Molecular mechanisms of stress resistance in sorghum: lmplications for crop improvement strategies

Hongxiang Zheng ,Yingying Dang ,Xianmin Diao ,Na Sui

1 Shandong Provincial Key Laboratory of Plant Stress/College of life Sciences, Shandong Normal University, Jinan 250014, China

2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Abstract Abiotic stresses,such as drought,salt,extreme temperatures,and heavy metal pollution,are the main environmental factors that limit crop growth and yield. Sorghum,a C4 grass plant with high photosynthetic efficiency,can grow in adverse environmental conditions due to its excellent stress resistance characteristics. Therefore,unraveling the stress-resistance mechanism of sorghum could provide a theoretical basis for developing and cultivating various stress-resistant crops. This understanding could also help to create a conducive environment for using marginal soil in agriculture and ensuring food security. In this review,we discuss the adaptation mechanisms of sorghum under drought,salinity,temperature,and soil heavy metal stresses,the specific response to stress,the screening of sorghum-resistant germplasm,and the identification and functional analysis of the relevant genes and quantitative trait loci (QTL). In addition,we discuss the application potential of different stress-tolerant sorghum germplasms reported to date and emphasize the feasibility and potential use in developing and promoting highly stress-tolerant sorghum in marginal soil.

Keywords: abiotic stress,C4 plants,QTL,sorghum,stress resistance,yield stability

1.Introduction

The expanding global population and rising food demand necessitate a significant increase in food production(Ranganathanet al.2016;Desa 2019). In natural environments,abiotic stresses like drought,salinity,and extreme weather limit crop growth and yield (Gillihamet al.2017;Bailey-Serreset al.2019). Industrial development has led to heavy metal contamination of cultivated land,threatening food security and human health (Mishraet al.2021). Conventional breeding primarily targets a controlled yield increase rather than crop stability under various stress conditions. Under natural conditions,the plant phenotype and yield are regulated by the genotypeenvironment relationship (G×E) (Boyleset al.2019).Hence,introducing stress-resistant genes through genetic engineering has limited effects on crop resistance and yield under natural conditions (Zhu 2016;Bailey-Serreset al.2019). Screening and cultivating stress-resistant crops can improve existing varieties,thereby enhancing overall production. Understanding the stress response mechanisms in highly stress-resistant crops can allow us to develop better crop varieties for sustainable,ecofriendly production.

Drought is a primary limitation in crop production.Approximately 50% of the world's land is arid or semiarid (Zika and Erb 2009). Climate change and extreme weather may worsen drought. Stomata closure during drought weakens photosynthesis,reducing the yield due to water loss-induced osmotic stress (Liet al.2022).Drought significantly reduces crop yield,especially during critical stages such as seedling,flowering,and grain filling(Varoquauxet al.2019).

Soil salinization is another key abiotic stress that limits crop yield (Zhanget al.2020;Liet al.2021). About onefifth of global irrigated land faces high salinity levels,and the affected area is growing. By 2050,approximately 50% of arable land worldwide may be salinized (Jamilet al.2011). Soil salinization induces ionic,osmotic,and oxidative stresses,and thus inhibiting plant growth and,in severe cases,causing plant death (Yuet al.2020). Alkaline lands are currently reclaimed through soil remediation and the cultivation of salt-tolerant plants;however,the former is costly. Widespread planting of stress-resistant species in arid regions,saline–alkali lands,and marginal terrains can enhance the amount of arable land,potentially ensuring food security (Gonget al.2020;Zelmet al.2020;Zou 2020).

Global climate change has increased the occurrence of extreme temperatures,and thus causing severe crop yield losses. Temperature stress during critical growth phases can substantially reduce yield. High temperatures can decrease pollen viability,reducing seed setting and grain yields. Low temperatures can hinder germination and cause plant death. Breeding temperature stress-resistant crop varieties is necessary to enhance crop stability under natural conditions.

Heavy industries have contaminated agricultural land with various heavy metals,such as cadmium,aluminum,and lead. Excessive metal accumulation generates reactive oxygen species,causing heavy metal toxicity and stunting crop growth. Elevated metal levels in the food chain pose a severe health threat. As just one example,consuming cadmium-contaminated rice can lead to itai-itai disease,which is potentially fatal (Wanget al.2021). Cultivating crops with a high metal ion accumulation capacity,tolerance to metal toxicity,and high biomass supports the rational use of contaminated land,increases economic benefits,and aids in achieving soil bioremediation goals.

After wheat,maize,rice,and barley,sorghum is the fifth most widely grown crop globally and has become a staple food in sub-Saharan Africa and South Asia (Bolotet al.2009). Sorghum has excellent stress-resistant characteristics,such as tolerance to salt,alkali,drought,flood,heat,and cold (Xie and Xu 2019). Sorghum has a strong absorption capacity and higher tolerance to many metal ions in soil such as Cd2+,Mn2+,Cu2+,and others(Jiet al.2014). Due to its excellent stress resistance properties,sorghum is ideally suited for arid and semiarid ecosystems,serving as a staple food source for approximately 500 million people in numerous regions of Africa and Southeast Asia (Belton and Taylor 2004;Maceet al.2013;Proiettiet al.2015). Sorghum stands out as one of the few crops capable of adapting to multiple abiotic stresses,complex climates,and various soil conditions (KhavaninZadeet al.2022). For instance,it has been successfully cultivated in the arid saline–alkali land of Yazd Province,Iran,the semi-arid land of the Indo-Gangetic Plain (Soniet al.2021),and the desert climate of central Egypt (Abd El-Mageedet al.2022).

In contrast to other cereal crops,such as maize,wheat,and barley,sorghum exhibits steeper shoot and root angles,as well as a higher number of primary and secondary roots in its root system. These features equip sorghum with the ability to draw water from deeper soil layers,thereby enhancing its resistance to drought stress(Robertsonet al.1993;Lamaouiet al.2018). Despite boasting the highest upper limit of water productivity among these cereal crops,sorghum has a lower water requirement. Under conditions of water deficit,sorghum’s water consumption is only half that of maize(Gudenet al.2021). Due to its higher comprehensive water use efficiency,sorghum is better suited for cultivation in water-deficient regions,and arid and semi-arid areas,and it exhibits a higher harvest index(Staggenborget al.2008;George-Jaeggliet al.2011).In regions susceptible to frequent high-temperature occurrences during the summer,maize shows various heat stress-related phenotypes,including plant death,leaf wilt,reduced pollen viability,and a subsequent reduction in final yield (Chenet al.2012). In sharp contrast,sorghum exhibits minimal to no damage from heat stress under analogous environmental conditions,underscoring its superior tolerance to high-temperature stress (Chenet al.2017). Moreover,certain sorghum accessions from temperate regions also display greater resilience to low-temperature stress compared to maize(Maulanaet al.2017;Hanet al.2020;Menget al.2021).Furthermore,sorghum demonstrates stronger tolerance to abiotic stresses such as salt and heavy metal stress compared to other crops (Mishraet al.2021;Shiet al.2022;Kazemiet al.2023).

For many years,investigations concerning the stress response mechanisms in sorghum predominantly hinged upon identifying natural variations within its genetically diverse population. This reliance on natural variations was driven by the challenges posed by genetic transformation in sorghum. Consequently,research into the stress resistance mechanisms of sorghum has lagged behind that in other essential food crops,such as rice,maize,and wheat. Several researchers have reviewed the research progress of plant resistance to abiotic stress(Zhu 2016;Gonget al.2020;Zelmet al.2020). In this review,we mainly summarize the specific responses of sorghum during abiotic stress resistance,including the physiological,biochemical,and molecular mechanisms.At the same time,we discuss the application prospects of the currently screened germplasm of resistant sorghum,and the feasibility and potential for developing and popularizing the transgenic sorghum with high tolerance in marginal soil and climatic conditions. Finally,we highlight the potential of sorghum,a model C4grass with high stress resistance,as a mainstream crop that can address both climate change and population growth.

2.Reaction of sorghum to salt stress

2.1.Physiological and biochemical reactions to salt tolerance in sorghum

Soil salinization results in ionic,osmotic,and oxidative stresses in plants,adversely inhibiting the normal growth of plants and leading to plant death. The cornerstone of plant salt tolerance lies in the maintenance of ion homeostasis within the cell cytoplasm under conditions of salt stress (Zhu 2016;Zelmet al.2020). In a high-salt environment,sodium ion (Na+) is one of the primary toxic ions in most plants (Demidchik and Maathuis 2007;Zelmet al.2020). A high concentration of Na+entering cells reduces potassium ion (K+) uptake by the cells,disrupting the intracellular ion balance. Salt-tolerant plants can maintain a low Na+concentration by rejecting,secreting,or diluting salts to reduce the ionic toxicity in a high-salt environment.

