Introduction

Rice, a leading staple food crop cultivated worldwide except in Antarctica, plays a critical role in ensuring food as well as nutritional security, particularly in Southeast Asian countries such as India1. The phrase “Rice is life” aptly captures its significance in India, where over 60% of the population relies on rice, which occupies around one-fourth of the country’s total cropping area2. In order to feed exponentially growing India’s population, numerous studies advocate the use of multidisciplinary approach to enhance rice production and mitigate losses caused by various environmental stresses3. Research indicates that rice production is extremely vulnerable to both abiotic and biotic stresses, with drought and submergence being major constraints4,5,6. The ongoing challenges posed by climate change have further exacerbated the situation7. Southeast Asia, including India, has experienced critical water shortages during key growth stages of rice, signaling potential future challenges for rice production6. In India, about 50% of the rice-growing regions depend on rainfed agriculture, and it is projected that the suitability of these areas for rice production could decline by 15–40% by 20508. The reliance on monsoon rains makes rainfed rice cultivation particularly vulnerable to environmental stresses such as drought and submergence9. A failure of the monsoon can lead to drought, hindering rice cultivation, while excessive rainfall can result in submergence stress9,10,11. Cultivation of rice is highly sensitive to water scarcity and flooding, leading to significant reduction in yield in majority of Asian countries9,12.

The onset of drought in rice cultivation causes a significant threat to yield stability particularly in rainfed areas13,14. It severely limits crop productivity by preventing them from achieving their full genetic yield potential15. Globally, drought conditions result in an estimated annual loss of 18 million tonnes of rice16. In Asia, around 23 million hectares are affected by drought, with 13.6 million hectares impacted in Eastern India alone17,18. Rice can suffer from drought at any growth stage including early seedling, vegetative, and reproductive stages causing considerable yield losses19. Among these, reproductive stage drought is particularly detrimental, leading to fewer grains per panicle, increased grain sterility, and lower grain weight20,21. The magnitude of yield loss during the reproductive stage varies with the severity of the drought15. Consequently, efforts have been made to select drought-tolerant lines during the reproductive stage, with the best performers being released in specific regions15. Sahbhagi Dhan, released in India in 2010, exemplifies a successful approach to improving rice varieties22. Numerous QTLs associated with drought stress have been reported, and efforts are underway to introduce these QTLs into popular rice genotypes. Nagina-22, a drought-tolerant donor variety, has been frequently used in breeding programs due to the presence of qDTY1.1, which influences grain yield during drought at the reproductive stage23,24. Conversely, excessive monsoon rains often lead to unpredictable flash floods, which can severely affect rice crops at various growth stages11. Yield losses can range from less than 10–100%, depending on factors such as water depth, submergence duration, water temperature, nitrogen levels, light conditions, and the plant’s age25. In South and Southeast Asia, early-stage flash floods can significantly reduce yields across more than 20 million hectares26. Submergence during the seedling phase is particularly damaging, with floods lasting 5 to 15 days causing major yield losses, sometimes up to 100%11. A key QTL, Sub1, has been identified as providing considerable tolerance up to 17 days of complete submergence during the seedling stage27,28,29. Swarna Sub1, a variety with the Sub1 gene, offers about a 45% increase in yield compared to conventional varieties when submerged for 10 days during flooding27,28,29.

