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A salt‐tolerant chloroplastic FBPase from Oryza coarctata confers improved photosynthesis with higher yield and multi‐stress tolerance to indica rice

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Abstract

C3 photosynthesis in rice is dependent on regeneration of Ribulose 1,5-bisphosphate (RuBP), the CO2 acceptor which is largely determined by the Fructose 1,6-bisphosphatase (FBPase) function in the chloroplast. Abiotic stress affects this function negatively impacting the decline in the photosynthetic potential of crop plants. In the present work the PcCFR gene, coding for a salt-tolerant chloroplastic FBPase, from Oryza coarctata (Roxb.) Tateoka, was introduced into the cultivated rice (Oryza sativa var. indica IR64). The homozygous transgenic PcCFR plants performed better than the untransformed lines in terms of overall plant growth, photosynthetic performances and grain yield under normal as well as under salt and different abiotic stress conditions. Under salinity, drought and cold stress PcCFR lines showed tolerance through emergence of new leaves, improved photosynthetic performance and overall growth rate. The cumulative results suggested that the overexpression of salt-tolerant FBPase (PcCFR) protein in the transgenic rice helps to keep the photosynthetic cycle by unabated generation of RuBP that retains better light harvesting capacity of the leaves under stress. It is presumed that this will provide an insight into the growth and development during abiotic stress by inducing an interaction among different sugars derived from photosynthetic carbon metabolism as well.

Key message

The transgenic IR64 plants, over-expressing a salt-tolerant chloroplastic FBPase from Porteresia coarctata, showed multi-stress tolerance, improved plant phenotype, electron transport rate, transpiration rate, photosynthetic efficiency and grain yield under salinity.

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Abbreviations

RuBP:

Ribulose 1,5-bisphosphate

FBPase:

Fructose 1,6-bisphosphatase

RuBisCO:

Ribulose bisphosphate carboxylase

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Acknowledgements

The authors thank Dr Rajeswari Mukherjee for her advice in rice transformation and preliminary analysis and Dr Sonali Sengupta for her helpful suggestions. Thanks are due to Prof Ashwani Pareek, Jawaharlal Nehru University, New Delhi for help in the Infra-Red Gas Analysis and to Prof Anup Misra, Bose Institute for his advice in carbohydrate analysis.

Funding

The project was supported by funds from the Department of Biotechnology (BT/AB/05/02/2007-III), Government of India and the Raja Ramanna Fellowship Project of the Department of Atomic Energy (DAE) (D.O No. 10/25/2010/RRF-R and D-II/3118) awarded to ALM, currently an INSA Senior Scientist. SM was supported by Fellowship from the Council of Scientific and Industrial Research (CSIR) and DAE. AM, SB and DC are supported by the DBT project. PD is supported by SERB Young Scientist Fellowship (YSS/2015/001872).

Author information

Author notes

  1. Sritama Mukherjee

    Present address: Department of Botany, Bethune College, Kolkata, India

  2. Subhendu Bandyopadhyay

    Present address: Department of Biochemistry, School of Life Sciences and Biotechnology, Adamas University, Kolkata, India

  3. Jolly Chatterjee

    Present address: C4 Rice Center, International Rice Research Institute, Los Baños, Philippines

  4. Sritama Mukherjee and Abhishek Mukherjee are equal contributors.

Authors and Affiliations

  1. Division of Plant Biology, Bose Institute (Centenary Building), P-1/12, C I T Scheme VII M, Kolkata, 700 054, India

    Sritama Mukherjee, Abhishek Mukherjee, Priyanka Das, Subhendu Bandyopadhyay, Debapriya Chattopadhyay, Jolly Chatterjee & Arun Lahiri Majumder

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  1. Sritama Mukherjee
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Contributions

ALM designed the project. SM, AM along with SB performed major experiments and analyses in the study. PD contributed to the photosynthesis and real time experiments. DC performed most of the transformation work. SM, PD and JC wrote the draft manuscript, ALM finalized the manuscript with input from all authors.

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Correspondence to Arun Lahiri Majumder.