Salt rejection is the most crucial salt tolerance mechanism in grasses such as sorghum. Salt rejection refers to the accumulation of salt ions in the xylem parenchyma of roots or rhizome junctions,limiting Na+inflow,increasing Na+efflux,or localizing Na+into vacuoles (Munns and Tester 2008). Monocotyledonous crops store the absorbed Na+in the developed organs and parenchymal tissues,such as roots,stem bases,nodes,and leaf sheaths,and regionalize Na+into the vacuoles. This process reduces Na+transport to leaves,which has been observed in wheat and barley (Munnset al.2006).

The salt overly sensitive (SOS) system plays a pivotal role as a defense mechanism for plants coping with Na+stress. Through the sequential activation of SOS3–SOS2–SOS1,excessive intracellular Na+is extruded from the cell,preserving the intracellular K+/Na+balance and regulating the adaptability of the plants to salt stress(Fig.1). The ion transport and photosynthetic changes of sorghum under salt stress have been described in detail in a recent review by Yanget al.(2020),and we are more concerned with the latest research progress in this review.

The apoplast barrier in sorghum roots,consisting of casparian bands and suberin lamellae,serves as the primary structure hindering ion transport to the aerial parts and leaves (Blumwald 2000;Krishnamurthyet al.2011;Yanget al.2020). Previous studies have shown that high-salt conditions result in the upregulation of apoplast barrier genes in sorghum (Suiet al.2015;Yanget al.2018) and the thickening of the apoplast barrier. Moreover,this high-salt-induced thickening of the apoplast barrier is more pronounced in salt-tolerant inbred lines than in salt-sensitive inbred lines. A similar phenomenon was found in salt-tolerant varieties of rice and barley.

The differential distribution of salt on the surface of saline–alkali soil produces different physiological responses in sorghum,and uneven salt stress can improve the stomatal conductance and transpiration efficiency of sorghum. The root system on the lowsalt side will have a compensatory effect and produce more fine roots,thereby alleviating the overall damage under salt stress (Zhang Het al.2019). Therefore,an inconsistent root salt content might improve the salt tolerance of sorghum. This phenomenon aligns with observations in other plant species,such as cotton and grapes,wherein uneven salt stress significantly diminishes Na+accumulation,bolsters K+absorption,and effectively mitigates salt stress,as compared to constant salt stress (Donget al.2010). Rainfall can exacerbate the irregular distribution of soil salinity and rapidly reduce surface soil salinity,making saline–alkali land with frequent rainfall conducive for sorghum cultivation. In addition,deliberately creating variable salinity conditions by constructing ridges or trenches can aid sorghum in surviving through the more salt-sensitive stages,including germination and seedling.

Elevated salt levels lead to the substantial accumulation of abscisic acid (ABA) in leaf tissues (Chenet al.2020). As a pivotal stress-responsive hormone in plants,ABA plays a crucial role in regulating various metabolic processes involved in plant adaptation to stress (Yoshidaet al.2019;Chenet al.2020).

Fig. 1 Regulatory mechanisms of sorghum under salt stress. Under salt stress,changes occur in cell membrane fluidity. Cell membrane Ca2+ sensors perceive changes in the extracellular Na+ concentration,leading to an influx of Ca2+. Ca2+ from the vacuole is released into the cytoplasm through TPC1,increasing the cytosolic Ca2+ concentration. Ion channels on the cell membrane respond to Ca2+ signals and regulate Na+/K+ transport. Simultaneously,the Ca2+-dependent SOS3–SOS2 signaling pathway is activated. The activity of the plasma membrane SOS1 antiporter is enhanced,promoting the efflux of Na+ out of the cell or its sequestration into the vacuoles. This process relies on the proton motive force provided by P-and V-type ATPases. Additionally,the concentration of abscisic acid (ABA) in cells increases in response to salt stress. In the ABA signaling pathway,activated SnRK2s phosphorylate and activate ABF (ABA-responsive element-binding factor)/AREB (ABRE-binding),which interacts with ABRE cis-elements to induce the expression of ABA-responsive genes. ABA-GE,ABA glucose ester;BGLU,β-glucosidase.

ABA is perceived by pyrabactin resistant protein (PYR),pyrabactin resistant-like protein (PYL),and regulatory component of ABA receptor (RCAR),which initiate ABA signal transduction through binding to ABA. This binding leads to the inactivation of protein phosphatase 2C(PP2Cs),relieving the inhibition of SNF1-related protein kinase 2 (SnRK2). The activated SnRK2 subsequently acts on target genes containing ABA-responsive elements (ABREs),thereby initiating the expression of ABA-responsive genes. Alternatively,ABA can activate transcription factors such as WRKY and MYB,which are associated with the salt stress response,to regulate processes like ion transport and ROS clearance in response to salt stress (Songet al.2022b;Xiaoet al.2023;Zhenget al.2023).

Calcium (Ca2+) is an essential component of cell walls and membranes that plays a crucial role in maintaining stem toughness and enhancing stem strength. Additionally,Ca2+acts as a significant second messenger in multiple metabolic processes involved in plant stress resistance. Under NaCl stress,Ca2+is released from vacuoles through the mediation of twopore channel 1 (TPC1). This leads to a rapid increase in the cytosolic Ca2+concentration,initiating calcium signal transduction through calcium-dependent protein kinases and calcium-binding proteins. In the SOS pathway,SOS3 and SOS3-like calcium-binding proteins (SCaBPs)sense the elevated Ca2+levels caused by Na+stress and interact with SOS2 to activate it. The activated SOS2 phosphorylates SOS1,enhancing its ability to transport Na+/H+in the reverse direction,thereby extruding Na+out of the cell or compartmentalizing it into vacuoles and reducing the intracellular Na+concentration (Maet al.2019).

2.2.ldentification of QTLs related to salt tolerance in sorghum

Salt tolerance in sorghum is a complex quantitative trait controlled by multiple genes. Therefore,constructing genetic populations with different levels of salt tolerance will help in identifying the quantitative trait loci (QTLs)related to the salt tolerance phenotype and elucidating the salt tolerance mechanism of sorghum (Table 1). Studies have identified several QTLs related to salt tolerance in sorghum seedlings (Wanget al.2020b). For example,six QTLs (qGP7-1,qSH1,qRL10-2,qSFW4,qTFW1,andqTFW4) affecting the germination rate biomass have more than 10% PVE (the largest phenotypic variation) in sorghum under salt stress at the seedling stage (Wanget al.2014). Under low salt stress,qSH7-2was associated with seedling height,and its expression pattern was highly context-specific (Wanget al.2020c).Meanwhile,Xtxp91andXtxp113were found to affect the fresh weight of sorghum at the seedling stage (Wanget al.2020c).

Table 1 QTLs related to different abiotic stresses,and their functional annotations and germplasm resources for the donors of tolerant QTLs in sorghum

Nonetheless,owing to the irregular distribution of rainfall and salinity in saline–alkali lands and variations in salt tolerance across different developmental stages of sorghum,it is imperative to identify the QTL governing salt tolerance throughout the entire growth period. In a recent study,Wanget al.(2020b) conducted an extensive exploration of QTL for salt tolerance spanning the entire growth period of sorghum. Notably,QTLs such asqBrix2,qTB6,qSFW9,qSTI-Brix9,qJW9,andqBrix10were identified as exerting some influence across the entire growth period (Wanget al.2020b). QTLs usually have additive effects;therefore,they may serve as target sites for marker-assisted selection (MAS) to improve salt tolerance throughout the growth period by combining molecular breeding methods. The potential functions of salt tolerance-related QTLs are summarized in Table 1 and Fig.2.

2.3.ldentification of genes related to salt tolerance in sorghum

The screening of salt-tolerant and salt-sensitive sorghum genotypes plays a pivotal role in the discovery of the key genes governing resistance (Donget al.2019;Zhang Fet al.2019;Chenet al.2022). Sui Na’s research group screened different sorghum germplasms for salt tolerance,and they screened out two sorghum inbred lines with differences in salt tolerance: the salt-tolerant inbred line M-81E and the salt-sensitive inbred line Roma (Suiet al.2015;Yanget al.2018). Of the two,M-81E has a better salt rejection ability and accumulates less Na+in its leaves than Roma (Suiet al.2015;Yanget al.2018).

