XlAO Qian-lin,HUANG Tian-hui,ZHOU Chang,CHEN Wei-xi,CHA Jian-kui,WEl Xi-mei,XlNG Fang-yu,QlAN Meng-ya,MA Qian-nan,DUAN Hong,LlU Zhi-zhai

College of Agronomy and Biotechnology,Southwest University,Chongqing 400715,P.R.China

Abstract Sucrose nonfermenting-related protein kinase 1 (SnRK1) is one of the critical serine/threonine protein kinases.It commonly mediates plant growth and development,cross-talks with metabolism processes and physiological responses to biotic or abiotic stresses.It plays a key role in distributing carbohydrates and sugar signal transporting.In the present study,eight SnRK1 coding genes were identified in sorghum (Sorghum bicolor L.) via sequences alignment,with three for α subunits (SnRK1α1 to SnRK1α3),three for β (SnRK1β1 to SnRK1β3),and one for both γ (SnRK1γ) and βγ (SnRK1βγ).These eight corresponding genes located on five chromosomes (Chr) of Chr1-3,Chr7,and Chr9 and presented collinearities to SnRK1s from maize and rice,exhibiting highly conserved domains within the same subunits from the three kinds of cereals.Expression results via qRT-PCR showed that different coding genes of SnRK1s in sorghum possessed similar expression patterns except for SnRK1α3 with a low expression level in grains and SnRK1β2 with a relatively high expression level in inflorescences.Results of subcellular localization in sorghum leaf protoplast showed that SnRK1α1/α2/α3/γ mainly located on organelles,while the rest four of SnRK1β1/β2/β3/βγ located on both membranes and some organelles.Besides,three combinations were discovered among eight SnRK1 subunits in sorghum through yeast two hybrid,including α1-β2-βγ,α2-β3-γ,and α3-β3-γ.These results provide informative references for the following functional dissection of SnRK1 subunits in sorghum.

Keywords: sorghum (Sorghum bicolor L.),SnRK1,expression analysis,combination pattern

1.lntroduction

SnRK1 (sucrose nonfermenting related protein kinase 1)plays important roles in balancing vivo metabolism and sugar signal transporting in plants,presenting as the orthologous proteins of SNF1 (sucrose non-fermenting-1)from yeast and AMPK (AMP-activated protein kinase) from mammal (Emanuelleet al.2016).As a protein kinase,SnRK1 cross-talks with different signal pathways mainly through the phosphorylating or physiological processes(Nietzscheet al.2016;Muralidharaet al.2021).The firstSnRK1in plants was isolated from rye endosperm (SecalecerealeL.) (Aldersonet al.1991),followed paralogs were identified fromArabidopsis thalianaand other plants,and the dissected function of these SnRK1s was continuously documented (Takanoet al.1998;Filipeet al.2018).The firstSnRK1in sorghum (Sorghum bicolorL.),SnRK1b,was identified to be associated with endosperm development (Jainet al.2010).In addition,some genes belonging to theSnRKfamily were also identified from the sorghum genome (Liet al.2010).A further document dissected that bothSnRK2andSnRK3in sorghum were downregulated by osmotic stress of PEG,and abiotic stresses of salt,cold,abscisic acid (ABA),and heat,exhibiting diverse functions responding to these unfavorable conditions (Dalal and Inupakutika 2014)

Plant SnRK1 exhibits as a heterotrimer consisting of three typical subunits of α,β,γ,and an atypical subunit of βγ (Emanuelleet al.2016).α contains a conserved N-terminal,presenting as the catalyzing domain of SnRK1 kinase.β presents a conserved C-terminal and forms a conserved C-terminal domain (CTD),containing a conserved carbohydrate-binding module (CBM) (Ruiz-Gayossoet al.2018).γ is mainly formed by four tandem cystathionine β-synthase (CBS),while βγ subunit contains both the CBM domain that belongs to the β-type subunit and four CBSs to the γ-type subunit at the N-terminal,forming a distinctive subunit structure of SnRK1 in plants(Emanuelleet al.2016).

α subunit in plants was encoded by different genes,such asSnRK1α1/2/3inArabidopsisandSnRK1aandSnRK1bin cereals (Baena-Gonzálezet al.2007).β and γ serve as important regulators,playing essential roles in maintaining the scaffold of heterotrimer,enzyme activity,and specific recognition of substrates(Emanuelleet al.2016).It was reported that β was encoded by three genes,i.e.,AtSnRK1β1/2/3,while γ by the atypical gene ofAtSnRK1βγinArabidopsis(Lionelet al.2004).In cereals,SnRK1ais expressed in most tissues,whileSkRK1bis only highly expressed in seeds(Avila-Casta?edaet al.2014).Similar trends were also observed among subunits of β,γ,and βγ (Avila-Casta?edaet al.2014).Differentiated expression patterns of subunits ofSnRK1suggested complicated profiles of subunit combinations and diverse biological functions ofSnRK1(Emanuelleet al.2016).

