Skip to main content
SpringerLink
Log in

Molecular characterization of allelic variation in spontaneous brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench)

  • Published:
Molecular Breeding Aims and scope Submit manuscript

Abstract

Modification of lignin composition and content are important to enhance the saccharification potential of lignocellulosic biomass. Brown midrib (bmr) mutants with altered lignin and enhanced glucose yields are a valuable resource for modification of the lignin biosynthetic pathway in sorghum (Sorghum bicolor (L.) Moench). Of the 38 bmr mutants reported in sorghum, some have been classified into four independent groups, namely bmr2, bmr6, bmr12 and bmr19, based on the allelic test, and a few have been characterized at the molecular level. The bmr2, bmr6 and bmr12 groups have mutations that impair 4-coumarate:coenzyme A ligase (4CL), cinnamyl alcohol dehydrogenase (CAD2) and caffeic O-methyltransferase (COMT), respectively. The molecular basis of bmr19 is unknown. In the present study, four spontaneous bmr mutants of sorghum were analyzed for allelic variation at two candidate gene loci. cDNAs of CAD2 and COMT genes were cloned and sequenced from these mutants. Sequence analysis revealed that two of these mutants, IS23789 and IS23253, share a new allele of CAD2. These mutants have a G-to-C transversion at position 3699 of the genomic sequence that leads to glycine-to-arginine (G191R) substitution in the CAD2 protein sequence. This mutation lies in the highly conserved glycine-rich motif 188G(X)GGV(L)G193 that participates in the binding of the pyrophosphate group of NADP+ cofactor and hence might impair the activity of CAD2. Phloroglucinol staining of midribs of these mutants also showed a dark wine-red color that is characteristic of the bmr6 group. These two mutants can be distinguished by an intron length polymorphic marker developed based on the COMT gene sequence in this study. Mutant IS23549, which has also been assigned to the bmr6 group, was found to have another new allele with alanine-to-valine (A164V) substitution in CAD2. Alanine-164 is highly conserved among MDR proteins in plants and hence may be necessary for the activity of the enzyme. In mutant IS11861, there was no mutation that led to a change in amino acid in CAD2, while a threonine-to-serine (T302S) substitution was found in COMT. This single nucleotide polymorphism (SNP) at position 2645 in the COMT gene was converted into a cleaved amplified polymorphic sequence marker that can be used for its identification. In addition, additional SNP- and/or indel-based markers were developed, which can be used for exploiting these alleles in the molecular breeding of sorghum for dedicated bioenergy feedstock.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Abramson M, Shoseyov O, Shani Z (2010) Plant cell wall reconstruction toward improved lignocellulosic production and processability. Plant Sci 178:61–72

    Article  CAS  Google Scholar 

  • Baucher M, Chabbert B, Pilate G, Van Doorsselaere J, Tollier MT, Petit-Conil M, Cornu D, Monties B, Van Montagu M, Inzé D, Jouanin L, Boerjan W (1996) Red xylem and higher lignin extractability by down-regulating a cinnamyl alcohol dehydrogenase in poplar (Populus tremula × P. alba). Plant Physiol 112:1479–1490

    PubMed  CAS  Google Scholar 

  • Betts MJ, Russell RB (2003) Amino acid properties and consequences of subsitutions. In: Barnes MR, Gray IC (eds) Bioinformatics for geneticists. Wiley, Chichester, pp 289–316

    Chapter  Google Scholar 

  • Bhuiyan NH, Selvaraj G, Wei Y, King J (2009) Role of lignification in plant defense. Plant Signal Behav 4:158–159

    Article  PubMed  CAS  Google Scholar 

  • Bittinger TS, Cantrell RP, Axtell JD (1981) Allelism tests of the brown midrib mutants of sorghum. J Hered 172:147–148

    Google Scholar 

  • Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54:519–546

    Article  PubMed  CAS  Google Scholar 

  • Bout S, Vermerris W (2003) A candidate-gene approach to clone the sorghum Brown midrib gene encoding caffeic acid O-methyltransferase. Mol Genet Genomics 269:205–214