Fig. 2 QTLs related to different abiotic stresses in sorghum. The periods and stages of the roles of QTLs with different abiotic stress tolerances in sorghum,the functional annotations and germplasm resources for the donors of tolerant QTL in sorghum are shown in Table 1.

Transcriptome analysis unveiled a multitude of differentially expressed genes under salt stress for both M-81E and Roma,implicating their involvement in various salt tolerance regulatory pathways,including ion transport,photosynthesis,and phenylpropane metabolism (Suiet al.2015;Yanget al.2018). These findings strongly suggest that the differential expression of these genes underlies the variation in salt tolerance between M-81E and Roma.Nevertheless,the specific reasons behind the disparate gene expression patterns remain unclear. Zhenget al.(2023) have identified several transcription factors that exhibit differential expression under salt stress in two inbred lines with disparate salt tolerance profiles,and these factors regulate the expression of numerous genes associated with salt tolerance. For example,SbMYBHv33negatively regulates the salt tolerance of sorghum by reducing the biomass accumulation and inhibiting the growth of sorghum at the seedling stage,by regulating the expression of ABA signaling pathway genes.SbWRKY50directly regulates the expression of ion transport-associated genes by binding to the W-box in theSbSOS1andSbHKT1promoter regions inArabidopsisand sorghum,thereby affecting plant salt tolerance (Songet al.2020).

On the other hand,SbWRKY55negatively regulates sorghum’s salt tolerance by inhibiting the expression of the ABA-GE hydrolysis geneSbBGLU22and modulating endogenous ABA levels (Songet al.2022b). Meanwhile,SbbHLH85regulates the expression of genes involved in the ABA and auxin signaling pathways and significantly enhances the number and length of root hairs,resulting in increased Na+accumulation. Furthermore,SbbHLH85 interacts with the phosphate transporter chaperone PHF1,leading to a reduction in the phosphate content inSbbHLH85-overexpressed sorghum. This alteration further contributes to reductions in both the number and length of root hairs under stress conditions,representing a potentially effective strategy for modulating salt tolerance in sorghum (Songet al.2022a). The potential functions of salt tolerance-related genes of sorghum are summarized in Table 2.

Table 2 Genes related to different abiotic stresses and their functional annotations and germplasm resources for the donors of tolerant genes in sorghum

2.4.Other factors affecting salt tolerance in sorghum

Physiological and biochemical regulatory processes in plants under stress usually depend on altered gene expression. While RNA-seq studies have effectively characterized changes in gene expression in sorghum under various environmental stresses,the realm of post-transcriptional regulation remains predominantly uncharted. At the post-transcriptional regulatory level,non-coding RNA and RNA modifications exert a pivotal influence on regulating gene expression in sorghum,particularly in the context of stress resistance(Yanet al.2022). In Roma and M-81E,Sunet al.(2020) reported five previously undisclosed lncRNAs,namely lncRNA11310,lncRNA13472,lncRNA26929l,lncRNA14798,and lncRNA2846. These lncRNAs may act as competitive endogenous RNA (ceRNA)regulators,regulating the transcription and expression of genes related to salt tolerance. Additionally,our recent investigation revealed intricate interactions among different miRNAs,plant hormones,and key transcription factors in Roma and M-81E,giving rise to intricate and diverse response networks (Sabadinet al.2012). These findings highlight the response mechanism of miRNA in salt-tolerant sorghum,which can be helpful for breeding high-quality sorghum germplasm with better abiotic stress tolerance in the future.

N6-methyladenosine (m6A) is an important epigenetic modification commonly found in eukaryotic mRNA andnon-coding RNAs. This modification plays a pivotal role in regulating RNA export,splicing,stability,and translation.m6A modification also exerts a multifaceted influence on various aspects of plant growth and development,encompassing embryonic development,stem cell fate,flowering transition,root development,tissue differentiation,flowering,fruit ripening,and stress tolerance(Shimet al.2020;Tanget al.2022). Mapping the m6A modification landscape has unveiled its sensitivity and dynamic nature in response to plant growth,development,and environmental stress. The transcriptome-wide m6A modification map of sweet sorghum with different salt tolerances under salt stress conditions showed that m6A modification was highly dynamic before and after salt stress treatment. The m6A modification trend and degree are closely related to genotype. Interestingly,m6A modification was more intense than altered gene expression after salt treatment. Besides,m6A is more sensitive than direct transcriptional regulation as far as salt resistance is concerned,and the altered level of m6A modification affects the mRNA stability of some salttolerance-related transcripts. Thus,these findings suggest that m6A modifications are involved in the salt stress response process in sweet sorghum (Zhenget al.2021).

Rhizosphere microorganisms may also play an important role in regulating salt tolerance in sorghum(Olanrewaju and Babalola 2022). Wuet al.(2022) have provided compelling evidence that the composition of sorghum rhizosphere microorganisms under salt stress is modulated by the sorghum genotype. Specifically,the bacterial community in salt-tolerant sorghum M-81E exhibited greater complexity compared to that in Roma’s rhizosphere. Concurrently,the core microbial components show a high correlation with the expression of some genes in sorghum (e.g.,SbHSP70). The mechanism of crop–microbial–soil interactions and functional enhancement technology may be the potential direction to further improve sorghum growth under saline–alkali stress(Caoet al.2021).

3.Reaction of sorghum to drought stress

3.1.Feasibility of growing sorghum in arid and semi-arid regions of the world

Due to its outstanding drought tolerance,sorghum is extensively cultivated in semi-arid tropical regions,primarily within sub-Saharan Africa and South Asia(Belton and Taylor 2004;Hadebeet al.2017). Sorghum is deemed an ideal crop for agricultural production in South Africa,given that sub-Saharan Africa experiences distinct dry and rainy seasons,often accompanied by flooding during the rainy period. Sorghum’s resilience to flooding renders it less vulnerable to losses from shortlived episodes of heavy rainfall. This resilience positions sorghum as a more suitable choice for cultivation in South Africa than other crops (Hadebeet al.2017).

In other drought-prone regions,such as Kansas and Nebraska in the United States,sorghum yields are higher and it offers economic advantages compared to maize (Staggenborget al.2008). Furthermore,sorghum has shown promise as a bioenergy source in semi-arid Mediterranean environments (Nazli 2020). In Europe,sorghum has been progressively replacing maize,since it is less susceptible to climate change than traditional maize crops and capable of delivering higher yields with lower input costs. Several European countries have embraced its cultivation to enhance crop yields in hot and arid regions(Popescuet al.2018;Przybylska-Balcereket al.2020).

Notably,some studies have highlighted variations in the extent of sorghum trait inhibition under diverse drought conditions. For instance,in China,the reductions in several trait indices of various sorghum varieties after drought stress was more pronounced in Hainan Province compared to Shanxi Province (Wanget al.2019). These distinctions may be attributed to variations in climate and rainfall between the regions. In Hainan,the drought period primarily occurs during winter,coinciding with the seedling and grain-filling stages of sorghum,thus considerably impacting the plants (Wanget al.2019).Similarly,under the same irrigation conditions,the yield and biomass of sorghum in northeastern Spain are lower than those in northeastern China (Farré and Faci 2007).This dissimilarity can be attributed to the disparate climatic conditions between the two regions. Northeastern China receives relatively higher annual rainfall,resulting in reduced crop evaporation during the growing season,and potentially less adverse effects on sorghum growth.

For grain sorghum,drought stress during the seedling phase significantly hampers development,while drought during the flowering and grain-filling stages directly impacts grain yield (Staggenborget al.2008). Although sorghum exhibits a relatively robust drought tolerance,considerable variability exists among varieties or genotypes under field conditions. Consequently,the key to harnessing the marginal soils in complex arid areas in actual production environments may lie in the screening and cultivation of germplasms that are better suited to local climate conditions during various developmental phases.

3.2.ldentification of QTLs related to drought tolerance in sorghum

Establishing a reasonable screening model for resistance indicators is the key to screening drought-resistant sorghum germplasm (Fig.2). Wanget al.(2019) showed that under production conditions,various traits of 165 grain sorghums were inhibited to varying degrees. Among these traits,leaf stay-green was most significantly affected by drought conditions. Compared with normal irrigation conditions,leaf stay-green of sorghum in different regions decreased by more than 30%. In addition,morphological indicators such as yield,plant height,and lodging resistance were also suppressed to varying degrees.The stay-green and yield of leaves can be used as the main morphological indicators for evaluating the drought resistance of sorghum after flowering. A similar trend was observed in prior studies by Zhouet al.(2014),which found that sorghum with higher leaf stay-green could maintain an elevated chlorophyll content and a heightened photosynthetic rate when exposed to drought stress. Additionally,leaf stay-green is co-regulated by the endogenous hormones CTK and ABA.

