A new allele in a *uzu* gene encoding brassinosteroid receptor

# D. Gruszka, J. Zbieszczyk, M. Kwasniewski, I. Szarejko and M. Maluszynski

Department of Genetics, Faculty of Biology and Environment Preservation,
University of Silesia, 40-032 Katowice, Poland

e-mail of main author: <wintergrun@op.pl>

Brassinosteroids are growth-promoting natural products, found at low
levels in pollen, seeds and young vegetative tissues throughout the
plant kingdom. They play an essential role in diverse developmental
processes including cell expansion, vascular differentiation, etiolation
and reproductive development (Clouse and Sasse, 1998). It has been found
that semidwarf barley accessions carrying the *uzu* gene are
non-responding to brassinosteroids. The synteny suggested that the *uzu*
gene may be homologous to rice *D61*, which is a homolog of *Arabidopsis
thaliana BR-insensitive1* (*BRI1*), encoding a BR-receptor protein. A
barley homolog of *BRI1, HvBRI1* (acc. no. AB088206) was isolated (Chono
et al., 2003). The sequence analysis showed that uzu phenotype was
caused by single-nucleotide substitution A \> G at the position 2612 of
the *HvBRI1* gene. This mutation resulted in an amino acid change at the
highly conserved residue (His-857 to Arg-857) of the kinase domain of
BRI1 receptor protein and caused the reduced sensitivity to BRs and
reduced plant height (Chono et al., 2003). We have found a semidwarf
mutant 093AR, produced by MNU (N-methyl-N-nitrosourea) treatment of
variety Aramir, that proved to be allelic to the uzu form. The aim of
this work was to determine and compare the sequences of *HvBRI1* gene
between spontaneous mutant uzu, induced mutant 093AR and its parent
Aramir variety in order to identify the putative mutation(s).

DNA extraction was performed according to "micro C-TAB" protocol (Doyle
and Doyle, 1987). Six primer pairs were designed on the basis of
*HvBRI1* sequence (GenBank acc. no. AB088206) utilizing the Primer3
program (<http://frodo.wi.mit.edu/cgi-bin/primer3>). PCR reactions were
performed using the "touch-down" strategy. Three independent PCR
reactions were performed for each primer pair and amplification products
were extracted from agarose gels using QIAEX II Gel Extraction Kit
(QIAGEN) and cloned into pGEM-T Easy vector (Promega). Plasmid
purification was carried out using QIAprep Spin Miniprep Kit (QIAGEN).
Inserts were sequenced using SequiTherm EXCEL II DNA Sequencing Kit
(EPICENTRE) utilizing LI-COR IR2 sequencer.

We determined the sequences of *HvBRI1* gene in the genotype uzu, 093AR
and Aramir variety. The single-nucleotide substitution has been
confirmed at the position 2612 (A-2612 to G-2612) in the spontaneous
mutant uzu, which proved to be specific for this genotype. This mutation
is localized in highly conserved kinase domain of receptor protein. The
comparison of sequences of *HvBRI1* gene between mutant 093AR and Aramir
variety, from which this mutant was obtained, led to the identification
of the C to A substitutions at the positions 1760 and 1761 in the mutant
(Fig. 1).

1 cgcttctcgcatggtctcaaggtagagcgagcgagctcccacatggattg

1701 gatgccgagcaagaagctgtgcaacttcacaaggatgtacatggggagca

1751
cagagtata**[cc]{.underline}**ttcaacaagaatggctccatgatatttctggatttgtca

1801 tttaatcagcttgactcggagatacccaaggagcttggcaatatgttcta

...

2551 gtgagtggccagggtgaccgggagttcacagcggaaatggagaccattgg

2601
caagatcaaac**[a]{.underline}**ccgcaaccttgttccgctcctcggctactgcaagatcg

2651 gcgaggagcggctgctgatgtatgacttcatgaagtatggcagcttggag

...

3501 gcctctcagatgattgatgatgatgtgatgataccattctgaggttccca

3551 cgcaagct

**Fig. 1**. The sequence of *HvBRI1* gene (acc. no. AB088206).
**[cc-]{.underline}** at the positions 1760 and 1761 indicate C \> A
substitutions in the 093AR sequence; **[a-]{.underline}** at the
position 2612 indicates A \> G substitution in spontaneous mutant uzu.

On the basis of *in silico* translation using Jellyfish program it was
postulated that this mutations lead to the replacement Thr-573 by
Lys-573 (Fig. 2).

1 mdclrlavaaaalllaalaaaaddaqlldefrmalpsqaplegwtarega

...

501 ippelaeqsgkmtvgliigrpyvylrndelssqcrgkgsllefssirsed

551 lsrmpskklcnftrmymgstey**[t]{.underline}**fnkngsmifldlsfnqldseipkelgn

601 mfylmimnlghnllsgaiptelagakklavldlshnrlegqipssfssls

...

1101 pgfgvmdmtlkeakeekd

**Fig. 2** The sequence of HvBRI1 protein (acc. no. BAD01654).
**[t]{.underline}** -- indicates the Thr-573 to Lys-573 conversion,
deduced by *in silico* translation, caused by C \> A substitutions at
the positions 1760 and 1761.

The threonine residue is localized between last LRR (Leucin-Rich Repeat)
domain and pair of cysteines participating in protein dimerization,
followed by transmembrane domain. It was shown utilizing ClustalW
program that Thr-573 residue is highly conserved among BRI1 homologs
from barley, rice, arabidopsis and tomato. As threonine and lysine have
different chemical properties, their substitution within the conserved
domain may lead to the change in protein activity.

**References:**

Chono, M., I. Honda, H. Zeniya, K. Yoneyama, D. Saisho, K. Takeda, S.
Takatsuto, T. Hoshino and Y. Watanabe. 2003. A semidwarf phenotype of
barley uzu results from a nucleotide substitution in the gene encoding a
putative brassinosteroid receptor. Plant Physiology. 133:1209-1219

Clouse, S. D. and J. M. Sasse. 1998. Brassinosteroids: essential
regulators of plant growth and development. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 49: 427-451.