Selected Reaction Monitoring (SRM) frente a Western Blot

We propose the SRM technology as a complementary method to the Western Blot for the detection and quantification of proteins in a sample. The technique Western Blot has its own limitations: i) only a protein-of-choice is detected, ignoring any non-relevant proteins, ii) the sensitivity of the technique depends on the specificity of the antibody and iii) Western Blot is expensive and time-consuming. The advantages of SRM with respect Western Blot are remarkable: i) you can detect up to hundreds of different proteins in a sample, ii) SRM is more sensitive, because just 50 copies of the target protein per cell are enough for the detection and iii) once it has been made an investment in the necessary machinery to develop this technique, the detection of proteins in a sample turns into a cheaper, faster, more specific and full-quantitative procedure, without the need of using antibodies. First of all, SRM requires the identification of little peptides, obtained by tryptic digestion, whose sequence must be unique for a single protein or isoform. There is software for that aim. Then, it’s necessary to create isotope-labeled peptides of that identified for acting as internal standards. That sample is introduced in a triple quadrupole mass spectrometer: it passes through a first quadrupole, which functions as a filter, where the fragments are selected, previously ionized, attending to the mass/charge (m/z) relation that correspond to that unique fragments of the protein of interest. In this first selection may be other peptides from other proteins, with the same m/z but with different sequence. To select those that are exclusive from the target protein, the fragments are moved to a second quadrupole, where they are fragmented again with a physical method, and so new smaller fragments are generated. All the new fragments are conduced to the third quadrupole, where just those which come from the protein of interest are selected, attending at their m/z again. The target peptide concentration is determined by measuring the observed signal response for the target peptide relative to that of the isotopic-labeled peptide, the concentration of which is calculated from a pre-determined calibration-response curve. Calibration curves have to be generated for each target peptide in the sample. Because SRM technology is increasing its use, there have been developed databases where the scientific community upload information about protocols and standards for each protein with the aim to facilitate the work to other researchers

Involvement of miRNAs in the short-term response of pine roots to ammonium nutrition

Post-transcriptonal gene regulation is an essential control point regarding gene expression programs. A class of small non-coding RNAs, microRNAs (miRNAs) play important roles during this process. They act on target mRNAs through post-transcriptional gene silencing, either by endonuclease mediated transcript cleavage or by translational repression of targeted mRNAs (Pattanayak et al., 2013). Thus, miRNAs are involved in the regulation mechanisms of important plant processes, including the regulation of the processes related to nitrogen nutrition (Gutiérrez, 2012). In the present work, maritime pine (Pinus pinaster Aiton) was used to study the role of miRNAs regarding NH4+ nutrition in conifers, a group of plants that exhibits tolerance to NH4+ nutrition compared to NO3- nutrition specially during seedling stage Ortigosa et al., 2022). This fact is of high interest since numerous NH4+ nutrition can negatively affect the growth and development of different crops. The global miRNA expression has been characterized in the roots of maritime pine seedlings after 2 h and 24 h from fertilization with two levels of NH4+ solutions (0.1 mM and 3 mM). The miRNA-seq analysis revealed 271 new miRNAs with an identified precursor although only 4 were differentially expressed. Gutiérrez RA. 2012. Science, 336:1673-1675. Ortigosa F, et al. 2022. Plant Cell and Environment, 45,915-935. Pattanayak D, et al. 2013. Plant Molecular Biology Reporter, 31, 493-506.Acknowledgements & Funding. This work was supported by the grants BIO2015-73512-JIN MINECO/AEI/FEDER, UE; P20_00036 PAIDI 2020/FEDER, UE, and B4-2021-01 (Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech). JMVM was supported by the grant FPU17/03517

Efecto combinado de la nutrición nitrogenada y concentración de CO2 en biomasa y perfiles de expresión génica en Pinus pinaster

Carbon dioxide (CO2) in high concentration is beneficial for crop development, but due to the reduction of photorespiration in C3 plants, there is less reducing power available for nitrate (NO3-) reduction and later nitrogen assimilation compared to ammonium (NH4+) nutrition. To overcome this problem, research is focusing on NH4+ nutrition, because its assimilation is less expensive in terms of energy (Bloom, 2015). Knowing how plants manage NH4+ toxicity by gene expression and enzymatic pathways activation, will result in a novel set of molecular mechanisms that could be applied in crops in the near future, solving the problem of photorespiration reduction and nitrogen use efficiency (South et al., 2018). In this work we studied the growth of Pinus pinaster (Aiton) seedlings in 400 and 720 ppm CO2 concentrations and under NO3- and NH4+ nutrition, due to the tolerance that conifers have for NH4+ (Marino et al., 2022). Results show that during the early development, the combination of high CO2 concentration and NH4+ leads to an increase in biomass of the seedling and growth rates. To further investigate the set of genes differentially regulated in these conditions of nitrogen nutrition and CO2 concentration, we analysed the data from RNA-seq experiments from different organs of 2 months seedlings growth in 400 and 720 ppm of CO2 and different nitrogen nutrition (8 mM NH4+ or 8 mM NO3-).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech