Expression of a P450 gene in barley (*Hordeum vulgare*)

Linh Nguyen and Robert J. Henry

Molecular Plant Breeding Cooperative Research Centre,

Centre for Plant Conservation Genetics, Southern Cross University, PO
Box 157, Lismore NSW 2480, Australia. <lnguye10@scu.edu.au>

The sequence reported here was submitted to GenBank (Accession number
DQ838490)

**Abstract**

A new member of the P450 gene family was identified in barley. The P450
gene was a member of the subfamily CYP72A. Northern analysis revealed
that various CYP72A39 transcripts were found in four barley cultivars at
a very early vegetative stage but no expression was detected at the
reproduction stage. This suggested that this P450 gene may be involved
in seedling development in barley. Comparison of expression profiles of
this gene and "digital expression" databases confirmed that this gene
was homologous to several cereal EST clones with tissue-specific
transcripts responding to various environmental stimuli. Among these,
many transcripts in barley were obtained from stressed tissues at the
vegetative stage, and two transcripts in wheat were expressed after
being challenged by barley powdery mildew pathogen (*Bumeria graminis*
f. sp. hordei). This suggested that the CYP72A39 gene may play a role in
defence in the barley seedling.

**Introduction**

Plant P450s belong to the cytochrome group of membrane-bound enzymes,
that are usually found in plant endoplasmic reticulum (Halkier, 1996).
Among 6008 P450 genes reported in all organisms including human, one
third of them have been reported in plant species. In plants, P450s play
a central role in numerous biosynthetic pathways such as production of
second metabolites (phenylpropanoids, alkaloids, lipids, sterols,
flavonoids and cyanogenic glycosides). In addition, plant P450s have a
significant response to stresses (Whitbred and Schuler, 2000) and
disease (Smigocki and Wilson, 2004; Takemoto et al., 1999). P450 enzymes
that are encoded by P450 genes are important components of the defense
mechanism of plants through their enzymatic reactions in various
biosynthesis pathways. Depending on the type of stimuli, specific
enzymes are produced under stress conditions. Many reports (Bolwell et
al., 1994; Halkier, 1996; Mizutani et al., 1998; Persans et al., 2001;
Schuler and Wreck-Reichhart, 2003; Toguri et al., 1993c) have emphasized
the affects of environmental factors such as stresses (salinity, low
temperature, pathogen elicitation) as well as developmental factors
(plant age, tissue specific) on the expression pattern of plant P450.
Here we report the identification and analysis of expression patterns of
a new P450 gene in barley.

**Materials and methods**

Randomly selected clones (512) from a cDNA library of four day old
seedling (cv Alexis) were sequenced. Details of the cDNA library
construction were previously described by Holton and co-workers (2002).
A clone (5E2) was identified showing 72% homology to a P450 (CYP72A21)
in rice.

This 1817bp full length sequence, consisting of 1578 nucleotide ORF, 189
nucleotides at the 3\' UTR and 50 nucleotides in the promoter region,
encoded a 526 amino acid polypeptide with molecular mass of 59.57 kDa
and isoelectric point of 8.59. Coding sequence was searched against the
P450 database from David Nelson\'s home page
(http://drnelson.utmem.edu/CytochromeP450.html) using BLASTP. P450
motifs and domains in the sequence were detected manually.

Four parental barley lines (Chebec, Harrington, Alexis and Sloop) which
have been used in many breeding programs were selected. Two growth
conditions, indoor and outdoor, were applied to each cultivar.
Observation and sample collection were based on the Zadok score, a
measurement of plant growth stage. For each cultivar, samples were
collected at different development stages, ranging from young seedling
to post-anthesis (Table 1). For all four cultivars, at vegetative stage,
both roots and shoots were collected for 4 day and 1 week old seedling
samples, and only shoot samples for 2 week old seedling (Parts A, B, C
of Figures 1 and 2). At the floral stage, only inflorescence parts were
sampled for two cultivars, Chebec and Harrington. Flowering was checked
daily and the first appearance of the flag leaf, indicating that the
plant was already in its floral stage, was marked. The floral stage
(Parts C, D of Figures 1 and 2) was divided into four sub-stages: stage
1 was when the flag leaf was first seen, stage two was when the first
awn appeared and stage three was recorded if the first spikelet was
seen. These three stages occurred before plants pollinated. When
pollination occurred, individual seeds that were pollinated on that day
were marked. Two weeks later, these marked seeds were harvested and
classified as at sub-stage four.

Total RNA was extracted from the tissues indicated in Table 1 using
either RNeasy Plant Mini kit (Qiagen) or CTAB method (Chang et al.,
1993). Probes were generated by using DIG-PCR probe synthesis kit
(Roche). Membrane transfer, blot hybridisation and detection were
carried out according to the manufacturer's protocol (Roche, 2000).
Barley Ubiquitin was used as house keeping gene. RNA extracted from
mix-tissues of shoot and root (4 day seedling) was used as positive
control. Primer sequences and PCR compositions to generate labelled
probes were listed in Tables 2 and 3.