The controllingviadiversified expression patterns ofSnRK1can function on different physiological pathways,including suppressing starch accumulation in barley (Zhanget al.2001),increasing starch content in potato,and controlling the starch quality,and even balancing nitrogen uptake and carbon assimilation in sweet potato (McKibbinet al.2010;Renet al.2018,2019).SnRK1 can also function on some special physiological pathways to mediate the phosphorylation of target substrates (Nietzscheet al.2016),or cross-talks with the transcription factors (TFs,i.e.,bZIP63,SOG1,and PTL of the NAC TF family) to control the transcription of downstream genes (Emanuelleet al.2016;Muralidharaet al.2021).In addition,the expression ofSnRK1could be induced by low temperature,drought,water logging,salt stress,and pathogens;it then responds to such biotic or abiotic stressesviacorresponding changes in expression patterns (Baena-González and Sheen 2008).

Sorghum is a worldwide important source of food,animal feed,and biofuel (Sakschewskiet al.2014).Though sorghum is more tolerable to environmental stresses than some other crops,the changing global climates still challenge its final performances of production and quality(Taoet al.2021).As SnRK1s play important roles in responding to both biotic and abiotic stresses in plants,the characterization ofSnRK1genes in sorghum might also provide potential guidance for sorghum varieties improvements.The present study isolated eight subunitcoding genes ofSnRK1in sorghum via sequence alignments.It characterized the eight sorghumSnRK1s,including gene expression patterns and functional characters of coding proteins,and dissected the subunit combinations.The results will provide informative references for the further functional profiling of sorghumSnRK1.

2.Materials and methods

2.1.Plant materials

Sorghum cultivar,BTx623,provided by Rice and Sorghum Institute,Sichuan Academy of Agricultural Sciences(Luzhou,Sichuan,China),was planted on a college farm(College of Agronomy and Biotechnology,Southwest University,Chongqing,China).All the samples used for gene cloning and expression pattern analysis were collected at jointing,flowering,and maturity stages.

2.2.Genome-wide screening and sequence alignment of SnRK1 in sorghum

Results fromArabidopsis,rice,and maize indicated that theSnRK1genes from these plant species possessed highly conserved sequences and coding domains,and a total of 30 coding genes for different subunits of SnRK1 were found,including seven inArabidopsis(Boulyet al.1999;Lionelet al.2004;Fragosoet al.2009),eight in rice (Takanoet al.1998;Luet al.2007),and 15 in maize (Schnableet al.2009).All these SnRK1 protein sequences were used as queries to search the sorghum genomeviaGramene (http://www.gramene.org/) and NCBI (https://www.ncbi.nlm.nih.gov/) through BLASTP.The phylogenic analysis of sorghumSnRK1s (SbSnRK1s)was carried outviaMEGA 5.10 (Tamuraet al.2011),while the synteny analysis ofSnRK1s from sorghum,rice,and maize was carried outviaMCScanX (Wanget al.2012).

2.3.Extraction of RNA and expression pattern dissection of SbSnRK1

RNA was extracted from tissues of seeding roots,leaves at the 5-developed-leaf stage (leaf-FLS),elongating stage (leaf-ES),and heading stage (leaf-HS),stem at the elongating stage (stem-ES) and heading stage (stem-HS),and developing seeds at 5,10,15,20,and 25 DAP (days after pollination).The corresponding cDNA was collected for quantitative real-time PCR (qRT-PCR) profiling with internal control of sorghumEiF4α.The relative transcription levels were calculatedviathe method of 2-ΔΔCt.

2.4.Preparation of sorghum leaf protoplasts and analysis of subcellular localization

The sub-cellular localization of SbSnRK1 subunits was confirmed by transient expression of a fusion construct containing eGFP in the protoplast of sorghum leaf.The pCAMBIA2300-35S-eGFP was used as the backbone vector,and the ClonExpress II One Step Cloning Kit(Vazyme,Nanjing,China) was used for the construction of pCAMBIA2300-35S-Gene-eGFP.All recombined genes were verified by both monoclonal bacterial fluid test and vector sequencing.All transferred protoplasts were incubated in a culture medium for 16 to 24 h at dark conditions,and then the eGFP were detectedviafluorescence microscope (ECLIPSE 80i).

