Quantitative proteomics of maize roots treated with a novel protein hydrolase: comparative study with transcriptomics highlights the molecular mechanisms responsive to biostimulants
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap
ORGANISM(S): Zea Mays (maize)
TISSUE(S): Root
SUBMITTER: 
Giorgio Arrigoni
LAB HEAD: Giorgio Arrigoni
PROVIDER: PXD017781 | Pride | 2020-11-16
REPOSITORIES: Pride
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Project description:Maize is a globally important food and feed crop, and a low-phosphate (Pi) supply in the soil frequently limits maize yield in many areas. MicroRNAs (miRNAs) play important roles in the development and adaptation of plants to the environment. In this study, the spatio-temporal miRNA transcript profiling of the maize inbred line Q319 root and leaf in response to low Pi was analyzed with high-throughput sequencing technologies, and the expression patterns of certain target genes were detected by real-time RT-PCR. Complex small RNA populations were detected after low-Pi culture and displayed different patterns in the root and leaf. miRNAs identified as responding to Pi deficiency can be grouped into ‘early’ miRNAs that respond rapidly, and often non-specifically, to Pi deficiency, and ‘late’ miRNAs that alter the morphology, physiology or metabolism of plants upon prolonged Pi deficiency. The miR827-Nitrogen limitation adaptation (NLA)-mediated post-transcriptional pathway was conserved in response to Pi availability of maize, but the miR399-mediated post-transcriptional pathway was different from Arabidopsis. Abiotic stress-related miRNAs engaged in interactions of different signaling and/or metabolic pathways. Auxin-related miRNAs (zma-miR393, zma-miR160a/b/c, zma-miR160d/e/g, zma-miR167a/b/c/d and zma-miR164a/b/c/d/g) and their targets play important roles in promoting primary root growth, inhibiting lateral root development and retarding upland growth of maize when subjected to low Pi. The changes in expression of miRNAs and their target genes suggest that the miRNA regulation/alterations compose an important mechanism in the adaptation of maize to a low-Pi environment; certain miRNAs participate in root architecture modification via the regulation of auxin signaling. A complex regulatory mechanism of miRNAs in response to a low-Pi environment exists in maize, revealing obvious differences from that in Arabidopsis. Maize (Zea mays L.) inbred line Q319 was used in this study. Seeds of the maize inbred line Q319 were surface sterilized and held at 28°C in darkness. Seedlings (4 days old) were transferred to a sufficient phosphate (SP, 1,000 μM KH2PO4) solution (Ca(NO3)2.4H2O 2 mM, NH4NO3 1.25 mM, KCl 0.1 mM, K2SO4 0.65 mM, MgSO4 0.65 mM, H3BO3 10.0 mM, (NH4)6Mo7O24 0.5 mM, MnSO4 1.0 mM, CuSO4.5H2O 0.1 mM, ZnSO4.7H2O 1.0 mM, Fe-EDTA 0.1 mM), allowed to grow for 4 days (plants with 2–3 leaves). After 2-3 days of re-culturing in SP nutrient solutions, half of the seedlings were transplanted into a low phosphate (LP, same composition as the SP solution, except that 5 μM KH2PO4 and 1 mM KH2PO4 were replaced with 1 mM KCl) nutrient solution. The plants were grown under a 32°C/25°C (day/night) temperature regime at a photon flux density (PFD) of 700 μmol m-2 s-1 with a 14 h/10 h light/dark cycle in a greenhouse with approximately 65% relative humidity. The roots and leaves were then harvested at 0, 1, 2, 4, 8 days and 8 days and cultured in SP solution (as a normal growth control) for small RNA analysis. The samples were frozen in liquid nitrogen immediately and stored at -80℃ for further analysis. Each biological repeat contains segments from 15~20 plants. Total RNA was extracted as previously described in Molecular Cloning (Sambrook and Russell David, 1989) and was then subjected to two additional chloroform washes prior to nucleic acid precipitation. The small RNA digitalization analysis based on HiSeq high-throughput sequencing takes the SBS-sequencing by synthesis. Then the 50nt sequence tags from HiSeq sequencing will go through the data cleaning first, which includes getting rid of the low quality tags and several kinds of contaminants from the 50nt tags. Length distribution of clean tags are then summarized. Afterwards, the standard bioinformatics analysis will annotate the clean tags into different categories and take those which can not be annotated to any category to predict the novel miRNA and base edit of potential known miRNA. Compare the known miRNA expression between two samples to find out the differentially expressed miRNA. The procedures are shown as below: (1)Normalize the expression of miRNA in two samples (control and treatment) to get the expression of transcript per million (TPM). Normalization formula: Normalized expression = Actual miRNA count/Total count of clean reads*1000000 (2)Calculate fold-change and P-value from the normalized expression. Then generate the log2ratio plot and scatter plot.