The temperature cycling condition for both probes were very similar.
They were 96^0^C for 2 min; followed by 25 cycles of 96^0^C for 1min,
45^0^C for 45s, 72^0^C for 2min, and a final extension at 72^0^C for
10min. The only exception was that the duration of extension at 72^0^C
was 1min shorter for the whole 25 cycles when the Ubiquitin primer was
used.

**Results and discussion**

**Sequence structure of CYP72A39**

Results of a BLAST search of CYP72A39 sequence against the Nelson\'s
P450 database (<http://132.192.64.52/p450.html>) showed 72% and 68%
identity to CYP72A21 and CYP72A33 in rice respectively. This indicated
that this new barley gene belong to the CYP72A subfamily and was
assigned as CYP72A39.

Several highly conserved regions which are consider as a benchmark of
P450s (Fig 3) are found in the gene structure of the new sequence such
as:

-   The membrane anchor (MVLLGVLASPTPATVLWTLLGLALL) is situated in the
    first 25 amino acids from the N terminal,

-   The C-helix motif (WVKHR) is located from residue 157 to161,

-   The I-helix motif (AGSET) required for oxygen binding, is located
    from residue 335 to 339,

-   The K-helix motif (EVLR) is located from residue 391 to 394,

-   The P450 signature PERF (also called domain C ) is located from
    residue 446 to 449,

-   The heme binding region PFGWGPRICIG (also called domain D) is
    located from residue 465 to 475 including the Cystein residue in
    position 473.

However, the proline rich motif (PPGP) which is usually located between
the C-helix and the membrane anchor, was not detectable in the barley
CYP72A39 sequence. Similarity, the absence of a proline rich motif in
the protein structure of CYP72A1 (accession L10081) was reported in
Catharanthus roseus (Irmler et al., 2000).

**Gene expression patterns of CYP72A39 by Northern blot analysis**

At the vegetative phase, under the same indoor condition; the CYP72A39
gene was expressed differently in all four cultivars. Overall, the
CYP72A39 transcript was found in root of 1 week old seedling of all four
cultivars (Figures 4, 5, 6). In cultivar Chebec, the gene was expressed
only in the root of 1 week old seedling (Figure 4). No transcript was
found in shoot at any stage of the vegetative phase of this cultivar. In
contrast, in cultivar Alexis, bands were found only in root at 4 day and
in 1 week old seedlings (Figure 6). Interestingly, the transcript of the
candidate gene hybridised strongly to RNA extracted from mixed tissues
(root and shoot at 4 day old seedling) of Alexis that were used as
positive probe control in all blots, but there was no evidence of this
candidate gene's transcriptions found in any shoot tissue of this
cultivar. Furthermore, as shown in Figures 5 and 6, CYP72A39 was
expressed in the shoots and roots of both Sloop and Harrington. At the
floral stage, no hybridised bands were detected in any reproductive
tissues of any cultivar, under both growth conditions, indoor and
outdoor (Figures 4, 5, 6). The house keeping gene, Ubiquitin, was
expressed in all tissues of the four cultivars at all developmental
stages as expected.

**Comparative analysis of gene expression of CYP72A39 and sequences from
"electronic expression" databases**

The gene expression patterns of CYP72A39 were compared to "electronic
expression" data from plant species by homology search. BLAST results
indicated that the CYP72A39 matched to numerous ESTs derived from
several cDNA libraries (Table 4). However, only a fraction of entries
(11 out of 171) are listed here.

Results of the Northern blot experiment showed that transcripts of
CYP72A39 were specifically found only in the vegetative tissues of the
four cultivars. This study also demonstrated that, in the two cultivars
Chebec and Harrington, there was no evidence of CYP72A39 transcripts in
any reproductive tissues, either from the growth cabinet or field grown
barley. Further more, the blast search against Plant Genome database
(Plant GDB) showed that CYP72A39 was highly homolog to EST sequences
isolated at seedling stages. These factors suggest that this gene may
play a role in seedling development of barley.

In most cases, the CYP72A39 showed significant homology to sequences
whose transcripts were induced in vegetative tissues. Blast searches
against the Plant GDB server showed several hits to the monocot EST
sequences that appeared under stressed or pathogenic challenging. To get
more specific sequence homology to each species, blast searches against
individual EST database was performed. Results from the searches against
the barley EST database showed that the CYP72A39 had 100% identity (over
812/812 nucleotides) at both coding and the 3-UTR region with the barley
contig20974 obtained from cold stressed shoots. In the coding region,
the three motifs, K helix, C domain and D domain containing the Heme
region that essential to a P450 gene, were identical to those found in
the CYP72A39 sequence.

