Genome-wide identification, characterization, and expression analysis of the sweet potato (Ipomoea batatas [L.] Lam.) ARF, Aux/IAA, GH3, and SAUR gene families.

BACKGROUND: Auxins are known to have roles in the tuberization process in sweet potato (Ipomoea batatas [L.] Lam.) and these effects are mediated by various auxin signalling gene families. In this study, an analysis of the sweet potato genome was performed to identify the ARF, Aux/IAA, GH3, and SAUR auxin signalling gene family members in this crop. RESULTS: A total of 29 ARF, 39 Aux/IAA, 13 GH3, and 200 SAUR sequences were obtained, and their biochemical properties and gene expression profiles were analysed. The sequences were relatively conserved based on exon-intron structure, motif analysis, and phylogenetic tree construction. In silico expression analyses of the genes in fibrous and storage roots indicated that many sequences were not differentially expressed in tuberizing and non-tuberizing roots. However, some ARF, Aux/IAA, and SAUR genes were up-regulated in tuberizing storage roots compared to non-tuberizing fibrous roots while many GH3 genes were down-regulated. Additionally, these genes were expressed in a variety of plant parts, with some genes being highly expressed in shoots, leaves, and stems while others had higher expression in the roots. Some of these genes are up-regulated during the plant's response to various hormone treatments and abiotic stresses. Quantitative RT-PCR confirmation of gene expression was also conducted, and the results were concordant with the in silico analyses. A protein-protein interaction network was predicted for the differentially expressed genes, suggesting that these genes likely form part of a complex regulatory network that controls tuberization. These results confirm those of existing studies that show that auxin signalling genes have numerous roles in sweet potato growth and development. CONCLUSION: This study provides useful information on the auxin signalling gene families in Ipomoea batatas and suggests putative candidates for further studies on the role of auxin signalling in tuberization and plant development.

Characterization and expression analysis of SnRK2, PYL, and ABF/ AREB/ ABI5 gene families in sweet potato.

Abscisic acid (ABA) signaling in plants is essential to several aspects of plant development, such as tolerance to environmental stresses and growth. ABA signaling is also important for storage organ formation in crops, such as sweet potato. However, the repertoire of I. batatas ABA signaling gene families has not yet been fully characterized, so that it is unclear which members of these families are necessary for tuberization. Therefore, genome-wide identification of the sweet potato ABF/ AREB/ ABI5, SnRK2, and PYL gene families was performed, along with phylogenetic, motif, cis-regulatory element (CRE), and expression analyses. Nine ABF, eight SnRK2, and eleven PYL gene family members were identified, and there was high sequence conservation among these proteins that were revealed by phylogenetic and motif analyses. The promoter sequences of these genes had multiple CREs that were involved in hormone responses and stress responses. In silico and qRT-PCR expression analyses revealed that these genes were expressed in various tissues and that IbABF3, IbABF4, IbDPBF3, IbDPBF4, IbPYL4, IbSnRK2.1, and IbSnRK2.2 were significantly expressed during storage root development. These results are an important reference that can be used for functional validation studies to better understand how ABA signaling elicits storage root formation at the molecular level.

Inversion induced Manihot esculenta stem tubers express key tuberization genes; Mec1, RZF, SuSy1 and PIN2.

Cassava (M. esculenta) gives rise to unique underground stem tubers when stem cuttings are planted in an inverted orientation. The nutritional profile of the stem and root tubers were similar except for protein content which was higher in stem than in root tubers. RT-PCR revealed that several key genes (Mec1, RZF, SuSy1 and PIN2) involved in root tuberization were also expressed in these stem tubers. At five weeks post planting, these genes were expressed in roots and underground stems as in the mature tubers. However at 15 weeks post planting, they were expressed in both root and stem tubers but not in adventitious roots or in the non-tuberized stems. Expression of, the root auxin efflux carrier gene PIN2 in the stem tubers indicate a role for auxin in the stem tuberization process.

Metabolic labeling with stable isotope nitrogen (15N) to follow amino acid and protein turnover of three plastid proteins in Chlamydomonas reinhardtii.

