Rice (Oryza sativa
L.) is an important food crop that feeds more than half of the world’s population [1
]. Global food security is being challenged by the convergence of multiple factors, including the continuously growing population, reduced arable land, demand for biofuel production, and abiotic stress [2
]. Using genetic engineering biotechnology to improve cold-stress resistance of rice is an alternative strategy. This includes using genetic engineering biotechnology to improve the cold resistance of rice.
Low temperature is one of several environmental stress factors that are applied in research aimed at improving the growth, productivity, and quality of crops [3
]. To adapt to the stress, plants have developed many ways to balance the effects of stress-induced damage, such as increasing the contents of proline, the activity of detoxifying substances or enzymes such as superoxide dismutase (SOD), and peroxidase (POD) [4
]. Cell membranes are major sites of freezing injury and cold acclimation. Membrane fluidity is important to sustain the functional activity of membrane proteins and the membranes themselves and is directly affected by temperature [5
]. Lipids are an essential component of cell membranes. Plant seeds store lipids as a food reserve for germination and seedling growth. The predominant component of seed lipids, triacylglycerols (TAGs) are not only essential for human nutrition, but also valuable as feedstocks for various industrial products and biofuels [6
]. In plants, the level of polyunsaturated fatty acids (PUFAs) are essential for cold acclimation and is essential for the regulation of cholesterol synthesis and transportation for the maintenance of cellular membranes [7
PUFAs are essential for cold stress. Regulating the expression of the PtFAD2 enzymes could potentially alter PUFAs content in membrane lipids [7
]. Genetic manipulation of the levels of PUFAs has led to the modification of cold sensitivity in tobacco plants [9
]. Changes in freezing tolerance in hybrid poplar was caused by up- or downregulation of PtFAD2
gene expression [7
]. PUFAs are the main component of soybean oil [10
]. Genes controlling the oleic acid and PUFA contents in soybean seed oil have been characterized, containing the two oleate desaturase genes GmFAD2-1A and GmFAD2-1B and three linoleate desaturase genes GmFAD3A, GmFAD3B, and GmFAD3C [11
]. Mutation of GmFAD3
resulted in lower linolenic acid content (from 7% to 10%) [10
]. A tobacco FAD3 expressed in rice could increase α-linolenic acid (ALA, C18:3) level up to 2.5-fold [14
]. The ALA content was increased up to a 13-fold when soybean FAD3 driven by the maize ubiquitin-1 promoter was introduced in rice [15
]. However, the function of GmFAD3A
to enhance rice to confer cold tolerance is still unclear.
In this study, the GmFAD3A was allogenous expressed in rice. The ectopic expression of GmFAD3A enhances cold stress tolerance in rice, including seed germination rates at low temperature (15 °C) and cold tolerance at the seeding stage. We also evaluated lipid content, the malondialdehyde (MDA), proline content, superoxide dismutase (SOD), and peroxidase (POD) activities to explain its cold stress tolerance.
Abiotic stress is a major concern for agriculture worldwide and is responsible for the loss of crop production [17
]. Low temperature is an important damaging factor affecting rice growth, development, and productivity in southern China [18
]. Conventional breeding of cultivated rice has generated increasing yield potential and yield stability. Ectopic expression of the RsICE1
gene enhanced tolerance to low-temperature stress in rice [20
]. In this study, we demonstrated that ectopic expression of GmFAD3A increased lipid contents, and resistance to cold stress in rice. Our results provide a new insight into plant-stress tolerance-related genes and will be useful for improving crop resistance.
In plants, the desaturation of linoleic acid (LA) to ALA occurs in plastids and endoplasmic reticulum (ER). Mutants of linoleate desaturase genes (GmFAD3), decreased the linolenic acid content in commercial soybeans by 98% [10
]. Ectopic expression of soybean oleosin genes significantly increased the lipid content in transgenic rice seeds [21
]. ER-type ω
-3 fatty acid desaturase catalyzes the conversion of 18:2 to 18:3 in phospholipids. The lipid transfer resulting from both sets of ω-3 fatty acid desaturases contributes to the total cellular 18:3 content [22
]. The ALA contents in the seeds of GmFAD3A
(an ER-localized gene) overexpressing lines increased significantly [23
]. In this study, we also detected a substantial increase in level of oleic acid (+5.6% and +21.5%) and linolenic acid (+25.1% and +47.2%) in GmFAD3A
transgenic lines compared with WT. However, whether the GmFAD3A protein has a function in rice growth and cold stress in transgenic rice remains a subject for further investigation.
