Plant physiology and proteomics reveals the leaf response to drought in alfalfa (Medicago sativa L.)

Abstract

Despite its relevance, protein regulation, metabolic adjustment, and the physiological status of plants under drought is not well understood in relation to the role of nitrogen fixation in nodules. In this study, nodulated alfalfa plants were exposed to drought conditions. The study determined the physiological, metabolic, and proteomic processes involved in photosynthetic inhibition in relation to the decrease in nitrogenase (N(ase)) activity. The deleterious effect of drought on alfalfa performance was targeted towards photosynthesis and N(ase) activity. At the leaf level, photosynthetic inhibition was mainly caused by the inhibition of Rubisco. The proteomic profile and physiological measurements revealed that the reduced carboxylation capacity of droughted plants was related to limitations in Rubisco protein content, activation state, and RuBP regeneration. Drought also decreased amino acid content such as asparagine, and glutamic acid, and Rubisco protein content indicating that N availability limitations were caused by N(ase) activity inhibition. In this context, drought induced the decrease in Rubisco binding protein content at the leaf level and proteases were up-regulated so as to degrade Rubisco protein. This degradation enabled the reallocation of the Rubisco-derived N to the synthesis of amino acids with osmoregulant capacity. Rubisco degradation under drought conditions was induced so as to remobilize Rubisco-derived N to compensate for the decrease in N associated with N(ase) inhibition. Metabolic analyses showed that droughted plants increased amino acid (proline, a major compound involved in osmotic regulation) and soluble sugar (D-pinitol) levels to contribute towards the decrease in osmotic potential (Ψ(s)). At the nodule level, drought had an inhibitory effect on N(ase) activity. This decrease in N(ase) activity was not induced by substrate shortage, as reflected by an increase in total soluble sugars (TSS) in the nodules. Proline accumulation in the nodule could also be associated with an osmoregulatory response to drought and might function as a protective agent against ROS. In droughted nodules, the decrease in N(2) fixation was caused by an increase in oxygen resistance that was induced in the nodule. This was a mechanism to avoid oxidative damage associated with reduced respiration activity and the consequent increase in oxygen content. This study highlighted that even though drought had a direct effect on leaves, the deleterious effects of drought on nodules also conditioned leaf responsiveness.

Fig. 1.

Water availability effect on (A) leaf soluble sugar (sucrose, glucose, fructose, pinitol, and total soluble sugar, TSS) and (B) free amino acid (Ala, alanine; Asp, aspartic acid; Glu, glutamic acid; Ser, serine; Asn, asparagine, and Pro, proline) content in Medicago sativa plants. Measurements were conducted at the end of the experiment. The y-axis scale for asparagine and proline was modified to make the text more understandable. In addition, since the proline values detected in control plants were low, the average value ±SE was added to the figure. Each value represents the mean ±SE (n= 4). Different letters indicate significant differences (P <0.05) between treatments.

Fig. 2.

Water availability effect on (A) nodule soluble sugar (sucrose, glucose, fructose, pinitol, and total soluble sugar, TSS) and (B) free amino acid (Ala, alanine; Asp, aspartic acid; Glu, glutamic acid; Ser, serine; Asn, asparagine, and Pro, proline) content in Medicago sativa plants. Measurements were conducted at the end of the experiment. Each value represents the mean ±SE (n=4). The different letters indicate significant differences (P <0.05) between treatments.

Fig. 3.

Silver-stained two-dimensional gel of proteins extracted from Medicago sativa leaves grown under control (A) and drought (B) conditions. In the first dimension, 125 mg of total protein was loaded on a 18 cm IEF strip with a linear gradient of pH 4–7. The second dimension was conducted in 12% polyacrylamide (w/v) gels (20×20 cm) (for details, see Materials and methods). The gel image analyses conducted with Progenesis SameSpots software v3.0 and the subsequent mass spectrometry analyses identified up to 26 proteins (marked by arrows) that, statistically, were involved in the plant response to drought.