BACKGROUND: The length of time that a protein remains available to perform its function is significantly influenced by its turnover rate. Knowing the turnover rate of proteins involved in different processes is important to determining how long a function might progress even when the stimulus has been removed and no further synthesis of the particular proteins occurs. In this article, we describe the use of 15N-metabolic labeling coupled to GC-MS to follow the turnover of free amino acids and LC-MS/MS to identify and LC-MS to follow the turnover of specific proteins in Chlamydomonas reinhardtii. RESULTS: To achieve the metabolic labeling, the growth medium was formulated with standard Tris acetate phosphate medium (TAP) in which14NH4Cl was replaced with 15NH415NO3 and (14NH4)6Mo7O24.4H2O was replaced with Na2MoO4.2H2O. This medium designated 15N-TAP allowed CC-125 algal cells to grow normally. Mass isotopic distribution revealed successful 15N incorporation into 13 amino acids with approximately 98% labeling efficiency. Tryptic digestion of the 55 kDa SDS-PAGE bands from 14N- and 15N-labeled crude algal protein extracts followed by LC-MS/MS resulted in the identification of 27 proteins. Of these, five displayed peptide sequence confidence levels greater than 95% and protein sequence coverage greater than 25%. These proteins were the RuBisCo large subunit, ATP synthase CF1 alpha and beta subunits, the mitochondrial protein (F1F0 ATP synthase) and the cytosolic protein (S-adenosyl homocysteine hydroxylase). These proteins were present in both labeled and unlabeled samples. Once the newly synthesized 15N-labeled free amino acids and proteins obtained maximum incorporation of the 15N-label, turnover rates were determined after transfer of cells into 14N-TAP medium. The t½ values were determined for the three plastid proteins (RuBisCo, ATP synthase CF1 alpha and beta) by following the reduction of the 15N-fractional abundance over time. CONCLUSION: We describe a more rapid and non-radioactive method to measure free amino acid and protein turnover. Our approach is applicable for determination of protein turnover for various proteins, which will lead to a better understanding of the relationship between protein lifetime and functionality.

Formamide-based RNA isolation at above zero temperatures from high starch cassava tubers.

INTRODUCTION: Cassava (Manihot esculenta) tubers are a main source of carbohydrate for a large percentage of people in the tropics. However, obtaining RNA from such high starch tubers proves problematic because gelation occurs during standard isolation procedures. In preliminary studies, formamide was used to homogenise tubers at room temperature and no gelation was observed. OBJECTIVES: To develop a simple, reproducible formamide-based procedure for RNA isolation from high starch tissues without the initial use of liquid nitrogen or lyophilisation. A second objective was to determine the impact of storage of formamide homogenates at 4°C on RNA integrity. METHODOLOGY: Tubers were homogenised in 100% formamide at room temperature and stored for 1, 4 or 7 days at 4°C. Homogenates were centrifuged at 14000 × g for 10 min and RNA recovered from the supernatants by isopropanol precipitation followed by dissolution in guanidinium buffer. Protein was removed by standard phenol-chloroform-isoamyl alcohol extraction and RNA recovered by isopropanol precipitation. For immediate RNA isolation, formamide homogenates can be processed without incubation at 4°C. RESULTS: In formamide homogenates a non-gelatinous white pellet was observed which was identified as starch by Lugol's staining. Intact RNA was observed by agarose gel electrophoresis. The RNA was successfully used in downstream RT-PCR reactions. CONCLUSION: Formamide maintained the integrity of the RNA during tissue processing at 25 ± 2°C and storage at 4°C. This room temperature formamide-based RNA isolation procedure met the criteria of simplicity, low-cost, reproducibility and addressed gelation problems associated with high starch tissues.

Induction of DREB2A pathway with repression of E2F, jasmonic acid biosynthetic and photosynthesis pathways in cold acclimation-specific freeze-resistant wheat crown.

Winter wheat lines can achieve cold acclimation (development of tolerance to freezing temperatures) and vernalization (delay in transition from vegetative to reproductive phase) in response to low non-freezing temperatures. To describe cold-acclimation-specific processes and pathways, we utilized cold acclimation transcriptomic data from two lines varying in freeze survival but not vernalization. These lines, designated freeze-resistant (FR) and freeze-susceptible (FS), were the source of crown tissue RNA. Well-annotated differentially expressed genes (p ≤ 0.005 and fold change ≥ 2 in response to 4 weeks cold acclimation) were used for gene ontology and pathway analysis. "Abiotic stimuli" was identified as the most enriched and unique for FR. Unique to FS was "cytoplasmic components." Pathway analysis revealed the "triacylglycerol degradation" pathway as significantly downregulated and common to both FR and FS. The most enriched of FR pathways was "neighbors of DREB2A," with the highest positive median fold change. The "13-LOX and 13-HPL" and the "E2F" pathways were enriched in FR only with a negative median fold change. The "jasmonic acid biosynthesis" pathway and four "photosynthetic-associated" pathways were enriched in both FR and FS but with a more negative median fold change in FR than in FS. A pathway unique to FS was "binding partners of LHCA1," which was enriched only in FS with a significant negative median fold change. We propose that the DREB2A, E2F, jasmonic acid biosynthesis, and photosynthetic pathways are critical for discrimination between cold-acclimated lines varying in freeze survival.

