Critical cellular functions such as photosynthesis, respiration, and membrane permeability are affected by cold temperatures (Rawat et al., 2021). Cold acclimation involves many physiological and biochemical changes, the principal changes being the reduction or cessation of growth (Achard et al., 2008). The reduced sorghum growth can be explained by the susceptibility of its C4 photosynthetic machinery to low temperatures, which, at the same time, can be modulated by the upregulation of C-repeat binding factor/dehydration-responsive element binding (CBF/DREB) that are required to activate cold-responsive genes, causing growth inhibition. These mechanisms have been studied in other plants, including Arabidopsis and rice (Ito et al., 2006). The mechanism of growth inhibition also involves the stimulation of gibberellin-inactivating enzymes in a process that promotes survival or escape from adverse environmental conditions (Achard et al., 2008). The reduction in growth could also be caused by an increase in reactive oxygen species (ROS) in plants, which affects growth and reduces crop yield (Devireddy et al., 2021; Wang et al., 2022).

Cold-sensitive plants usually have low leaf water potentials, whereas cold-tolerant plants preserve water potentials by closing their stomata and preventing water loss through transpiration during chilling (Wilkinson et al., 2001). Stomatal conductance and transpiration rates have been suggested as traits that can be used for marker-assisted selection in sorghum (Ortiz et al., 2017). The ability of the plants to maintain gas exchange is vital for biomass production (Khanthavong et al., 2022). Under waterlogging and low temperature, the environmental responses of stomatal conductance in sorghum showed a decrease in stomatal conductance, compared with the combination of moderated soil moisture or gradual soil drying with high temperature (Khanthavong et al., 2022).

Light and water use are more efficient in C4 plants than that in C3 plants (Zhu et al., 2008b). However, only a few C4 plants are able to maintain photosynthetically competent leaves at chilling temperatures (<15°C) (Du et al., 1999; Wang et al., 2008). The ability for active photosynthesis during exposure to cold is vital for sorghum or any other crop bred for cold tolerance (Hurry et al., 2002). The leaf greenness indicates photosynthetic activity, which is a necessary trait for selection of chilling tolerance.

Under cold conditions, pyruvate phosphate dikinase (PPDK) instability is associated with a reduced photosynthetic rate in leaves (Naidu et al., 2003; Wang and Li, 2010). PPDK is a cold-labile enzyme that plays a critical role in the carboxylation of pyruvate to phosphoenolpyruvate in C4 metabolism plants. However, some C4 plants such as Miscanthus giganteus are adapted to cold conditions (Naidu et al., 2003), which has a high level of expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and PPDK under cold stress, contrasting to maize and other cold susceptible C4 plants (Ortiz et al., 2017). The reduction in Rubisco activity under low temperatures is another limitation to carbon assimilation in C4 plants compared with the photosynthetic rates of C3 plants (Sage, 2002). The lower quantum yield of photosynthesis (ΦPSII) under cold stress indicates a reduction in the linear electron transport rate (Ortiz et al., 2017). Genetic variation in the photosynthetic response, the ability to recover, and the carbon fixation capacities of sorghum exposed to cold stress has been reported (Ortiz et al., 2017). Further analysis of sorghum varieties could include the selection of varieties with better carbon fixation under cold conditions, helping to improve yield and biomass.

Cold stress enhance the accumulation of soluble sugars, which are originated from starch metabolism ( Figure 2 ) (Lin et al., 2019; Shi et al., 2022). Sugars are an energy source, but they also have other functions as substrates for polymers, carbon precursors, storage as reserves, and signaling molecules during cold stress (Yamada and Osakabe, 2018). Trehalose and trehalose-6-phosphate are disaccharides that have a protective role in proteins and membranes during abiotic stress (Elbein et al., 2003; Iordachescu and Imai, 2008). Sorghum transgenic lines with overexpression of alpha-trehalose-phosphate synthase (TSP1) were able to tolerate high salinity and develop higher root growth and biomass (Yellisetty et al., 2015). Still, tolerance to cold stress has not been tested yet. Rice seedlings treated with chitooligosaccharides (COS) showed cold tolerance and increased contents of proline and glutamate, revealing that COS significantly induced genes associated with the glutamate and proline biosynthesis pathway (Zhang et al., 2019) ( Figure 2 ). This suggests that COS might be a key regulator through the accumulations of glutamate and proline (Zhang et al., 2019) and that it could be explored in sorghum. Glucose and fructose levels increased under chilling in sorghum, and sucrose was slightly lower (Marla et al., 2017). The small starch content and the increase in simple sugars during chilling exposure to sorghum suggest an essential role for the B-amylases in cold acclimation (Marla et al., 2017).