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Supplementary Information

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11240_2021_2026_MOESM1_ESM.tif

Supplementary file 1 Supplementary Fig. 1. Production of rice transgenic PcCFR and OsCFR. Schematic diagram of binary vector pCAMBIA construct used in transformation and probe designing strategy for DNA gel blot hybridization (a). Gene of interest (PcCFR or OsCFR) was transcriptionally fused with tobacco chloroplastic transit peptide (Tp) sequence and placed under the constitutive expression of CaMV35S promoter and NOS terminator. Hygromycin resistant gene (hptII) is present in the vector backbone as plant selection and GUS is present as reporter gene. LB: Left Border, RB: Right Border. Probe 1 is designed at the GUS region, probe 2 is designed at the gene (end to end) region and probe 3 is designed from the gene to the NOS terminator region. Stages of seed to transgenic plant production (b-f). Dehulled seeds of rice cultivar IR64 was kept on the callus initiation medium (CIM), the MS supplied with 2 mg / L 2, 4-D (b), Mature scutellar calli after 4 weeks of culture, in a selection media [MS+(Cefotaxime-250 mg/L + Hyg-50 mg/L)] after Agrobacterium transformation (c) regeneration of infected callus in selection media (d). The mature PcCFR transgenic plant shows viable healthy panicle and florets (e). Healthy PcCFR T0 plants ready for harvesting T1 seeds (f). (TIF 21891 kb)

11240_2021_2026_MOESM2_ESM.tif

Supplementary file 2 Supplementary Fig. 2. Molecular verification of the PcCFR and OsCFR transgene introduction in IR64. PCR analysis of PcCFR and OsCFR T2 transgenic plants (a). The positive plants show ~ 1Kb band for hptII. P: Positive control, M: Marker. The copy number of the introduced PcCFR/OsCFR gene in IR64 genome was further checked by DNA gel blot analysis of different T2 transgenic plants (b). Genomic DNA was digested with HindIII and charged either with GUS probe1 (blot1), gene probe 2 (blot 2), or gene + NOS terminator probe 3 (blot3 and blot 4). N: Negative control (WT, IR64 plant), P: Positive control is the plasmid construct. Arrows indicate the number of extra bands, seen in the gel, other than the 2 endogenous bands that are present in WT, using Probe 2 and 3, and thus confirm the copy number of the introduced gene. Probe designing strategy is provided in Supplementary Fig. 1a. (TIF 21624 kb)

11240_2021_2026_MOESM3_ESM.tif

Supplementary file 3 SupplementaryFig. 3. Identification of specific integration site of the transgene in rice genome. Amplification of T-DNA flanking sequences from transgenic rice plants by high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) according to the protocol of Liu and Chen, 2007. Three Hygromycin PCR positive T2 PcCFR lines are used to invent the transgene integration site in the rice IR64 genome. The gel pictures show the preamplification using primer LAD 1, 2, 3, and 4 (left). Preamplification product of LAD 3 and 4 are used for the primary amplification with AC1 and RB1 (right). M: 500 bp ladder; WT: Wild Type (a). The integration sites are identified after sequencing the desired PCR product followed by similarity checking of that sequence using NCBI BLAST tool. The cloned sequence for Pc3.2 shows alignment with the Oryza sativa Japonica Group DNA, chromosome 5, complete sequence, cultivar Nipponbare (b). The cloned sequence for Pc7.3 shows alignment with the Oryza sativa Japonica Group DNA, chromosome 5, complete sequence, cultivar Nipponbare (c). The cloned sequence for Pc1.4 shows alignment with the Oryza sativa Japonica Group DNA, chromosome 8, complete sequence, cultivar Nipponbare (d). The red marked letters are the sequences of the vector region. (TIF 23245 kb)