Results from the searches against the wheat EST database indicated that
the CYP72A39 was strongly homologised to five wheat ESTs from the same
cultivar, Fidel. These five wheat ESTs were listed as pathogenic
inducible genes. Of these, two EST sequences AJ888598 and AJ890237
showed significant identity (93% over 594/638 and 627/674 nucleotides
respectively) to the CYP72A39. Sequence comparison between these two and
the CYP72A39 showed that they shared a 168 identical amino acid sequence
included the stop codon (ATG). Similar to the barley contig20974
sequence, these two wheat ESTs also contained the three motifs (K helix,
C and D domains) identical to those found in the CYP72A39 sequence
(Figure7). Ten amino acid substitutions were found between CYP72A39 and
the two wheat ESTs. Attention was focused on the AJ888598 sequence, the
3'UTR of which matched to that of the CYP72A39 sequence (data not
shown). However at present, information about the wheat EST (AJ888598)
was very limited. Overall significant sequence identity at both coding,
particularly at the three important motifs, and non-coding regions
between the CYP72A39 and several monocot ESTs suggested that the
CYP72A39 gene appears to be a good candidate for stress and disease
resistant study.

**Conclusion**

Various expression patterns of CYP72A39 genes were found in four barley
cultivars by Northern analysis. These patterns detected in vegetative
but not in any reproductive tissues suggested that this gene may be
involved in seedling development of barley. In addition, significant
homology between this gene and cereal EST samples expressed under
stresses and particularly pathogen challenges leads to a speculation
that functionally this barley gene is likely to be involved in defence
in barley.

**Acknowledgments**

The authors wish to thank Dr David Nelson for assigning the gene, Dr
Agnelo Furtado for technical advice on the hybridisation blot, Dr
Timothy Holton for constructing the cDNA library, Toni Spark (Hermitage
Research Station) and Dr David Moody (VIDA) for providing the barley
seeds. This study was funded by Molecular Plant Breeding CRC.

**References**

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*Phytochemistry*. 37:1491-1506.

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9:63-71.

Irmler, S., G. Schroder, B. St-Pierre, N.P. Crouch, M. Hotze, J.
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*Plant Journal*. 24:797-804.

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  --------------- ----------------------- ------------- -------------- -------------
  Table 1.                                                             
  Developmental                                                        
  stages, type of                                                      
  collected                                                            
  tissues and                                                          
  growth                                                               
  conditions of                                                        
  barley                                                               
  materials used                                                       
  in the gene                                                          
  expression                                                           
  experiment.                                                          

  Developmental   Morphological           Type of       Growth         Cultivar^b^
  stage           appearance              tissue        condition^a^   
                                          collected                    

  Vegetative      Four day old seedling   Shoot, Root   I              C, H, A, S
  stage                                                                

                  One week old seedling   Shoot, Root   I              C, H, A, S

                  Two week old seedling   Shoot         I              C, H, A, S

  Floral stage    Appearance of flag leaf Head          I/O            C, H

                  Appearance of first awn Head          I/O            C, H

                  Appearance of first     Head          I/O            C, H
                  spikelet                                             

                  Two weeks after         Individual    I/O            C, H
                  pollination             seed                         
  --------------- ----------------------- ------------- -------------- -------------

^a^I: Indoor; O: Out door;

^b^C: Chebec, H: Harrington A: Alexis, S: Sloop

  ------------------- --------------------------------- ------------------
  Table 2. Primer                                       
  sequences                                             

  Primer ID           Primer sequence (5\'-3\')         Probe length (bp)

  CYP72A39 forward    TCAAGCACCGGAGGATCCTCA             1023

  CYP72A39 reverse    CAAGGTCGAATTCGAAGCGTTGA           

  Ubiquitin forward   CGACAACGTCAAGGCGAAGAT             314

  Ubiquitin reverse   CCAAAGCCACGGCACAAGTT              
  ------------------- --------------------------------- ------------------

  ----------------------------- ---------- ------------ ---------- ------------
  Table 3. PCR components of                                       
  labelled and unlabelled                                          
  samples                                                          

                                For                     For house  
                                CYP72A39                keeping    
                                probe                   gene probe 

                                Labelled   Unlabelled   Labelled   Unlabelled
                                fragment   fragment     fragment   fragment

  10x PCR buffer (1.5mM MgCl2,  2μl        2μl          2μl        2μl
  Roche)                                                           

  Taq Polymerase (5 units,      0.4μl      0.4μl        0.4μl      0.4μl
  Roche)                                                           

  10mM dNTP\'s (Promega)        \_         0.4μl        \_         0.4μl

  DMSO                          1μl        1μl          \_         \_

  50% glycerol                  1.2μl      1.2μl        \_         \_

  PCR DIG mix (10x              2μl        \_           2μl        \_
  concentration, Roche)                                            

