Medicago truncatula root nodule proteome analysis reveals differential plant and bacteroid responses to drought stress

Abstract

Drought is one of the environmental factors most affecting crop production. Under drought, symbiotic nitrogen fixation is one of the physiological processes to first show stress responses in nodulated legumes. This inhibition process involves a number of factors whose interactions are not yet understood. This work aims to further understand changes occurring in nodules under drought stress from a proteomic perspective. Drought was imposed on Medicago truncatula 'Jemalong A17' plants grown in symbiosis with Sinorhizobium meliloti strain 2011. Changes at the protein level were analyzed using a nongel approach based on liquid chromatography coupled to tandem mass spectrometry. Due to the complexity of nodule tissue, the separation of plant and bacteroid fractions in M. truncatula root nodules was first checked with the aim of minimizing cross contamination between the fractions. Second, the protein plant fraction of M. truncatula nodules was profiled, leading to the identification of 377 plant proteins, the largest description of the plant nodule proteome so far. Third, both symbiotic partners were independently analyzed for quantitative differences at the protein level during drought stress. Multivariate data mining allowed for the classification of proteins sets that were involved in drought stress responses. The isolation of the nodule plant and bacteroid protein fractions enabled the independent analysis of the response of both counterparts, gaining further understanding of how each symbiotic member is distinctly affected at the protein level under a water-deficit situation.

Figure 1.

Influence of the extraction method on bacteroid protein contamination in the plant fraction of M. truncatula root nodules. Homogenization using liquid nitrogen was compared to homogenization using an extraction buffer (adapted from Saalbach et al., 2002). Plant and bacteroid fractions were separated by centrifugation and aliquots (10 μg of protein) were loaded onto a SDS gel. Immunoblot was performed using antibodies against bacterial NifDK as a marker for bacteroid contamination. Fractions 1 and 2 are the plant and bacteroid fractions obtained when using homogenization buffer. Fractions 3 and 4 are the plant and bacteroid fractions obtained when nodules were homogenized using liquid nitrogen. Molecular mass markers are shown on the left. Predicted values for S. meliloti NifD and NifK are 56.5 and 57.6 kD, respectively.

Figure 2.

Diagram of the experimental work flow. Two types of analyses have been performed: The M. truncatula plant nodule proteome was profiled using FPLC and 2D-LC/MS/MS and a quantitative study of plant and bacteroid responses to drought stress at the protein level was carried out using 1D-LC/MS/MS. SCX, Strong cation exchange; RP, reverse phase.

Figure 3.

Functional classification of the identified proteins in the 2D-LC/MS/MS analysis of M. truncatula nodule plant fraction. Proteins were functionally classified using Mapman, which is based on Gene Ontology Consortium and Kyoto Encyclopedia of Genes and Genomes database. OPP, Oxidative pentose pathway.

Figure 4.

Effect of drought stress on nodule water potential (A) and SNF as a measure of ANA (B) in M. truncatula ‘Jemalong’ plants. Control plants are represented in white, drought-stressed plants in black. For each parameter, an asterisk represents statistically significant differences with the corresponding control value at P ≤ 0.05. Bars indicate the se (n = 6 biological replicates).

Figure 5.

Graphical representation of ICA results on the nodule proteome during drought. Plant analysis is represented in A, bacteroid analysis in B. C represents the result of the integration of both plant and bacteroid data sets. C3 and D3 stand for control and drought at day 3 of treatment. C6 and D6 represent control and drought at day 6 of treatment. The ICA graphs allow for sample pattern recognition. Each point in the graph represents the whole protein profile of one biological sample.