Conventional breeding and marker-assisted introgression of QTLs associated with water stress have led to significant reductions in yield losses occurred owing to drought and submergence10,30,31. Despite these advancements, the insight of the mechanisms behind drought and submergence responses remains limited. While the incorporation of QTLs has helped to reduce the yield gap between stress-exposed and control plants, complete closure of this gap has not been achieved32. A deeper understanding of the mechanisms behind these stresses is essential for further bridging this gap32. Plants respond to various stress stimuli by activating specific signaling pathways. Among these, calcium ions (Ca2+) play a central role as key signaling molecules, serving as convergence points for different stress-related pathways21,33. Abiotic stresses often lead to alterations in cytosolic free Ca2 + concentrations ([Ca2+]cyt), generating distinct calcium signatures34. Calmodulin (CaM), a calcium-binding protein, detects these changes in [Ca2+]cyt. Although CaM lacks intrinsic enzymatic activity, it partners with various CaM-binding proteins (CBPs)35. Among CBPs, members of CaM binding protein 60 (CBP60s) family generally act as transcription factors regulated by CaM, with most members of the AtCBP60 family containing a CaM-binding domain (CBD)36,37. In contrast to the typical C-terminal location of the CBD in most AtCBP60 proteins, the CBD in AtCBP60g is positioned at the N-terminus38,39. This domain in AtCBP60g facilitates CaM binding, whereas AtSARD1, which lacks a CBD, does not interact with CaM38,40. Similarly, the OsSARDL proteins have been found not to have a predicted CBD41. Numerous CBPs have been studied in plants, many of which are implicated in stress adaptations42,43,44,45. The CBP60 family is notable for containing only the CaM-binding domain46. In Arabidopsis, the CBP60 (AtCBP60) family includes eight members, which regulate responses to various stresses47,48. For instance, genetically modified plants with enhanced expression AtCBP60gdemonstrated greater resilience to drought48.

Research on the CBP60 family pertaining to different stresses in plants remains limited. In tomato (Solanum lycopersicum), 11 SlCBP60genes have been identified that regulate responses to pathogens and temperature stress, indicating their involvement in both disease resistance and temperature vulnerability49. For example, SmCBP60A1 in eggplant (Solanum melongena) serves as a positive regulator under salt stress, increasing the plant’s tolerance to high salinity. This highlights the dual function of CBP60s in managing both biotic and abiotic stress50. In cotton (Gossypium hirsutum), GhCBP60B recognized as a pivotal role in improving salt and drought stress51. In soybean (Glycine max), GmCBP60A−1plays a pivotal role in improving drought and salt stress tolerance, as demonstrated by the enhanced stress resilience in transgenic plants overexpressing this gene51,52. Recently, we have identified and characterized 15 OsCBP60 gene families in rice, which play significant roles in pathogen defense41. Specifically, OsCBP60g−3 and OsCBP60g−4are upregulated in response to the economically important pathogens of rice41. These genes are also responsive to salicylic acid (SA) and brassinosteroids (BRs), which are key regulators of plant immune responses41. In present investigation, we explored the roles of various OsCBP60 genes in response to different abiotic stresses in rice. Our study showed that OsCBP60bcd−2 and OsCBP60g−1/OsSARD1 were consistently upregulated during reproductive drought stress. OsCBP60g−5 and OsCBP60g−6 were found to be consistently upregulated in rice seedlings subjected to submergence stress. Notably, OsCBP60g−4 showed consistent upregulation under both reproductive drought stress (with the exception of 3rd day) and submergence stress. Interestingly, in our previous findings, OsCBP60g−4 showed enhanced expression in response to pathogen and phytohormone (SA and BRs) treatments41. These findings suggest the potential of CBP60 genes in crop improvement strategies aimed at increasing tolerance to different environmental stresses. Our results indicate that OsCBP60g−4 has a broad function in regulating rice responses to various biotic and abiotic stresses, making it a valuable candidate for molecular breeding and genetic engineering efforts to enhance stress resilience in rice.

Materials and methods

Planting materials

All the rice genotypes used in this investigation were collected from BAU, Sabour, Bhagalpur, Bihar, India. Rice genotypes viz. Rajendra Kasturi, Sahbhaghi Dhan, and Nagina-22 were used for the reproductive drought stress experiment. For the submergence stress, Swarna and Swarna-Sub1, submergence susceptible and tolerant rice genotypes, respectively, were used in this study. These rice genotypes were previously used in different stress experiments23,30,53,54,55. Before sowing, seeds were surfaced sterilized by washing 70% ethanol (v/v) followed by washing thrice with autoclaved distilled water. Further, seeds were treated with treated HgCl2 (0.1%) for 20 min with intermittent shaking followed by washing five times with autoclaved distilled water.

Drought stress treatment

Surface sterilized seeds of different rice genotypes were grown in earthen pots containing a mixture of soil and coco-peat (2:1 ratio) in two sets with 5 replications in completely randomized design (CRD). Plants were grown in a greenhouse with temperatures maintained at 28 °C during the day and 23 °C at night. All pots were irrigated alternate day with equal amount of water until the onset of drought. To induce reproductive-stage drought stress, irrigation was withheld fifteen days earlier to booting stage as previously described56,57. The moisture level was reduced to 60% of field capacity (FC) in stressed pots using gravimetric method as described previously58. In contrast, properly-watered rice plants acted as the control. Leaf tissues from drought-stressed and control plants were collected at 3rd, 6th, 9th and 15th day after the initiation of the drought treatment. Collected leaf tissues were quickly frozen using liquid nitrogen and kept at −86 °C for future use.

Submergence stress treatment

For submergence stress, 30 days old seedlings of different genotypes were submerged in a submergence tank (concrete tank) at a depth of 1 m for fifteen days as described earlier11. A control set was also maintained along with submergence stress11. Leaf tissues were collected from both the submerged and control plants. The collected samples were rapidly frozen with liquid nitrogen and kept at −86 °C freezer for future analysis.

Quantitative real-time RT-PCR (qRT-PCR) analysis

Total RNA was isolated from rice samples using the SV Total RNA Isolation kit (Promega, Madison, WI, USA) as per manufacturer’s guidelines. Total RNA (1ug) was then utilized for cDNA synthesis using random hexamer primers in 20 ul system, following the manufacturer’s instructions (GoScript™ Reverse Transcriptase kit, Promega, Madison, WI, USA). The cDNA was diluted with nuclease-free water (1:5) and used for qRT-PCR analysis. qRT-PCR was performed on a Light Cycler system (Agilent Technologies) using SYBR Green dye. Each gene’s expression was performed in triplicate, using gene specific primers (Table S1). The qRT-PCR amplification conditions were as follows: 95 °C for 3 min; 40 cycles at 95 °C for 20 s; 55 °C for 30 s; and 72 °C for 45 s. A melting curve analysis was performed by gradually heating the amplicon from 65 °C to 95 °C to confirm primer specificity. Actin expression was used as a reference to normalize the expression data, and the fold change was calculated using the 2−∆∆Ctmethod relative to the control59.

Statistical analysis

The data were statistically analysed using SPSS software (Version 16.0, SPSS Inc.). One-way analysis of variance (ANOVA) was conducted to determine the significance of differences. Treatment means were compared using the Least Significant Difference (LSD) multiple-comparison test60.

Results

OsCBP60  Expression analysis in response to reproductive drought

In this study, expression analysis of OsCBP60s was carried out in both drought-tolerant and drought-susceptible rice genotypes during the reproductive drought stage using qRT-PCR. Rice genotypes namely Rajendra Kasturi, Sahbhagi Dhan, and Nagina-22 were used in this study. Sahbhagi Dhan and Nagina-22 were chosen for their drought tolerance, while Rajendra Kasturi was used as a drought-susceptible genotype. To simulate reproductive drought conditions, irrigation was withheld for 15 days before the booting stage. The expression levels of OsCBP60 were examined at 3, 6, 9, and 15 days after withdrawal of water.

The expression profiling of OsCBP60a gene showed a similar downregulation pattern in Rajendra Kasturi and Sahbhagi Dhan on the 3rd, 9th, and 15th day of reproductive drought. In Rajendra Kasturi, the gene was significantly downregulated ~ 16-fold on the 3rd and 15th days, and ~ 7-fold on the 9th day of drought stress. Conversely, OsCBP60a was significantly upregulated in Nagina-22 in all the time point studied compared to Rajendra Kasturi. The significantly higher upregulation of OsCBP60a was also observed in Nagina-22 compared to Sahbhagi Dhan except 15th days of drought treatment (Fig. 1a).

Fig. 1
figure 1figure 1

Expression analysis of OsCBP60s in rice under reproductive drought stress. Transcript levels of CBP60s were assessed using qRT-PCR in drought- susceptible and -resistant genotypes at 3rd, 6th, 9th, and 15th days of reproductive drought. The standard error (SE) of the mean for three replicates is represented by error bars. The results represent three independent biological experiments. Values with the same superscript letter are not significantly different (p < 0.05), as determined by a one-way ANOVA followed by the LSD test.

