Unknown,Transcriptomics,Genomics,Proteomics

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Phosphate Starvation of Maize Inhibits Lateral Root Formation and Alters Gene Expression in the Lateral Root Primordium Zone

ABSTRACT: 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.

ORGANISM(S): Zea mays

SUBMITTER: juren zhang

PROVIDER: E-GEOD-36368 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:Previous work showed that the maize primary root adapts to low water potential (-1.6 MPa) by maintaining longitudinal expansion in the apical 3 mm (region 1), whereas in the adjacent 4 mm (region 2) longitudinal expansion reaches a maximum in well-watered roots but is progressively inhibited at low water potential. To identify mechanisms that determine these responses to low water potential, transcript expression was profiled in these regions of water-stressed and well-watered roots. In addition, comparison between region 2 of water-stressed roots and the zone of growth deceleration in well-watered roots (region 3) distinguished stress-responsive genes in region 2 from those involved in cell maturation. Responses of gene expression to water stress in regions 1 and 2 were largely distinct. The largest functional categories of differentially expressed transcripts were reactive oxygen species and carbon metabolism in region 1, and membrane transport in region 2. Transcripts controlling sucrose hydrolysis distinguished well-watered and water-stressed states (invertase vs. sucrose synthase), and changes in expression of transcripts for starch synthesis indicated further alteration in carbon metabolism under water deficit. A role for inositols in the stress response was suggested, as was control of proline metabolism. Increased expression of transcripts for wall-loosening proteins in region 1, and for elements of ABA and ethylene signaling were also indicated in the response to water deficit. The analysis indicates that fundamentally different signaling and metabolic response mechanisms are involved in the response to water stress in different regions of the maize primary root elongation zone. Keywords: water stress response, root growth Three pair-wise comparisons were made of transcripts from water-stressed and well-watered tissues in the different root tip regions. In the first comparison (C1), transcripts from region 1 of water-stressed seedlings were compared with those from region 1 of well-watered seedlings. The second comparison (C2) was made between transcripts from region 2 of the two treatments. We expected a larger number of genes to be differentially expressed in region 2 because its elongation rate decreased greatly under water-stressed compared with well-watered conditions. To prioritize the differentially expressed genes revealed in this comparison, a distinction was made between those genes that are associated with growth inhibition in region 2 specifically as a response to water stress, and those genes that are involved in root cell maturation whether under stress or control conditions. A hypothetical example of the former might be genes involved in auxin response since water stress can increase maize root auxin content [8] and application of exogenous auxin can shorten the root growth zone [9]. An example of the latter might involve genes for secondary wall synthesis, e.g., [10]. To experimentally make this distinction a third pair-wise comparison (C2/3) was included to compare expression of genes between water-stressed region 2 and well-watered region 3 as these are both regions of cell maturation. Genes differentially expressed in both C2 and C2/3 are more likely to cause growth inhibition at low water potential and are not likely to be part of the maturation program itself, whereas genes differentially expressed only in C2 are more likely related to maturation. Four biological replicates were included each with dye-swap. The maize oligonucleotide array comes in two slides, A and B. Thus for each of the three comparisons made there were 16 slides (4 biological reps, 2 for dye-swap, 2 parts to an array).

Project description:This model is from the article: The influence of cytokinin-auxin cross-regulation on cell-fate determination in Arabidopsis thaliana root development Muraro D, Byrne H, King J, Voss U, Kieber J, Bennett M. J Theor Biol.2011 Aug 21;283(1):152-67. PMID: 21640126, Abstract: Root growth and development in Arabidopsis thaliana are sustained by a specialised zone termed the meristem, which contains a population of dividing and differentiating cells that are functionally analogous to a stem cell niche in animals. The hormones auxin and cytokinin control meristem size antagonistically. Local accumulation of auxin promotes cell division and the initiation of a lateral root primordium. By contrast, high cytokinin concentrations disrupt the regular pattern of divisions that characterises lateral root development, and promote differentiation. The way in which the hormones interact is controlled by a genetic regulatory network. In this paper, we propose a deterministic mathematical model to describe this network and present model simulations that reproduce the experimentally observed effects of cytokinin on the expression of auxin regulated genes. We show how auxin response genes and auxin efflux transporters may be affected by the presence of cytokinin. We also analyse and compare the responses of the hormones auxin and cytokinin to changes in their supply with the responses obtained by genetic mutations of SHY2, which encodes a protein that plays a key role in balancing cytokinin and auxin regulation of meristem size. We show that although shy2 mutations can qualitatively reproduce the effect of varying auxin and cytokinin supply on their response genes, some elements of the network respond differently to changes in hormonal supply and to genetic mutations, implying a different, general response of the network. We conclude that an analysis based on the ratio between these two hormones may be misleading and that a mathematical model can serve as a useful tool for stimulate further experimental work by predicting the response of the network to changes in hormone levels and to other genetic mutations.

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Phosphate Starvation of Maize Inhibits Lateral Root Formation and Alters Gene Expression in the Lateral Root Primordium Zone

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