The stay-green trait of sorghum,an indicator of drought tolerance,is regulated by multiple genes. Previous studies have identified several QTLs regulating the staygreen trait by constructing genetic populations using the typical green-keeping sorghum variety B35 as the parent (Table 1). Among them,Stg1,Stg2,andStg3are closely related to the stay-green trait. Of these three,Stg2significantly correlates with the chlorophyll content in leaves (Subedi and Ma 2005) and is the most important control site for the stay-green trait. These QTLs were validated in genetic populations constructed from different parents. For example,the green-holding QTLs,Stg2,Stg3,andStg4,of the B35xTx7000 screen were nearly identical to the green-holding QTLs,StgA,StgD,andStgJselected from the B35xTx430 population(Crastaet al.1999;Xuet al.2000). Using two combined RIL populations,Haussmannet al.(2002) mapped five to eight QTLs regulating the stay-green trait in sorghum,and these QTLs explained 31 to 42% of the phenotypic variation of stay-green sorghum. Furthermore,other QTLs contribute to the observed differences in drought resistance. For example,Kebedeet al.(2001)constructed a genetic population using the droughttolerant SC56 and the drought-sensitive cultivar Tx7000,mapping the QTLs to chromosome 7,which accounted for 15 to 37.7% of the differences in drought resistance prior to flowering in sorghum (Haussmannet al.2002).

In comparison to other crops,sorghum possesses an extensive root system that enhances water absorption during drought stress. Root length and structure,notably the nodal root angle,are crucial factors influencing drought tolerance during the seedling stage. Under water-deficient conditions,a narrow root angle facilitates greater root depth and vertical root growth,enabling the absorption of water from deeper soil layers (Maceet al.2012). Previous studies have identified several QTLs that regulate stem components and biomass,such asbrown midrib 2(bmr2,encode 4-coumarate coenzyme A ligase),bmr6(encode cinnamyl alcohol dehydrogenase),bmr12(encode caffeic acid O-methyltransferase),andbmr19,which control lignin synthesis and,in turn,affect stem height and composition (Walkeret al.2013). Recent studies have shown thatbmr12regulates water uptake in sorghum under drought conditions by regulating the root structure (Salujaet al.2021). Thebmr12premature termination mutant displayed reduced lignin deposition in roots,resulting in reduced lateral roots and altered node root angles. Consequently,these changes in the root structure resulted in limited water uptake inbmr12mutants under well-watered conditions (Salujaet al.2021).

3.3.ldentification of genes related to drought tolerance in sorghum

The advent of high-throughput sequencing has enabled the screening and cloning of differentially expressed genes in sorghum varieties with varying drought tolerance,a critical step in the analysis of drought resistance (Fig.3).At the gene transcription level,drought stress induces the expression of more than 40% of the genes in sorghum.Genotype differences represent the primary driver affecting the rate of recovery during the drought recovery stage,thereby influencing the expression of numerous genes related to photosynthesis (Gaoet al.2020).

Heat shock proteins (HSPs) play a crucial role in plant responses to various stresses,including drought and high temperatures. Zhang Det al.(2019) conducted a differential gene expression analysis of sorghum under different levels of drought treatment,leading to the identification of six upregulated HSP genes in response to drought stress. Additionally,the precise genomic locations of these HSP genes in the sorghum genome were determined. These findings highlight the significant involvement of HSP proteins in enhancing drought tolerance in sorghum. The MAPK cascade is involved in regulating various stress responses in plants.The MAPK signaling pathway consists of three specific kinases: MAPK kinase kinase (MAPKKK),MAPK kinase(MAPKK),and MAPK (Bailloet al.2019). They are sequentially activated through phosphorylation,and the activated MAPK phosphorylates various downstream target genes,thereby modulating their activities and expression. This cascade is involved in the regulation of plant drought tolerance and responses to hormones such as ABA. MAPK may act as a negative regulator in drought stress responses. For example,Zhouet al.(2022)identified and analyzed the sorghum MAPK cascade genes and found that several MAPK family genes in sorghum respond to drought stress. Overexpression ofSbMPK14inArabidopsisand maize leads to increased drought sensitivity,possibly due to the reduced activity of ERF and WRKY transcription factors mediated bySbMPK14. In addition,an analysis of the expression profiles of drought-responsive genes in different sorghum germplasms revealed the upregulation ofSbMAPKKK7andSbMAPK10under drought conditions (Abou-Elwafa and Shehzad 2018). Furthermore,bothMAPKKK7andMAPK10in sorghum,under drought stress,are responsive to ABA signaling and contribute to the ABAmediated stomatal closure in response to drought stress(Prasadet al.2021).

Fig. 3 Regulatory mechanisms of sorghum under drought stress. Under drought stress,the cell membrane sensor OSCA1(reduced hyperosmolality-induced calcium increase 1) perceives changes in the osmotic pressure,leading to the opening of Ca2+gated channels and an increase in the intracellular Ca2+ concentration. The expression of drought-responsive genes,particularly those involved in ion transport,is regulated by CDPKs/CPKs,which also participate in the regulation of the activity of the stomatalrelated protein SLAC1 (slow anion channel 1),thereby mediating stomatal closure during drought stress. Simultaneously,under drought stress,sorghum accumulates abscisic acid (ABA) in the roots and shoots,which is subsequently transported to guard cells. Activated SnRK2 interacts with SLAC1,promoting anion efflux and facilitating stomatal closure in response to drought stress.KUP6/8 (K+ uptake transporter 6/8) is also regulated by SnRK2s and contributes to stomatal regulation.

Calcium-dependent protein kinases (CDPKs) play important regulatory roles in plant responses to various stresses,including drought. Under drought stress,multiple CDPK genes in sorghum leaves and roots are upregulated or downregulated to varying degrees. For example,SbCDPK16andSbCDPK6are upregulated in sorghum leaves,whileSbCDPK80is downregulated in both leaves and roots. Some authors have speculated that different CDPKs may perform distinct roles in regulating drought tolerance in various sorghum tissues(Shikhaet al.2017). The CDPKs participate in ABA and MAPK cascade responses by perceiving upstream Ca2+signals and phosphorylating downstream proteins.They transmit drought signals to regulate droughtresistant genes through interactions between different CDPKs. An increase in the ABA concentration triggers Ca2+signal transduction,and specific CDPKs respond to ABA signals to regulate the expression of ABA-related genes. A comparative analysis of CDPK families in different species revealed thatSbCDPK6,the homolog ofAtCDPK46inArabidopsis,participates in the ABA signaling pathway under drought stress and activates the transcription factors involved in ABA responses (Shikhaet al.2017). Similarly,SbCDPK6,the homolog ofOsCDPK75in rice,also plays a role in the ABA signaling pathway thereby enhancing plant drought tolerance (Saijoet al.2000).

Liuet al.(2016) cloned theSbSKIPgene,and overexpression ofSbSKIPin tobacco improved drought resistance.SbER2-1enhanced maize drought tolerance by increasing the net photosynthetic rate and water-use efficiency (Liet al.2019). Several other genes,such asSbSNAC1,SbNAC0584(Zuet al.2015),SbWRKY1,andSbWRKY2(Xuet al.2017),exhibited differential expression under drought and various abiotic stresses,identifying them as potential components in the sorghum drought resistance regulatory network. At the posttranscriptional level,genes encoding the argonaute proteinsSbAGO1-1,SbAGO-3,andSbAGO5-2were also differentially expressed under drought stress and high temperature (Linet al.2019). AGO proteins may play a role in regulating the degradation or translational repression of mRNAs related to drought tolerance at the post-transcriptional level by forming complexes with miRNAs (Linet al.2019). Nonetheless,due to the limitations in genetically transforming sorghum,the verification of the functions of these drought resistancerelated genes and the research on their regulatory mechanisms are mostly carried out in other plants. In the future,efforts should be made to directly overexpress the genes that positively regulate drought tolerance or knockout the genes that negatively impact sorghum’s drought toleranceviagenetic engineering,thereby improving drought resistance (Doet al.2016;Aregawiet al.2022).

3.4.Other factors affecting the drought tolerance of sorghum

Recent studies have illuminated the capacity of rhizospheric microbiota to alleviate the damage caused by drought stress in plants,so regulating the structure of the rhizospheric microbiota can provide a new direction for stress resistance in crop production.