2015-07-08 | E-GEOD-70612 | biostudies-arrayexpress
Project description:As maize seedlings germinate into the soil, they encounter an environment teeming with insects seeking rich sources of nutrition. Maize presumably has developed a number of molecular mechanisms to ensure survival at the beginning of its life cycle. Bioassays indicated maize seedlings were more toxic to caterpillars than shoots from 3 or 4 leafed plants. Microarray technology was utilized to document the expression of a number of genes with potential defensive functions in seedling tissue. In addition to elevated levels of the genes involved in the biosynthesis of DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), an anti-insect resistance molecule, other highly expressed genes in the seedling encode the following putative proteins: defensin, hydroxyproline and proline-rich protein, thaumatin-like protein, lipase, cystatin, protease inhibitor, and a variety of proteases. Changes in the levels of gene expression by the microarray technology were well correlated to results using quantitative RT-PCR. The potential resistance genes identified occurred mainly on chromosomes 1 and 5 in the B73 genome. Analysis of promoters of four DIMBOA biosynthetic genes determined that a Dof transcription factor, two of which were differentially expressed in three vegetative stages, is possibly involved in regulation of the DIMBOA biosynthetic pathway. The results indicate that maize employs a wide variety of potential resistance mechanisms in seedling tissue to resist a possible insect attack. Three different Zea mays plant (B73) life stages were sampled: a) seedlings, which had grown just above the soil line, which also served as reference tissue (reference tissue was always labeled Cy5 and consisted of leaf shoots pooled from 12 plants); b) leaf shoots from plants with four leaves; and c) leaf shoots from plants with six leaves. There were 3 samples (biological replications) of each treatment (6 plants per sample) and the samples were labeled Cy3 and which were combined with a reference chip for a total of 9 chips: (3 biological replicates of seedling, 4 leaf stage and 6 leaf stage).
2011-11-03 | E-GEOD-27709 | biostudies-arrayexpress
Project description:Low phosphate concentrations are frequently a constraint for maize growth and development, and therefore, enormous quantities of phosphate fertilizer are expended in maize cultivation, which increases the cost of planting. Low phosphate stress not only increases root biomass but can also cause significant changes in root morphology. Low phosphate availability has been found to favor lateral root growth over primary root growth by dramatically reducing primary root length and increasing lateral root elongation and lateral root density in Arabdopsis. While in our assay when inbred line Q319 subjected to phosphate starvation, The numbers of lateral roots and lateral root primordia were decreased after 6 days of culture in a low phosphate solution (LP) compared to plants grown under normal conditions (sufficient phosphate, SP), and these differences were increased associated with the stress caused by phosphate starvation. However, the growth of primary roots appeared not to be sensitive to low phosphate levels. This is very different to Arabidopsis. To elucidate how low phosphate levels regulate root modifications, especially lateral root development, a transcriptomic analysis of the 1.0-1.5 cm lateral root primordium zone (LRZ) of maize Q319 treated after 2 and 8 days by low phosphate was completed respectively. The present work utilized an Arizona Maize Oligonucleotide array 46K version slides, which contained 46,000 maize 70-mer oligonucleotides designated by TIGR ID, and the sequence information is available at the website of the Maize Oligonucleotide Array Project as the search item representing the >30,000 identifiable unique maize genes (details at http://www.maizearray.org). Keywords: low phosphate, Lateral Root Primordium Zone, maize Two-condition experiment, low phosphate treated lateral root primordium zone of maize root vs. normal cultrued lateral root primordium zone. Biological replicates: 9 control, 9 treated, independently grown and harvested. One replicate per array.
2012-03-09 | E-GEOD-36368 | biostudies-arrayexpress
Project description:Not much is known about the molecular processes involved during gravitropism in monocot plants such as maize. A microarray based study on the expression of genes after a gravity stimulating the maize pulvinus will provide us with valuable information and a better understanding of the underlying molecular processes involved in monocot gravitropism. Objectives for this study included the identification of genes that were regulated at the transcriptional and translation level during gravitropism in the maize pulvinus. This was achieved by microarray analysis of total RNA versus polyribosome associated RNA during a time-course of gravity stimulation of the maize pulvinus. Experiment Overall Design: Six week old maize plants were gravity stimulated by 90º reorientation. Upper (slow elongation) and lower (fast elongation) halves of the most gravity competent pulvini were harvested over a time course ranging from 2 minutes up to one hour (2min, 5 min, 15min, 30min, 60min). Pulvini samples from control (vertical, no gravity stimulation) plants were harvested and labeled as left and right. For each time point, total mRNA and polyribosome-associated mRNA were purified and the transcript profiles analyzed using Affymetrix GeneChip® Maize Genome Arrays. The experiment was repeated twice (two growing seasons) and represent two biological repetitions.
