Cold is among the key environmental stresses impacting crop production as it limits growth, yield, and quality in crop species30. Plants, as sessile organisms, have evolved different physiological, biochemical, and molecular mechanisms to respond to cold. These mechanisms are adjusted by a complex of transcription factors and proteins to raise plant tolerance31. Cold tolerance has a quantitative property controlled by several genes. This work results provide insights into the expression profiles of cold-responsive genes in two contrasting chickpea genotypes32.
Metabolic Pathways Engaged In Cold Stress Response Of Chickpea
According to the GO enrichment analysis of the genes exclusively up-regulated in the cold-tolerant genotype (Saral), the phenylpropanoid metabolic process was significantly enriched under the cold stress condition. Likewise, mapping the DEGs of Saral under cold stress to the secondary metabolites pathway indicated that phenylpropanoids were exclusively enriched. The phenylpropanoid pathway is the main metabolites pathway involved in synthesizing the majority of secondary metabolites, including lignin, lignans, flavonoids, hydroxycinnamic acid amides, phenylpropanoid esters and sporopollenin33,34. Accumulation of phenolic compounds, including suberin or lignin, caused the thickness of cell wall to be increased, prohibiting cold stress injury and cell collapse35,36. Phenolic biosynthesis enhancement under cold stress is caused by up-regulation of PAL (Phenylalanine ammonia-lyase), CAD (cinnamyl alcohol dehydrogenase), and HCT (hydroxycinnamoyl transferase) expression37. In the present research, while significant up-regulation of three genes coding for cinnamyl alcohol dehydrogenase was observed in Saral, only one gene was significantly induced in ILC533. In addition, the up-regulation of the common CaCAD gene in response to cold stress was much higher in Saral compared to ILC533.
GO enrichment analysis of the genes exclusively down-regulated under cold stress in the cold-sensitive genotype (ILC533) indicated that photosystem II, chloroplast part and photosystem process were significantly enriched under cold stress conditions. Photosynthesis, as a principal plant metabolic process, is severely sensitive to cold stress. Low temperature disturbs almost all key components of the photosynthesis apparatus, including Photosystems I and II, photosynthetic pigments, CO2 reduction pathways, and electron transport systems, inhibiting overall photosynthesis38–40.
Furthermore, the ILC533 DEGs mapping to the secondary metabolites pathway indicated that the isoprenoid pathway was enriched, and most involved genes significantly were down-regulated under cold treatments. Isoprenoids are belonged to a huge and diverse category of volatile organic compounds, which are synthesized from terpenes and have essential functions, including lipids in cell membranes, quinones in the electron transport chain and signal transduction, as well as antioxidants and hormones41,42. Isoprene (simplest Isoprenoid) protects plants from different extreme conditions, including drought43,44, heat45–47 and oxidative stresses48. It protects the photosynthetic system through thylakoid membrane stability49,50 enhancement and ROS quenching. High destruction resilience of thylakoid membrane in isoprene-emitting plants preserves the better status for molecular diffusion, electron transport, dynamic lumen swelling, and molecular/structural reorganization under heat stress48.
Metabolic Pathways Engaged In Cold Stress Response Of Chickpea
The current research identified many transcription factors (TFs) among the DEGs. TFs have a vital role in cold stress response through transcription adjustment of the downstream genes engaged in plants cold stress tolerance51. The APETALA2/Ethylene responsive factor (AP2/ERF), NAC, MYB, TCP4, and Zn-finger have been identified as important TFs engaged in the plant cold stress16,52,53 response regulation; such stress-responsive TFs may be significant targets for developing crops with improved cold stress tolerance.
The AP2/ERF is among the large TF families engaged in stress response pathways and developmental processes in plants54,55. Several genes from this family were found exclusively cold-responsive in the tolerant genotype (e.g., ethylene-responsive transcription factor RAP2-1-like (LOC101512420), ethylene-responsive transcription factor-like protein (LOC105851094), ethylene-responsive transcription factor TINY-like (LOC101506537), AP2-like ethylene-responsive transcription factor (LOC101498533), dehydration-responsive element-binding protein 1E-like (LOC101505186). C-repeat binding factors (CBFs), recognized as Dehydration responsive element binding proteins (DREBs), are the most popular members of the AP2/ERF family56,57. DREBs have a key role in plant stress tolerance and act as the vanguard of plant regulatory networks57–59. They can activate the expression of COR (cold-related), RD (Responsive to Dehydration), LTI (Low-temperature Induced), and other cold-regulated genes16,60. The CBFs' overexpression enhances cold tolerance by increasing antioxidant enzymes such as catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), as well as proline and reducing MDA, H2O2, and O− 2 content61–63. The overexpression of the BpERF13 gene in white birch significantly improves cold tolerance via up-regulation of CBF genes and decrease in reactive oxygen species accumulation64.
