Gene Selection and characterization
OsRuvBL1a gene has been selected based on our preliminary studies in which it was found to be an abiotic stress-responsive gene (Saifi et al. 2018) and thus, make it a potential candidate gene mediating abiotic stress tolerance in rice. OsRuvBL1a encodes for a 55 kDa protein (Fig. 1 a & b). Recently, it has been reported that purified recombinant protein showed nucleic acid independent ATPase (Fig. 1 c & d) and ATP dependent DNA unwinding activity (Fig. 1e) (Saifi et al. 2018).
Development of in-planta transformation method for rice with mature seeds
OsRuvBL1a overexpressing rice transgenics were produced by using improved in-planta transformation method, which is less labour-intensive, easy and fast, using the mature rice seeds as explant. This report briefly describes the OsRuvBL1a overexpression transgenic of rice by using this improved in-planta method and its optimization for various factors.
(i) Effect of in-planta transformation and hygromycin concentration on germination percentage
The hygromycin concentration of 20 mg/L was observed as lethal (Minimum Inhibitory Concentration, MIC) as more than 50% of seeds failed to germinate (Table S1). Later same concentration (20 mg/L) was used for selecting the putative transformed seedlings (Fig. 2a). Wild type control and non-transgenic seedlings died on hygromycin selection (Fig. 2ai and 2aiii) whereas putative transgenic plants survived and were selected (Fig. 2aii and 2aiv).
(ii) Optimization of acetosyringone concentration, co-cultivation duration and effect of different strains on in-planta transformation efficiency
Different concentrations of acetosyringone (0, 40, 100, 200 and 400 mg/L) were supplemented with Agrobacterium inoculum to study the effect of acetosyringone concentration on transformation efficiency of this method. Among the different concentrations and co-cultivation duration, Agrobacterium strain LBA4404 with 200 mg/L of acetosyringone incubated for 24 hrs showed maximum transformation of 32.7±0.9% after PCR with hptII gene specific primers and CaMV35S forward and gene specific reverse primers, respectively (Fig. 2bi and 2bii) and Gus screening (Fig. 2c) (Table S2). Similarly, for Agrobacterium strain EHA105 mediated transformation, the same concentration of acetosyringone (200 mg/L) resulted in maximum transformation of 36.0±2.0% when incubated for 24 hrs (Table S3).
(iii) Influence of pre-culture duration on in-planta transformation efficiency
The transformation efficiency was observed for different durations of pre-culture (0, 3, 12, 18, 24 and 48 hrs). A pre-culture period of 24 hrs was found to be the most suitable for T-DNA delivery, resulting in a significant increase in the transformation frequency from 32.7±0.9% to 41.33±3.5% for LBA4404 strain (Table S4).
(iv) Optimization of co-cultivation media for in-planta transformation method
- Agrobacterium inoculum concentration: Highest percentage of transformed plants was observed when optical density (O.D.) of Agrobacterium inoculum was kept 0.4 at 600nm. Further increase in O.D. resulted in a decrease in transformation efficiency.
- Optimization of pH: pH of co-cultivation medium is the authoritative divisor for the expression of vir genes and T-DNA transfer. In this study we obtained the highest efficiency at pH 5.8.
- Surfactants: Best standard conditions (24 hrs incubation with 200 mg/L acetosyringone concentration) were chosen to test the effect of different surfactants on transformation efficiency. Two surfactants (Tween 20 and Triton X-100) were applied with three different concentrations (0.05, 0.1 and 0.2 %). The application of surfactants at higher concentration showed continuous decline in the transformation efficiency.
- Growth Regulator: A growth regulator, GA3 in co-cultivation medium, did not show any significant change in transformation efficiency.
This in-planta method is strain-independent (Table S5) because both the strains of Agrobacterium (LBA4404 and EHA105) showed maximum transformation efficiency by 24 hrs co-cultivation. We have optimized that rice seeds pre-cultured for 24 hrs in MS medium with pH-5.8 and co-cultivated with Agrobacterium cells (at O.D. = 0.4) for 24 hrs in the presence of 200 mg/L acetosyringone showed maximum transformation efficiency. The transformed seeds were selected on 20 mg/L hygromycin supplemented MS medium to narrow down the selection process.
Copy number
Results of both the qPCR and Southern blot hybridization methods for copy number analysis of putative transgenic lines (L-1 to L-5) showed that L-1, L-3, L-4 and L-5 have single copy insertion whereas L-2 showed two copies of target gene in rice genome (Fig. 2d and 2e). Based on these results single copy lines L-1, L-3, L-4 and L-5 were used for further analysis.
