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Expression of chickpea CIPK25 enhances root growth and tolerance to dehydration and salt stress in transgenic tobacco.

Meena MK, Ghawana S, Dwivedi V, Roy A, Chattopadhyay D - Front Plant Sci (2015)

Bottom Line: Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions.Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants.Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Plant Genome Research New Delhi, India.

ABSTRACT
Calcium signaling plays an important role in adaptation and developmental processes in plants and animals. A class of calcium sensors, known as Calcineurin B-like (CBL) proteins sense specific temporal changes in cytosolic Ca(2+) concentration and regulate activities of a group of ser/thr protein kinases called CBL-interacting protein kinases (CIPKs). Although a number of CIPKs have been shown to play crucial roles in the regulation of stress signaling, no study on the function of CIPK25 or its orthologs has been reported so far. In the present study, an ortholog of Arabidopsis CIPK25 was cloned from chickpea (Cicer arietinum). CaCIPK25 gene expression in chickpea increased upon salt, dehydration, and different hormonal treatments. CaCIPK25 gene showed differential tissue-specific expression. 5'-upstream activation sequence (5'-UAS) of the gene and its different truncated versions were fused to a reporter gene and studied in Arabidopsis to identify promoter regions directing its tissue-specific expression. Replacement of a conserved threonine residue with an aspartic acid at its catalytic site increased the kinase activity of CaCIPK25 by 2.5-fold. Transgenic tobacco plants overexpressing full-length and the high active versions of CaCIPK25 displayed a differential germination period and longer root length in comparison to the control plants. Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions. Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants. Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.

No MeSH data available.


Related in: MedlinePlus

Effect of salt stress on transgenic tobacco seedlings. (A) Control (vector only), CaCIPK25- and CaCIPK25T/D- expressing tobacco seeds were germinated on media without (left) and with (right) 200 mM sodium chloride for 15 days. (B) 8-day-old seedlings of control, CaCIPK25− and CaCIPK25T/D-expressing lines were transferred to media without (left) and with (right) 250 mM sodium chloride for 10 days and then transferred to normal medium for 8 days. (C) Leaves of the seedlings of two representative transgenic lines (L1 and L2) exposed to salt were scored for chlorosis and bleaching and presented as relative to total number of leaves. (D) Fresh weights of the seedlings from the same experiment were presented relative to the fresh weights of corresponding seedlings grown in normal medium for the same period.
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Figure 7: Effect of salt stress on transgenic tobacco seedlings. (A) Control (vector only), CaCIPK25- and CaCIPK25T/D- expressing tobacco seeds were germinated on media without (left) and with (right) 200 mM sodium chloride for 15 days. (B) 8-day-old seedlings of control, CaCIPK25− and CaCIPK25T/D-expressing lines were transferred to media without (left) and with (right) 250 mM sodium chloride for 10 days and then transferred to normal medium for 8 days. (C) Leaves of the seedlings of two representative transgenic lines (L1 and L2) exposed to salt were scored for chlorosis and bleaching and presented as relative to total number of leaves. (D) Fresh weights of the seedlings from the same experiment were presented relative to the fresh weights of corresponding seedlings grown in normal medium for the same period.

Mentions: To investigate the effect of the CaCIPK25 expression on germination efficiency of the transgenic seeds under high salinity condition, seeds were sown on normal growth medium supplemented with 200 mM sodium chloride and allowed to germinate for 15 days. The tobacco seeds transformed with the empty vector and both the constructs of CaCIPK25 germinated simultaneously and showed similar growth in normal growth medium. Both the CaCIPK25-transformed seeds took 6 days more to germinate on the salt-supplemented medium as compared to their germination on the normal growth medium. Only 7% of vector-control seeds germinated in comparison to 60 and 64% seed germination of CaCIPK25− and CaCIPK25T/D-transformed lines, respectively, after 15-day exposure on high-salt medium (Figure 7A). To assess salt-tolerance of the seedlings, 8-day-old seedlings were exposed to medium supplemented with 250 mM sodium chloride. After 10 days of exposure, the seedlings were transferred to the normal growth medium for recovery for 8 days (Figure 7B). Leaves were scored for chlorosis and the relative fresh weights for each line were assessed by comparing the fresh weights of the corresponding lines continuously grown on the normal growth medium. Approximately 80% leaves of the control plants have undergone chlorosis in contrast to about 36 and 26% chlorosis in the lines expressing CaCIPK25 and CaCIPK25T/D, respectively (Figure 7C). The relative fresh weight of the control seedlings grown in high salinity was about 20% of those grown in normal medium, while the relative fresh weights of the CaCIPKL25− and CaCIPK25T/D− expressing plants were 53 and 57%, respectively (Figure 7D). Enhanced tolerance to salt and drought was also observed in plants grown in pots. Fifty days-old plants of one line for each of the constructs, three in a pot in duplicate, were irrigated with 300 mM sodium chloride twice a week for 2 weeks. All the vector-control plants were etiolated, displayed severe chlorosis and died. The plants expressing both the forms of CaCIPK25 showed a moderate level of chlorosis and etiolation. Similarly, 50-day-old plants in pots were not irrigated for 25 days. All the control plants died within this period while all the plants expressing the either form of CaCIPK25 showed a moderate level of etiolation (Figure 8) and nine out of 12 plants recovered upon further irrigation for 2 weeks.