One of the recognized candidate genes in the present study was dehydration-responsive element-binding protein 1E-like (CaDREB1E, LOC101505186), which was highly up-regulated in the tolerant genotype in response to cold stress; however, its induction was not significant in the sensitive line (Fig. 4b). Previous studies also have indicated that the overexpression of AtDREB1 enhances freezing tolerance in transgenic Arabidopsis65, potato66, and tobacco67. Overexpression of the DREB/CBF genes results in biochemical variations related to cold tolerance68,69. The OsDREB1A, OsDREB1B, and OsDREB1C interaction with the GCC box increase the cold tolerance of the rice plants70. Chen et al. (2008) stated that the overexpression of rice DREB1E enhanced plant survival rate under water-deficient conditions71.
Based on the results of the current study, CaMYB4 (LOC101508022) was significantly up-regulated in both genotypes but higher increase was observed in the susceptible line (Fig. 4a). The MYB superfamily, one of the most abundant classes of TFs in plants, holds a substantial quota in cold stress response72. The MYBs' role in cold stress response has been further recognized by functional studies using overexpression and knock-out systems73. Transgenic Arabidopsis plants with overexpression of Osmyb4 have shown improved cold stress tolerance74. The overexpression of Osmyb4 in Arabidopsis leads to multiple metabolic changes (free amino acids) commonly observed in plants during cold acclimation75,76. Furthermore, an increase in soluble sugars, leaf chlorophyll content, and superoxide dismutase activity, as well as a reduction in malondialdehyde (MDA) content, under chilling stress have been reported in LcMYB4-overexpressing Arabidopsis. Indeed, LcMYB4 overexpression enhances soluble sugar content and cold-inducible gene expression and attenuates oxidative and membrane damage, resulting in cold tolerance77.
Based on the results, up-regulation of CaNAC47 (XM_004503844) was observed in both genotypes, while its induction was more in the tolerant genotype (Fig. 4c). NAC transcription factors have a fundamental role in responses to stresses in plants78. The role of NACs has been considered and recognized in different plants, including Arabidopsis79, rice80, peppers81, and Medicago truncatula82, under cold stress conditions. ABA hypersensitivity and improved tolerance to salt, drought, and freezing have been demonstrated in transgenic Arabidopsis plants with overexpression of TaNAC47. In addition, increased soluble sugars and proline contents have been reported in TaNAC47 overexpressing plants after exposure to drought and cold treatments79.
In the present study, cold stress led to up-regulation of CaTCP4 (LOC101506032) in both cultivars; however, more increase was observed in Saral genotype (Fig. 4d). TCP transcription factors are a plant-specific category with fundamental roles during the development of plants and their responses to cold stress83–85. The overexpression of MeTCP4 of Cassava (Manihot esculenta) in Arabidopsis led to enhanced cold tolerance by increasing proline content and reducing cell membrane damage. Furthermore, much higher expression of ROS-scavenging-related genes such as GSTF7, GSTU12, and FRO3 was detected in MeTCP4 overexpressing plants as compared with the wild type under cold stress conditions86. Glutathione S-transferases (GSTs), recognized as ubiquitous and multifunctional proteins, inhibit oxidative damage87. They are involved in cold, drought, salt, and oxidative stress tolerance in Arabidopsis88. The up-regulation of GSTs (LOC101508652, LOC113783892) was also observed in the tolerant genotype in the current investigation.
Based on the present research results, CaWRKY33 (LOC101509113) was substantially up-regulated in the tolerant genotype in response to cold stress, while its induction was not statistically significant in the sensitive cultivar(Fig. 4e). The WRKY TF family is among the important transcription factor families in higher plants89,90. WRKY TFs are recognized as essential regulators in various physiological and developmental processes89 as well as abiotic stress responses, including cold stress91,92. The overexpression of CsWRKY46 from cucumber in Arabidopsis resulted in higher seedling survival rates under freezing stress compared to the wild type. This overexpression enhanced cold tolerance in Arabidopsis via expression regulation of stress-induced genes such as RD29A and COR47 in the ABA-dependent manner. The up-regulation of COR47 (LOC101512214) and a chloroplastic early responsive to dehydration (LOC101495575) were also observed in present study.
Furthermore, the expression of a regulatory gene called probable protein phosphatase2C6 (CaPP2C6, LOC101510725), which negatively affects stress tolerance, decreased under cold stress in both genotypes. However, its down-regulation was greater in Saral compared to ILC533 under cold stress (Fig. 4m). Type 2 C protein phosphatases (PP2Cs), the main class of plant protein phosphatases, have converse functions in stress signaling pathways in various plant species93–95. The negative regulatory functions for ZmPP2C-A10 have been demonstrated in maize and Arabidopsis under drought stress96,97. Moreover, the suppression of AtPP2CA expression caused cold acclimation and enhanced freezing tolerance in Arabidopsis98. Certain PP2C genes are engaged in the ABA signaling cascade regulation by changing the kinase activity, MAPK or SnRK, under abiotic stress conditions97.