Full size image

The significant downregulation of the OsCBP60bcd−1 gene was observed in the drought-susceptible genotype Rajendra Kasturi on the 3rd and 15th days, whereas significant upregulation was observed on the 6th and 9th days of drought treatment in the drought-tolerant genotype Nagina-22 (Fig. 1b). Notably, OsCBP60bcd−1 showed significant upregulation on day 9 in both genotypes, with a particularly strong 24-fold increase observed in Nagina-22 (Fig. 1b). The expression of OsCBP60bcd-2 was observed to be upregulated at all these time points studied. However, the significant upregulation of OsCBP60bcd-2 was observed in Nagina-22 compared to all the genotype at all the time point studied. The significant upregulation of Nagina-22 was also observed in 9th day of drought treatment compared to Rajendra Kasturi and Sahbhagi dhan (Fig. 1c). Except 3rd day, the upregulation of OsCBP60bcd-3 was recorded in most of the time point studied in drought-susceptible and tolerant genotypes (Fig. 1d). On 6th and 9th day of drought, OsCBP60bcd−3 expression increased in both types of genotypes, with a notable ~ 27-fold upregulation in Nagina-22 on day 9. By day 15, OsCBP60bcd−3 was downregulated in Rajendra Kasturi, but showed significantly upregulation in both the drought-tolerant genotypes (Fig. 1d).

The expression of OsSARDL-1, 2, 3 was significantly downregulated in the drought-susceptible genotype Rajendra Kasturi during most of the time points studied. However, significant upregulation of OsSARDL-1, 2, 3 was observed in Nagina-22 on the 3rd, 6th, and 9th days of drought stress (Fig. 1e-g). Drought stress induced maximum upregulation of approximately ~ 86, ~63, and ~ 20-fold in OsSARDL-2, OsSARDL-1, and OsSARDL-3, respectively. In Sahbhagi Dhan, significant upregulation of OsSARDL-1 was observed on the 6th and 9th days of drought treatment. The expression of the OsSARDL-3 gene was found to be down-regulated in all genotypes studied on the 15th day of drought stress (Fig. 1g).

Except OsCBP60g−6, all the OsCBP60g genes showed up-regulation at 6th and 9th day of drought stress treatment in both drought-susceptible and tolerant rice varieties (Fig. 1h-o). In Nagina-22, OsCBP60g−1/OsSARDL, OsCBP606g-3, 7 and 8 showed up-regulation in all the time point studied (Fig. 1h-o). Interestingly, significantly up-regulation of all OsCBP60g genes was observed in Nagina-22 at 6th day of drought stress. Further, significant up-regulation of OsCBP60g−1/OsSARDL, OsCBP606g-3, 4, 7 and 8 were detected in Nagina-22 during 6th and 9th day of drought stress. Significantly up-regulation of OsCBP60g−1/OsSARDL and OsCBP60g−4 were determined during 3rd, 6th and 15th day of drought stress treatment in Sahbgagi Dhan, compared to Rajendra Kasturi (Fig. 1h-o). OsCBP60g−3 and 4 showed significant up-regulations at 9th and 15th day whereas OsCBP60g-7 and 8 at 6th and 9th day of drought stress treatment in Sahbhagi Dhan compared to Rajendra Kasturi. OsCBP60g-3 and 4 showed significant up-regulation on the 9th and 15th days, whereas OsCBP60g-7 and 8 were up-regulated on 6th and 9th days of drought stress treatment in Sahbhagi Dhan compared to Rajendra Kasturi. At majority of time points studied, OsCBP60g genes were observed to be significantly down-regulated in Rajendra Kasturi, a drought-susceptible rice genotype (Fig. 1h-o).

OsCBP60s expression analysis in response to Submergence stress

.