Membrane lipids remodeling is key for cold tolerance. The fatty acid unsaturation helps preserve membrane integrity during chilling by lowering the melting temperature and increasing fluidity (Marla et al., 2017). The ability to adjust membrane fluidity to low temperature change is associated with regulating membrane fatty acid desaturation. The desaturation of both phospholipids and sphingolipids is essential for remodeling the plasma membrane under cold stress (Barrero-Sicilia et al., 2017). In chilling-sensitive plants at low temperatures, the galactolipase (GAlP) is significantly active, leading to an increase in free fatty acid in chloroplasts ( Figure 2 ) (Kaniuga, 2008).

Chilling causes a decrease in the concentrations of mono-galactosyl-diacyl-glycerols (MGDGs) and sulpho-quinovosyl-diacyl-glycerols ( Figure 2 ), which are significant lipids in the thylakoid (Marla et al., 2017; Liu et al., 2018). Lipid metabolism gene analysis in sorghum has identified lipids deregulated in response to chilling in sorghum seedlings that may cause chilling tolerance in this crop (Marla et al., 2017). Genes that are involved in fatty acid biosynthesis (i.e., acetyl coenzyme A (CoA) synthetase and acyl-Acetyl coenzyme A (CoA) oxidase 1), thylakoid lipid synthesis (MGDG synthase MGD2 and DGFGI synthase 1 DGD1), and phospholipids metabolism (G-3-phosphatase acyltransferase 5, phosphatidiylserine synthase, and phospholipase D delta) were upregulated in response to chilling (Marla et al., 2017). An increase in the unsaturation index of total phospholipid PC and LysoPC also has observed under chilling in sorghum (Marla et al., 2017).

During cold stress, certain amino acids and amine compounds adjust its metabolism, particularly those associated with proline biosynthesis ( Figure 2 ) (Kaplan et al., 2004). The proline amino acid can act as a regulatory or signaling molecule capable of altering the expression of antioxidants-associated genes in plant response to environmental constraints (De Carvalho et al., 2013; Ben Rejeb et al., 2014). During dehydratation, proline accumulates and contributes to ROS formation in mitochondria, playing a role in the hypersensitive response in plants (Ben Rejeb et al., 2014). Proline is usually used as an osmolyte, but it can also function as a powerful antioxidant (Das and Roychoudhury, 2014). Proline accumulation is an adaptive mechanism that allow plants tolerate cold or chilling stress (Teh et al., 2016). Proline accumulation in stress situations will depend on both the activation of its biosynthesis and the inhibition of its degradation ( Figure 2 ) (Ben Rejeb et al., 2014). In cold-tolerant sorghum plants, a higher proline accumulation is related to a better tolerance (Vera-Hernández et al., 2018). There are two biosynthesis pathways for proline, glutamate, and ornithine ( Figure 2 ). The enzymes Δ¹-pyrroline-5-carboxylate synthetase (P5CS), Δ¹-pyrroline-5-carboxylate reductase (P5CR), and ornithine-δ-aminotransferase (δ-OAT) are involved in the proline biosynthesis ( Figure 2 ) (Bagdi et al., 2015; Vera-Hernández et al., 2018). A transcription factor binding site analysis of these genes indicated the presence of a low-temperature response element in the promoter region of ɗ-OAT, suggesting that the proline accumulation in cold response in sorghum may be using the Orn pathway (Vera-Hernández et al., 2018). P5CS2 expression is associated with high proline levels in response to drought stress in sorghum, resulting in an exciting gene for cold tolerance analysis. γ-Aminobutyric acid (GABA) is another amine metabolite that can be rapidly accumulated in plant tissues and is associated with cryoprotection in monocots (Mazzucotelli et al., 2006), which could be involved in tolerance to chilling stress in sorghum ( Figure 2 ).