Signal perception and transduction, as well as the expression of stress-responsive genes, are the basic ingredients in stress responses99. In the current research, cold stress led to significant up-regulation of calcium-dependent protein kinase 4 (CaCDPK4, LOC101492192) in the tolerant genotype; however, its induction was not significant in the susceptible line (Fig. 4f). CDPK4 is a calcium-dependent protein kinase (CDPK) gene family member. Several CDPK genes are transcriptionally altered by cold stress100. The overexpression of PeCPK10 resulted in more proline accumulation and caused freezing tolerance of transgenic Arabidopsis101.
In the present research, CaHSFA3 (XM_004497545) was up-regulated in both genotypes under cold conditions, more in the tolerant genotype (Fig. 4g). Plant Heat-Shock Factors (HSFs) coded by extensive gene families are divergent from expression, function, and structure points of view. HSFs are members of complex signaling systems that regulate responses to different abiotic stresses, including cold, high temperatures, salinity, drought and oxidative stress102. They are engaged in increasing the expression of HSPs, such as HSP90s, HSP70s, and some small HSPs103,104. Genes encoding HSP70/90 and HsfA3/A8 are not principally only regulated by temperature stress, but also interact with chlorophyll synthesis and peroxide scavenging processes under cold stress105. The overexpression of TaHSF3 seriously increased resilience to freezing and heat stresses by inducing HSP70s in transgenic Arabidopsis plants106. Additionally, OsHsfA3 is particularly induced in both the shoot and root tissues of rice under cold stress107.
The present study showed that mitogen-activated protein kinase 4-like (CaMKK2, XM_004492727) was up-regulated in the tolerant genotype under cold conditions, whereas its induction was not significant in the sensitive line (Fig. 4h). Mitogen-activated protein kinase (MAPK) cascades are popular signal transduction pathways in all eukaryotes with fundamental roles108,109. The MAPK cascade controls plant tolerance to temperature stresses by phosphorylating downstream targets to directly alter related gene expression and cellular metabolism (enhancing compatible solutes and antioxidative enzyme activities)110,111. Transgenic tobacco plants overexpressing SlMPK3 from tomato exhibited enhanced antioxidant activity, raised proline and soluble sugars content, and improved cold tolerance112. MEKK1-MKK2-MPK4/6 pathway positively controls cold response and freezing tolerance in Arabidopsis113. Under low temperatures, MEKK1 is activated and subsequently phosphorylates MKK2114. Phosphorylated MKK2 activates MPK4 and MPK6 involved in regulating downstream components to cope with low-temperature stress conditions113. The mkk2 mutant plants exhibited enhanced susceptibility to freezing, while transgenic plants that expressed a constitutively active form of MKK2 showed enhanced freezing tolerance by increasing the CBF genes' expression113.
The present study indicated a greater down-regulation for the gene coding polygalacturonase 1 beta-like protein 3 (CaPGL3, LOC101490440) in the tolerant genotype as compared with the sensitive genotype (Fig. 4n). Polygalacturonases (PGs) are enzymes necessary for the degradation of cell wall pectin115. It was shown that the overexpression of OsBURP16, a member of the PG1β-like subfamily, increased sensibility to cold, drought and salinity stresses compared to controls in rice. The OsBURP16 overexpression led to pectin degradation, affecting the integrity of cell wall and transpiration rate, and caused abiotic stress tolerance to be reduced116. Instead, it has been shown that cold acclimation increases cell wall pectin content and enhances freezing tolerance117.
Based on the obtained results, cold stress led to the up-regulation of CaLEA3 (LOC101508885) in both genotypes, mostly in the tolerant genotype (Fig. 4l). Late embryogenesis abundant (LEA) proteins, recognized as small molecule-specific peptides, are created in the late step of seed development, helping plants deal with diverse abiotic stresses118. Members of the LEA gene family are regulated and expressed under various stress conditions. Different studies show the involvement of LEA proteins in cold stress tolerance in different plants. The overexpression of the wheat LEA gene (WCOR410) increased cold tolerance in transgenic strawberry plants118. Salt and drought stress tolerance simultaneously increased in wheat and rice plants overexpressing barley LEA (HVA1) gene. The ZmLEA3 overexpression in tobacco resulted in increased cold tolerance119.
Another candidate gene identified in the current study is dehydrin (CaCOR47, LOC101512214), playing a role in the cold tolerance of chickpeas. CaCOR47 was up-regulated in both genotypes under cold stress; however, more rise in its expression was observed in the tolerant genotype under cold stress (Fig. 4k). COR47 is a member of the group II LEA proteins120,121. COR (cold-responsive) genes are quickly induced by cold stress during cold acclimation122. They are generally up-regulated by numerous abiotic stresses through binding of CBFs to the related cis-elements located in their promoters. Simultaneous overexpression of COR47 and RAB18 genes increased freezing tolerance in Arabidopsis, which could be partly due to their protective effect on membranes123.