Comparative Physiological and Transcriptional Analyses of Two Contrasting Drought Tolerant Alfalfa Varieties

Abstract

Drought is one of major environmental determinants of plant growth and productivity. Alfalfa (Medicago sativa) is a legume perennial forage crop native to the arid and semi-arid environment, which is an ideal candidate to study the biochemical and molecular mechanisms conferring drought resistance in plants. In this study, drought stress responses of two alfalfa varieties, Longdong and Algonquin, were comparatively assayed at the physiological, morphological, and transcriptional levels. Under control condition, the drought-tolerant Longdong with smaller leaf size and lower stomata density showed less water loss than the drought-sensitive Algonquin. After exposing to drought stress, Longdong showed less severe cell membrane damage, more proline, and ascorbate (ASC) contents and less accumulation of H2O2 than Algonquin. Moreover, significantly higher antioxidant enzymes activities after drought treatment were found in Longdong when compared with Algonquin. In addition, transcriptional expression analysis showed that Longdong exhibited significantly higher transcripts of drought-responsive genes in leaf and root under drought stress condition. Taken together, these results indicated that Longdong variety was more drought-tolerant than Algonquin variety as evidenced by less leaf firing, more lateral root number, higher relative aboveground/underground biomass per plant and survival rate.

Keywords: alfalfa; drought stress; lateral root; physiological changes; stomata density; transcriptional expression.

FIGURE 1

Quantitative comparison of water status and electrolyte leakage (EL) of two alfalfa varieties differing in drought tolerance. (A) At drought stress 12 days, water loss of Algonquin and Longdong was compared under control condition. (B) LWC under control and drought conditions. (C) EL under control and drought conditions. The results shown are means ± SE (n = 5). Asterisk symbols indicate significant differences from Algonquin at P < 0.05 (Student’s t-test).

FIGURE 2

Evaluation of stomatal density. (A) The upper epidermis and lower epidermis of 4-week-old terminal leaflets were photographed by fluorescence microscope. Bars = 50 μm. (B) Stomatal density of well-watered Longdong and Algonquin (n = 15). The results shown are means ± SE. Asterisk symbols indicate significant differences at P < 0.05 (Student’s t-test).

FIGURE 3

Effect of drought stress on leaf size of two alfalfa varieties. (A) The same part leaves of plant were collected from control and stress conditions after drought treatment 12 days. (B,C) Terminal leaflet length and width of two alfalfa varieties under control and drought conditions (n = 8). The results shown are means ± SE. Asterisk symbols indicate significant differences at P < 0.05 (Student’s t-test).

FIGURE 4

Quantitative analysis of root growth in two alfalfa varieties. (A,B) Roots of Longdong and Algonquin. The root systems of the two varieties were collected from control and stress conditions after drought treatment for 6 days. (C–E) Lateral root number, main root length and lateral root density between two varieties (n = 15). The results shown are means ± SE. Asterisk symbols indicate significant differences from Algonquin at P < 0.05 (Student’s t-test).

FIGURE 5

Plants response to drought stress. (A) Four-week-old plants of Algonquin and Longdong treated with drought stress for 18 days. Plants were photographed after 18 days stress treatment. (B) Survival rates of two varieties after re-watering 7 days were calculated from the results of three independent experiments (n = 20). (C,D) Relative plant height and relative main root length were compared after drought treatment 18 days relative to control (n = 8). (E,F) After drought treatment 18 days, the relative aboveground and underground biomass (DW, dry weight) per plant were analyzed under drought stress relative to control (n = 8). The results shown are means ± SE. Asterisk symbols indicate significant differences from Algonquin at P < 0.05 (Student’s t-test).

FIGURE 6

The contents of proline and ASC of two varieties affected by drought. (A,B) The accumulation of proline and ASC of two alfalfa varieties during drought stress. The results shown are means ± SE (n = 3). Asterisk symbols indicate significant differences from Algonquin at P < 0.05 (Student’s t-test).

FIGURE 7

Changes of H₂O₂ level in two alfalfa varieties after drought treatment. The results shown are means ± SE (n = 3). Asterisk symbols indicate significant differences from Algonquin at P < 0.05 (Student’s t-test).

FIGURE 8

Effect of drought stress on antioxidant enzyme activities. (A–C) SOD, POD, and CAT activities of two varieties during drought stress. The results shown are means ± SE (n = 3). Asterisk symbols indicate significant differences from Algonquin at P < 0.05 (Student’s t-test).

FIGURE 9

Transcriptional expression of drought responsive genes in leaf and root after drought stress. (A–H) Changes in expression of MtP5CS, MtProDH, MtCorA1, MtDehyd, MsNAC, MtCBF4, MtRD2, and MsHSP23 genes in leaf and root of alfalfa plant after 12 days of drought treatment. The results shown are means ± SE (n = 3). Asterisk symbols indicate significant differences at P < 0.05 (Student’s t-test).

FIGURE 10

A model depicting drought tolerance of two contrasting alfalfa varieties.