The kinase CIPK11 functions as a positive regulator in cadmium stress response in Arabidopsis

Highlights

• CIPK11 expression activated by cadmium (Cd) stress and played a positive regulatory role in tolerance to Cd stress.

• ABA marker genes were higher up-regulated in CIPK11-overexpression plants.

• Exogenous ABA made plants more tolerant to Cd.

• ABA-producing genes were activated in CIPK11-overexpression plants.

• ABA inhibited the expression of IRT1 by inhibiting the expression of FIT and bHLH039.

Abstract

Cadmium (Cd) pollution in agricultural soil has always been a knotty problem, which made it necessary to find the mechanism related to Cd transport in plant. In this study, we found a novel character of the CIPK11 modulating the transport of Cd in Arabidopsis thaliana. Over-expression of CIPK11 (CIPK11OE#1-7, CIPK11OE#8-5) resulted in the increased tolerance to Cd stress, which embodied in higher fresh weight, lower Cd enrichment and reactive oxygen species (ROS) than the wild-type (WT) plants. qRT-PCR results showed a collective down-regulation of the expression of IRT1 and transcription factor genes FIT, bHLH039 in the CIPK11-overexpression plants after Cd stress. Overexpression of CIPK11 significantly increased the expression of ABA marker genes in Arabidopsis after Cd stress. With different concentrations of ABA treatment, the root length differences caused by Cd stress could be recovered. However the transcription levels of FIT and bHLH039 decreased in WT and cipk11 mutant when treated with ABA which indicated that ABA can inhibit the transcription of IRT1 by repressing FIT and bHLH039 expression. Taken together, our results demonstrated that the kinase CIPK11 responses to Cd stress by ABA signaling pathway.

Introduction

Cd pollution is one of the most serious problem in the world-wide agricultural soil, which may arise from natural sources and anthropogenic sources such as rock weathering, soil erosion, industrial and agricultural activities, adversely affecting human health through the food chain (Fasani et al., 2018). Cd stress usually leads to aberration of chromosomal, inhibition of growth, eventually causing plant death. Plants are not only under direct stress of Cd ions, but other stresses caused by Cd, such as reactive oxygen species (ROS) (Sharma and Dietz, 2009). There are many methods carried out for lowering the pollution risk of Cd in crops; such as encapsulation of the contaminated soil and chemical immobilization of Cd. However, problems such as high cost and possible secondary pollution still remain. Using the plant genetic engineering to improve soil and water contaminated by heavy metals is a new research field. Two tactics that are gaining popularity are to increasing the tolerance of heavy metals in plant or the capacity of stabilize and accumulate metals (Fasani et al., 2018). Overexpression of the Arabidopsis thaliana metal transporter gene HMA4 reduced the uptake of Cd by strengthening the apoplastic barrier in the epidermis and first cortical layer (Siemianowski et al., 2014). Phytoremediation as a green restoration method has the advantages of low cost and easy to operate. Expressing the endogenous zinc finger transcription factor AtZAT6 in Arabidopsis, resulting in the accumulation of phytochelatins, made the plants more tolerance to Cd and accumulated 23% more Cd than wide-type plants (Chen et al., 2016).

IRON-REGULATED TRANSPORTER1 (IRT1) is a well-known broad-spectrum transporter in plant root, driving the uptake of iron from soil, as well as the main entry route of toxic metals such as Cd (Barberon et al., 2011, Vert et al., 2002). The irt1 loss-of-function mutant is chlorotic severely and dies early, unless fertilized with massive amounts of iron. In addition, irt1 mutant fails to accumulate manganese, zinc, and cobalt. It indicated that although iron served as the first substrate of IRT1, IRT1 also transports the non-iron metals due to its poor selectivity (Vert et al., 2002). FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) plays an important role in uptaking Fe and maintaining cellular iron balance in plants. FIT could enhance Fe transport by upregulating the expression of FERRIC REDUCTASE-OXIDASE2 (FRO2) and IRT1 (Colangelo and Guerinot, 2004). In Arabidopsis, FIT exists mostly in an inactive form, and overexpression of AtFIT does not activate downstream gene expression. FIT activation is realized through protein interaction. Four subgroup Ib bHLH (Ib basic helix-loop-helix) proteins (bHLH038, bHLH039, bHLH100 and bHLH101) could physical interact with FIT, then FIT phosphorylated and activated by upstream kinases, induces the expression of its target genes (Wang et al., 2013).

