CDPK1 from ginger promotes salinity and drought stress tolerance without yield penalty by improving growth and photosynthesis in Nicotiana tabacum

Abstract

In plants, transient changes in calcium concentrations of cytosol have been observed during stress conditions like high salt, drought, extreme temperature and mechanical disturbances. Calcium-dependent protein kinases (CDPKs) play important roles in relaying these calcium signatures into downstream effects. In this study, a stress-responsive CDPK gene, ZoCDPK1 was isolated from a stress cDNA generated from ginger using rapid amplification of cDNA ends (RLM-RACE) - PCR technique and characterized its role in stress tolerance. An important aspect seen during the analysis of the deduced protein is a rare coupling between the presence of a nuclear localization sequence in the junction domain and consensus sequence in the EF-hand loops of calmodulin-like domain. ZoCDPK1 is abundantly expressed in rhizome and is rapidly induced by high-salt stress, drought, and jasmonic acid treatment but not by low temperature stress or abscissic acid treatment. The sub-cellular localization of ZoCDPK1-GFP fusion protein was studied in transgenic tobacco epidermal cells using confocal laser scanning microscopy. Over-expression of ginger CDPK1 gene in tobacco conferred tolerance to salinity and drought stress as reflected by the high percentage of seed germination, higher relative water content, expression of stress responsive genes, higher leaf chlorophyll content, increased photosynthetic efficiency and other photosynthetic parameters. In addition, transgenic tobacco subjected to salinity/drought stress exhibited 50% more growth during stress conditions as compared to wild type plant during normal conditions. T3 transgenic plants are able to grow to maturity, flowers early and set viable seeds under continuous salinity or drought stress without yield penalty. The ZoCDPK1 up-regulated the expression levels of stress-related genes RD21A and ERD1 in tobacco plants. These results suggest that ZoCDPK1 functions in the positive regulation of the signaling pathways that are involved in the response to salinity and drought stress in ginger and it is likely operating in a DRE/CRT independent manner.

Figure 1. Amino acid sequence alignment and phylogenetic tree of ginger CDPK1.

(A) The amino acid sequence alignment of Zingiber CDPK1 [GenBank ID: KC544003] with Datura CDPK, DmCPK1 [GenBank ID: 163658596] and Arabidopsis CDPK, AtCPK30 [GenBank ID: 30699042]. Identical amino acids are indicated by asterisks, and similar amino acids are marked with single dots and colons. Catalytic domains (I–XI), Junction domain, and EF hand loops of CaM-LD domain of ZoCDPK1 are marked. The 15 invariant amino acid residues for eukaryotic Ser/Thr protein kinase were highlighted. Protein kinase ATP-binding site is shown by broken lines. Active site is shown in red box. (B) Phylogenetic analysis of ZoCDPK1 with all Arabidopsis CDPKs in the database. The tree was constructed using NJ method of Mega4 software. The numbers on branches showed bootstrap probabilities determined for 1000 re-samplings. The database accession numbers are indicated in parantheses after CDPK gene names.

Figure 2. Domain analysis of ZoCDPK1.

(A) Multiple sequence alignment of the JDs of CDPKs containing a bipartite NLS as a subdomain. The NLS is shown in the black box. The relative positions of autoinhibitory and CaM-LD binding sub-domains are indicated. The determinant amino acids for the bipartite NLS are shaded. (B) Multiple sequence alignment of Ca²⁺-binding EF hand loops of the above CDPKs [DX(DNS)(ILVFYW) (DENSTG)(DNQGHRK)(GP)(LIVMC)(DENQSTAGC)X(2)(DE)(LIVMFYW]. The residue numbers important for coordination with Ca²⁺ in the respective Ca²⁺-binding loops are indicated.

Figure 3. Quantitative real time PCR analysis of ZoCDPK1 transcripts in ginger.