Polyunsaturated fatty acids (PUFAs) are essential for cold acclimation [7
]. Genetic manipulation of the levels of PUFAs has led to the modification of cold tolerance in tobacco and poplar trees [7
]. The unsaturation of lipids has been shown to protect the photosynthetic machinery from photoinhibition at low temperatures [24
]. The total polyunsaturated fatty acids levels in GmFAD3A
transgenic lines (OE4-3: 4.271 mg/g and OE8-5: 4.983 mg/g) were largely changed compared with WT (3.945 mg/g; Table 1
). The seed germination rates of GmFAD3A
transgenic lines increased significantly under low temperature condition (15 °C) compared with WT (Figure 3
). Furthermore, the survival and growth rates of GmFAD3A
-OE seedlings increased significantly compared with WT at 4 °C. This evidence supported the function of GmFAD3A in cold stress.
Cold stress mediates a series of physiological and metabolite changes, such as alterations in chlorophyll fluorescence, electrolyte leakage, reactive oxygen species (ROS), malondialdehyde (MAD), sucrose, lipid peroxides, proline, and other metabolites [19
]. The MDA contents in GmFAD3A
transgenic lines were lower (by −0.03 and −0.04 μmol/mg) than WT under cold stress after 2 d and 4 d, respectively. It was suggested that ectopic expression of GmFAD3A in rice reduced the membrane caused lipid peroxidation caused by cold stress. At the same time, the proline content and antioxidant enzymes activity were observed as being higher than that in the WT under cold stress, which might serve as the frontline of defense against oxidative stress. Thus, we suggested that the ectopic expression of GmFAD3A
in rice could protect the rice from oxidative damage under cold stress by increasing polyunsaturated fatty acids, antioxidant enzyme activities, and the proline content.
In summary, the data presented here demonstrated that GmFAD3A plays a critical role in cold tolerance and low-temperature germination in rice.
4. Materials and Methods
4.1. Plant Materials and Growth Conditions
cDNA of soybean (Glycine max) was used as template to amplify the full-length coding sequence of GmFAD3A. Oryza sativa ssp. japonica cv. Zhonghua 11 (ZH11) was used as the background. WT (ZH11) and GmFAD3A transgenic rice were used for the phenotypic trait analyses. WT and transgenic materials were respectively grown under natural field conditions in Nanchang University, Nanchang, China.
4.2. Structure and Sequence Analysis of GmFAD3A
Amino acid sequences of 14 FAD proteins were obtained from rice and soybean. The multiple alignments of amino acid sequences and the neighbor-joining (NJ) phylogenetic tree construction were performed according to previous research [25
4.3. Plasmid Construction and Rice Transformation
The full-length coding region of GmFAD3A was amplified with specific primers FAD3A-C (Table S1
) and cloned into a pCAMBIA1301 binary vector. The construction was sequenced to ensure its integrity. The recombinant vector was maintained in Escherichia coli
DH5α, and then introduced into Agrobacterium tumefaciens
EHA105. The construction was transferred into Zhonghua 11 by Agrobacterium
-mediated transformation, as previously described [26
4.4. RNA Isolation and Quantitative RT-PCR Analyses
The leaves from WT and GmFAD3A transgenic plants used for RNA isolation were frozen in liquid nitrogen and were then stored at −80 °C. Total RNAs were extracted using TRIzol reagent (TransGen, Beijing, China) according to the manufacturer’s protocol. First-strand cDNA was synthesized using PRIME Script Reverse Transcriptase (TaKaRa, Dalian, China, http://www.takara.com.cn/
). Quantitative real-time PCR (qRT-PCR) was carried out using an ABI StepOne™ Real-time PCR instrument (Applied Biosystems, Carlsbad, CA, USA, http://www.appliedbiosystems.com/
) and Maxima SYBR Green qPCR Master Mix (Thermo, Waltham, MA, USA). Relative expression levels were calculated via the 2−ΔΔCT
]. The gene-specific primers used for qRT-PCR are listed in Table S1
4.5. Analysis of Lipid Content
The crude lipid content was studied using the SoxtecTM 2050 Auto Fat Extraction (Foss®
Analytic, Hilleroed, Denmark) according to the Soxtec method [1
]. Dehulled rice grains were ground to powder. A quantity of 3.3 g powder was added into the extraction unit, while solvent was added to the extraction cups in a closed system. The extraction consisted of four steps: boiling, rinsing, solvent recovery, and pre-drying. The results were calculated as total amount of fat (g) per 100 g powder. All measurements were performed with three replicates.
