Comparative Physiological and Molecular Analyses of Two Contrasting Flue-Cured Tobacco Genotypes under Progressive Drought Stress

Abstract

Drought is a major environmental factor that limits crop growth and productivity. Flue-cured tobacco (Nicotiana tabacum) is one of the most important commercial crops worldwide and its productivity is vulnerable to drought. However, comparative analyses of physiological, biochemical and gene expression changes in flue-cured tobacco varieties differing in drought tolerance under long-term drought stress are scarce. In this study, drought stress responses of two flue-cured tobacco varieties, LJ851 and JX6007, were comparatively studied at the physiological and transcriptional levels. After exposing to progressive drought stress, the drought-tolerant LJ851 showed less growth inhibition and chlorophyll reduction than the drought-sensitive JX6007. Moreover, higher antioxidant enzyme activities and lower levels of H₂O₂, Malondialdehyde (MDA), and electrolyte leakage after drought stress were found in LJ851 when compared with JX6007. Further analysis showed that LJ851 plants had much less reductions than the JX6007 in the net photosynthesis rate and stomatal conductance during drought stress; indicating that LJ851 had better photosynthetic performance than JX6007 during drought. In addition, transcriptional expression analysis revealed that LJ851 exhibited significantly increased transcripts of several categories of drought-responsive genes in leaves and roots under drought conditions. Together, these results indicated that LJ851 was more drought-tolerant than JX6007 as evidenced by better photosynthetic performance, more powerful antioxidant system, and higher expression of stress defense genes during drought stress. This study will be valuable for the development of novel flue-cured tobacco varieties with improved drought tolerance by exploitation of natural genetic variations in the future.

Keywords: ROS; antioxidant enzymes; drought; gene expression; tobacco.

Figure 1

Effects of drought stress on dry weight (A), chlorophyll content (B), relative leaf water content (C) and leaf water potential (D) in both flue-cured tobacco varieties. LJ851-CK stands for LJ851 under control conditions; LJ851-TR stands for LJ851 under drought conditions; JX6007-CK stands for JX6007 under control conditions; JX6007-TR stands for JX6007 under drought conditions. Bar indicates SE. ^*t-test, with P < 0.05; ^**t-test, with P < 0.01.

Figure 2

Dynamic changes of MDA content (A), Ion leakage (B), and H₂O₂ (C) in both genotypes of flue-cured tobacco plants during 14 days of drought stress. LJ851-CK stands for LJ851 under control conditions; LJ851-TR stands for LJ851 under drought conditions; JX6007-CK stands for JX6007 under control conditions; JX6007-TR stands for JX6007 under drought conditions. Bar indicates SE. ^*t-test, with P < 0.05; ^**t-test, with P < 0.01.

Figure 3

Dynamic changes of SOD (A), CAT (B), and POD (C) activities in both types of tobacco plants during 14 days of drought stress. LJ851-CK stands for LJ851 under control conditions; LJ851-TR stands for LJ851 under drought conditions; JX6007-CK stands for JX6007 under control conditions; JX6007-TR stands for JX6007 under drought conditions. Bar indicates SE. ^*t-test, with P < 0.05; ^**t-test, with P < 0.01.

Figure 4

Dynamic changes of Nicotine (A) and Reducing sugar (B) contents in both types of flue-cured tobacco plants during 14 days of drought stress. LJ851-CK stands for LJ851 under control conditions; LJ851-TR stands for LJ851 under drought conditions; JX6007-CK stands for JX6007 under control conditions; JX6007-TR stands for JX6007 under drought conditions. Bar indicates SE. ^*t-test, with P < 0.05; ^**t-test, with P < 0.01.

Figure 5

Dynamic changes of photosynthetic parameters in both types of tobacco plants during 14 days of drought stress. (A) Net photosynthetic rate; (B) Stomatal conductance; (C) Intercellular CO₂ concentration. LJ851-CK stands for LJ851 under control conditions; LJ851-TR stands for LJ851 under drought conditions; JX6007-CK stands for JX6007 under control conditions; JX6007-TR stands for JX6007 under drought conditions. Data are means ± SE calculated from three replicates. Bar indicates SE. ^*t-test, with P < 0.05; ^**t-test, with P < 0.01.

Figure 6

Expression profiles of genes for photosynthesis-related proteins in both types of tobacco plants after drought stress. RNA was extracted from stressed leaves sampled after 10 days of drought treatment and reverse-transcribed to synthesize cDNA, which was used for qPCR analysis with primers specific for six photosynthesis-related genes psbA (A), psbB (B), psbC (C), psbD (D), RBCL (E), and clpP1(F). Data represented means ± SE of three biological replicates. Bar indicates SE. ^**t-test, with P < 0.01; ^*t-test, with P < 0.05.

Figure 7

Transcriptional expression of osmolyte biosynthesis and dehudrin-type genes in leaf and root under drought stress. RNA was extracted from 10-day stressed leaves and roots and reverse-transcribed to synthesize cDNA, which was used for qPCR analysis with primers specific for four genes P5CS (A), ADC2 (B), LEA5 (C), and ERD10C (D). Data represented means ± SE of three biological replicates. Bar indicates SE. ^**t-test, with P < 0.01; ^*t-test, with P < 0.05.

Figure 8

Transcriptional expression of antioxidant-related and chaperon proteins in leaf and root under drought stress. RNA was extracted from 10-day stressed leaves and roots and reverse-transcribed to synthesize cDNA, which was used for qPCR analysis with primers specific for four genes SOD1 (A), CAT1 (B), HSP70-1 (C), and HSP23 (D). Data represented means ± SE of three biological replicates. Bar indicates SE. ^**t-test, with P < 0.01; ^*t-test, with P < 0.05.

Figure 9

Transcriptional expression of signaling and transcription factor genes in leaf and root under drought stress. RNA was extracted from 10-day stressed leaves and roots and reverse-transcribed to synthesize cDNA, which was used for qPCR analysis with primers specific for four genes AREB (A), NAC1 (B), DREB2 (C), and CDPK2 (D). Data represented means ± SE of three biological replicates. Bar indicates SE. ^**t-test, with P < 0.01; ^*t-test, with P < 0.05.

Figure 10

Scatter plot of the top two principal components (including 15 physiological biochemical and gene transcript parameters) under drought stress. The proportion of variance for principal component analysis based on the drought stress index (DSI) of 15 physiological traits is shown in the figure, showing that PC1 and PC2 explain 92.48% of total variation.