Expression analysis of OsRuvBL1a in transgenic plant
Four transgenic plants (L-1, L-3, L-4 and L-5) overexpressing OsRuvBL1a gene were screened to identify the transgenic lines which showed higher expression for OsRuvBL1a gene by qPCR method. Out of 4 transgenic lines, transcript level quantification showed that line L-1, L-4 and L-5 have the highest expression for the transgene with around 13, 23 and 15 folds’ increase, respectively (Fig. 2f).
Transgene inheritance
The segregation pattern for T-DNA was tested as a single dominant Mendelian gene segregation method. About 45 seeds were taken from each line (L-1, L-4 and L-5) and the results were validated using the classical chi-square (ᵪ2) test taking 1 as degree of freedom. According to χ2 test value T1 population did not follow the Mendelian segregation pattern because average chi square value was much higher than table value (20.90) (Table S6). Gus histochemical assay was also performed in T1 seeds and seedlings for the positive transgenics and it showed the formation of blue coloration in transgenic seeds and seedlings (Fig. 2g).
Homozygous transgenic line
The identification of homozygous lines was performed with qPCR in T1 transgenic plants for the lines L-1, L-4 and L-5 for 14 plants. Out of 14 plants in the line L-1, four plants (namely, L1.1, L1.2, L1.6 and L1.10) possessed 2 copies (Fig. 2h). In the line L-4, three plants (namely, L4.5, L4.7 and L4.10) possessed 2 copies of OsRuvBL1a (Fig. 2i). Similarly, in the line L-5,3 three plants (L5.2, L5.6 and L5.9) showed 2 copies for the transformed gene and hence considered as homozygous (Fig. 2j).
Physiological analysis of OsRuvBL1a transgenic plants
To examine the role of overexpression of OsRuvBL1a gene in conferring the abiotic stress tolerance in rice, comparative analysis of several physiological tests was performed in WT type and OsRuvBL1a overexpressing transgenic lines (L1, L4 and L5) under salinity and drought stress conditions. Germination assay revealed that under stress-free control conditions there was no significant difference in germination pattern in WT as compared to OsRuvBL1a transgenic lines (Fig. 3a-d), however, when grown under salinity (200 mM NaCl) and drought (150 mM mannitol) stress the transgenic lines were germinated earlier than the corresponding WT. The seeds exposed to salinity stress had the germination percentage of ~60%, 75% and 55% (Fig. 3d); whereas, under drought stress the germination rate was 68%, 80% and 62% for lines L-1, L-4 and L-5, respectively (Fig. 3d). The WT seeds exhibited only ~15% germination under both the stress conditions (Fig. 3d). High root/shoot ratio and strong root architecture helps plant to overcome the salinity (Fig. 3e) and drought stress conditions (Fig. 3f). Transgenic lines (L-1, L-4 and L-5) showed root/shoot ratio of 1.2, 1.3 and 1.5, respectively under salinity stress (Fig. 3g) and 0.7, 0.8 and 0.8, respectively under drought stress (Fig. 3g). Whereas WT plants showed root/shoot ratio of 0.2 and 0.6 under salinity and drought stress (Fig. 3g). Higher dry weight of plant indicates lower water retention capacity. Lines L-1, L-4 and L-5 showed 1.7, 1.8 and 1.5-fold lesser dry weight, respectively than WT plants under the salinity stress conditions (Fig 3h). Under drought stress, all the three transgenic lines showed 1.8-fold lesser dry weight as compared to WT plants (Fig. 3h), whereas under the normal control conditions, transgenic lines and WT plants did not have significant difference in their respective dry weight (Fig. 3h). Stress conditions affected the photosynthetic machinery of plants and caused chlorophyll degradation (Fig. 3i-l). Total chlorophyll content measured in WT and OsRuvBL1a transgenic lines showed about 5-fold high chlorophyll content in all the three transgenic lines (L-1, L-4 and L-5) as compared to WT plants during salinity stress (Fig. 3j). Under drought stress, lines L-1and L-5 showed about 2.5-fold higher retention of chlorophyll content as compared to WT plants, whereas L-4 line showed 1.5-fold higher chlorophyll retention (Fig. 3l).
