Isolation and Sequence Analysis of BoCRP1 gene
Previously we have performed proteomic study to compare the gene expression analysis in Brassica oleracea var. capitata under both normal and cold conditions (4°c). Under cold conditions, a novel low molecular weight protein called cold induced protein 1 (BoCRP1) was found to be highly induced. The CDS of of BoCRP1 (Accession no.GQ461800.1 ) consists of 198 bps which encodes a low molecular weight protein of 65amino acids a molecular weight of 6.5 KDa PI approaching to 9.1. Various sequence alignment tools revealed a strong homology between BoCRP1 protein and Kin proteins of B. napus, B. rapa and A. thaliana annotated cold-resistant proteins (Fig. 1a). On performing the phylogenetic analysis, it was clear that BoCRP1 has a close semblance to homologue from A. thaliana, B. rapa and B. napus, (Fig. 1b). As shown in Fig. 1, LEA proteins are also similar stress related proteins which are homologous to BoCRP1 protein.
However, BoCRP1 is closer to Kin1/Kin2 of Arabidopsis than to LEA-related protein from Actinidia chinensis. We found that the closest relative plant to B. oleracea on STRING database is Brassica rapa. It has a close relative (ortholog) of BoCRP1. In B. rapa, the gene (Bra008661) is connected to BRA000263 (COR15B Cold regulated gene), which suggests that Bra008661 (and hence BoCRP1 too) is involved in the cold response. Homology models showed a folded alpha-helix structure of BoCRP1 similar to that of KIN2 of A. thaliana25. All the above findings strongly advocate the involvement of BoCRP1 protein in cold resistance.
Transcript analysis of BoCRP1 in B. oleracea
To examine the tissue specific mRNA levels of BoCRP1 in B. oleracea var. capitata, plants were exposed to cold (4°C) for varying time periods. Expression studies indicated that the BoCRP1 transcript levels were highly up-regulated and reached a maximum up to 8 fold after 12hrs. After that, the expression shows a gradual decline (Fig. 2b). These results strongly suggest that BoCRP1 plays an important role by offering an early response to cold stress.
Tissue-specific expression analysis exhibited enhanced mRNA levels of BoCRP1 in the leaf tissues when exposed to cold. However, comparatively lower levels of BoCRP1were observed in the stem and the root tissues under cold compared to normal conditions (Fig. 2a).
Transformation and molecular Analysis
To validate and characterize the function of BoCRP1 in cold susceptible tomato variety, (Shalimar 1) the plant binary vector pcambia2301 was selected to clone the entire ORF of BoCRP1 gene under a stress-inducible promoter of Rd29A gene (Fig. 3a).The transformation of the recombinant vector was executed in tomato cultivator Shalimar 1 (Fig. S1a-d) and obtained 20 kanamycin-resistant tomato lines independently (T0 generation).
Using NPTII and BoCRP1 specific primer sequences, a total of 10 transgenic lines were identified (Fig. 3b). Among them, four stably transgenic lines, OE1, OE2, OE8 and OE11 were confirmed to contain single-copy insertion and segregated in 3:1 ratio for antibiotic selection possibly do to single T-DNA insert (Fig. 3c). In order to assess the transgene expression, q-PCR was performed to analyse different transgenic lines. We obtained three putative independent transgenic lines OE1, OE2 and OE11 with significant proportion of transgene expression under cold stress and were considered for further investigations (Fig. 3d). These selected transgenic lines were allowed to grown for 2 to 3 generations to obtain homozygous (T3) lines.
Overexpression of BoCRP1 in tomato improved the seedling growth and seed germination
To assess the cold tolerance, it was imperative for us to study the germination rate and seedling growth in both transgenic lines as well as in WT under normal (25˚C) and cold conditions (4˚C). At 25˚C, we observed a similar germination rate both in WT as well as transgenic lines, confirming that both the set of seeds are 100% viable, however, the rate of seed germination went significantly up in transgenic lines relative to the WT under chilling stress (4˚C) (Fig. 4a). The germination rate of transgenic lines OE1, OE2, and OE11 was approximately 80%, 79%, and 84% respectively compared to about 30% in WT under cold stress (Fig. 4b).
Furthermore, to understand whether BoCRP1 over-expression influenced the seedling growth in transgenic lines, seedlings after germination were placed at 10˚C for a period of 2 weeks, following which the hypocotyl length along with main root length was measured with a ruler. In BoCRP1 expressing plants, no obvious difference in the root and hypocotyl length were observed compared to wild-type at 25˚C. Interestingly, at 10˚C both the root as well as hypocotyl length was suppressed in WT as compared to BoCRP1 expressing lines (Fig. 4c). Which at 10˚C displayed a marked increase in root and hypocotyl length. (Fig. 4d & e). The above results declared that the cold-induced expression of BoCRP1 can modulate the ability of a plant to resist low temperatures as was observed in early seedling and germination stage of transgenic tomato.