An investigation into the rhizospheric microbiota of sorghum at various developmental stages and under different drought stress conditions revealed that the early stages of drought caused more substantial harm to the plant’s rhizospheric microbiome. Consequently,the abundance of rhizosphere actinomycetes declined as the duration of drought stress was prolonged (Xuet al.2018).

Following drought stress and subsequent rehydration,the composition of the rhizospheric microbiota partially recovered but only reached approximately half of its predrought composition level. Simultaneously,colonization of roots and the rhizosphere by drought-stressedStreptomycesparvumisolates bolstered root development during drought,thus modulating the plant’s responses under drought conditions. An integrated analysis of the sorghum microbiome and persistent transcriptome in the natural environment by Varoquauxet al.(2019)demonstrated that the total biomass of arbuscular mycorrhizal (AM) decreased during drought. However,after the drought,the pre-flowering drought-tolerant genotype RTx430 was able to restore its symbiotic relationship between mycorrhizae more quickly compared to the post-flowering drought-tolerant cultivar BTx642.The composition of rhizosphere microbes appears to result from the interplay between plant genotype and the environment,and it is influenced by the developmental stage of the plant (Gaoet al.2020).

At the post-transcriptional level,non-coding RNA,particularly miRNAs,plays a pivotal role in regulating the response of sorghum to drought stress. miRNAs are abundant and diverse in drought-tolerant plants,and Hamzaet al.(2016) identified eight miRNAs: sbi-miR160,sbi-miR166,sbi-miR167,sbi-miR168,sbi-miR393,sbimiR396,sbi-miR397-5p,and sbi-miR398. These miRNAs have the potential to mitigate the damage caused by drought stress in sorghum,and some have postulated that these eight miRNAs are linked to the drought tolerance mechanism in sorghum. This indicates that sorghum miRNAs may hold promise for enhancing its drought resistance.

4.Reaction of sorghum to high-temperature stress

4.1.Physiological and biochemical reactions of heat tolerance in sorghum

Sorghum primarily thrives in hot and arid regions,such as tropical and subtropical areas,where droughts often coincide with elevated temperatures. Therefore,sorghum predominantly experiences warmer temperatures during most of its growth cycle,as influenced by the planting location and season.

The principal factor contributing to a reduction in sorghum yields under high-temperature conditions is heat stress during the flowering and grain-filling stages.For example,Tacket al.(2017) analyzed the field yield data for 400 sorghum germplasms over the past 30 years,and established a direct link between reduced sorghum yield and the intensity and duration of hightemperature stress both before and after the flowering stage. Their regression model revealed that the threshold for high-temperature stress in sorghum growth is 33°C. Beyond that point,yields decrease with a 20%reduction occurring for every 2°C rise in temperature(Tacket al.2017).

High temperatures during the flowering period diminish pollen vigor,leading to reduced seed setting and grain yield (Prasadet al.2015). Even mild heat stress (35/22°C)reduced the spikelet fertility by 5–20% relative to normal conditions (32/22°C) (Chiluwalet al.2020). Besides,moderate high-temperature stress (38/22°C) reduced the fertility of spikelets by 18–45%. Under extremely high temperatures (40/22°C),the sorghum of different genotypes failed to form any seeds.

Detailed microscopic analysis of ovarian anatomy revealed that elevated temperatures led to cytoplasmic content shrinkage in the ovarian tissue adjacent to the micropylar region and the destruction of nucleoli and nuclei. This damage was further exacerbated at 40/22°C,resulting in 100% spikelet sterility. In sorghum,reduced pollen germination and impaired ovarian structure could be major factors in heat-stress-induced sterility (Sunojet al.2017;Tacket al.2017;Chiluwalet al.2020).

Meanwhile,heat stress from the booting stage to late maturity of sorghum negatively affected pollen germination,grain yield,photosynthesis,chlorophyll index and PSII efficiency,and it increased leafFo/Fm(Sunojet al.2017;Chiluwalet al.2020). Although heat stress inflicts widespread damage on sorghum flowers,the extent of this damage exhibits variability among sorghum genotypes. For example,the pollen of certain potential heat-tolerant genotypes (e.g.,Maica and SC155) maintained high pollen germination capacity under heat stress conditionsin vitro(Chopraet al.2017;Sunojet al.2017). The rate of pollen germination at different temperatures primarily hinged on the growth and incubation temperatures as well as the genotype (Chopraet al.2017;Sunojet al.2017;Tacket al.2017).

Nonetheless,in comparison to other major crops like rice,the flowering time of sorghum exhibits lower sensitivity to environmental conditions. Sorghum’s flowering time is primarily governed by specific genes and other environmental factors. In contrast to rice,where peak flowering typically occurs around noon,90% of sorghum spikelets bloom within a concentrated timeframe of 30 min following dawn (6:00–6:30). The flowering model of sorghum under heat stress and water stress was similar to that under normal conditions,primarily focused on the completion of flowering within 1 hour after dawn(6:00–7:00) (Chiluwalet al.2020). The unique earlymorning flowering phenomenon (EMF) in sorghum plays a crucial role in safeguarding pollination and fertilization events from exposure to the sun,thus protecting the reproductive organs from high temperatures. This phenomenon likely accounts for sorghum’s ability to complete the grain production process under hightemperature stress.

4.2.ldentification of genes related to high-temperature tolerance in sorghum

There is a scarcity of research on high-temperature stressrelated genes in sorghum,with most studies focusing on functional research related to drought tolerance genes.It is worth noting that some of the genes associated with drought resistance traits may also have relevance for high-temperature resistance. For example,the staygreen trait’s QTLs confer tolerance to both drought and high-temperature stress (Maoet al.2017;Chadalavadaet al.2021;Ndlovuet al.2021). Furthermore,several other genes are also induced by both drought and heat stress. For instance,the expression levels of several members of the ARF gene family are upregulated under high temperatures (Moritaet al.2009).SbHSF1is induced by heat stress (Liuet al.2011),whileSbHSF5andSbHSF13are concurrently induced by drought and heat stress (Reddyet al.2016). These genes may play a role in the heat stress response by regulating downstream gene expression.

Furthermore,plants typically produce heat shock proteins (HSPs) in response to stimuli such as high temperatures. These proteins rapidly accumulate to high levels and function as molecular chaperones,influencing plant responses to heat stress by regulating metabolic reactions. For example,SbHSP90in sorghum exhibits significant expression in response to heat stress,with the highest levels observed in the Honey Graze variety,suggesting its potential utility in future breeding programs(Pavliet al.2011). Moreover,an analysis of the small heat shock protein (sHsps) gene family in sorghum reveals that theSbHsp20gene responds to both drought and heat stress,and exhibits high expression in both roots and leaves. This indicates the involvement ofSbHsp20in regulating multiple resistances in sorghum (Markaet al.2020). These genes may participate in high-temperature stress responses by regulating the expression of downstream genes.

In the context of traditional breeding,drought-tolerant sorghum varieties are often considered as potential sources of heat-tolerant germplasm. For example,SC35 is a drought-tolerant sorghum cultivar,but its pollen germination is severely reduced under heat stress,leading to lower grain yield (Sunojet al.2017). Earlier research has indicated that different genotypes may respond to various biotic stresses in different ways.Therefore,solely enhancing drought tolerance may not be sufficient to ensure the yield stability of sorghum under natural conditions,as drought frequently coincides with elevated temperatures. As a result,it is imperative to specifically target heat tolerance in sorghum,particularly during the critical flowering stage.

5.Reaction of sorghum to low-temperature stress

5.1.Physiological and biochemical reactions of cold tolerance in sorghum

As a tropical and subtropical crop,sorghum exhibits adaptability to arid and high-temperature environments.Many studies have predominantly concentrated on scrutinizing sorghum’s response to drought and heat stress conditions. Nevertheless,owing to constraints imposed by the planting season,sorghum routinely encounters low-temperature stress during its early germination and seedling growth phases.

Temperatures consistently below 15°C reduced the germination rate and seedling emergence in sorghum by 50% (Chiluwalet al.2018). Thus,15°C is considered the threshold for studying the response of sorghum seedlings to low temperatures. Low temperature mainly damages the embryos in the young coleoptiles of the germinating seeds and hampers normal photosynthesis in newly emerged seedlings,resulting in the cessation of growth or death. When the temperature dropped below 10°C,sorghums of different genotypes did not germinate normally,and seedling emergence was also halted(Chiluwalet al.2018). Reduced seedling emergence rates can translate into a lower sorghum density compared to the intended planting density,consequently diminishing crop yields. Enhancing sorghum’s cold tolerance can optimize the utilization of residual water before the dry season,mitigate the impacts of unfavorable conditions like drought and high temperatures during later growth stages,enhance planting season flexibility,and expand the planting areas.

