An S-domain receptor-like kinase, OsSIK2, confers abiotic stress tolerance and delays dark-induced leaf senescence in rice

Abstract

Receptor-like kinases play important roles in plant development and defense responses; however, their functions in other processes remain unclear. Here, we report that OsSIK2, an S-domain receptor-like kinase from rice (Oryza sativa), is involved in abiotic stress and the senescence process. OsSIK2 is a plasma membrane-localized protein with kinase activity in the presence of Mn(2+). OsSIK2 is expressed mainly in rice leaf and sheath and can be induced by NaCl, drought, cold, dark, and abscisic acid treatment. Transgenic plants overexpressing OsSIK2 and mutant sik2 exhibit enhanced and reduced tolerance to salt and drought stress, respectively, compared with the controls. Interestingly, a truncated version of OsSIK2 without most of the extracellular region confers higher salt tolerance than the full-length OsSIK2, likely through the activation of different sets of downstream genes. Moreover, seedlings of OsSIK2-overexpressing transgenic plants exhibit early leaf development and a delayed dark-induced senescence phenotype, while mutant sik2 shows the opposite phenotype. The downstream PR-related genes specifically up-regulated by full-length OsSIK2 or the DREB-like genes solely enhanced by truncated OsSIK2 are all induced by salt, drought, and dark treatments. These results indicate that OsSIK2 may integrate stress signals into a developmental program for better adaptive growth under unfavorable conditions. Manipulation of OsSIK2 should facilitate the improvement of production in rice and other crops.

Figure 1.

OsSIK2 gene expression, protein kinase activity, and subcellular localization. A, Schematic representation of the OsSIK2 protein. SP, Signal peptide; TM, transmembrane domain. B, Expression of OsSIK2 in response to NaCl, drought, cold, and ABA by quantitative reverse transcription (qRT)-PCR. Each column is the average of three repeats, and error bars indicate sd. C, Expression of OsSIK2 in different organs by qRT-PCR. Each column is the average of three repeats, and error bars indicate sd. D, Phosphorylation assay of OsSIK2-KINASE DOMAIN (KD). The OsSIK2 kinase domain was expressed and purified from E. coli by affinity resin. The MBP fusion protein was stained with Coomassie blue (left) and confirmed by western blot using an anti-MBP monoclonal antibody (middle). OsSIK2-KD (2 μg) was incubated with [γ-³²P]ATP in the presence of 5 mm Mg²⁺, 5 mm Ca²⁺, or 5 mm Mn²⁺; OsSIK2-KD was also incubated with Mn²⁺ and 10 mm EDTA and used as a control (right). Numbers on the left indicate protein sizes in kD. E, Subcellular localization of OsSIK2 protein. OsSIK2-f indicates the full-length OsSIK2 protein. OsSIK2-t indicates truncated OsSIK2 without most of the extracellular region. GFP was used as a control.

Figure 2.

Generation of OsSIK2-overexpressing plants, mutant sik2 identification, and phenotype analysis. A, Schematic of mutant sik2 insertion and the OsSIK2-truncated protein (OsSIK2-t). SP, Signal peptide; TM, transmembrane domain. The arrow indicates the Tos17 insertion site. The full length of OsSIK2 is represented by OsSIK2-f below. B, The sik2 mutant has no expression of OsSIK2 as revealed by reverse transcription-PCR. The ACTIN gene was amplified as a control. C, OsSIK2 expression in OsSIK2-overexpressing lines and the mutant sik2 by qRT-PCR. OsSIK2-t indicates a transgenic line with a truncated OsSIK2 gene. OsSIK2-f indicates a transgenic line with a full-length OsSIK2 gene. OX indicates OsSIK2-overexpressing transgenic lines. For each column, error bars indicate sd (n = 3). D, Comparison of the phenotypes of young seedlings. Fourteen-day-old plants were used. E, Comparison of various plant lines. Nine-day-old plants grown in soil were used for photography. F, Shoot length of 9-d-old plants. G, Root length of 9-d-old plants. H, Ratio of root length to shoot length in 9-d-old seedlings. I, Third leaf emergence rates in various plants. In total, 30 seedlings for each line were surveyed at the indicated time points. J, Phenotypes of field-grown plants. Field-grown 50-d-old (top) and 120-d-old (bottom) plants were regrown in pots for photography. K, Measurement of plant height. For F, G, H, and K, each column represents an average of at least 15 plants. Error bars indicate sd. Asterisks indicate significant differences from the corresponding controls (Nip or TP309) at *P < 0.05 and **P < 0.01.

