present a model suggesting that AtbHLH112 is a transcriptional activator that regulates the expression of genes via binding to their GCG- or E-boxes to mediate physiological responses, including proline biosynthesis and ROS scavenging pathways, to enhance stress tolerance.

title: Two distinct high-affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots.
abstract: Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment. Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively. We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate. A novel sulfate transporter gene, Sultr1;2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (K(m) for sulfate = 6.9 +/- 1.0 microm). Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1;2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface. Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50-100 microm sulfate), whereas Sultr1;2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50-1500 microm sulfate), indicating that Sultr1;2 is less responsive to changes in sulfur conditions. Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots. The level of Sultr1;2 mRNA was not influenced under these conditions. Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.