(A) ZoCDPK1 expression was studied in leaf, stem and rhizome. (B) Total RNA was isolated from leaves collected at successive intervals of 0, 3, 6, 9, 12, 15 and 24 h post salinity (400 mM NaCl). (C) Total RNA was isolated from leaves collected at successive intervals of 0, 3, 6, 9, 12, 15 and 24 h post drought (dehydration) treatment. (D) Total RNA was isolated from leaves collected at successive intervals of 0, 3, 6, 9, 12, 15 and 24 h post ABA (100 μM). (E) Total RNA was isolated from leaves collected at successive intervals of 0, 3, 6, 9, 12, 15 and 24 h post low temperature (4°C) treatment. (F) A time course expression profile of ZoCDPK1 against JA (100 µM) was studied at regular intervals of 0, 24, 48, 72 and 96 h post treatment. The EF1α gene was used as endogenous control in all experiments and relative gene expression was calculated by the equation 2^-ddCt. Data are presented as mean + SE (n=3) and error bars represent SE.

Figure 4. Schematic representation of the T-DNA and molecular analysis of ZoCDPK1-expressing tobacco.

(A) Gateway recombination reaction between pCR^®8/GW/TOPO-ZoCDPK1 entry vector and pMDC85 destination vector and resultant T-DNA region of the vector pMDC85-ZoCDPK1-GFP. RB, right T-DNA border; LB, left T-DNA border; 35S, cauliflower mosaic virus 35S promoter; nos T, NOS terminator; Hyg^r, hygromycin phosphotransferase II gene. (B) RT-PCR analysis using ZoCDPK1 specific primers and EF1α specific primers of wild type and five independent T3 transgenic lines. (C) Western blot analysis of wild type and and transgenic tobacco (CD-1 and CD-6) lines. Proteins (20 µg per lane) were fractionated by 12.5% SDS-PAGE and immunobloted with GFP-specific antibody (D) Quantitative real time PCR analysis of ZoCDPK1 transcripts in wild type and transgenic tobacco lines (CD-1 and CD-6) under normal, salinity and drought conditions. The EF1α gene was used as endogenous control in all experiments. Relative gene expression was given as the fold expression change using the equation 2^-ddCt. Data are presented as mean + SE (n=3) and error bars represent SE.

Figure 5. Sub-cellular localization of ZoCDPK1-GFP fusion protein.

Transient expression of the ZoCDPK1-GFP in the plasmolyzed onion epidermal cells. Onion epidermal peels were incubated in IM sucrose for 10 minute before staining and visualization. GFP fluorescence, PI fluorescence and merged images are respectively the first, middle and last panels.

Figure 6. Leaf disk senescence assay for salinity and drought tolerance in transgenic tobacco plants (T3).

(A) Representative pictures to show phenotypic differences in leaf disks of wild type (WT), vector control (VC) and T3 plants (CD-1 and CD-6) after incubation in 0, 100, 200, 300, 400 and 500 mM NaCl or 500 mM mannitol solutions. (B) Representative diagram to show the chlorophyll content from leaf disks of wild type, vector control and T3 transgenic plants after incubation in 200 mM NaCl or 300 mM mannitol solutions. Data are presented as mean + SE (n=3) and error bars represent SE. Data shows a significant difference of chl.a or chl.b content between transgenic and wild type controls at P<0.0001, by Student’s t-test.

Figure 7. Analysis of ZoCDPK1 transgenic (T3) plants under salinity and drought.

(A) Representative pictures to show the germination of transgenic (CD-1 and CD-6), vector control (VC) and wild type (WT) seeds under normal conditions (non-stress), 200 mM NaCl (salinity) and 300 mM mannitol (drought). (B) Representative seedlings of WT, VC and T3 homozygous lines (CD-1 and CD-6) taken after 14 days of germination on normal (non-stress), 200 mM NaCl (Upper) and 300 mM mannitol (Lower) conditions.

Figure 8. Quantitative real time PCR analysis of stress responsive genes in wild type, vector contorl and T3 transgenic plants after induction with 200 mM NaCl.

Relative gene expression was given as the fold expression change using the equation 2^-ddCt. Data are presented as mean + SE (n=3) and error bars represent SE.

Figure 9. Physiological assessment of transgenic plants (T3), vector control and wild type plants.

(A) Net CO₂ uptake rate measured at a PPFD of 20 and 2000 µmol quanta m^-2 sec^-1 and 400 p.p.m. CO₂. Effect of salinity and drought stress on the net photosynthetic rate (B), Transpiration rate (C), inter-cellular CO₂ concentration (D), leaf conductance (E) and photosynthetic efficiency (F) of transgenic and wild type plants.