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Quantitative proteomics of maize roots treated with a novel protein hydrolase: comparative study with transcriptomics highlights the molecular mechanisms responsive to biostimulants

ABSTRACT: Protein hydrolysates (PHs) - based biostimulants offer a cost-effective, and sustainable approach for the regulation of physiological processes in plants to stimulate growth, and improve stress tolerance. Understanding the mode of action of PHs is challenging however, it is indispensable to improve existing candidates and to develop novel molecules with enhanced stimulatory effects. Hence, the present study aimed to understand the proteome level responses in the B73 maize roots treated with APR® at two increasing concentrations and to compare and integrate it with the transcriptomic data obtained previously under identical experimental conditions. Results indicate APR® induced dose-dependent global changes in the transcriptome and proteome of maize roots. APR® treatment altered the expression and abundance of several genes and proteins related to redox homeostasis, stress response, glycolysis, tricarboxylic acid cycle, pentose phosphate pathway and other metabolic pathways of carbohydrates, amino acids, and lipids. Further, metabolic processes of phytohormone, secondary metabolites especially phenylpropanoids, flavonoids, and terpenoids and transport, and cytoskeletal reorganization associated mechanisms were stimulated. Our results suggest that APR® treatment altered the redox homeostasis and thus triggered an oxidative signal. This could be one of the key regulators of the cascade of downstream events involving multiple signaling, hormonal, and metabolic pathways, resulting in an altered physiological and metabolic state which consequently could lead to improved growth, and stress adaptation observed in biostimulant - treated plants.

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.

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).

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.

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 FiskeÂ® Micro-Osmometer (FISKEÂ® 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.

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Quantitative proteomics of maize roots treated with a novel protein hydrolase: comparative study with transcriptomics highlights the molecular mechanisms responsive to biostimulants

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