Cloning, expression and phylogenetic analyses of WRI1-like gene from yellow nutsedge
A 490 bp fragment corresponding to the conserved region of nutsedge WRI1-like gene was amplified by using degenerated primers (Additional file 1). Subsequently, a 1423bp full length fragment was obtained by using primers based on the 5’- and 3’-RACE results (Additional file 1). This gene contained an ORF of 1098 bp, while the sequences before the start codon and after the termination codon being the 5’UTR and 3’UTR, respectively (Fig. 1).
The expression analysis of WRI1-like gene showed the highest expression in the leaves, followed by that in the roots, while the lowest expression was found in tuber (Additional file 2). Conserved domain analysis (https://www.ncbi.nlm.nih.gov/Structure/cdd/) indicated that this WRI1-like gene contained two AP2 domains. Multiple alignment analysis indicated that the second AP2 domain was more conserved than the first one (data not shown). Phylogenetic analyses indicated that AP2, ANT and WRIs clustered into three major groups and the nutsedge WRI1-like peptide was more closely related to WRI3 and WRI4 (Fig. 2). Therefore, this gene is named as nutsedge WRI3/4 (CeWRI3/4).
CeWRI3/4 improves drought tolerance in transgenic Arabidopsis
Tolerance to PEG-simulated drought stress
For functional characterization of the cloned gene, Arabidopsis thaliana plants were transformed with CeWRI3/4 gene and the drought tolerance of the transgenic plants was determined. Seed germination of both wild type and transgenic Arabidopsis was higher than 90% under non-stress and PEG-simulated stress conditions (data not shown). We also determined drought tolerance at seedling stage. Without PEG stress the growth of 10-day-old wild type plants was not significantly different from the transgenic lines B1 and K2 (Fig. 3A). On the other hand, 14-day-old wild type seedlings grown on PEG-medium had only two very small yellow true leaves and the growth of primary roots was severely inhibited with much fewer lateral roots, while the transgenic seedlings contained 4 much larger true leaves and the primary and lateral roots were not significantly inhibited (Fig. 3A). The root length and seedling fresh weight (FW) data also confirmed that the transgenic lines had better tolerance to PEG-simulated drought stress (Fig. 3B-C).
Tolerance to real dehydration
The growth of wild type and transgenic Arabidopsis plants was not different before the onset of dehydration stress (Fig. 4). After 15 days of dehydration, the wilting frequency reached 63.6-81.8% in the wild type, while in the transgenic lines it was only 18.2-25% (Fig. 4-5). After 22 days of dehydration, the stressed plants were re-watered, and the extent of recovery was determined 1 day after resumption of the watering. The transgenic lines showed significantly higher recovery frequency i.e 58.3-63.6% compared with 18.1-36.3% in wild type. (Fig. 4-5). Based on these data, it can be concluded that CeWRI3/4 improves plant drought stress tolerance.
Expression of key genes involved in fatty acid and cuticular wax biosynthesis is modulated in transgenic lines
Keeping in view the role of CeWRI3/4, and to further understand the drought tolerance phenotype, we attempted to explore the expression of key genes involved in fatty acid and cuticular wax biosynthesis. Quantitative RT-PCR data indicated that under unstressed conditions the expression of key genes involved in fatty acid biosynthesis such as PIPK-β1 (At5g52920), BCCP2 (At5g15530) and PDHE1α (At1g01090) [19], was not significantly different between wild type and transgenic Arabidopsis lines. However, 10hr after 5% PEG treatment, the expression of above genes was slightly lower in the wild type compared to their unstressed counterparts. While in the transgenic lines the expression of PIPK-β1, BCCP2 and PDHE1α was increased by 130-230%, 50-100% and 130-220% respectively, compared with that without PEG treatment (Fig. 6A-C).
Without PEG treatment, the expression of TAG1 (At2g19450), a gene involved in TAG accumulation [21], was 10-40% lower in transgenic lines compared with the wild type. After PEG treatment, the expression of TAG1 was only slightly decreased in the wild type, while in the transgenic lines this decrease was 20-70% compared with that without PEG treatment (Fig. 6D) and was significantly lower than wild type.
Without PEG treatment the expression of LACS1 (At2g47240), WSD1 (At5g37300),KCS1 (At1g01120),CER1 (At1g02200) and CER4 (At4g33790), the genes involved in cuticular wax biosynthesis [20], was 10-160%, 50-140%, 30-70%, 50-270% and 90-130% higher in the transgenic lines compared with that in the wild type. After PEG treatment the expression of above mentioned genes was slightly decreased compared with that without PEG treatment in the wild type, while in the transgenic lines the expression of LACS1, WSD1,KCS1,CER1 and CER4 was 130-300%, 30-80%, 20-80%, 10-110%, 80-140% higher than that without PEG treatment (Fig. 6E-I). Over all, these results show that expression of key genes involved in fatty acid and cuticular wax biosynthesis is differentially modulated in transgenic lines.
Soluble sugars, free proline and MDA content in wild type and transgenic Arabidopsis
After two weeks of real dehydration, the concentration of soluble sugars, free proline and MDA in the wild type was found to be 27.1mg/g (Fresh Weight, FW), 69.4 μg/g (FW) and 5.3 mmol/g (FW), respectively, while in the transgenic lines the soluble sugars, free proline and MDA content was only 14.3-29.9% (Fig. 7A), 26.9-50.6% (Fig. 7B), 36.5-45.5% (Fig. 7C) of the wild type, respectively.
Cuticular wax and oil content in the wild type and transgenic Arabidopsis lines
In this study it was found that in both wild type and transgenic Arabidopsis leaves the cuticular wax was mainly composed of alkanes and primary alcohols. C29 and C31 of the alkanes amounted to about 68% of the total cuticular wax, while for alkanes C31 was the predominant composition (Fig. 8A-B). In transgenic lines the contents of total cuticular wax, C26, C28 and C30 of primary alcohols, and C27 and C31 of alkanes, were all significantly higher than those in the wild type, while the contents of C33 of alkanes and C32 of primary alcohols, were not significantly different (Fig. 8A-C). The content of C29 in alkanes was significantly different between wild type and transgenic line B1, but not between wild type and transgenic line K2 (Fig. 8A). The oil content in Arabidopsis leaves was 6.32-6.51mg/g and there was no significant difference between wild type and transgenic lines (Additional file 3).