2.5.lnteraction dissecting of SnRK1 subunits

Protein-protein interactions among the SnRK1 subunits were revealed by the yeast two-hybrid (Y2H) system of GAL4.All SnRK1 subunits were subcloned into the yeast expression vectors of pGBKT7 and pGADT7 vectors,and all constructed vectors were verified through the PCRbased colony test and vector sequencing.Yeast strain of AH109 was used as a receptor,and the competent cells were preparedviathe PEG-LiAc method.The salmon essence was used to carry the plasmids.

3.Results

3.1.Characterization of SbSnRK1s

A total of eightSbSnRK1s were identified to locate on five chromosomes (Chrs)viasequence alignment (Table 1).All these eightSbSnRK1s exhibited diverse length ranges of both cDNA and coding products (Table 1).Results of synteny analysis showed that 26 orthologous gene pairs were identified among rice,maize,and sorghum,including α,β,γ,and βγ subunits (Fig.1-A).In addition to the sorghum γ subunit coding gene,only collinear genes were found in maize,and all other remaining subunit coding genes were found orthologs in rice and maize.The phylogenetic results indicated that eight SbSnRK1s were divided into four different clusters.Cluster I contained all theαsubunits of SnRK1 fromArabidopsis,rice,maize,and sorghum.Cluster II corresponded toβ3andγfrom rice,maize,sorghum,andβfromArabidopsis.Cluster III consisted ofβ1 andβ2 from three types of cereal,while allβγwere gathered in Cluster IV (Fig.1-B).According to the phylogenetic results,the eight SbSnRK1s were named as SbSnRK1α1/α2/α3,SbSnRK1β1/β2/β3,SbSnRK1γ,and SbSnRK1βγ (Table 1;Fig.1-B).

Fig.1 Collinearity and phylogenetic analysis of SnRK1s.A,collinearity analysis of SnRK1s in rice,maize,and sorghum.Sb,Zm,and Os refer to Sorghum bicolor L.,Zea mays L.,and Oryza sativa L.,respectively.B,evolutionary analysis of SnRK1s in Arabidopsis thaliana,rice,maize,and sorghum.At,Sb,Zm,and Os correspondingly refer to A. thaliana,S. bicolor L.,Z. mays L.,and O. sativa L.

Table 1 Summary information of SnRK1s in sorghum

3.2.Subcellular localization of SbSnRK1s

Subcellular localization resultsviasorghum leaf protoplasts showed that the GFP signals of empty vector,pCAMBIA2300-35S-eGFP without any SbSnRK1s could be detected at both cell membrane and organelle(Fig.2-A).While the inflorescent signals only were detected at organelles when the vector carriedsubunits of SbSnRK1α1,SbSnRKα2,SbSnRKα3,and SbSnRK1γ.For those confused vectors carried subunits of SbSnRK1β1,SbSnRK1β2,SbSnRK1β3,and SbSnRK1βγ,in addition to organelles,green inflorescent signals were also detected on cell membranes,similar to that observed via the empty vector (Fig.2-A).

3.3.Dissection of expression pattern of SbSnRK1s

Relative expression resultsviaqRT-PCR showed that allSbSnRK1s expressed in all 12 assayed tissues of root,leaf,stem,inflorescence,and grains of sorghum,exhibiting non-distinctive expression patterns (Fig.2-B).Among eight coding genes,SbSnRK1α3presented relatively lower expression levels in grains,whileSbSnRK1β2exhibited the highest levels of inflorescence than the other genes (Fig.2-B).Besides,allSbSnRK1s possessed relatively higher expression levels in stem tissues at the elongation stage (Fig.2-B).

3.4.Dissection of interaction patterns of SbSn-RK1s

Y2H results also indicated different and complex interaction patterns among the eight SbSnRK1s (Fig.2-C).Among three α-type subunits of SbSnRK1,SbSnRK1α1 interacted with β2 and βγ subunits in yeast;SbSnRK1α2 interacted with both β3 and γ;SbSnRK1α3 only interacted with SbSnRK1β3 (Fig.2-C).Relatively simple interactions were detected among three β-type subunits and SbSnRK1βγ: SbSnRK1β1 and SbSnRK1β3 could interact with SbSnRK1γ,while SbSnRK1β2 only interacted with SbSnRK1βγ (Fig.2-C).