2008-10-18 | E-GEOD-8320 | biostudies-arrayexpress
Project description:Maize kernels are susceptible to infection by the opportunistic pathogen Aspergillus flavus. Infection results in reduction of grain quality and contamination of kernels with the highly carcinogenic mycotoxin, aflatoxin. To understand host response to infection by the fungus, transcription of approximately 9,000 maize genes were monitored during the host-pathogen interaction with a custom-designed Affymetrix GeneChip® DNA array. More than 1,000 maize genes were found differentially expressed at a fold change of 2 or greater. This included the up regulation of defense-related genes and signaling pathways. Transcriptional changes also were observed in primary metabolism genes. Starch biosynthetic genes were down regulated during infection, while genes encoding maize hydrolytic enzymes, presumably involved in the degradation of host reserves, were up regulated. These data indicate that infection of the maize kernel A. flavus induced metabolic changes in the kernel, including the production of a defense response, as well as a disruption in kernel development. Maize kernels were mock inoculated at the blister (R2) or dough (R4) stage or inoculated with A. flavus at the blister (R2), milk (R3), dough (R4), or dent (R5) stage, and harvested 4 days later. Each treatment consisted of three biological replications. For each biological replication, 8 kernels were ground and RNA was isolated and further processed.
2014-05-14 | E-GEOD-57629 | biostudies-arrayexpress
Project description:Nitrate is the major source of nitrogen available for many crop plants and is often the limiting factor for plant growth and agricultural productivity especially for maize. Many studies have been done identifying the transcriptome changes under low nitrate conditions. However, the microRNAs (miRNAs) varied under nitrate limiting conditions in maize has not been reported. MiRNAs play important roles in abiotic stress responses and nutrient deprivation. Root is the organ that plants transport nitrate. we used the microarray systems to perform a genome-wide search to detect miRNAs responding to the chronic and transient nitrate limiting conditions in maize. MicroRNAs (miRNAs) are small, endogenous RNAs that are regulators of gene expression in plants and animals. And, miRNAs have been known for years to be important for phosphate, sulphate and copper deprivation responses in plants. In Arabidopsis, small RNA deep sequencing associated with nitrate response had been analyzed. However, information about the way by which miRNA are regulated by abiotic stresses in general and by low nitrate in particular is unavailable for maize. In this study, we used GeneChip® microarray systems to perform a genome-wide search to detect miRNAs responding to the chronic and transient nitrate limiting conditions in roots in maize.
2011-08-31 | E-GEOD-31231 | biostudies-arrayexpress
Project description:Periods of soil water deficit could occur at any time during the crop season, but maize is particularly sensitive to water stress around flowering time. At this time the stress usually causes remarkable yield loss. Heading time, which is just before tassel flowering, is one of the most important stages that maize productivity would be affected severely if plants encounter water stress. The whole-genomic gene expression changes of maize plants in response to water deficit stress at this stage have not been studied. The present work utilized an Arizona Maize Oligonucleotide Array Version 1.9ï¼which was consisted of A and B slides carrying with a total of 57,452 maize 70-mer oligonucleotides, was used to monitor gene expression profile changes in maize leaves subjected to 1 day and 7 days water-deficit stress respectively at the heading stage. Keywords: stress response Maize (Zea mays L.) inbred line DH4866 was used in this study. Plants grew in flowerpots containing field soil under natural conditions. When the tassel spread out of the uppermost leaf, some plants were treated with drought stress for 7 days, and others were left under normal-watered conditions as control. In the period of the stress, the middle part of the top fully expanded leaves of the plants under water stress treatment and the control were taken to measure the leaf osmotic potential at different times by the Fiske® Micro-Osmometer (FISKE® ASSOCIATES, USA). And the stressed plants were watered every day to maintain a similar water-deficiency state by adjusting water supply, according to the osmotic potential of the fully expended leaf of the plants measured at 8:00 am each day. During the stress, the leaf osmotic potential of the stressed plants was hold to -0.4 to -0.5 Mpa, while the osmotic potential was -0.2 to -0.25 Mpa in the leaves of the control plants. And there were visible signs of water deficiency such as leaf rolling during the daytime, though at night the rolled leaves spread out. Maize plants were stress-treated at the beginning of heading stage. After 7 days of water deficiency by withholding water, the plants began to enter the flowering stage. The maize flag leaves were harvested after 24h (1d) and 168h ï¼7dï¼ of stress treatment, frozen in liquid nitrogen immediately and stored at -80â for further analysis. Unstressed plants as controls were harvested at the same time as the stressed plants. One sample consisted of the leaves from three independent plants under the same condition. Total RNA was isolated as McCarty (1986) described from the frozen samples. For each sample, 100ïg of total RNA was transcribed into cDNA and fluorescently labeled with Cy3 and Cy5 dyes using the SuperScript indirect cDNA labeling system (INVITROGEN, USA) according to the manufacturerâs instructions. The Cy3 and Cy5 labels were swapped between sample and control cDNA to minimize any possible impact of inequalities in DNA incorporation and photobleaching of the fluorescent dyes. Hybridizations were performed according to the protocols at the website of the Maize Oligonucleotide Array Project. Two technical replicates (dye-swap experiments) for each biological sample from two independent treatments were performed.