Ectopic expression of BoCRP1 in transgenic tomato enhances tolerance to cold
To further our understanding, we investigated the functional significance and physiological effect of BoCRP1 under chilling stress (4˚C) in both WT as well as transgenic lines. For this we incubated plants at 4˚C in the growth chamber for 4 days and then shifted to recovery at 25˚C. At ambient temperatures (25˚C), we observed no significant differences at any stage, neither in transgenic lines nor in WT tomato plants (Fig. 4f). However, transgenic lines showed a survival rate of about 73% (OE1), 68% (OE2), and 80% (OE11), respectively, while only 26% of the WT plants survived during recovery (Fig. 4e), suggesting an increased tolerance in BoCRP1 expression lines under the chilling stress.
BoCRP1 transgenic plants accumulate increased osmo-protectants under cold conditions
Under normal growth conditions, the WT and BoCRP1 expressing lines showed similar content of osmo-protectants (proline and soluble sugar). However, we observed significant accumulation of both the osmo-protectants at 4˚C in BoCRP1 lines compared to WT. Further, soluble sugar content increased by 2.4, 2.6 & 2.7 fold in transgenic line OE1, OE2 and OE11 respectively compared to WT under cold stress (Fig. 5a). Similarly, proline content shot up by 1.5 fold in OE1, 1.6 fold in OE2 and OE11 respectively, compared to WT (Fig. 5b).
Overexpression of BoCRP1 improves membrane stability in transgenic lines
In the context of Abiotic stress(cold, drought and salt) the level of malondialdihyde (MDA) and and relative electrolyte leakage (REL) is considered one of the vital parameter in evaluating the effect on lipid peroxidation and cytomembrane penetrability 26. While evaluating the levels of MDA and REL in wild-type and BoCRP1 expressing plants, we observed similar no obvious differences in MDA and REL content in wild-type and BoCRP1 lines under control conditions (25 ˚C) (Fig. 5c & d). However, under cold conditions both the WT and BoCRP1 lines displayed an increase in REL and MDA content relative to the controls grown under ambient temperatures (25 ˚C). Cold stressed transgenic seedlings showed significantly reduced content of MDA and REL levels. Above results concluded that in WT plants cold treatment induces 2.9 fold increase in MDA content and only 1.8, 2.1 and 1.7 fold increase was observed in BoCRP1 lines OE1, OE2 and OE11 respectively (Fig. 5c) and the REL increased by almost 2.3 fold in WT and only 1.5, 1.5 and 1.3 fold increase in OE1, OE2 and OE3 respectively (Fig. 5d).
Overexpression of BoCRP1 improves the ROS scavenging capacity to enhance tolerance to cold stress.
To evaluate the extent of ROS accumulation under cold and normal temperatures in both WT and BoCRP1 transgenic lines, hydrogen peroxide (H2O2) staining of leaves was carried out. The staining pattern was almost similar in WT and transgenic leaves grown under normal temperature (25 oC). However, after 3 days of cold treatment (4˚C) , we observed significantly higher staining (dark brown spots) in WT plants relative to transgenic lines (Fig. 6a). The reduced staining pattern observed in transgenic lines depicts improved detoxification of H2O2 in the transgenic lines. These attributes were well linked with reduced levels of REL and MDA content in BoCRP1 expression lines, indicating reduced oxidative damage under cold stress. Under cold treatment, the BoCRP1 transgenic lines showed nearly 2.1, 3.0, and 3.1 fold increase in SOD, APX and CAT activity respectively compared to 1.8, 1.3 and 1.7 fold increase of these genes in WT maintained at 25 oC (Fig. 6b-d). These results suggest that BoCRP1 over-expression led to decrease in the levels of MDA and enhanced antioxidant capacity resulting in reduced oxidative injury to the transgenic tomato plants.
BoCRP1 over-expression enhanced Stress Responsive genes in transgenic tomato
To further widen our understanding of the molecular mechanism that governs the enhanced tolerance to cold in BoCRP1 transgenic lines, we analysed the mRNA expression levels of six ROS associated/stress response gene transcripts in both control as well as transgenic lines maintained under cold stress. Following the exposure to cold treatment (4˚C) for 3 days, the mRNA levels of ROS detoxification enzymes(POD, CAT and Cu-Zn SOD), significant regulatory protein (DREB1), proline Transporter 1 (ProT1), , Lipid transfer protein (LTP1) and stress defensive proteins (EDR15-2 and LEA) was significantly up-regulated BoCRP1 expressing lines compared to WT (Fig. 7a-h). These findings therefore, led to the conclusion that the enhanced stress responsive in BoCRP1 lines is a consequence of elevated expression of stress-associated genes.