Figure 3.

Performance of 35S-OsSIK2 transgenic plants and the sik2 mutant under salt stress. A, Comparison of seedling growth under salt stress. For 35S-OsSIK2 transgenic plants, 14-d-old seedlings in pots were soaked in 0.6% NaCl for 14 d and then recovered for 7 d. For sik2, 14-d-old seedlings were soaked in 0.5% NaCl for 18 d and then allowed to recover for 10 d. Numbers below the images indicate survival rates. B, Chlorophyll content in 14-d-old plants treated with 200 mm NaCl for 24 h. Each column represents an average of three replicates. Error bars indicate sd. Asterisks indicate significant differences from the corresponding controls (Nip or TP309) at *P < 0.05 and **P < 0.01. fw, Fresh weight.

Figure 4.

Performance of OsSIK2-overexpressing transgenic plants and the sik2 mutant under drought stress. For 35S-OsSIK2 transgenic plants, 21-d-old seedlings were withheld from water for 5 d and then exposed to water for 7 d. For sik2, 14-d-old seedlings were withheld from water for 10 d and then exposed to water for 10 d. Numbers below the images indicate survival rates.

Figure 5.

Salt tolerance of OsSIK2-overexpressing transgenic plants under field conditions. A, Growth of various rice seedlings. One-month-old field-grown seedlings were transferred to the controlled system and grown for 1 week before salt treatment. B, Plant growth for 3 weeks under salt stress. The above seedlings were exposed to 0.5% NaCl for 3 weeks and then photographed. C, Plant growth for 4 weeks under salt stress. The seedlings in A were exposed to 0.5% NaCl for 4 weeks and then photographed for comparison. D, Comparison of plant growth after recovery from salt stress. The seedlings exposed to 0.5% NaCl for 4 weeks were recovered in water for 3 weeks.

Figure 6.

Antioxidative capacity was enhanced in transgenic plants overexpressing OsSIK2. Two-week-old plants grown in pots were soaked in water or in 200 mm NaCl for 4 d, and then flag leaves were detached for DAB staining or measurement of POD activity. Brown regions or spots on leaves indicate the presence of H₂O₂. A, DAB staining of flag leaves. B, POD activity in flag leaves of various rice seedlings. Each column represents an average of three replicates. Error bars indicate sd. Asterisks indicate significant differences from the corresponding controls (Nip or TP309) at *P < 0.05 and **P < 0.01. C, Expression of the POD genes POX-1 and POX-2. Leaves from 2-week-old plants were pooled for RNA extraction, and expression was determined by qRT-PCR. Error bars indicate sd (n = 3).

Figure 7.

Delayed leaf senescence in OsSIK2 transgenic plants in the dark. A, Comparison of plants after dark treatment. Top left, 14-d-old plants before treatment; top right, second leaves from top left plants; bottom left, plants in the dark for 6 d; bottom right, second leaves from bottom left plants. B, Chlorophyll (Chl) contents in the second leaves of rice seedlings. Each column represents an average of three replicates. Error bars indicate sd. Asterisks indicate significant differences from the corresponding controls (Nip or TP309) at *P < 0.05 and **P < 0.01. FW, Fresh weight. C, Expression of OsSIK2 in response to dark treatment. Two-week-old TP309 plants were treated, and leaves were pooled for RNA extraction. Expression was determined by qRT-PCR. D, Expression pattern of OsSIK2 in a senescing leaf. One-month-old leaves showing approximately 20% senescence were dissected into three parts as indicated, and OsSIK2 expression was measured. For C and D, error bars indicate sd (n = 3).

Figure 8.

OsSIK2 alters the expression of downstream genes. Two-week-old plants were treated, and gene expression was determined by qRT-PCR. Each column represents an average of three replicates. Error bars indicate sd. A, Genes specifically up-regulated in OsSIK2-f transgenic plants (OX-15 and OX-17). B, Genes specifically up-regulated in OsSIK2-t transgenic plants (OX-72 and OX-74). C, Induction of gene expression under NaCl stress in TP309 seedlings. D, Induction of gene expression with PEG (20%) treatment in TP309 seedlings. E, Dark-induced gene expression in TP309 seedlings.