2010-05-26 | E-GEOD-10596 | biostudies-arrayexpress
Project description:Poly- and perfluorinated alkyl substances (PFAS) are a group of persistent organic pollutants. Plants can accumulate PFAS but their effect on plant physiology at the molecular level is not understood yet. We used hydroponically-grown maize plants treated with a combination of eleven different PFAS (each at 100 µg L-1) to investigate their bioaccumulation and effects on the growth, physiology and their impact on the root proteome. From the root proteome analysis, we identified 75 differentially abundant proteins, mostly involved in cellular metabolic and biosynthetic processes, translation and cytoskeletal reorganization. Results were validated using quantitative real-time PCR and further substantiated using amino acid and fatty acid profiling.
2023-02-08 | PXD029993 | Pride
Project description:Maize kernels are susceptible to infection by the opportunistic pathogen Aspergillus flavus. Infection results in reduction of grain quality and contamination of kernels with the highly carcinogenic mycotoxin, aflatoxin. To understand host response to infection by the fungus, transcription of approximately 9,000 maize genes were monitored during the host-pathogen interaction with a custom-designed Affymetrix GeneChip® DNA array. More than 1,000 maize genes were found differentially expressed at a fold change of 2 or greater. This included the up regulation of defense-related genes and signaling pathways. Transcriptional changes also were observed in primary metabolism genes. Starch biosynthetic genes were down regulated during infection, while genes encoding maize hydrolytic enzymes, presumably involved in the degradation of host reserves, were up regulated. These data indicate that infection of the maize kernel A. flavus induced metabolic changes in the kernel, including the production of a defense response, as well as a disruption in kernel development.
2014-05-14 | GSE57629 | GEO
Project description:In an effort to confirm binding of SOMAmer®s to their respective targets in an endogenous matrix, we have conducted a series of experiments using SOMAmer®s to enrich target proteins followed by measurement using two mass spectrometry techniques: data dependent analysis (DDA) and multiple reaction monitoring (MRM). For data dependent analysis, the library of 4783 SOMAmer®s were multiplexed in sets of 8 and used for enrichment of target proteins from cell lysate and conditioned media, as well as human plasma and serum. A subset of the SOMAmer®s were screened in urine by this global profiling technique as well. Cell lines were selected from Cancer Cell Line Encyclopedia for screening by comparing gene expression of the target proteins measured by RNA sequencing across the CCLE.1 Using the criterion of FPKM value greater than 5 for the target protein, the minimum number of cell lines was grown to maximize protein coverage across the SOMAmer® library. For a given cell line, the presence of at least 8 target proteins with FPKM values greater than 5was required for inclusion. Approximately 400 target proteins were not covered by this strategy were screened in serum and plasma only. For enrichment from biological matrices, SOMAmer®s were combined into sets of 8 such that the potential interaction with similar proteins or binding partners was minimized. Non-specific binding of the SOMAmer®s was assessed in each matrix by using a SOMAmer® generated against the bacterial protein CysH. Target protein spectral counts in the SOMAmer® enriched samples were compared to the respective CysH control. A positive hit is defined as target protein detection with a minimum of 2 spectral counts and signal over CysH background greater than 10X (if applicable).
2018-08-03 | PXD008822 | Pride