Computational protocol: Is the Inherent Potential of Maize Roots Efficient for Soil Phosphorus Acquisition?

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Protocol publication

[…] We used Olsen-P as the indicator of plant available P in the calcareous soils used here and CaCl2-P as the indicator of P loss risk . Soil Olsen-P level was determined by the molybdo-vanadophosphate method based on extraction of air-dried soil with 0.5 M NaHCO3 (pH 8.5) at 25°C . Soil CaCl2-P was measured by extracting air-dried soil with 0.01 M CaCl2 according to Hesketh and Brookes . Plant P concentration was measured by the molybdo-vanadophosphate method after samples were digested with concentrated H2SO4 and H2O2 . Plant P uptake was then calculated from plant dry weight and P concentration.To measure root morphological traits, cleaned root samples were dispersed in water in a transparent array (30 cm×20 cm×2 cm) and imaged with a scanner (Epson Expression 1600, Seiko Epson, Nagano, Japan) at a resolution of 800 dpi. The images were analyzed by WinRhizo software (Regent Instrument Inc., Quebec, QC, Canada) to determine root length. Root dry weight was determined by weighing the oven-dried samples after scanning. Specific root length was calculated from root length and root dry weight, and root/shoot ratio was assessed from root dry weight and shoot dry weight, respectively.AM colonization was measured in 1-cm fine root segments that had been thoroughly mixed. Roots were cleared with 10% KOH at 90°C for 1 h and stained with Trypan blue . The percent root colonization by AM fungi was assessed by examining 30 randomly selected stained root segments at 100–400 × magnification with a light microscope according to Trouvelot et al. .Six Pi transporters of the Pht1 family have been reported for maize: from ZEAma;Pht1;1 to ZEAma;Pht1;6 . For simplification, these genes are presented as ZmPht1;1 to ZmPht1;6 here. Expression of these Pi transporter genes was analyzed in maize root samples by real-time quantitative RT-PCR (qRT-PCR) . Total RNA was isolated from frozen root tissue using the Trizol reagent (cat. no. 15596018, Invitrogen, USA). The isolated RNA was treated with the RNase-Free DNase Set (cat. no. 79254, Qigen, Germany) to eliminate genomic DNA contamination before it was cleaned further with the RNeasy Plant Mini Kit (cat. no. 74904, Qigen, Germany). The first-strand cDNA was synthesized using the PrimeScript® RT reagent Kit Perfect Real Time (cat. no. DRR037A, Takara, Dalian) according to the manufacturer’s protocol. Then the qRT-PCR was performed on a Mastercycler Realplex4 Real Time PCR System (Eppendorf, Germany) based on the protocol of the SYBR® Premix EX TaqTM (cat. no. DRR041A, Takara) in 20 µl reaction volume, which contained 10 µl of SYBR Green PCR mix, 0.4 µM of each forward and reverse primer, 0.4 µg of diluted cDNA template, and the appropriate amounts of sterile double distilled water. The applied program was set as initial polymerase activation at 95°C, 30 s, then 40 cycles at 95°C, 5 s; 60°C, 35 s. The specificity of the PCR amplification was evaluated with a melt curve analysis from 60°C to 95°C following the final cycle of the PCR. All reactions were set up using four biological replicates. We used UBQ2 as the internal control gene as reported by Calderon-Vazquez et al. , and the transcription levels of each gene were normalized to that of UBQ2 by the 2−△△Ct method. The sequences of the gene-specific primers used for the six transporters and UBQ2 were provided by L. Z. Long, pers. comm.: ZmPht1;1 primers: - 5′-GACCCAGATGGTGTAGAATCGAACAT-3′, and- 5′-TCACTTACTTTCCCGCCTATAACACACA-3′. ZmPht1;2 primers: - 5′-GTCTGGTGAGGCTGAAGACTCAGAGG-3′, and- 5′-ACATGATAGCCCACCATGTGCAGTGC-3′. ZmPht1;3 primers: - 5′-TGTTTCCGTTCTGTCTGGTGCTTGTG-3′, and- 5′-TCCCGACGGTGACCTCCGATTATTTA-3′. ZmPht1;4 primers:- 5′-GAGACCCAGATGGTGTAGAGAATCG-3′, and- 5′-CATCAAAACACAGCCAGGGTTGACT-3′. ZmPht1;5 primers: - 5′-CCAAAGGTAAGTCGCTGGAAGAGAT-3′, and- 5′-CCATTGCGTGCAACAAACAGTGAC-3′. ZmPht1;6 primers: - 5′-CGGACGTGAGCAAGGATGACAA-3′, and- 5′-GGATTCCACACCCCCTGTGTAGT-3′. ZmUBQ2 primers: -5'- CTTTGCTGCTGCACGGGAGGAATG- 3', and-5'- ATGGACGCACGCTGGCTGACTA-3'. [...] Data are presented as means and standard errors (SE). One-way ANOVA (SPSS 13.0, USA) was conducted, and significant differences among means were determined by LSD at the p < 0.05 probability level. To explore the relationship between shoot dry weight and soil Olsen-P, we used relative shoot dry weight by normalizing shoot dry weight data according to the maximum value obtained at each sampling time for each experiment; the data were then fitted to a linear-plateau model using SAS statistical software (SAS 8.1, USA) . The relationships between soil CaCl2-P, root morphological traits, AM colonization, expression of the six Pi transporter genes and soil Olsen-P were plotted using SigmaPlot statistical software (SigmaPlot 10.0, USA). […]

Pipeline specifications

Software tools WinRhizo, SPSS, SigmaPlot
Applications Miscellaneous, Macroscope & basic digital camera imaging
Organisms Zea mays
Chemicals Phosphates, Phosphorus