Transcription factors (TFs) are a category of proteins that bind to cis-regulatory DNA sequences and are responsible for either positively or negatively affecting the transcription of certain genes. They determine whether a specific gene will be turned “on” or “off” (Philips and Hoopes, 2008). Currently, the plant’s most understood cold-signaling pathway is the CBF/DREB transcriptional regulatory cascade (Shi et al., 2015; Wang et al., 2022). The CBF genes respond rapidly to chilling temperatures and induce the expression of several proteins that protect plants from freezing temperatures. These proteins play a crucial role in cold acclimation (Agarwal et al., 2006; Nakashima and Yamaguchi-Shinozaki, 2006; Achard et al., 2008). SbCBF6 is highly expressed during chilling in tolerant sorghums (Chopra et al., 2015; Marla et al., 2017). CBF expression is controlled through jasmonic acid (JA) ( Figure 3 ). In this sense, allene oxidase synthase and 12-oxoyphytodienic acid reductase, which are important genes in JA biosynthesis, are upregulated in chilling-tolerant sorghum. This suggests that JA biosynthesis contributes to chilling tolerance (Marla et al., 2017). DELLA proteins (a critical negative regulator of gibberellin signaling) may contribute to the CBF1-mediated freezing tolerance (Achard et al., 2008). DREBs (a significant subgroup of AP2/ERF TF) induce the expression of genes involved in abiotic stresses. Genes that code for DREB proteins regulate the transcription of many genes involved in the plant response to cold stress. Fifty-two ERF (ethylene response factor) genes encoding DREB proteins have been predicted in sorghum (Mathur et al., 2020). Bioinformatic analysis showed that the motifs within sorghum DREB promoters are principally involved in abscisic acid (ABA), light, and calcium-mediated regulation (Srivastav et al., 2010). However, the regulation of DREB genes is not well understood in sorghum. In sorghum, the CBF/DREB and ERF proteins have similar structures and evolutionary relationships (Mizoi et al., 2012; Rashid et al., 2012). In sorghum, CBF/DREB and ERF proteins have groups that contain an LWSY motif at the C-terminus, which respond to cold treatment, suggesting that such motif may be involved in cold plant tolerance (Yan et al., 2013). Sorghum genes that are predicted to be involved in ethylene biosynthesis, which regulates cold signaling pathways, were upregulated (Marla et al., 2017). Recently, it was indicated that TaMYC2 plays a critical role in cold stress in many plants and activates the ICE (inducer of CBF expression)–CBF-COR (cold responsive) cold resistance pathway through interaction with ICE in Arabidopsis (Wang et al., 2022), and some MYC2 (A/B/D) improved cold tolerance through ROS metabolic.

Another critical group of TF is NF-Ys (plant nuclear factors), which show differential stress response gene expression in several tissues and interact with abiotic stress signaling pathways to regulate the plant stress responses (Kavi et al., 2022). In sorghum, 24 NF-Y genes are expressed in cold stress (Maheshwari et al., 2019). Sixteen NF-Ys (NF-YA2/4/6/8, NF-YB2/7/10/11/12/14/16/17, and NF-YC4/6/12/13) are induced by both cold and high temperatures (Maheshwari et al., 2019). These suggest a crosstalk among both stresses and microRNA (miRNA) association in the regulation of SbNF-Ys (Maheshwari et al., 2019), denoting that miRNAs may also participate in gene networks controlled by TFs like NF-Ys (Maheshwari et al., 2019).