Biochemical analysis of OsRuvBL1a transgenic plants
Abiotic stresses affect ROS machinery in plants and it was detected and quantified by some biochemical tests such as malondialdehyde (MDA) accumulation, proline, H2O2 content and cell membrane stability in T2 generation (Fig. 4a-l). Generation of H2O2 and MDA in a cell is the marker of negative effects of stress since these molecules are result of cell degradation. This study showed significantly less H2O2 and MDA accumulation in transgenic line L-4 under both the salinity (Fig. 4 a & b) as well as the drought stress (Fig. 4 g & h) as compared to the corresponding WT plants. To overcome the water deficit condition imposed by the salinity and drought stress, plants are known to accumulate osmolytes such as proline. OsRuvBL1a transgenic lines showed higher level of proline content as compared to WT with line L-4 showing maximum accumulation of proline under both salinity (Fig. 4c) and drought stresses (Fig. 4i). All transgenic lines showed higher electrolyte retention as compared to the WT plants with line L-4 showing maximum electrolyte retention under salinity stress (Fig. 4d) and line L-1 showed maximum electrolyte retention under drought stress (Fig. 4j). Overall these observations suggest the reduced effect of stress and damage to cell membrane in transgenic lines and confirm the role of OsRuvBL1a in maintenance of the cell membrane stability under the abiotic stresses. The activity of ROS scavenging enzymes- ascorbate peroxidase (APX) and catalase were also estimated. Both the enzymes APX and catalase use H2O2 as substrate to reduce it into water. Under salinity stress condition transgenic line L-4 showed higher APX and catalase activities as compared to WT plants (Fig. 4 e & f). Under drought stress the transgenic line L-4 showed maximum activity of APX and catalase (Fig. 4 k & l). These biochemical analyses showed improved ROS scavenging machinery and maintenance of cell integrity in transgenic lines as compared to WT plant under stress conditions.
Determination of ion content and cell death in response to abiotic stresses
The quantification of ions accumulation and cell death in plant tissue is another method to observe the effect of stress conditions on plants. In this study, we measured the effect of salinity stress and drought stress on rice seedlings. Sodium ion (Na+) accumulation in root tissue during salinity stress was studied as a method for quantification of Na+ ion imbalance in plants during stress conditions. CoroNa green dye was used for the non-destructive monitoring of relative accumulation of Na+ ion in roots of WT and OsRuvBL1a overexpressing transgenic lines by using the confocal microscopy. The transgenic line L-5 showed the least fluorescence and lines L-1 and L-4 also showed lesser fluorescence as compared to WT roots under salinity stress (Fig. 5 a & b). These results suggest that more Na+ ion accumulation occurred inside WT roots during salinity stress as compared to the transgenic lines. For the study of calcium ion (Ca2+) accumulation an esterified form of Fluo-4, Fluo4-AM was used. Fluo4-AM is a Ca2+ sensitive fluorescent probe indicator which shows increase in fluorescence upon binding with cytosolic Ca2+. Confocal microscopy of WT and transgenic roots under salinity stress was used to observe the cytosolic Ca2+ accumulation. Higher fluorescence in WT roots as compared to transgenic line L-1 suggested an accumulation of more cytosolic Ca2+ in WT roots under stress conditions (Fig. 5 c & d). Stress conditions also cause ionic imbalance and oxidative stress that make the cells inviable. Cell viability can be studied in plant root in non-destructive manner by using propidium iodide (PI) dye. PI is a membrane impermeant dye and hence viable cells show less or no fluorescence whereas it penetrates in dead cells and intercalates in double stranded DNA and provides florescence on excitation. The cell viability study of rice root tissue under salinity and drought conditions showed higher fluorescence in roots of WT plants as compared to transgenic lines under salinity stress (Fig. 5 e & f) and drought stress (Fig. 5 g & h). These results showed that stress conditions caused higher cell death in WT plants as compared to overexpressing transgenic lines. These studies suggest the role of OsRuvBL1a in providing stress tolerance under stress conditions at ionic and cell viability level.
Salinity and drought stress tolerance under in vivo conditions
WT and the transgenic plants (L-1, L-4 and L-5) overexpressing OsRuvBL1a gene showed significantly different behaviors under salinity and drought stress (Fig. 6). At day-1, all the plants of same age were exposed to salinity stress (200 mM NaCl) as well as drought stress (non-availability of water) as shown in Fig. 6 a & c. On day 20 salinity stress, the WT plant could not survive salinity stress till 20 days, on the other hand, the OsRuvBL1a transgenic plants withstood the stressed condition (Fig. 6b). Among the transgenics, the L-4 line was found to be the most tolerant line. For drought stress, WT plant died after 15 days while OsRuvBL1a-overexpressing transgenic plants were thriving (Fig. 6d).