The influence of low temperature on sorghum photosynthesis primarily stems from the unique photosynthetic characteristics of sorghum as a C4plant. The photosynthetic mechanism in C4plants is contingent on rubisco activity. When the temperature falls below 20°C,rubisco activity and capacity in C4plants significantly lag behind their C3counterparts,thus impeding photosynthesis. Concurrently,low temperatures undermine the stability of pyruvate phospho-dikinase(PPDK),further exacerbating the photosynthetic limitations (Ortizet al.2017).

Nonetheless,certain local accessions at high latitudes,which are more resistant to cold,can also germinate at low temperatures. For instance,the Chinese sorghum variety Shan qiu red (SQR) has better germination and seedling rates than other sorghum genotypes under lowtemperature conditions (Chiluwalet al.2018). Leveraging cold-tolerant germplasm to screen potential lowtemperature resistance genes presents a viable approach for cultivating cold-tolerant varieties and elucidating the regulatory mechanisms governing sorghum’s cold tolerance.

In temperate climates like Central Europe,sorghum encounters low-temperature stress during the reproductive phase. Nonetheless,most studies have predominantly emphasized low-temperature stress during the germination and seedling stages of sorghum.Low-temperature stress,both before and after flowering,detrimentally impacts pollen vigor and,ultimately,seed setting rates in sorghum. Notably,F1hybrid sorghum displays enhanced cold tolerance during the seedling phase compared to inbred sorghum (Schaffaszet al.2019). Moreover,the hybrid sorghum has more viable pollen,resulting in improved spikelet fertility and yield(Schaffaszet al.2019). Therefore,using heterosis to improve the cold resistance of sorghum is a feasible approach for cultivating cold-tolerant sorghum.

5.2.ldentification of genes related to low-temperature tolerance in sorghum

Most studies on this aspect have focused on sorghum emergence and seedling growth in low-temperature environments. For example,Moghimiet al.(2019)performed a genome-wide association analysis from hundreds of sorghum accessions with different levels of cold tolerance,and screened three regulatory genes related to cold tolerance at the seedling stage. These genes included a DnaJ/Hsp40 motif-containing protein (Sobic.007G005400),an m6A-modified binding protein on RNA containing a YTH domain (Sobic.007G005500),and an alpha/beta hydrolase domain protein ABHD (Sobic.007G037000). ABHD enhances the cold tolerance of roots and shoots under lowtemperature stress by regulating lipid synthesis. The YTH domain protein is a type of m6A-binding protein,and studies have shown the role of m6A modification in regulating plant development under various types of stress. Consequently,the YTH family protein Sobic.007G005500 may exert a regulatory influence by participating in signal transduction pathways or binding to m6A-modified transcripts associated with cold tolerance.

Furthermore,several other QTLs have been linked to low-temperature resistance in sorghum during the seedling stage,includingXtxp34,Xtxp88,Xtxp319,andQTL57on chromosome 6 (Parra-Londonoet al.2018).These QTLs are primarily related to the germination rate of seeds exposed to low-temperature conditions.Additionally,Xtxp20,Xtxp211,andXtxp304have been associated with seedling development (Maulanaet al.2017;Parra-Londonoet al.2018).

Meanwhile,certain unavoidable trade-offs may exist between the beneficial and detrimental traits in sorghum.Some sorghum varieties,such as the hardy varieties from China growing in temperate conditions,are highly coldresistant. However,the seeds of these varieties have high tannin contents,and many QTLs related to cold tolerance are associated with tannin content. Tannin in sorghum seeds is undesirable since it reduces protein digestibility.On the other hand,appropriate tannin levels might be beneficial for ruminants and reduce bird foraging and pre-harvest losses (Wuet al.2019;Xieet al.2019). In addition,sorghum germplasm from other regions has also shown cold tolerance,such as the varieties from the highaltitude areas of Africa,which can grow in environments below 10°C (Rutayisireet al.2021). Screening tanninfree sorghum germplasm from other cold regions may be a novel approach for screening new cold tolerance-related genes and QTLs (Maulanaet al.2017). Therefore,future sorghum breeding efforts must consider the trade-offs between different uses.

6.Reaction of sorghum to heavy metal stress

6.1.Physiological and biochemical reactions of heavy metal tolerance in sorghum

The escalation of heavy metal (HM) concentrations in soil,including Cd,Pb,As,Al,and Hg,is primarily attributable to anthropogenic activities such as sewage irrigation and the utilization of low-quality fertilizers. The accumulation of these heavy metals in soil,due to the limited capacity of microorganisms to break them down,leads to soil contamination by toxic metals. The ramifications of heavy metal pollution extend beyond the degradation of agricultural land quality and the impediment of crop production;they also exert deleterious effects on plant growth and pose a threat to human health through the food chain.

The toxic effects of heavy metals on plants are reflected in their morphology,physiology,biochemistry,and molecular genetics. Heavy metals affect normal plant growth and development,damage root cell structure,reduce both aboveground and underground biomass,and severely affect crop yields. The competitive binding of HM with the reducing proteins (containing -SH groups)that are absorbed by plant roots affects normal redox reactions,increases ROS levels in the plant cells,and causes oxidative stress reactions and damage to the plants. It also interferes with enzyme-catalyzed reactions,photosynthesis,and other normal biochemical metabolic processes. Heavy metals that enter the cell nucleus can cause mutagenic damage,such as DNA double-strand breaks.

Sorghum,distinguished by its substantial biomass,efficient heavy metal enrichment and transport capabilities,as well as its notable tolerance,emerges as a promising candidate for phytoremediation. It can be extensively cultivated in heavily contaminated land,harvested before reaching the flowering stage,and serve the dual purpose of contributing to bioethanol production while remediating the soil. Sweet sorghum exhibits a remarkable capacity to absorb Hg2+,Cd2+,Mn2+,Zn2+,and Cu2+from the soil. When compared to common crops such as corn and wheat,it demonstrates higher resilience to heavy metal-induced stress (Fig.4) (Zhuanget al.2009;Metwaliet al.2013;Jiet al.2014). Furthermore,sweet sorghum can accumulate a significant quantity of metal ions while maintaining a higher biomass. For instance,an analysis of data from the harvesting period of two sorghum varieties,H18,known for its high Cd2+accumulation,and L69,a low Cd2+accumulator,in Cd2+-contaminated land revealed that the Cd2+contents in the stems and roots of H18 significantly exceeded those in L69. However,H18 managed to maintain an equivalent fresh weight to L69 (Jiaet al.2021).

The robust tolerance of sorghum to heavy metals hinges on its capacity to absorb and transport metal ions,as well as a series of detoxification mechanisms. In response to heavy metal toxicity,the root epidermal cells of sorghum release various chemicals,such as malic acid and citric acid,which form complexes with metal ions in the soil,thereby facilitating their excretion or adsorption onto the cell wall,and reducing metal ion uptake (Ghori 2019).

Fig. 4 The absorption,transport,and detoxification mechanisms of heavy metals (HM) in sorghum. Toxic metals in the soil can form complexes with chemical substances released by the roots,such as citric acid and malic acid,or become immobilized in the cell wall,thereby reducing metal uptake by the roots. The entry of heavy metal ions into the cell relies on the proton motive force provided by H+/ATPase. Metals that enter root cells can be actively extruded to the apoplast or transferred to other tissues through various metal transport proteins,including ZIP,HM-ATPase,NRAMP,cation diffusion facilitator (CDF),and cation exchanger (CAX).Metal ions in the cytoplasm form metal chelates with organic acids,amino acids,and phytochelatins predominantly composed of glutathione (GSH),which are sequestered into vacuoles to reduce metal toxicity.

However,when the concentration of metal ions in the soil surpasses a certain threshold,the adsorption and chelation compounds on the cell wall prove insufficient to prevent metal ion ingress. In such instances,metal ions penetrate the root epidermal cells through specific metal transport proteins,initiating a sequence of detoxification mechanisms. These mechanisms include the formation of chelates with amino acids,organic acids,phytochelatins,metallothioneins,and others,sequestering the metal ions within vacuoles for detoxification. Furthermore,the cells synthesize a substantial quantity of antioxidant enzymes and reductases,such as glutathione reductase,to mitigate the toxicity of the metal ions. The plants can also activate other stress-related response mechanisms,including plant hormones and signal molecules,to combat metal toxicity (Mishraet al.2021).