ABA is an important hormone implicated in the cold stress response by regulating specific stress-responsive genes (Xue-Xuan et al., 2010). The genes that are predicted to be involved in the biosynthesis of ABA were upregulated in sorghum during cold treatment (Marla et al., 2017). Regarding the amino acid sequences of ABA receptor (ABARs) family genes from Arabidopsis, eight candidate genes were identified in the sorghum genome (Dalal and Inupakutika, 2014). ABA binds to ABARs and brings conformational changes that facilitate ABA-mediated interaction of PYL with PP2C. Dalal and Inupakutika (2014) identified these eight genes as members of the PYL family in sorghum, namely, SbPYL1–SbPYL8. In addition, nine functional SbPP2C genes were predicted in the sorghum genome (Dalal and Inupakutika, 2014). Among ABARs receptors, the SbPYL4 gene was found to be specific for cold stress, with higher expression observed in leaf exposed to cold stress. PhyA and phyB genes have been reported to function antagonistically to regulate cold tolerance through ABA-dependent jasmonate signaling (Yang et al., 2019). In tomatoes, the genes HY3 and HY5 enhance cold tolerance by integrating myoinositol and light signaling (Yang et al., 2019). Lin et al. (2021) found that HY5 can be activated by photoreceptors to promote photomorphogenesis (Lin et al., 2021).

During chilling, genes encoding components of photosystem I (PSI/P700) and photosystem II (P680), responsible for the light reactions phase of photosynthesis, are downregulated (Marla et al., 2017). Sobic.003G370000, an ortholog of AtNPQ4 (nonphotochemical quenching), was increased under chilling and could be implicated in preventing damage to the photosynthetic apparatus due to photoinhibition (Marla et al., 2017), as was reported in Arabidopsis (Havaux et al., 2000). ROS detoxification in plants has been mediated by the ascorbate-glutathione cycle (Foyer and Noctor, 2011). Marla et al. (2017) showed that the monodehydroascorbate reductase 1 (Sobic.007G171000) and the ascorbate biosynthesis gene vitamin C defective 5 (VTC5, Sobic.008G064700) of the ascorbate–glutathione cycle were highly expressed during cold stress in sorghum. Similarly, some glutathione S-transferase (GST) enzymes have antioxidant properties. Some GST genes were highly upregulated in chilling-tolerant sorghum but not in chilling-sensitive sorghum in cold treatments. Other genes involved in sugar breakdown as hexokinase (Sobic.009G06980) and fructokinase (Sobic.003G386000) increased during cold in sorghum. Several starch synthase genes were induced, which correlated with the composition of the carbohydrate in sorghum during cold stress (Marla et al., 2017).

C2H2-type zinc finger proteins are one of the best-studied TF associated with abiotic stress in plants (Liu et al., 2022). In S. bicolor, 145 SbC2H2-ZFP members have been predicted (Cui et al., 2022). In sorghum, Sobic.005G121100 was significantly upregulated in cold stress, and Sobic.008G088842 was activated by cold and inhibited in drought in stems and leaves (Cui et al., 2022). In tomatoes, multiple B-box proteins (BBxs) play a role in responses to light quality and cold stress (Bu et al., 2021). In SlBBX7-, SlBBX9-, and SlBBX20-silenced tomato plants, cold tolerance was suppressed. Moreover, Bu et al. (2021) found a photosynthetic response instantly after cold stress by the impairment of non-photochemical quenching, and the consequent excess photon energy excited by low temperature is not consumed, leading to the over-reduction of electron carriers and damage of the photosystem (Bu et al., 2021). Twenty-four SbBBX genes have been identified in sorghum (Shalmani et al., 2019), suggesting that SbBBX studies in response to cold tolerance may improve the current understanding of this trait.

Differential expression of K+ transport genes might also be involved in sorghum’s cold response. Anil Kumar et al. (2022) found higher expression levels of potassium transport genes (SbAKT1, SbHAK7, SbHKT5, SbHAK25, and SbAKT7) in cold stress treatments. In this context, genes with differential expression could be proposed as good candidates for functional analysis and further use in genetic engineering, and traditional breeding programs increase the cold tolerance of sorghum.

