Luca

Espen

Co-tutor

Characterization of changes in root membrane

proteomes in maize (Zea mays L.) in responses

to nitrogen sources

PhD School on Agriculture, Environment and

Bioenergy

(XXXV cycle, 2019-2021)

Chiara

Muratore PhD Student Bhakti Prinsi Tutor Luca Espen Co-Tutor

• Current position

1st-year PhD Student in Agriculture, environment and bioenergy.

Field of interest: Agricultural chemistry, plant mineral nutrition,

plant proteomics.

• Academic studies

Master’s degree in Food Technology 2017-2019

University of Milan

Department of Food Environmental and Nutritional Sciences, (DeFENS)

Thesis title: "Effects of the debittering process on the Tarwi (Lupinus

mutabilis) protein component”

Thesis supervisor: Scarafoni Alessio

Bachelor’s degree in Food technology 2014-2017

University of Turin

Department of Agricultural, Forest and Food Sciences (Disafa)

Thesis title: "In-shell nuts: microbiologial quality and food safety”

Thesis supervisor: Dolci Paola

Chiara Muratore | chiara.muratore@unimi.it

Nitrogen (N)

Is an essential element throughout plant

growth and development

The low bio-availability of N in the

pedosphere with respect to the request of

cereal crops prompt the use of fertilizers

Major N forms present in soils

‘Nitrogen cycle’ (Lappalainen, H. K., et al., 2005)

RELEVANCE OF THE TOPIC AND STATE OF THE ART

Plants elaborate responses to enhance

nitrogen use efficiency such as uptake

activity and root architecture

Inorganic

(Nitrate, NO3- ;

Ammonium, NH4+)

Organic

(amino acids, peptides, urea)

Inorganic

fertilizers

Organic

fertilization,

biostimulants

THE ROLE OF ROOTS IN N-NUTRITION

WHY FOCUS ON MEMBRANE PROTEOMES?

➢ Perception of N sources

➢ Nutrient uptake

➢ Metabolism (respiration, protein synthesis …)

➢ Communication among organelles (transport,

storage, regulation …)

THE KEY ROLES OF ROOT IN N NUTRITION

➢ PERCEPTION

➢ ADPTATION

➢ SIGNALLING

Taiz et al., (2013)

Britto & Kronzucker, (2002) Forde, G., (2013)

Hordeum vulgare Arabidopsis thaliana L.

➢ How root perceives and adapt to different N-availabilities?

➢ How membrane proteome vary in response to different N-sources?

➢ What is the correlation at physiological and metabolic level?

AIMS OF THE PROJECT

Characterization of changes in

root membrane proteomes in

maize (Zea mays L.)

in responses to nitrogen sources

INTEGRATIVE APPROACH

➢ PHENOTIPICAL CHARACTERIZATION

➢ PHYSIOLOGICAL AND BIOCHEMICAL EVALUATIONS

➢ PROTEOMIC PROFILING

First year Second year Third year

▪ Literature review

▪ Focus on inorganic N-

sources

▪ Optimization of the

hydroponic nutritional

treatments and analytical

procedures for proteome

study

▪ Study of the root

responses to NO3- e

NH4+:

▪ Proteomics

▪ Phenotyping of RSA

▪ Physiology

▪ Focus on of N-organic

sources (amino acids)

▪ Study of the root

responses to amino acids

(Glu, Gln, biostimulants..):

▪ Proteomics

▪ Phenotyping of RSA

▪ Physiology

▪ Visiting PhD exchange

PROJECT SCHEDULE AND MAJOR STEPS

▪ Different timings (plant

growth, nutritional

treatments..)

▪ Validation of the results by

means of bioinformatic

tools, targeted analyses

and complementary

approaches

▪ Thesis write up

• Marschner H: Mineral Nutrition of Higher Plants London: Academic PressLimited; 1995.

• Britto, D. T., & Kronzucker, H. J. (2002). NH4+ toxicity in higher plants: acritical review. Journal of plant physiology, 159(6), 567-584.

• Miller, A. J., & Cramer, M. D. (2005). Root nitrogen acquisition andassimilation. In Root physiology: From gene to function (pp. 1-36).Springer, Dordrecht.

• Forde, B. G., & Lea, P. J. (2007). Glutamate in plants: metabolism,regulation, and signalling. Journal of experimental botany, 58(9), 2339-2358.

• Xu, G., Fan, X., & Miller, A. J. (2012). Plant nitrogen assimilation and useefficiency. Annual review of plant biology, 63, 153-182

• Andrews, M., Raven, J. A., & Lea, P. J. (2013). Do plants need nitrate? Themechanisms by which nitrogen form affects plants. Annals of appliedbiology, 163(2), 174-199.

• Forde BG. Glutamate signaling in roots. J Exp Bot (2014), 65:779-787.DOI: doi:10.1093/jxb/ert335.