11240_2021_2026_MOESM4_ESM.tif

Supplementary file 4 Supplementary Fig. 4. Salt tolerance analysis of PcCFR transgenic plants showing growth and photosynthetic performance compared to OsCFR and WT at early growth stage. Growth of 7 days old seedling of the transgenic PcCFR (Pc1.4, Pc7.3), OsCFR (Os3.6) and WT lines (IR64) after 7 days of 200mM salt treatment (a); comparative analysis of shoot length (b) root length (c), fresh weight (d) and dry weight (e) showing significant protection to salt effect in PcCFR lines compared to WT and OsCFR lines; photosynthetic parameters like Fv/Fm (f), PI(abs) (g) and ET0/CSm (h) collectively represent the photosynthetic efficiency of the plants. Both the PcCFR lines (Pc1.4, Pc7.3) show better photosynthetic performance in terms of the absolute performance index (PIabs) and electron transport under normal and salt stressed condition with a marked difference in the photosystem II yield (Fv/Fm) in 200mM NaCl. Scale bar = 1 cm. Data in the graphs are the average ± SE of six independent sets of experiments with six plants per line. Different letters denote statistically significant differences (P < 0.01).Values are compared separately for the normal or the stressed condition. (TIF 16089 kb)

11240_2021_2026_MOESM5_ESM.tif

Supplementary file 5 Supplementary Fig. 5. Analysis of photosynthetic parameters in 15 days old WT and transgenic lines (Os3.6, Pc1.4, Pc7.3) under control (0mM NaCl) as well as saline (200 mM NaCl) conditions. Non Photo chemical Quenching (NPQ) (a), Electron Transport Rate (ETR) (b), CO2 assimilation rate (c), transpiration rate (d) and stomatal conductance (E) respectively in WT, Os 3.6, Pc 1.4 and Pc 7.3 plants under control (0mM NaCl) and under salt (200mM NaCl) . (TIF 9585 kb)

11240_2021_2026_MOESM6_ESM.tiff

Supplementary file 6 Supplementary Fig. 6. Salt tolerance analysis of some more PcCFR plants. Photographs of one month old plants were taken after 10 days of salt treatment (0, 100, 200, 300mM) (a). Scale bar = 1 cm. A comparative analysis of shoot, root length, fresh weight and dry weight showed better performance of PcCFR plants (Pc2.7, Pc1.2, Pc3.2) than OsCFR (Os3.4)(b – e).Overall performance of PcCFR are better in terms of Fv/Fm, PI(abs) and ET0 / CSm (f-h). Line Pc 7.3 showed emergence of new leaves under severe salt stress for 10 days (300mM; a) Data in the graphs are the average ± SD of six independent sets of experiments with six plants per line. Different letters denote statistically significant differences (P < 0.01). (TIF 3495 kb)

11240_2021_2026_MOESM7_ESM.tiff

Supplementary file 7 SupplementaryFig. 7. Drought and Cold tolerance analysis of some more PcCFR plants. Photographs of one month old plants were taken after 10 days of stress treatment (drought/cold) (a). Scale bar = 1 cm. A comparative analysis of shoot, root length, fresh weight and dry weight showed better performance of PcCFR plants (Pc2.7, Pc1.2, Pc3.2) than OsCFR (Os3.4) for drought stress (b.i.- e.i.) and cold stress (b.ii.- e.ii.) respectively. Overall performance of PcCFR are better in terms of Fv/Fm, PI(abs) and ET0 / CSm in drought (f.i.-h.i.) and cold stresses (f.ii.-h.ii.). Data in the graphs are the average ± SD of six independent sets of experiments with six plants per line. Data in the graph are the average ± SE of six independent sets of experiments with six plants per line. Different letters denote statistically significant differences (P < 0.01). (TIF 141 kb)

11240_2021_2026_MOESM8_ESM.tif

Supplementary file 8 Supplementary Fig. 8 . List of the primers (a) and the PCR conditions (b) applied to the hi-TAIL PCR according to (Liu and Chen, 2007) (TIF 11456 kb)

Supplementary file 9 (DOCX 226 kb)

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Mukherjee, S., Mukherjee, A., Das, P. et al. A salt‐tolerant chloroplastic FBPase from Oryza coarctata confers improved photosynthesis with higher yield and multi‐stress tolerance to indica rice. Plant Cell Tiss Organ Cult 145, 561–578 (2021). https://doi.org/10.1007/s11240-021-02026-1

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  • DOI: https://doi.org/10.1007/s11240-021-02026-1

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