Once metal ions or metal chelates are taken up by root epidermal cells,they are transported to the endodermisviasymplastic and apoplastic routes. Within the endodermis,the Casparian strip forms a barrier that prevents the further transport of metal ions. However,certain metal ions such as Cd2+can bypass the weak points in the barrier or utilize metal transport proteins (e.g.,Cr2+and Ni2+) to enter the xylem and be transported to the aerial parts of the plant (Fenget al.2018). In summary,sorghum roots effectively take up metal ions through different transport proteins and sequester toxic metals in the form of metal complexes within vacuoles or immobilize them within cell walls. Furthermore,they alleviate metal toxicity by generating substantial quantities of antioxidant and reducing enzymes.

6.2.ldentification of genes related to heavy metal tolerance in sorghum

Transcriptome analysis has revealed that sorghum exhibits elevated expression levels of genes associated with the synthesis of metallothioneins and plant chelators when exposed to Cd2+stress. When absorbed by the roots,the Cd2+ions form complexes with these substances and accumulate in the root tissue,and they are subsequently mitigated by the action of glutathione reductase. Meanwhile,the activity of catalase is enhanced,reducing the oxidative damage caused by Cd2+. In high-Cd2+-accumulating sorghum H18,the root cells efficiently absorb Cd through different ZIP metal transport proteins,and the Cd2+with a weak binding affinity for the root cell wall can pass through the weak outer layer of the root endodermis and enter the xylem to be transported to the aboveground parts,since the xylem has a high loading capacity for Cd2+ions (Fenget al.2018)

Within the Zn-regulated transporters,the iron-regulated transporter-like protein (ZIP) transporters participate in the transmembrane transport of various metal cations such as Mn2+,Co2+,Cu2+,and Cd2+. Among the ZIP family members,ZIP1 and ZIP3 are mainly present in the roots of sorghum,maintaining a steady state under Zn stress (Ghori 2019). ZIP3 can also respond to Cd stress and upregulate its expression (Jiaet al.2021).Metal transporters play important roles in maintaining a steady state under heavy metal stress and increasing the tolerance to heavy metal stress in sorghum. In addition to the ZIP transporters,heavy metal P1B-type ATPases(HMA) use the energy of ATP hydrolysis to transport metal ions across membranes and play important roles in the transport of Cd2+,Cu2+,Pb2+,and Zn2+(Hussainet al.2004). Through a comprehensive analysis of the expression patterns of 11 P1B-type ATPase genes related to heavy metals in sorghum,Eet al.(2018) identified significant upregulation ofSbHMA3a(Sb02g006940)after treatment with 500 μmol L–1CuSO4,whileSbHMA8(Sb01g045340) exhibited high expression levels in response to both 500 μmol L–1CuSO4and CdCl2treatments. These findings suggest that these genes play a crucial role in enhancing sorghum’s tolerance to heavy metal stress. Furthermore,the expression of P1B-type ATPase transporters is regulated by ABA signaling under heavy metal stress and is further induced by ABI4 (Darabiet al.2017).

Natural resistance-associated macrophage protein(NRAMP) plays a role in transporting metal ions in almost all organisms. In plants,NRAMP proteins are mainly expressed in the plasma membrane or vacuolar membrane of the root and stem cells,and participate in the transport of various metal ions such as Cd2+,Zn2+,Co2+,Cu2+,and Pb2+(Thomineet al.2000). The sorghum NRAMP protein-related geneSbNrat1is involved in the absorption and transport of aluminum in sorghum roots and shoots,and is associated with enhancing sorghum’s tolerance to Al (Thomineet al.2000;Luet al.2017). In addition,under Al stress,theSbMATEencoding citrate transporters in the roots are activated,inducing the formation of Al-citrate complexes,which are isolated in the root vacuoles and play a major role in sorghum’s Al tolerance (Magalhaeset al.2007;Meloet al.2019). Moreover,the β-1,3-glucanase geneSbGlu1(Sb03g045630.1) can improve sorghum’s Al tolerance by affecting the degradation of calluses in sorghum roots(Zhanget al.2015).

At the post-transcriptional level,microRNAs (miRNAs)also participate in regulating sorghum’s tolerance to heavy metal stress (Jiaet al.2021). Jiaet al.(2021)identified 61 miRNAs involved in establishing sorghum’s Cd2+tolerance. They participate in the synthesis of metal transporters in Cd2+-accumulating sorghum and affect the accumulation of Cd2+,as well as participating in the clearance of reactive oxygen species,thereby enhancing sorghum’s tolerance to Cd2+(Jiaet al.2021). Twentyfive of these miRNAs participate in the restructuring of the root cell wall structure,leading to changes in cell wall components such as cellulose,hemicellulose,and pectin,while the root endodermal cells of Cd2+-accumulating sorghum also undergo changes,indicating that changes in root cell structure are related to sorghum’s Cd2+tolerance.

7.Regulation and responses of sorghum to complex abiotic stress

In contrast to common crops like wheat and maize,sorghum is known for its robust stress resistance,and it is often cultivated worldwide under adverse abiotic conditions such as saline–alkali soils,drought,and extreme climates. Therefore,in future research on sorghum resistance,the establishment of rapid and effective resistance evaluation methods and screening of sorghum varieties with excellent resistance to stress should be carried out. These steps are very important for the breeding and application of sorghum. In natural settings,plants routinely encounter a myriad of abiotic stress factors. For instance,high-temperature stress in tropical and subtropical regions often coincides with drought stress. Consequently,the prevailing plant breeding approaches tailored to individual stress conditions fall short of addressing these multidimensional issues. The QTL analysis of plants under drought and high-temperature stress has indicated that certain cultivars with drought stress resistance might be sensitive to high temperatures. Thus,it may be necessary to breed for a combination of stress conditions in the future to improve yield stability.

In semi-arid regions like Africa,drought frequently occurs in tandem with elevated temperatures.Concurrently,elevated temperatures intensify leaf transpiration rates,compounding the detrimental effects of drought stress. The combined impact of drought and heat stress on sorghum growth and development surpasses that of the individual stressors (Chaves and Oliveira 2004). During seed germination and seedling emergence,increased soil temperatures resulting from the elevated heat exacerbate the inhibitory effect of drought stress on germination rates,leading to reduced germination or seed dormancy. In the seedling stage,both drought and heat stress result in reduced stem elongation. In the reproductive growth stage,drought and heat stress reduce partitioning vigor and grain filling,and ultimately lead to reduced sorghum yield. However,sorghum is a highly stress-tolerant crop with a unique EMF mechanism that minimizes the damage to pollen viability caused by high temperatures compared to other crops (Jagadish 2020).Furthermore,several greenness-related QTLs identified in sorghum exhibit high correlations under drought and heat stress conditions,and they facilitate the maintenance of photosynthesis in sorghum under drought and heat stress(Sukumaranet al.2016;Awikaet al.2017).

High salt stress concurrently induces ionic and osmotic stresses in plants,while erratic rainfall patterns subject sorghum to drought stress throughout its developmental stages,thereby compounding the damage caused by salt stress. Presently,numerous critical QTLs and genes have been pinpointed in sorghum,and they govern its response to both salt and drought stress. ABA,as the most important non-biological stress-responsive hormone,rapidly accumulates under salt and drought stress. Increased levels of ABA simultaneously regulate multiple pathways involved in the salt and drought stress responses,enhancing sorghum’s resistance under these conditions. Additionally,both salt and drought stress induce osmotic stress in plants. Proline,as an osmoprotectant,plays a crucial role under salt and drought stress conditions,and the proline-regulating genesSbP5CS1andSbP5CS2in sorghum respond to both salt and drought stress (Suet al.2011). Aquaporins,which regulate osmotic balance,also play a significant role in controlling the water status under osmotic and salt stress. In sorghum,both salt stress and PEGinduced drought stress alter the expression of aquaporinregulating genes (Yanget al.2018;Hostetleret al.2021).Furthermore,several gene families,such as nuclear factor Y (NF-Y) and the trihelix genes,exhibit responses to multiple non-biological stressors,including salt,mannitol,high and low temperatures,and others (Kumariet al.2018;Maheshwariet al.2019;Li Het al.2021;Kumaret al.2022).