  Primer forward (10μM)         3μl        3μl          3μl        3μl

  Primer reverse (10μM)         3μl        3μl          3μl        3μl

  Plasmid DNA                   10ng       10ng         100pg      100pg

  And MiliQ water was added to  20μl       20μl         20μl       20μl
  a total volume                                                   
  ----------------------------- ---------- ------------ ---------- ------------

  ---------------- --------- ---------- --------------- --------------------- ------------ -------
  Table 4.                                                                                 
  Representative                                                                           
  sequences that                                                                           
  homolog to                                                                               
  CYP72A39                                                                                 

  Accession        Species   Cultivar   Expressed       Description of        Nucleotide   E value
                                        tissue          treatment and sample  identity %   
                                                        source (if known)                  

  Contig 20974\*   Barley    Morex      5 day old shoot Cold stress (EST and  100          00.0
                                        seedling        Chip)                 (812/812)    

  BF623161         Barley    Morex      5 day old shoot Cold stress (EST and  98 (581/591) 0.00
                                        seedling        Chip)                              

  AJ888598         Wheat     Fidel      Leaf of 7 day   Pathogenic elicitor   93 (594/638) 00.0
                                        old seedling    (EST)                              

  AJ890237         Wheat     Fidel      Leaf of 7 day   Pathogenic elicitor   93 (627/674) 00.0
                                        old seedling    (EST)                              

  CB871128         Barley    Sloop      3 day old       Unknown (EST)         97 (412/423) 00.0
                                        coleoptile                                         

  CB867923         Barley    Sloop      3 day old       Unknown (EST)         97 (420/430) 00.0
                                        coleoptile                                         

  BE587940         Rye       Blanco     Root tip of     Unknown EST           92 (432/469) e-180
                                        seedling                                           

  BU050675         Maize     NA         NA              Unknown (EST, Unigen) 85 (372/433) e-101

  CN137181         Sorghum   BTx623     Root and leaf   Methyl viologen       84 (393/467) 2e-90
                                        of 8 day old    treatment, oxidative               
                                        seedling        stressed leaves &                  
                                                        roots (EST)                        

  BQ743931         Wheat     Chinese    Root at         Salt stress (EST)     81 (414/508) 4e-67
                             Spring     tillering stage                                    

  CB927177         Sorghum   IS3620C    Seedling        Abscisic acid (ABA)   84 (307/364) 1e-69
                                                        treatment (EST)                    
  ---------------- --------- ---------- --------------- --------------------- ------------ -------

Asterisk (\*) indicated blast results obtained from Barley1contig20974
of 3 members (HVSMEa0012A09f2, HVSMEa0019H07r2, HVSMEa0012A09r2).
Unknown means not enough specific information to determine if tissues
were treated or not.

![](media/image1.jpeg){width="5.222916666666666in"
height="3.5909722222222222in"}

Figure 1. Development and appearance of cultivar Chebec at various
sample collection stages. A, B, and C: seedlings at vegetative stage. D:
inflorescence parts at floral stage of indoor plants.

![](media/image2.jpeg){width="5.145833333333333in"
height="3.954861111111111in"}

Figure 2. Development and appearance of cultivar Harrington at various
sample collection stages. A, B, and C: seedlings at vegetative stage. D:
inflorescence parts at floral stage of indoor plants.

![](media/image3.jpeg){width="5.401388888888889in"
height="4.789583333333334in"}

Figure 3. Deduced amino acid sequence of CYP72A39 is shown in capital
letters. Motifs and domains are underlined. Stop codon is indicated by
asterisk (\*).

![](media/image4.jpeg){width="5.49375in" height="3.34375in"}

Figure 4. Expression patterns of CYP72A39 in cultivar Chebec by Northern
blot analysis. Asterisk (\*) indicates expressed tissues.

![](media/image5.jpeg){width="5.7659722222222225in"
height="4.061111111111111in"}

Figure 5. Expression patterns of CYP72A39 in cultivar Harrington by
Northern blot analysis. Asterisk (\*) indicates expressed tissues.

![](media/image6.jpeg){width="5.766666666666667in"
height="3.277083333333333in"}

Figure 6. Gene expression patterns of CYP72A39 in cultivars Sloop and
Alexis by Northern blot analysis. Asterisk (\*) indicates expressed
tissues. Double asterisk (\*\*) indicates expressed tissue of 1 week old
seedling root of Harrington (as shown in Figure 4) used as an extra
internal control.

![](media/image7.jpeg){width="5.768055555555556in"
height="4.446527777777778in"}

Figure 7. Alignment of CYP72A39 and the three EST sequences (barley
contig 20974, wheat AJ888598 and AJ890237). Motifs are indicated by
arrows. Mismatched sequences are indicated in colon (:). Stop codon is
indicated by asterisk (\*).