The fatty acid (FA) in seeds was extracted using a chloroform-methanol method according to previous study [27
]. Quantification of the FA content was performed using GC-MS referring to the method [28
]. C13:0 was used as the internal standard. All measurements were performed with three replicates.
4.6. Seed Germination Assays
To determine seed germination, 30 seeds of ZH11 and GmFAD3A T2 homozygous line (OE8-5) were sterilized and spread on 1/2MS medium. Seeds were placed in a growth chamber with light/dark cycle (12 h/12 h) at 15 °C or 28 °C. Germination was defined as the emergence of the radicles through the seed coat. The germination rate was calculated from the results of three independent experiments.
4.7. Cold Stress Tolerance Experiment
For low-temperature treatment, all rice seedlings including ZH11 and GmFAD3A homozygous line (OE8-5) were grown in 1/2 MS medium and normal conditions (28 °C, 16 h light and 8 h dark). 14-day-old seedlings were transferred to a growth chamber at 4 °C for 7 days. The survival ratio, fresh weight, and plant root or shoot length were calculated after stress or recovery in each pot according to the reported methods [29
]. 20 independent transgenic plants and 20 ZH11 seedlings were tested in each replicate. Cold treatments were applied in three independent biological replicates.
4.8. Physiological and Biochemical Measurements
Samples for physiological and biochemical measurements assay were collected from non-stressed and cold stressed plants. To measure the free proline (Pro) and malondialdehyde (MDA) contents, 0.2 g and 0.5 g of leaves were used for assaying, respectively. All measurements were performed in three biological replicates.
The free proline content was measured by a sulfosalicylic acid method [30
]. Quantities of 200 mg (fresh weight) of leaf tissue and 5.0 mL 3% sulfosalicylic acid were added to a mortar for grinding. After centrifugation, 200 µL of supernatant was mixed with 400 µL of glacial acetic acid and 600 µL of 25% ninhydrin and boiled at 100 °C for 40 min. After adding toluene, the absorbance was measured at A520.
The MDA level was studied in reference to the thiobarbituric acid-reactive-substances (TBARS) assay in previous reports [31
]. Leaf tissues were frozen in liquid nitrogen and ground to powder. A volume of 1.2 mL of 0.1 (w
) trichloroacetic acid (TCA) was added into a tube containing 500 mg of leaf powder, incubated at room temperature for 10 min, then centrifuged at 12,000 rpm for 20 min. An aliquot of the supernatant (0.3 mL) was mixed with 0.3 mL of 0.5% (w
) thiobarbituric acid (TBA), and was incubated at 100 °C for 20 min. Then, it was quickly cooled, and was centrifuged at 12,000 rpm for 10 min. The A440, A532, and A600 values of the supernatant were recorded.
4.9. Enzyme Activity Assay
For the estimation of antioxidant enzyme activities, 1 g of fresh leaves was ground to powder used liquid nitrogen and then homogenized in 4 mL of chilled buffer. The homogenate was centrifuged for 15 min at 12,000 rpm and the supernatant was collected for various enzymatic assays as the described [32
]. The enzymatic activity levels of SOD, CAT, and POD were determined using the method given by Wang [30
4.10. Statistical Analysis
All experimental data were the mean of at least three independent replicates, and comparisons between transgenic and WT plants were performed using one-way ANOVA with Duncan’s multiple range test. All the statistical analyses were performed using SPSS 12.0 software.