The expression of OsCBP60s were analysed using qRT-PCR in two rice genotypes, Swarna and Swarna Sub-1, which are submergence-susceptible and submergence-tolerant, respectively. The majority of the OsCBP60 (OsCBP60a, OsCBP60bcd−2, OsCBP60bcd−3, OsSARDL−2, OsSARDL−3, OsCBP60g-1/OsSARDL, OsCBP60g−2, 3, 7 and 8) showed down-regulation on the 3rd, 6th and 9th days of submergence stress in the submergence-susceptible rice genotype Swarna (Fig. 2). However, in Swarna, OsCBP60g-6 was found to be up-regulated at all the time points studied, but the expression was non-significant compared to Swarna Sub-1, except on the 3rd day of submergence (Fig. 2m). In contrast, most of the OsCBP60 genes were upregulated in Swarna Sub-1, a submergence-tolerant rice genotype. However, down-regulation of OsSARDL−2, OsSARDL−3 and OsCBP60g−1/OsSARDL were observed in both the genotypes at all the time point studied during submergence treatment. The expression of OsCBP60a, OsCBP60g−2, OsCBP60g−3 OsCBP60g−4 and OsCBP60g−8 were significantly increased in Swarna-Sub1 compared to Swarna in at least two time point studied during submergence stress (Fig. 2).

Fig. 2
figure 2figure 2

Expression study of OsCBP60s in rice under submergence stress. Transcript levels of CBP60s were assessed using qRT-PCR in submergence susceptible (Swarna) and submergence tolerant (Swarna Sub1) genotypes. Samples were collected at 3rd, 6th and 9th day of submergence stress. Y-axis represents fold change. The standard error (SE) of the mean for three replicates is represented by error bars. The results represent three independent biological experiments. Values with the same superscript letter are not significantly different (p < 0.05), as determined by a one-way ANOVA followed by the LSD test.

Full size image

Discussion

Rice, a staple crop in many Asian countries including India, is particularly vulnerable to both drought and flooding, leading to significant yield losses23,61. These challenges make it vital to understand the mechanisms involved in water stress for developing rice varieties capable of increased tolerance against such stresses while maintaining high yields62,63. Over recent decades, several drought- and submergence-tolerant rice varieties were developed. Notable among these is the introgression of the Sub1 QTL into popular Indian rice varieties like Swarna Sub1 and Sambha Mahsuri Sub164, which have gained widespread acceptance among farmers due to their ability to reduce yield variability in flood-prone areas65,66,67. The Sub1 locus is responsible for up to 70% of the phenotypic variation68. Similarly, various drought-tolerance QTLs (e.g., DTY1.1, DTY2.1, and DTY3.1) have been identified, contributing significantly to yield stability under drought conditions69. While the identification and introgression of QTLs for drought and submergence tolerance have been beneficial, the molecular mechanisms behind these traits remain largely unknown. The present study aims to explore the molecular mechanisms that contribute to water stress tolerance in rice, with the goal of developing even more resilient rice varieties.

Plants are exposed to various environmental stresses, and due to their immobile nature, they rely on internal defense mechanisms to counter these challenges. Calcium ions (Ca2+) play a key role in signaling pathways that help plants respond to abiotic stresses33. When plants experience stress, changes in cytosolic free Ca2+ concentration ([Ca2+]cyt) occur, forming a “calcium signature” that triggers various responses70. Calmodulin (CaM) proteins detect these changes but rely on interactions with Calmodulin-Binding Proteins (CBPs) to initiate specific responses35. Among the known CBPs, the CBP60 family is notable as its members play important roles in stress responses different crop plants29,30,36.

The Arabidopsis CBP60 (AtCBP60) family consists of eight members28, which are involved in regulating various stress responses47,48. Studies have shown that transgenic plants overexpressing AtCBP60g exhibit improved tolerance to drought stress48. Inspired by these findings, we explored the role of rice CBP60s (OsCBP60s) in managing drought and submergence stresses in rice. Recently, 15 CBP60 proteins were identified in rice, with their roles linked to disease resistance41. Despite this, there has been no prior report on the involvement of OsCBP60s in water stress tolerance. This study aims to investigate the function of these 15 OsCBP60s in response to water stress through gene expression analysis.

In this study, expression profiling of 15 OsCBP60 genes revealed varied expression patterns under reproductive drought stress. Rice genotypes, namely Sahbhagi Dhan and Nagina-22, which are drought-tolerant varieties, and Rajendra Kasturi, which is drought-sensitive, were used in this study. Sahbhagi Dhan, a drought-resistant rice variety widely cultivated in India, Bangladesh (as ‘BRRI Dhan 56’), and Nepal (as ‘Sookha Dhan 3’), can yield 1–2 t/ha even in severe drought conditions when other high-yielding varieties fail67,71,72,73,74,75. Nagina-22, a rice landrace, is often used as a donor for drought tolerance due to its mapped drought-resistant QTLs, such as qDTY1.1 and qDTY3.215,18. The use of both drought-tolerant and susceptible genotypes helped clarify the role of OsCBP60 genes under reproductive-stage drought conditions.