Expression of chickpea CIPK25 enhances root growth and tolerance to dehydration and salt stress in transgenic tobacco.

Meena MK, Ghawana S, Dwivedi V, Roy A, Chattopadhyay D - Front Plant Sci (2015)

Effect of salt stress on transgenic tobacco seedlings. (A) Control (vector only), CaCIPK25- and CaCIPK25T/D- expressing tobacco seeds were germinated on media without (left) and with (right) 200 mM sodium chloride for 15 days. (B) 8-day-old seedlings of control, CaCIPK25− and CaCIPK25T/D-expressing lines were transferred to media without (left) and with (right) 250 mM sodium chloride for 10 days and then transferred to normal medium for 8 days. (C) Leaves of the seedlings of two representative transgenic lines (L1 and L2) exposed to salt were scored for chlorosis and bleaching and presented as relative to total number of leaves. (D) Fresh weights of the seedlings from the same experiment were presented relative to the fresh weights of corresponding seedlings grown in normal medium for the same period.
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Related In: Results  -  Collection

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Show All Figures
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Figure 7: Effect of salt stress on transgenic tobacco seedlings. (A) Control (vector only), CaCIPK25- and CaCIPK25T/D- expressing tobacco seeds were germinated on media without (left) and with (right) 200 mM sodium chloride for 15 days. (B) 8-day-old seedlings of control, CaCIPK25− and CaCIPK25T/D-expressing lines were transferred to media without (left) and with (right) 250 mM sodium chloride for 10 days and then transferred to normal medium for 8 days. (C) Leaves of the seedlings of two representative transgenic lines (L1 and L2) exposed to salt were scored for chlorosis and bleaching and presented as relative to total number of leaves. (D) Fresh weights of the seedlings from the same experiment were presented relative to the fresh weights of corresponding seedlings grown in normal medium for the same period.
Mentions: To investigate the effect of the CaCIPK25 expression on germination efficiency of the transgenic seeds under high salinity condition, seeds were sown on normal growth medium supplemented with 200 mM sodium chloride and allowed to germinate for 15 days. The tobacco seeds transformed with the empty vector and both the constructs of CaCIPK25 germinated simultaneously and showed similar growth in normal growth medium. Both the CaCIPK25-transformed seeds took 6 days more to germinate on the salt-supplemented medium as compared to their germination on the normal growth medium. Only 7% of vector-control seeds germinated in comparison to 60 and 64% seed germination of CaCIPK25− and CaCIPK25T/D-transformed lines, respectively, after 15-day exposure on high-salt medium (Figure 7A). To assess salt-tolerance of the seedlings, 8-day-old seedlings were exposed to medium supplemented with 250 mM sodium chloride. After 10 days of exposure, the seedlings were transferred to the normal growth medium for recovery for 8 days (Figure 7B). Leaves were scored for chlorosis and the relative fresh weights for each line were assessed by comparing the fresh weights of the corresponding lines continuously grown on the normal growth medium. Approximately 80% leaves of the control plants have undergone chlorosis in contrast to about 36 and 26% chlorosis in the lines expressing CaCIPK25 and CaCIPK25T/D, respectively (Figure 7C). The relative fresh weight of the control seedlings grown in high salinity was about 20% of those grown in normal medium, while the relative fresh weights of the CaCIPKL25− and CaCIPK25T/D− expressing plants were 53 and 57%, respectively (Figure 7D). Enhanced tolerance to salt and drought was also observed in plants grown in pots. Fifty days-old plants of one line for each of the constructs, three in a pot in duplicate, were irrigated with 300 mM sodium chloride twice a week for 2 weeks. All the vector-control plants were etiolated, displayed severe chlorosis and died. The plants expressing both the forms of CaCIPK25 showed a moderate level of chlorosis and etiolation. Similarly, 50-day-old plants in pots were not irrigated for 25 days. All the control plants died within this period while all the plants expressing the either form of CaCIPK25 showed a moderate level of etiolation (Figure 8) and nine out of 12 plants recovered upon further irrigation for 2 weeks.

Bottom Line: Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions.Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants.Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Plant Genome Research New Delhi, India.

ABSTRACT
Calcium signaling plays an important role in adaptation and developmental processes in plants and animals. A class of calcium sensors, known as Calcineurin B-like (CBL) proteins sense specific temporal changes in cytosolic Ca(2+) concentration and regulate activities of a group of ser/thr protein kinases called CBL-interacting protein kinases (CIPKs). Although a number of CIPKs have been shown to play crucial roles in the regulation of stress signaling, no study on the function of CIPK25 or its orthologs has been reported so far. In the present study, an ortholog of Arabidopsis CIPK25 was cloned from chickpea (Cicer arietinum). CaCIPK25 gene expression in chickpea increased upon salt, dehydration, and different hormonal treatments. CaCIPK25 gene showed differential tissue-specific expression. 5'-upstream activation sequence (5'-UAS) of the gene and its different truncated versions were fused to a reporter gene and studied in Arabidopsis to identify promoter regions directing its tissue-specific expression. Replacement of a conserved threonine residue with an aspartic acid at its catalytic site increased the kinase activity of CaCIPK25 by 2.5-fold. Transgenic tobacco plants overexpressing full-length and the high active versions of CaCIPK25 displayed a differential germination period and longer root length in comparison to the control plants. Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions. Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants. Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.

No MeSH data available.


Related in: MedlinePlus