    PubMed  CAS  Google Scholar 

  • Calvino M, Messing J (2011) Sweet sorghum as a model system for bioenergy crops. Curr Opin Biotechnol 23:1–7

    Google Scholar 

  • Carpita NC, McCann MC (2008) Maize and sorghum: genetic resources for bioenergy grasses. TIPS 13:415–420

    CAS  Google Scholar 

  • Chen F, Dixon RA (2007) Lignin modification improves fermentable sugar yields for biofuel production. Nat Biotechnol 25:759–761

    Article  PubMed  CAS  Google Scholar 

  • Cherney JH, Axtell JD, Hassen MM, Anliker KS (1988) Forage quality characterization of a chemically induced brown midrib mutant in pearl millet. Crop Sci 28:783–787

    Article  Google Scholar 

  • Coleman HD, Samuels AL, Guy RD, Mansfield SD (2008) Perturbed lignifications impacts tree growth in hybrid poplar—a function of sink strength, vascular integrity, and photosynthetic assimilation. Plant Physiol 148:1229–1237

    Article  PubMed  CAS  Google Scholar 

  • Dai Z, Hooker B, Anderson D, Thomas S (2000) Improved plant-based production of E1 endoglucanase using potato: expression optimization and tissue targeting. Mol Breed 6:277–285

    Article  CAS  Google Scholar 

  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15

    Google Scholar 

  • Fu C, Mielenz JR, Xiao X, Gea Y, Hamilton CY, Rodriguez M, Chen F, Foston M, Ragauskas A, Bouton J, Dixon RA, Wang ZY (2011) Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass. Proc Natl Acad Sci USA 108:3803–3808

    Article  PubMed  CAS  Google Scholar 

  • Gupta SC (1995) Allelic relationships and inheritance of brown midrib trait in sorghum. J Hered 86:72–74

    Google Scholar 

  • Jorgenson LR (1931) Brown midrib in maize and its linkage relations. J Am Soc Agron 23:549–557

    Article  Google Scholar 

  • Kuc J, Nelson OE (1964) The abnormal lignins produced by the brown-midrib mutants of maize. 1. The brown-midrib-1 mutants. Arch Biochem Biophys 105:103–113

    Article  PubMed  CAS  Google Scholar 

  • Oraby H, Venkatesh B, Dale B, Ahmad R, Ransom C, Oehmke J, Sticklen M (2007) Enhanced conversion of plant biomass into glucose using transgenic rice-produced endoglucanase for cellulosic ethanol. Transgenic Res 16:739–749

    Article  PubMed  CAS  Google Scholar 

  • Porter K, Axtell JD, Lechtenberg V, Colenbrander V (1978) Phenotype, fiber composition, and in vitro dry matter disappearance of chemically induced brown midrib (bmr) mutants of sorghum. Crop Sci 18:205–208

    Article  CAS  Google Scholar 

  • Ransom C, Balan V, Biswas G, Dale B, Crockett E, Sticklen M (2007) Heterologous Acidothermus cellulolyticus 1,4-b-endoglucanase E1 produced within the corn biomass converts corn stover into glucose. Appl Biochem Biotechnol 137:207–219

    Article  PubMed  Google Scholar 

  • Rao PS, Deshpande S, Blummel M, Reddy BVS, Hash T (2012) Characterization of brown midrib mutants of Sorghum (Sorghum bicolor (L.) Moench). Eur J Plant Sci Biotech 6:71–75

    Google Scholar 

  • Saballos A, Vermerris W, Rivera L, Ejeta G (2008) Allelic association, chemical characterization and saccharification properties of brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench). BioEnergy Res 1:193–204

    Article  Google Scholar 

  • Saballos A, Ejeta G, Sanchez E, Kang C, Vermerris W (2009) A genomewide analysis of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L.) Moench] identifies SbCAD2 as the Brown midrib6 gene. Genetics 181:783–795

    Article  PubMed  CAS  Google Scholar 

  • Saballos A, Sattler SE, Sanchez E, Foster TP, Xin Z, Kang CH, Pedersen JF, Vermerris W (2012) Brown midrib2 (Bmr2) encodes the major 4-coumarate:coenzyme A ligase involved in lignin biosynthesis in sorghum (Sorghum bicolor (L.) Moench). Plant J 70:818–830