Overall, most OsCBP60 genes were upregulated in Nagina-22 during at least one time point under reproductive-stage drought stress in rice showed the highest expression levels for the majority of OsCBP60s, followed by Sahbhagi Dhan, while Rajendra Kasturi exhibited either lower or negatively regulated expression compared to the other two genotypes. Our findings revealed varying expression patterns for different OsCBP60s during reproductive drought. Specifically, OsCBP60g−6 and OsCBP60a were generally downregulated, while OsCBP60bcd−2 was consistently upregulated across all genotypes.

Recent research has increasingly underscored the crucial role of CBP60 genes in regulating responses to both abiotic and biotic stresses across various crops41,48,51,76. In Arabidopsis, CBP60 proteins, particularly CBP60g, are key players in salicylic acid (SA)-mediated defense against biotic stress, but they also contribute to abiotic stress responses48. Mutations in AtCBP60g result in higher sensitivity to drought48, indicating a role similar to that of OsCBP60 in rice. In other crops, CBP60  genes have been shown to manage both biotic and abiotic stresses50,77. In tomato (S. lycopersicum), SlCBP60 genes help regulate pathogen response and temperature stress, enhancing the plant’s resilience49. Similarly, in eggplant (S. melongena), SmCBP60A1 acts as a positive regulator of salt stress, improving the plant’s tolerance to high salinity by influencing stress-responsive pathways50. In cotton (G. hirsutum), GhCBP60B is crucial for regulating both salt and drought stress, enhancing resilience under challenging environmental conditions51. Likewise, GmCBP60A−1plays an important role in managing drought and salt tolerance in soybean51,52. In another study, it has been shown that drought stress can suppress the expression of CBP60g and SARD1, leading to reduced production of SA and other defense compounds, weakening the plant’s defense and increasing susceptibility to bacterial pathogens during drought conditions78. These findings highlight the broader significance of CBP60s in abiotic stress also.

For submergence stress studies, two rice genotypes namely Swarna Sub-1, a submergence-tolerant variety, and Swarna, a submergence-sensitive variety were analysed. Expression profiling of OsCBP60 genes in Swarna and Swarna Sub-1 revealed distinct responses to submergence stress. The expression of OsCBP60g−4 was significantly upregulated in Swarna Sub-1 in all the time point studied under submergence stress compared to Swarna. Similarly, upregulation of OsCBP60g−4 was also observed in Rajendra Kasturi challenged with bacterial and fungal pathogens as well as phytohormones (SA and 24-epibrassinolide)41. In Swarna, all other CBP60 genes were downregulated under submergence stress, while in Swarna Sub 1, several genes, including OsCBP60g-1/OsSARDL, OsCBP60g-6, OsCBP60g-8, OsCBP60bcd-1, OsCBP60g-5, and OsSARDL-1, were consistently upregulated. These contrasting gene expression patterns likely reflect the inherent differences between the tolerant and susceptible rice genotypes.

Taken together, the consistent upregulation of gene members, although to different levels, in response water stress and pathogens and SA in previous study41 strongly advocates that the OsCBP60 family is involved in stress responses in rice. The expanded and diversified OsCBP60s gene family appears to be involved in both abiotic and biotic stress tolerance in rice.

Conclusions

A plethora of reports suggest the expanding nature of immunity related CBP60s in different plants. Previously we have identified 15 OsCBP60s in rice and shown their involvement in disease resistance. In this study, we have shown that the expansion of OsCBP60 genes are not only limited to biotic stress but also involved in abiotic stress tolerance in rice. The differential expression of CBP60s in response to drought and submergence stress in contrasting (resistant vs. susceptible) rice genotypes strongly suggests their involvement water stress tolerance. The substantial expansion of rice CBP60 genes in biotic (our previous report30), and abiotic stresses strongly suggests that the OsCBP60 gene family has evolved for conferring broad spectrum resistance in rice.