Heat shock proteins (HSP) function as chaperons and protect proteins from the harmful effect of different types of abiotic stress besides heat, including cold (Sabehat et al., 1998; Swindell et al., 2007). Transcriptomic analysis showed that the HSP Sb03g027330 was highly abundant under cold stress (Chopra et al., 2015). GST enzymes have functions in detoxifying xenobiotic compounds and ROS and are abundant under cold stress in sorghum (Chopra et al., 2015). Late embryogenesis abundant proteins are essential for membrane stabilization when the cytoplasm becomes dehydrated (Hanin et al., 2011) and might be expressed under cold conditions by a crosstalk signaling process with ABA (Li et al., 2017). To understand better how cold stress signals are perceived and integrated into gene regulation by changes at multiple levels, it is necessary to know the TFs, miRNAs, and the expression of gene products that might be involved in different degrees of tolerance to low temperatures, resulting in a change in metabolites in plants.

miRNAs are small non-coding RNAs, approximately 18–24 nucleotides in length, that can control gene expression at the post-transcriptional level (Fire et al., 1998). miRNAs act as regulators of gene expression through the degradation or inhibition of target gene translation (Djami-Tchatchou et al., 2017). The upregulation of miRNAs is associated with reduced expression of its target gene (Megha et al., 2018). In contrast, the downregulation of a miRNA can increase its target genes expression. MiRNAs may mediate the gene regulation network under cold stress in two ways: regulating stress-related signal transduction pathways or modulating the expression of cold-responsive TFs (Huang et al., 2022). Target genes and miRNAs involved in cold stress responses as a complex gene network may influence cold tolerance.

Some miRNAs participate in plant responses to cold stress (Thiebaut et al., 2012; Liu et al., 2018; Martínez Núñez et al., 2021; Satyakam et al., 2022). MiR397 is involved in different abiotic stress, including cold (Huang et al., 2020). For example, under normal conditions, miR397 is expressed at high levels, altering the abundance of its target genes laccases, which play an essential role in anthocyanin biosynthesis and abiotic stress responses (Xu et al., 2022; Zaman et al., 2022). Under cold stress conditions, miR397 is downregulated, leading to the accumulation of laccases (Xu et al., 2022). This association between miR397 and its target genes under cold treatments has been observed in plants including wheat (Gupta et al., 2014) and grapevine (Sun et al., 2015). In addition, miR397 overexpression improved the tolerance to cold stress in Arabidopsis (Dong and Pei, 2014; Satyakam et al., 2022). The core regulator in cold acclimation ICE1 (Chinnusamy et al., 2007) was identified as a target of miR397 in cold adaption in wheat. In sorghum, a homolog of miR397 was identified (Dash et al., 2022).

MiR319 and its target genes have been analyzed in the monocot sugarcane under cold stress (Thiebaut et al., 2012). Thiebaut et al. (2012) found differences in the timing and intensity of regulation of miR319 and its target genes PCF5, PCF6, and GAMyb. The Myb family genes are known to be CBFs suppressors; therefore, the upregulation of miR319 can prevent its repression effect, leading to the expression of COR genes ( Figure 3 ), possibly contributing to cold tolerance. The overexpression of miR319 can enhance cold tolerance, after chilling acclimation, in transgenic rice seedlings (Yang et al., 2013; Wang et al., 2014; Iqbal et al., 2021). Another example of this is the family of miR398; they control Cu/Zn SODs under oxidative stress caused accumulation of ROS during cold or some other kind of abiotic stress such as heat and salinity stress. SODs play an essential role in converting superoxide to H₂O₂ and molecular oxygen and thus reduce oxidative stress in plant cells. Downregulation of miR398 is vital for the SODs expression and ROS detoxifying. This association between miR398 and its target genes has been observed in cold-stress plants, including Arabidopsis (Sunkar and Zhu, 2004) and grapevine (Sun et al., 2015).

In sorghum, miRNA–target gene interactions generated with degradome analysis have been reported in cold response, and some interactions as miR169-NF-Y network were identified (Franke et al., 2018). Validation of miRNA–target gene networks will screen their roles in cold stress tolerance, which will also help develop plants with stress tolerance.

Understanding the mechanisms of cold stress response at the molecular level is crucial for improving crops against stresses without affecting the yield (Adhikari et al., 2022). Integration of signal perception, signal transduction, gene expression, and molecules produced is essential to understand the cold stress response and cold plant acclimation. Several molecules, including calcium (Ca2+), receptors, ROS, and signaling proteins, have been identified in plants. TFs, miRNAs, and proteins also play an essential role, and several have been identified in sorghum’s cold response. In summary, an illustration of the cold response and its overlapping dehydration response–mediated regulatory networks in sorghum is proposed using the evidence previously described ( Figure 3 ).