• Forde, B. G., & Roberts, M. R. (2014). Glutamate receptor-like channels inplants: a role as amino acid sensors in plant defence?. F1000primereports, 6.

• Buchanan, B. B., Gruissem, W., & Jones, R. L. (Eds.). (2015). Biochemistryand molecular biology of plants. John Wiley & Sons.

• Kiba, T., & Krapp, A. (2016). Plant nitrogen acquisition under lowavailability: regulation of uptake and root architecture. Plant and CellPhysiology, 57(4), 707-714

• Prinsi, B., & Espen, L. (2018). Time-course of metabolic and proteomicresponses to different nitrate/ammonium availabilities in roots andleaves of maize. International journal of molecular sciences, 19(8), 2202.

…

• Hodges, T. K., & Mills, D. (1986). Isolation of the plasma membrane.

In Methods in Enzymology (Vol. 118, pp. 41-54). Academic Press.

• Larsson, C., Widell, S., & Kjellbom, P. (1987). [52] Preparation of high-

purity plasma membranes. In Methods in Enzymology (Vol. 148, pp. 558-

568). Academic Press.

• Alexandersson, E., Saalbach, G., Larsson, C., & Kjellbom, P. (2004).

Arabidopsis plasma membrane proteomics identifies components of

transport, signal transduction and membrane trafficking. Plant and

Cell Physiology, 45(11), 1543-1556.

• Zhang, Z. J., & Peck, S. C. (2011). Simplified enrichment of plasma

membrane proteins for proteomic analyses in Arabidopsis

thaliana. Proteomics, 11(9), 1780-1788.

• Wang, X., Bian, Y., Cheng, K., Zou, H., Sun, S. S. M., & He, J. X. (2012). A

comprehensive differential proteomic study of nitrate deprivation in

Arabidopsis reveals complex regulatory networks of plant nitrogen

responses. Journal of proteome research, 11(4), 2301-2315

• Takahashi, D., Kawamura, Y., & Uemura, M. (2013). Detergent-resistant

plasma membrane proteome to elucidate microdomain functions in

plant cells. Frontiers in plant science, 4, 27

• Yadeta, K., Elmore, J. M., & Coaker, G. (2013). Advancements in the

analysis of the Arabidopsis plasma membrane proteome. Frontiers in

plant science, 4, 86..

• Szymanski, W. G., Zauber, H., Erban, A., Gorka, M., Wu, X. N., & Schulze,

W. X. (2015). Cytoskeletal components define protein location to

membrane microdomains. Molecular & Cellular Proteomics, 14(9), 2493-

2509.

…

LITERATURE REVIEW: some examples

Nitrogen nutrition

in model plants and crops

Subcellular and membrane

proteomics

Proteomics has the potential to

effectively profile membrane proteins

and to provide unprecedented

insight into the protein composition

of a given sample

Subcellular and membrane

proteomics are challenging:

In comparison to soluble proteins,

There is still a lack of knowledge about

membrane proteomes due to protein

chemo-physical peculiarities:• heterogeneity,

• hydrophobicity,

• low abundance

Overview of typical PM proteomics experiments (Yadeta et al., 2013)

LITERATURE REVIEW

Plant nutritional status

Determination of the contents of Nitrate and Ammonium

WORK IN PROGESS

Root system architecture (RSA)

Ongoing evaluation through ImageJ

Values are the means ± standard error (SE) (n = 3). The statistical significance was

assessed by ANOVA (p < 0.05, Tukey post hoc method).

Experimental design & nutritional treatments

Hybrid line P1817

WORK IN PROGRESS

EXTRACTION YELD

REPRODUCIBILITY

PURITY OF MEMBRANE FRACTIONS

(Immunoblotting analysis)

Ongoing evaluations

Workflow for the enrichment of different fraction of membrane proteins

Muratore, C., Espen, L., Prinsi, B. (2020, February).Characterization

of changes in root membrane proteomes in maize (Zea mays L.) in

responses to nitrogen sources. Poster session presented at

Agricultural Chemistry Winter School, “Plant-soil-microbe interactions

and ecosystem dynamics in a changing environment”, University of

Turin, Turin.

Courses & Seminars

“Introduzione all’esplorazione dati in R”

(2019, November)

By Kater Martin

WORK IN PROGRESS

EDUCTIONAL &TRAINING ACTIVITIES

PhD Schools

Muratore, C., Espen, L., Prinsi, B. (2020, February).Characterization

of changes in root membrane proteomes in maize (Zea mays L.) in

responses to nitrogen sources. Poster session presented at

Agricultural Chemistry Winter School, “Plant-soil-microbe interactions

and ecosystem dynamics in a changing environment”, University of

Turin, Turin.

Courses & Seminars

“Introduzione all’esplorazione dati in R”

(2019, November)

By Kater Martin

WORK IN PROGRESS

EDUCTIONAL &TRAINING ACTIVITIES

PhD Schools

Thank you for your attention!