    Article  PubMed  CAS  Google Scholar 

  • Sattler SE, Saathoff AJ, Haas EJ, Palmer NA, Funnell-Harris DL, Sarath G, Pedersen JF (2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is responsible for the sorghum brown midrib6 phenotype. Plant Physiol 150:584–595

    Article  PubMed  CAS  Google Scholar 

  • Sattler SE, Palmer NA, Saballos A, Greene AM, Xin Z, Sarath G, Vermerris W, Pedersen JF (2012) Identification and characterization of four missense mutations in Brown midrib 12 (Bmr12), the caffeic O-methyltranferase (COMT) of sorghum. BioEnergy Res. doi:10.1007/s12155-012-9197-z

    Google Scholar 

  • Solomon BD, Barnes JR, Halvorsen KE (2007) Grain and cellulosic ethanol: history, economics, and energy policy. Biomass Bioenergy 31:416–425

    Article  Google Scholar 

  • Vogler R, Ejeta G, Johnson K, Axtell JD (1994) Characterization of a new brown midrib sorghum line. Agronomy abstracts Am Soc Agron, Madison

  • Wagner A, Donaldson L, Kim H, Phillips L, Flint H, Steward D, Torr K, Koch G, Schmitt U, Ralph J (2009) Suppression of 4-coumarate-CoA ligase in the coniferous Gymnosperm pinus radiate. Plant Physiol 149:370–383

    Article  PubMed  CAS  Google Scholar 

  • Xin Z, Wang ML, Burow G, Burke J (2009) An induced sorghum mutant population suitable for bioenergy research. BioEnergy Res 2:10–16

    Article  Google Scholar 

  • Youn B, Camacho R, Moinuddin SGA, Lee C, Davin LB, Lewis NG, Kang CH (2006) Crystal structures and catalytic mechanism of the Arabidopsis cinnamyl alcohol dehydrogenases AtCAD5 and AtCAD4. Org Biomol Chem 4:1687–1697

    Article  PubMed  CAS  Google Scholar 

  • Zhang K, Qian Q, Huang Z, Wang Y, Li M, Hong L, Zeng D, Gu M, Chu C, Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice. Plant Physiol 140:972–983

    Article  PubMed  CAS  Google Scholar 

  • Ziegelhoffer T, Raasch J, Austin-Phillips S (2001) Dramatic effects of truncation and sub-cellular targeting on the accumulation of recombinant microbial cellulase in tobacco. Mol Breed 8:147–158

    Article  CAS  Google Scholar 

  • Ziegler M, Thomas S, Danna K (2000) Accumulation of a thermostable endo-1,4-β-D-glucanase in the apoplast of Arabidopsis thaliana leaves. Mol Breed 6:37–46

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The study was funded by the Fast Track Scheme for Young Scientist from Science and Engineering Research Council (SERC), Department of Science and Technology (DST), India. The authors acknowledge Dr. HD Upadhyaya, ICRISAT, Patancheru 502 324, Andhra Pradesh, India, for providing the seeds of bmr mutants. We thank Dr. Viswanathan Chinnusamy for critical reading of the manuscript.

Author information

Authors and Affiliations

  1. Directorate of Sorghum Research, Rajendra Nagar, Hyderabad, 500 030, Andhra Pradesh, India

    Sunita Gorthy, Karthikeyan Mayandi & Monika Dalal

  2. National Research Center on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, 110 012, India

    Dhara Faldu & Monika Dalal

Authors
  1. Sunita Gorthy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Karthikeyan Mayandi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dhara Faldu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Monika Dalal
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Monika Dalal.

Additional information

Sunita Gorthy, Karthikeyan Mayandi: Joint First Authors.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gorthy, S., Mayandi, K., Faldu, D. et al. Molecular characterization of allelic variation in spontaneous brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench). Mol Breeding 31, 795–803 (2013). https://doi.org/10.1007/s11032-012-9834-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11032-012-9834-4

Keywords

Search

Navigation