A Reverse-Genetics Mutational Analysis of the Barley HvDWARF Gene Results in Identification of a Series of Alleles and Mutants with Short Stature of Various Degree and Disturbance in BR Biosynthesis Allowing a New Insight into the Process.
Brassinosteroids (BRs) are plant steroid hormones, regulating a broad range of physiological processes. The largest amount of data related with BR biosynthesis has been gathered in Arabidopsis thaliana, however understanding of this process is far less elucidated in monocot crops. Up to now, only four barley genes implicated in BR biosynthesis have been identified. Two of them, HvDWARF and HvBRD, encode BR-6-oxidases catalyzing biosynthesis of castasterone, but their relation is not yet understood. In the present study, the identification of the HvDWARF genomic sequence, its mutational and functional analysis and characterization of new mutants are reported. Various types of mutations located in different positions within functional domains were identified and characterized. Analysis of their impact on phenotype of the mutants was performed. The identified homozygous mutants show reduced height of various degree and disrupted skotomorphogenesis. Mutational analysis of the HvDWARF gene with the "reverse genetics" approach allowed for its detailed functional analysis at the level of protein functional domains. The HvDWARF gene function and mutants' phenotypes were also validated by measurement of endogenous BR concentration. These results allowed a new insight into the BR biosynthesis in barley.
Department of Genetics, Faculty of Biology and Environment Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland. damian.gruszka@us.edu.pl.
Damian
D
Gruszka
Department of Genetics, Faculty of Biology and Environment Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland. malgorzata.a.gorniak@gmail.com.
Malgorzata
M
Gorniak
Department of Genetics, Faculty of Biology and Environment Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland. ewelina.glod1@wp.pl.
Ewelina
E
Glodowska
Department of Genetics, Faculty of Biology and Environment Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland. e.wierus@gmail.com.
Ewa
E
Wierus
Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, CZ-783 71 Olomouc, Czech Republic. jana.oklestkova@upol.cz.
Jana
J
Oklestkova
Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Krakow, Poland. anna.janeczko@poczta.fm.
Anna
A
Janeczko
Department of Genetics, Faculty of Biology and Environment Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland. miroslaw.maluszynski@us.edu.pl.
Miroslaw
M
Maluszynski
Department of Genetics, Faculty of Biology and Environment Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland. iwona.szarejko@us.edu.pl.
Iwona
I
Szarejko
D000483:Alleles; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D060406:Brassinosteroids / Q000096:biosynthesis*; D002777:Cholestanols / Q000378:metabolism; D005091:Exons; D006720:Homozygote; D001467:Hordeum / Q000235:genetics* / Q000502:physiology; D007438:Introns; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010937:Plant Growth Regulators / Q000096:biosynthesis*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
D029681:Arabidopsis Proteins; D060406:Brassinosteroids; D002777:Cholestanols; D010937:Plant Growth Regulators; D010940:Plant Proteins; C083545:castasterone
barley; biosynthesis; brassinosteroids; mutant; reverse-genetics; semi-dwarf; splicing