Morpho-Physiological and Proteome Level Responses to Cadmium Stress in Sorghum

Abstract

Cadmium (Cd) stress may cause serious morphological and physiological abnormalities in addition to altering the proteome in plants. The present study was performed to explore Cd-induced morpho-physiological alterations and their potential associated mechanisms in Sorghum bicolor leaves at the protein level. Ten-day-old sorghum seedlings were exposed to different concentrations (0, 100, and 150 μM) of CdCl2, and different morpho-physiological responses were recorded. The effects of Cd exposure on protein expression patterns in S. bicolor were investigated using two-dimensional gel electrophoresis (2-DE) in samples derived from the leaves of both control and Cd-treated seedlings. The observed morphological changes revealed that the plants treated with Cd displayed dramatically altered shoot lengths, fresh weights and relative water content. In addition, the concentration of Cd was markedly increased by treatment with Cd, and the amount of Cd taken up by the shoots was significantly and directly correlated with the applied concentration of Cd. Using the 2-DE method, a total of 33 differentially expressed protein spots were analyzed using MALDI-TOF/TOF MS. Of these, treatment with Cd resulted in significant increases in 15 proteins and decreases in 18 proteins. Major changes were absorbed in the levels of proteins known to be involved in carbohydrate metabolism, transcriptional regulation, translation and stress responses. Proteomic results revealed that Cd stress had an inhibitory effect on carbon fixation, ATP production and the regulation of protein synthesis. Our study provides insights into the integrated molecular mechanisms involved in responses to Cd and the effects of Cd on the growth and physiological characteristics of sorghum seedlings. We have aimed to provide a reference describing the mechanisms involved in heavy metal damage to plants.

Fig 1. Schematic representation of the experimental setup used to compare Cd-treated sorghum seedlings with untreated plants (Control).

The sorghum seedlings (BTX 623) were grown in growth chambers for 10 days in Hoagland solution. Ten-day-old sorghum seedlings were exposed to different concentrations of cadmium (0 μM, 100 μM, 150 μM CdCl₂) for 5 days. The samples were collected from the control and Cd-induced leaves for measurement of morphological and physiological parameters (shoot lengths, fresh weights, relative water content, confocal and ion analysis). For proteome analysis, leaves were excised, pooled, rinsed with de-ionized water, rapidly frozen in liquid nitrogen, and stored at -80°C. Molecular changes were investigated in S. bicolor using two-dimensional gel electrophoresis (2-DE) in samples derived from the leaves of both control and Cd-treated seedlings.

Fig 2. Responses induced by cadmium stress in morphological alterations in sorghum seedlings (A. Shoot length, B. Root length, C. Fresh weight, D. Relative water content) exposed to different concentrations of cadmium.

Ten-day-old sorghum seedlings (BTX 623) were exposed to different concentrations of cadmium (0 μM, 100 μM, 150 μM CdCl₂) for 5 days. After 5 days of Cd stress, the leaves and roots were collected from both the control and Cd-treated seedlings. Prior to measuring morphological and physiological parameters, the seedlings were washed with de-ionized water. Three plants were randomly selected for measurements at each time point for each replicate, and the experiment was biologically replicated 3 times. Each bar represents the average ± SE for 3 plants. Significant differences between the control and the cadmium-induced seedlings were determined by performing a one-way analysis of variance (ANOVA) with Tukey’s all pairs of column comparison test. Asterisks indicate significant differences at p < 0.05.

Fig 3. Ion concentrations and cadmium accumulation in sorghum leaves (A. Cd²⁺, B. Zn²⁺, C. Ca²⁺, and D. Fe²⁺) exposed to cadmium stress.

Ten-day-old sorghum seedlings (BTX 623) were exposed to different concentrations of cadmium (0 μM, 100 μM, 150 μM CdCl₂) for 5 days. Values (means ± SD) were determined for 3 independent experiments (n = 3).

Fig 4. Cadmium distribution and fluorescence intensities in sorghum seedling leaves treated with dithizone staining.

A. Control, B. 100 μM CdCl₂, C. 150 μM CdCl₂, D. Effect on the fluorescence intensity of cadmium-dithizone complexes. Samples obtained from different cadmium-treated leaves were placed on slides, mounted in mounting solution and observed using confocal microscopy (LSM 410; Carl Zeiss, Jena, Germany).

Fig 5. Cluster analysis of cadmium-responsive leaf proteins.

Ten-day-old sorghum seedlings were exposed to cadmium stress for 5 days. Samples from non-treated control plants and treated plants were collected on the same day. Differences in the intensity of labeling associated with the proteins in both the control and the cadmium-treated samples are shown as clusters. Any statistically significant difference (p < 0.05) in labeling intensity was considered to be positive. The protein spot numbers are indicated on the right side of the cluster. The clusters were determined using Genesis software (ver. 1.7.6).

Fig 6. Representative images of gels used in 2-DE analysis of S. bicolor leaves exposed to 0 μM, 100 μM and 150 μM CdCl₂.

Leaf tissues were extracted using TCA-acetone precipitation method as described in the Materials and Methods section. Proteins were extracted from the leaves of 15-day-old seedlings that were treated with cadmium for 5 days. For IEF, 100 μg of proteins was loaded onto pH 3–10 NL IPG strips (7 cm). SDS-PAGE was performed on 12% gels, and the proteins were separated using 2-DE and then stained with silver staining. The differentially expressed protein spots (>1.5-fold difference) are indicated by circles on the 2-D gel map. These spots were found to be statistically significant at a level of 95% per group (Student’s t-test) using biological and analytical replicates (n = 3). The MW of each protein was determined using standard protein markers.

Fig 7. Magnified views of some of the differentially expressed protein spots that correspond to the identified proteins.

The protein spots that were identified using mass spectrometry are indicated by squares and labeled in the figure.

Fig 8. Protein encoding gene functions of 33 differentially expressed proteins identified in the leaves of S. bicolor.

The frequency distribution for the identified proteins within functional categories was determined based on their molecular functions (A), cellular localization (B), and their involvement in biological processes (C). Classifications were made using iProClass databases, and the assignment of functions was based on gene ontology.

Fig 9. A model of the putative subcellular localization of the leaf proteins identified in S. bicolor seedlings to be affected by exposure to Cd.

The Cd-responsive proteins are indicated as follows: up-regulated proteins are indicated by “↑”, and down-regulated proteins are indicated by “↓”. The proteins were categorized with regard to their localization in cellular components using iProClass databases, and the assignment of functions was based on gene ontology.