Isolation and identification of interacting partners of OsRuvBL1a through yeast two-hybrid method
Physiological and biochemical analysis of overexpressing transgenic lines showed better performance as compared to WT under salinity and drought stress. To understand the working mechanism of OsRuvBL1a, its interacting partners were identified by using yeast two-hybrid method. Sequential selection of transformed yeast on two drop outs (2-DO) (-Leu, -Trp), 3-DO (-His, -Leu, -Trp) and 4-DO (-Ade, -His, -Leu, -Trp) media plates followed by filter lift assay showed positive interacting clones. The colonies found positive on X-gal assay were sequenced and analyzed using rice genome to find the interacting partners of OsRuvBL1a enlisted as Table 1. Y2H (one-to-one interaction) as well as BiFC studies showed that OsRuvBL1a does not self-interact whereas, it exhibits strong interaction with another member of its family, OsRuvBL2a. The OsRuvBL1a-OsRuvBL2a interaction led to the formation of a hetro-oligomeric structure by these proteins. BiFC based one-to-one interaction study of OsRuvBL1a with few selected partners showed positive interactions and validate the Y2H results (Figure S2). These validated interacting partners may have some direct or indirect role in conferring the stress tolerance to the plants.
Table 1
List of interacting partners of OsRuvBL1a
Class
|
Locus ID
|
Gene Name
|
Putative Function
|
Hormonal Signaling
|
LOC_Os08g44510
|
UDP-N-acetylglucosamine–peptide N-acetylglucos-
aminyltransferase SPINDLY
|
Negative regulator of GA signaling
|
LOC_Os10g31770
|
START domain containing protein
|
ABA signaling. Biotic and Abiotic stress tolerance
|
LOC_Os01g13030
|
OsIAA3 - Auxin-responsive Aux/IAA gene family member
|
Auxin signaling
|
LOC_Os07g28480
|
glutathione S-transferase
|
Auxin and oxidative stress- signaling
|
LOC_Os03g20790
|
ethylene-insensitive 3
|
Me-JA and SA signaling
|
LOC_Os12g43600
|
RNA recognition motif containing protein
|
Response to stress and RNA silencing and splicing
|
Gene Regulation
|
LOC_Os03g60080
|
NAC domain-containing protein 67
|
Stress-responsive transcription factor
|
LOC_Os06g39700
|
DNA-directed RNA polymerase subunit alpha
|
Transcription
|
LOC_Os01g63980
|
ZOS1-17 - C2H2 zinc finger protein
|
Transcription
|
LOC_Os01g54100
|
CK1_CaseinKinase_1a.2
|
Transcription elongation
|
LOC_Os05g41110
|
Ribosomal protein L7Ae
|
Translation
|
LOC_Os01g24690
|
60S ribosomal protein L23A
|
Translation
|
LOC_Os07g40580
|
eukaryotic translation initiation factor 5A
|
Translation
|
LOC_Os06g43210
|
zinc finger, C3HC4 type domain containing protein
|
Ubiquitination pathway
|
LOC_Os01g62230
|
Core histone H2A/H2B/H3/H4 domain containing protein
|
Chromatin remodeling
|
LOC_Os06g45390
|
Expressed protein
|
RNA binding, post transcriptional gene regulation and translation
|
LOC_Os01g50100
|
ABC transporter, ATP-binding protein
|
Multiple drug resistance, Telomerase reverse transcriptase activity, Activation of double strand break machinery
|
Cellular Mechanism and Development
|
LOC_Os05g50370
|
DnaJ domain containing protein
|
Chaperon and protein transportation by vesicle coat proteins
|
LOC_Os06g12580
|
Pro-resilin precursor
|
Chaperon
|
LOC_Os11g47970
|
AAA-type ATPase family protein
|
Rubisco Activase, A chaperon for Rubisco under Heat stress
|
LOC_Os07g05810
|
Glycine-rich protein
|
Peroxisome biogenesis, transport mechanism
|
LOC_Os12g19381
|
Ribulose bisphosphate carboxylase small chain
|
Involved in CO2 metabolism
|
LOC_Os08g15170
|
ATP synthase epsilon chain
|
ATP synthesis
|
Stress Responsive
|
LOC_Os08g03290
|
glyceraldehyde-3-phosphate dehydrogenase
|
glycolytic pathway, response to ER stress, heat, H2O2, oxidative, salt stress, sucrose and temperature stimuli
|
LOC_Os08g19980
|
NBS-LRR disease resistance protein
|
R-gene involved in biotic stress regulation
|
LOC_Os03g28940
|
ZIM domain containing protein
|
Jasmonate Signalling
|
LOC_Os07g48500
|
Stress-responsive protein
|
Stress-responsive and ROS scavenging
|