Cbf genes of the Fr-A2 allele are differentially regulated between long-term cold acclimated crown tissue of freeze-resistant and - susceptible, winter wheat mutant lines.

BACKGROUND: In order to identify genes that might confer and maintain freeze resistance of winter wheat, a comparative transcriptome analysis was performed between control and 4 wk cold-acclimated crown tissue of two winter wheat lines that differ in field freeze survival. The lines, generated by azide mutagenesis of the winter wheat cultivar 'Winoka' were designated FR (75% survival) and FS (30% survival). Using two winter lines for this comparative analysis removed the influence of differential expression of the vernalization genes and allowed our study to focus on Cbf genes located within the Fr-A2 allele independent of the effect of the closely mapped Vrn allele. RESULTS: Vernalization genes, (Vrn-A1, B1 and D1), and the transcription factor gene, TaVrt-2, were up-regulated to the same extent in FR and FS lines with cold acclimation thus confirming that azide mutagenesis had not modified the winter habitat of the lines. One category of Cbf genes, (Cbf-2, -A22 and B-22) reflected an increase in level of expression with cold acclimation in both FR and FS lines. Another category of Cbf genes (Cbf-3, -5, -6, -12, -14 and -19) were differentially expressed between cold-acclimated FR and FS lines relative to the non-acclimated controls. Comparison of expression patterns of the two categories of Cbf genes with the expression patterns of a set of ABA-dependent and -independent Cor/Lea genes revealed similar patterns of expression for this sample of Cor/Lea genes with that for Cbf-2 and -22. This pattern of expression was also exhibited by the Vrn genes. CONCLUSION: Some Cor/Lea genes may be co-regulated by the Vrn genes during cold acclimation and the Vrn genes may also control the expression of Cbf-2, -A22 and -B22. The increased freeze survival by the FR line and the increase in expression levels of wheat Cbf genes, Cbf-3, -5, -6, -12, -14 and -19 with cold acclimation in the FR line suggests a possible gain of function mutation resulting in higher levels of expression of these Cbf genes and increased freeze survival.

Sequential CaCl2, polyethylene glycol precipitation for RNase-free plasmid DNA isolation.

Functional genomics is facilitated by the ability to express genes in heterologous systems. In some cases function can be assayed by generation of in vitro transcripts of the unknown genes and expressing those transcripts in various expression systems. Plasmids bearing phage promoters are used to generate in vitro transcripts. Therefore, it is important to ensure that the template plasmid DNA is not contaminated with RNase from the isolation procedure. We have developed a plasmid purification protocol that does not utilize RNase yet yields pure plasmid DNA. The protocol combines the selective precipitation of RNA with 1.4M CaCl2, followed by a final selective precipitation of the plasmid DNA in a 10% polyethylene glycol (PEG), 250 mM NaCl solution. Purity of the resulting plasmid DNA was determined spectrophotometrically and by gel electrophoresis. No detectable contaminating RNA was observed in the plasmid DNA preparations. Inhibitory effects of the protocol were assayed by performing restriction analyses, sequencing, PCR, and in vitro transcription. These procedures were successful. The in vitro transcripts visualized by gel electrophoresis were found to be full length, thus indicating no significant endogenous RNase activity associated with the procedure.

Plant water uptake by hard red winter wheat (Triticum aestivum L.) genotypes at 2 degrees C and low light intensity.

BACKGROUND: Hard red winter wheat (HRWW; Triticum aestivm L.) plants from genotypes selected in the Northern Great Plains of the U.S. have less tissue water after exposure to cool autumn temperatures than plants from the Southern Great Plains. It is generally assumed that the reduced tissue water content of northern compared to southern cultivars is due to an impedance to water uptake by northern plants as a result of the low autumn temperatures. We hypothesize that if low temperature impedes water uptake then less soil water would be removed by northern than by southern-selected cultivars. This hypothesis was tested by comparing plant water uptake of a northern (FR) and a southern (FS) cultivar in relation to their foliage water content at 2 degrees C. RESULTS: At 2 degrees C foliage water content of FR plants decreased more rapidly than that of FS plants, similar to field results in the fall. During 6 wk, foliage water content of FR plants decreased 20 to 25% of the pre-treatment value, compared to only 5 to 10% by FS plants. Plant water uptake was about 60 g H2O*g FDW(-1) by FS plants, while FR plants maintained plant water uptake in excess of 100 g H2O*g FDW(-1) during the 6 wk period at 2 degrees C. When four other northern genotypes of equal freeze resistance were studied, foliage water content and plant water uptake change were similar to FR plants. CONCLUSION: In these northern-selected HRWW cultivars foliage water content reduction resulting from cold acclimation is not due to impedance to plant water uptake.