The mechanism of sensing low temperatures in plants is scarce, and it is still unknown whether there is a cold signaling receptor; possibly, with a similar complex as in rice (Ma et al., 2015), Chilling Tolerance Divergence1 (COLD1) encodes a regulator of G protein signaling coupled with rice G protein A subunit 1 that participates in cold stress signaling via Ca2+ signals (Ma et al., 2015; Zhu et al., 2022), regulating the cold stress–driven influx of intracellular Ca2+. Low temperatures can change the membrane fluidity; the potential sensors of cold include Ca2+ influx channels, transmembrane stress-sensing histidine kinases proteins (Fedurayev et al., 2018), and receptors associated with G proteins (Zhu et al., 2022), which could allow identify the low temperatures and induce a signal to subsequently transduce it to the nucleus (Chinnusamy et al., 2007). A decrease in cell membrane fluidity leads to conformational changes in membrane proteins and lipids, which generate second messengers IP3, Ca2+, and ROS, which are necessary to transduce signals through protein kinases or TF cascades ( Figure 3 ). Ca2+ is the most common secondary messenger in plants. Within only a few seconds under low temperatures, cytosolic Ca2+ can liberate from vacuoles. The Ca2+ release is upstream to the expression of CBFs and COR genes in the cold signaling pathways (Hiraki et al., 2019); in the same manner, ROS and IP3 serve as second messengers to be integrated into a genetic response that initiates variations in gene expression, leading to physiological and metabolic changes in the cell, and culminates in response and tolerance (Pareek et al., 2017).

Plant hormone JA also affects the low-temperature stress response. The JA signaling pathway activates MYC2, which is involved in most JA-mediated responses; in the same way, MYC2 is capable of activating the expression of ICE in ICE-CBF-COR cold resistance pathway. CBFs and AREB TFs regulate COR genes containing CRT/DRE and ABRE motifs in their promoters, respectively ( Figure 3 ). CBFs are activated by ICE1 and calmodulin-binding transcription activators (CAMTA) TFs but are suppressed by Myb family TFs. MiR319 can suppress TCP-like and MYB-like factors, stopping its repression effect and possibly contributing to cold tolerance. HOS1 and SIZ1 encode protein ligases that regulate the abundance of ICE1 protein (Zhou et al., 2011). Cold activates ICE1, and AREB is induced by ABA-mediated dehydration signaling. In response to cold stress, DRE element is recognized by CBF1 TFs, which control the expression of COR genes. At the same time, some COR genes are regulated by miRNAs, such as SODs by miR398, and laccases by miR397 and miR408. Consequently, the downregulation of these miRNAs is necessary for COR genes expression. There is a correct coordination between the different elements, and different elements are conserved in both biotic and abiotic stress responses. The more information that we have, the more precise the mechanisms of response and tolerance in sorghum will be.

The sorghum genome of ~730 Mb has been sequenced (Paterson et al., 2009). Quantitative trait locus (QTL) is the most comprehensive tool for marker‐assisted selection. In sorghum, co‐localization of different QTLs close to each other is known as hotspots, highly heritable genes map irregularly to the 10 sorghum chromosomes, and several are physically clustered together in chromosomes. In parallel, there are other vital tools, such as cold stress–induced transcriptome profiling (Chopra et al., 2015) and analysis of single-nucleotide polymorphisms (Chopra et al., 2015; Parra-Londono et al., 2018). Cold acclimatization is a process conserved between species where many genes are induced (Chinnusamy et al., 2007). Histone marks like acetylation and deacetylation are central for activation and repression during cold acclimation. The HOS15 gene product, which works as a histone deacetylation, interacts explicitly with histone H4 during cold acclimatization in Arabidopsis (Zhu et al., 2008a; Park et al., 2018). The chromatin remodeling derived from epigenetic changes during cold acclimatization has not been studied yet in sorghum. Nevertheless, there are some clues about epigenetic memory to cold stress; sorghum exposed to photoinhibition treatment changes the levels of the carotenoids and then restores during a recovery period, and, when photoinhibited, plants showed better protection (Sharma and Hall, 1996).