As an important second messenger in plants, calcium (Ca²⁺) is stimulated by various abiotic stresses, such as drought and salt. Kinases CBL-INTERACTING PROTEIN KINASE (CIPK), together with calcineurin B-like protein (CBL), are a class of Ca²⁺ signal decoders catalyzing serine/threonine protein phosphorylation elicited by environmental stimuli (Batistič and Kudla, 2012, Edel et al., 2017). 10 CBLs and 26 CIPKs have been found in Arabidopsis and part of the CBL-CIPK working models have been well characterized in recent years (Kolukisaoglu et al., 2004, Kanwar et al., 2014). The first revealed CBL-CIPK working model is CBL4-CIPK14, also known as SOS3-SOS2, which functions as positive regulators in salt stress through activating the plasma membrane (PM)-located Na⁺/H⁺ exchanger SOS1 in Arabidopsis (Qiu et al., 2002, Shi et al., 2002). CBL2/3 and CIPK2/3/23/26 formed a multivalent interaction network to regulate the chelation of Mg²⁺ in vacuoles, thereby alleviating the Mg²⁺ toxicity (Tang et al., 2015). The CBL1/9-CIPK23 complex could actives potassium transporter HAK5 and AKT1 when the plants suffer low potassium environment and facilitate potassium uptake (Ragel et al., 2015). In addition, CBL1/9-CIPK23 could modulate the stomatal movement by participating in abscisic acid (ABA) pathway (Geiger et al., 2009). However, the downstream components of most CIPKs are still unknown, and how CIPKs regulate plant response to biotic and abiotic stress remains unclear, which requires further study. Protein kinase CIPKs target enzymes, transcription factors and membrane transporters (Xu et al., 2006, Fuglsang et al., 2007, Ho et al., 2009, Drerup et al., 2013, Dubeaux et al., 2018, Gratz et al., 2019). CIPK23 phosphorylates the plant metal transporter IRT1, triggers its K63 polyUb by IDF1 and vacuolar degradation when subjected to excess non-iron metal substrates, limites the highly reactive metals accumulate in plants Dubeaux et al. (2018). CIPK11 mediates the drought stress negatively by regulating the transcription factor Di19-3. The Arabidopsis overexpression of the AtCIPK11 were more sensitive to drought stress, exhibited higher leaf water loss and content of ROS after drought treatment (Ma et al., 2019). In iron-deficient environment, the kinase CIPK11 activated by CBL1/9 and further phosphorylate FIT at Ser272 and formed heterodimer with bHLH039, activating the expression of IRT1 to take in Fe under iron deficiency condition (Gratz et al., 2019).

ABA plays an important role in plant growth and development, such as seed dormancy, germination and seedling growth. In addition, it is necessary for plants to respond and adapt to various biotic and abiotic stresses mediating by ABA signaling (Zhao et al., 2014, Rodrigues et al., 2017, Jiang et al., 2010). In the absence of ABA, group A protein phosphatases type 2C (PP2Cs) combine and inhibits the activity of subclass III SNF1-related protein kinases (SnRK2.2, 2.3 and 2.6) by dephosphorylating the activation loop (Soon et al., 2012). In the presence of ABA, by forming the ABA-PYLs-PP2C complex, SnRK2s kinase activity are released. The activated SnRK2s activate ABA signaling by phosphorylating downstream factors (Melcher et al., 2009). Recent research has shown that SnRK2 kinases activated by MAPKK-kinases at a specific SnRK2/OST1 site. The mechanism is necessary for ABA signal transduction and rapid osmotic stress response (Takahashi et al., 2020). Previous study has demonstrated that under Cd exposure, SnRK2s (for SNF1-related protein kinase2) were transiently activated and involved in the metabolic regulation under Cd stress (Kulik et al., 2012).It is known that CIPK11 can regulate ABA-responsive gene expression through phosphorylation a serine (Ser42) residue in ABI5. It is important for the ABA regulation of gene expression in Arabidopsis (Zhou et al., 2015). External application of ABA or co-cultivation with ABA-generating bacteria could reduce Cd uptake in Arabidopsis by down-regulating the expression of IRT1 (Xu et al., 2018, Fan et al., 2014). Therefore, ABA plays a key role in plant response to heavy metal stress.

Kinase CIPK11, a SOS2-like protein kinase, also known as SOS3-interacting protein 4 (SIP4) and PROTEIN KINASE SOS2-LIKE 5 (PKS5). Is a member of the CBL-interacting protein kinase family. The mRNA length of CIPK11 is 1865 nt, encoding 435 amino acids. In this study, we reported that CIPK11-overexpression plants were more tolerable to Cd. The expression of IRT1, and its transcription factors FIT, bHLH039 decreased significantly in CIPK11OE# plants after Cd treatment. Our study suggested that ABA marker genes ABA1, AAO3 significantly higher up-regulated in CIPK11OE# plants. The phenotypic differences of all plants caused by Cd stress could be recovered with the application of ABA. Further research found that adding ABA to Cd stress environment could significantly reduce the expression of FIT and bHLH039 in wild-type and cipk11 mutant plants. Hence, our analysis demonstrated that kinase CIPK11 regulated the ABA signaling pathway to develop tolerance to Cd stress partly.