Given the complexity of the practical production environment,identifying the genes that exhibit multiple responses to varying types of non-biological stress is challenging. Woldesemayatet al.(2018) have devised an integrated assessment method to identify complex trait QTLs and regulatory genes related to multiple stressors in sorghum. This method amalgamates diverse expression profile datasets and deploys multi-omics analysis to identify regulatory genes under multifaceted conditions.Among these genes,50% of the drought stressregulated genes also respond to multiple stress types,such as salt,cold,heat,and oxidative stress. Some genes concurrently govern two or more stress types,underscoring the possibility that certain stress-responsive genes in sorghum may mediate cross-talk among the various stress types through shared regulatory pathways or metabolic processes.

8.Germplasm of sorghum with tolerance to different abiotic stresses

Currently,limited research exists on the breeding of sorghum for stress resistance,with most research primarily focusing on the screening of sorghum germplasm resistant to individual stressors. However,it is imperative to investigate sorghum stress resistance breeding,given sorghum’s remarkable stress tolerance which gives it great significance for addressing extreme environmental conditions and marginal soil utilization. Here we discuss the currently reported germplasm resources of sorghum with tolerance to different abiotic stresses (Fig.5).

Salt tolerance in different salt-tolerant sorghum varieties exhibits variation across distinct developmental stages. The germination stage,marking the onset of the crop growth period,is crucial for achieving high sorghum yields in saline-alkali soils. Kausaret al.(2011) found that Sandalbar and JS-2002 always maintained the longest root length under 0–200 mmol L–1NaCl treatments,and still had a higher germination rate after a 200 mmol L–1NaCl treatment. A screening by Penget al.(2022)found that 09305R,363C,67B,and 07221 had higher germination potential and percentages under salt stress.Dinget al.(2018) showed that Chinese sorghum Jitianza 7 was the most salt-tolerant variety at the germination stage. High germination rates in sweet sorghum germplasm under salt stress significantly impact seedling emergence and overall yield,so these salt-tolerant sorghum germplasms during the germination stage hold promise as foundational resources for developing salttolerant sorghum throughout its entire growth cycle.

Fig. 5 Germplasm resources of sorghum tolerant to different abiotic stresses. The sorghum genotypes with different abiotic stress tolerances were screened in the laboratory or field according to the results of different physiological parameters of stress tolerance.

The seed setting rate has been used as a screening index for the identification of high temperature tolerant sorghum germplasm. For example,Singhet al.(2015)found that PI609489,Tx623,AQL33/QL36,CCH2,IS8525,and R9403463-2-1 can maintain high seed setting rates under the threshold temperature of 38°C. Among them,IS8525 from Ethiopia and PI609489 from Mali also have the characteristics of a low stalk and lodging resistance,so these varieties can maintain a higher yields in the future warmer climate.

The germination stage is particularly sensitive to low temperatures throughout the sorghum growth period,with the relative germination rate serving as a pivotal screening index. Noteworthy varieties such as Zhongshuhui 1,GPR148,IS-160,re sown sorghum and PI610727 with seed germination in low temperature environments showed strong cold tolerance,so they can be planted in cold areas to reduce the risk of planting in early spring (Fernandezet al.2014). Given that most cold-tolerant sorghum germplasm at the germination stage originates in China,Chinese sorghum germplasm presents a valuable resource for screening cold-tolerant sorghum and identifying the beneficial genes related to low-temperature tolerance.

Currently,excellent sorghum varieties have been screened for extracting heavy metals from contaminated soil. For example,the sorghum variety Keller has strong accumulation abilities for Zn2+,Cu2+,and Cd2+in heavily polluted soil (Zhuanget al.2009),and the variety Roma has a high tolerance to Al (Zhanget al.2015).Muhammadet al.(2020) demonstrated that the sorghum variety JS-2002 has an efficient antioxidant defense system and can tolerate high concentrations of Cd.Tsuboiet al.(2016) analyzed the QTLs related to Cd2+accumulation in the aboveground parts of sorghum and selected two sorghum varieties,SDRS47 and SDRS48,with strong Cd2+absorption and transport capabilities.Furthermore,the pH value and organic matter of the rhizosphere soil also affect the absorption of metal ions by root cells (Adamczyk-Szabelaet al.2015). The utilization of ammonium fertilizers (containing ammonium nitrate or ammonium sulfate) can increase the accumulation of Zn2+and Cd2+in sorghum by changing the pH of the soil(Zhuanget al.2009). A comprehensive understanding of the heavy metal accumulation mechanisms in sorghum and its adaptation to heavy metal stress,and the development of sorghum varieties with heightened tolerance to heavy metal stress are instrumental in advancing sustainable soil remediation and energy production.

Global sorghum germplasm resources are abundant and offer valuable genetic diversity for developing varieties that are tailored to diverse conditions. Leveraging highthroughput sequencing data has emerged as a practical approach for elucidating the molecular mechanisms underpinning stress resistance (Xinet al.2021;Silvaet al.2022). Many sorghum-specific high-throughput omics databases have been established,including the gene expression databases MOROKOSHI (Makitaet al.2015) and CATchUP (Nakamuraet al.2017),the sorghum genomics database SorghumFDB (Tianet al.2016),the sorghum genome SNP database (Luoet al.2016),and the sorghum cultivated and wild germplasm pan-genome database (Taoet al.2021). In Table 3,we provide detailed information on these different types of high-throughput sorghum databases. Some sorghum resistance genes have been identified by mining the highthroughput genomic data and expression profiles (Liet al.2019;Zhouet al.2022). Therefore,by establishing and integrating sorghum-specific high-throughput data,phenotype data,gene expression data,and phenotype databases,data mining in sorghum can be carried out,especially for specific resistance genes and their regulatory mechanisms (Luoet al.2016;Taoet al.2021;Wu Xet al.2022).

Table 3 Sorghum specific high-throughput omics databases

9.Summary

Sorghum is a versatile C4crop with a high photosynthetic efficiency that holds significant promise as a cereal,energy source,and silage crop. Nevertheless,it is imperative to address several specific issues in sorghum research.For instance,the current research on grain sorghum is focused on improving yield under ideal conditions rather than yield stability under more realistic stress conditions.In addition,the current directions of sorghum breeding mainly focus on yield,dwarfing,reducing tannin content and increasing the sugar content of energy sorghum.Regrettably,scant attention has been directed towards sorghum stress tolerance breeding and specialized stress tolerance breeding within the realm of sorghum.

Due to the challenges involved in constructing a transformation system,research on sorghum growth and stress regulation often lags behind other crops. A few indepth analyses have focused on the mining,utilization,and regulatory functions of key genes associated withabiotic stress in sorghum. Although some studies have mined resistance genes by identifying the homologous genes in close species,they are not completely accurate(Menget al.2021). For example,maize and sorghum are closely related,but some orthologous genes show different response patterns under the same abiotic stress conditions.

Furthermore,given sorghum’s inherently high stress resistance,unveiling its molecular mechanisms can potentially pave the way for enhancing stress resistance in other major crops such as rice and wheat.

Several researchers have explored sorghum’s resistance genes with the aim of bolstering stress resistance in other plants. For instance,the overexpression of sorghum genesSbMPK14andSbER2-1in maize has demonstrated augmentations in net photosynthetic rate and drought tolerance (Liet al.2019;Zhouet al.2022). TheSbSKIP-overexpressing tobacco also exhibited high drought tolerance. Zhanget al.(2023)recently identified and cloned a major locus associated with alkaline tolerance in sorghum (SbAT1) using a genome-wide association analysis. Sorghum plants with a modifiedSbAT1gene showed 20–30% increases in both yield and biomass. Moreover,the AT1/GS3 allele of this gene improved the yield and biomass in rice,maize,and foxtail millet grown in saline–alkali soils. These findings imply that employing sorghum in breeding and resistance gene exploration can offer valuable insights into augmenting the stress resistance and yield stability of other crops under environmental stress.

The integration of current bioinformatics resources and a comprehensive assessment approach to screen for non-biological stress-responsive genes under complex stress conditions is particularly noteworthy. Such an approach promises to be beneficial for the selection and breeding of sorghum varieties with enhanced yield stability (Woldesemayatet al.2018;Kazemiet al.2023).Moreover,as numerous resistance genes in plants exhibit conservation across diverse species,the resistance genes and molecular markers identified in sorghum can serve as benchmarks for the investigation of potential resistance genes in other crops. Consequently,this approach will facilitate the selection and enhancement of crop productivity under complex stress conditions.

Acknowledgements

We are grateful for financial support from the National Key R&D Program of China (2022YFD1201702),the National Natural Science Foundation of China (32272040),and the Agricultural Fine Seed Project of Shandong Province,China (2021LZGC006).

Declaration of competing interest

The authors declare that they have no conflict of interest.