4.Discussion

SnRK1 is a vital kinase in regulating sugar metabolism and sugar signal transduction in plants (Smeekenset al.2010).In plant species,SnRK1s also respond to development processes,hormonal,energy metabolism and transformation,quality performance,and stress conditions (Hulsmanset al.2016).The present study identified eightSbSnRK1s through the documented references,and the corresponding products of SbSnRK1s presented similarly to those reported orthologs.InArabidopsis,both three α-and β-type subunits were reported,similar to those in rice and sorghum in the present study (Crozetet al.2014;Emanuelleet al.2015).Besides these two major types of subunits,only one βγ subunit coding gene was discovered in bothArabidopsis thalianaand rice,like what the present study identified;five were reported in maize,suggesting more complex statuses of SnRK1 proteins in different plant species.

It is known that the functions and interactions are usually associated tightly with the diverse expression patterns of target genes (Xiaoet al.2021).While for SnRK1s in plant species,no tissue-specific expressed patterns were documented for all subunits coding genes(Luet al.2007).For example,inArabidopsis,SnRK1a3presented low expression levels on both pollen and seeds (Reviewed by Margalhaet al.2019),two typicalSnRK1genes ofSnRK1a(orBKIN2) andSnRK1b(orBKIN12) mainly expressed in the seeds of barley (Zhanget al.2001).In maize,Wanget al.(2019) also observed expression signals ofZmSnRK1s in almost all 13 assayed reproductive and vegetative tissues.Similar trends were also discovered among eightSbSnrk1s in the present study (Fig.2-B),implying the interactions ofSbSnRK1s in the biological development progresses and energy metabolism and transformation in sorghum.

Besides,three different subunit combinations were revealed in sorghum in the present study,and such complexes of SnRK1s were also documented by previous reports (López-Pazet al.2009).The localization trends of SbSnRK1s are quite different from those reported in the other plants.It was reported that in maize,ZmSnRK1.1/1.2/1.3 located in both tissues of nucleus and cytoplasm,while in barley,HvSnRK1.2 and HvSnKR1.4 localized to chloroplast and mitochondrion,respectively (Wanget al.2019;Chenet al.2021).Subcellular localization usually links with the function of the corresponding genes.

Documented results ofSnRK1s in plants suggest diverse functions associated with growth and stress responses,such as leaf senescence in Arabidopsis,and infertility in barley (Zhanget al.2001;Wanget al.2019).Besides,SnRK1 proteins in wheat could enhanceFusariumtoxin tolerance,while overexpression ofOsSnRK1ain rice was observed to increase disease resistance with a wide range (Filipeet al.2018;Perochonet al.2019).

In addition to the independent functional profile,some most recent publications reviewed the increasing discovery of complex cross-talks of SnRK1s with other pathways,i.e.,TOR (target of rapamycin) and Tre6P(trehalose 6-phosphate),in plants (Margalhaet al.2019;Baena-González and Lunn 2020).In plant species,SnRK1 and TOR might serve as the major regulators to balance defense and growthviaboth mutual inhibition and regulating down processes independently (Margalhaet al.2019).Besides,Tre6P in plant tissues can bind to theαsubunit,inhibiting the activation activity of SnRK1 in plants,or serve as an unidentified protein factor to suppress the SnRK1 complex in developing tissues of plants (Baena-González and Lunn 2020).

The coming thorough profiling of plantSnRK1independently or interacting with other pathways contributes to well understanding plant growth and development under unfavorable conditions.In sorghum,the genome-wide identification and characterization ofSbSnRK1s could provide informative references for the following functional profiling of these genes and then serve as potential candidates for biotic or abiotic enhancement of sorghum germplasm.

5.Conclusion

A total of eightSbSnRK1s were identified in the present study,coding three α-type,three β-type,one γ,and one βγ subunits,with corresponding syntenic orthologs in both rice and maize.Among the eight genes,SnRK1α3exhibited low levels in grains,SnRK1β2showed high levels in inflorescences,while the other sixSbSnRK1s shared moderate and similar expression patterns among all assayed tissues in sorghum.All SbSnRK1s located on both organelles and cell membranes and formed three major complexes of α1-β2-βγ,α2-β3-γ,and α3-β3-γ.The identification and characterization ofSbSnRK1s in the present study provide informative references for the following functional dissections of these genes in sorghum.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (32001607) and the Fundamental Research Funds for the Central Universities of Southwest University,China (SWU118087).

Declaration of competing interest

The authors declare that they have no conflict of interest.