3. Results
3.2. Cloning of the gene LAXATUM-A
3.2.8. Global analysis of gene expression
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stages did not cluster as expected. The different tissue stages as well as the mutant and wild-type samples did not clustere separately from each other (Figure 21a). Considering only significantly differentially expressed genes leads to the expected genotype specific clustering for all samples of BW457 and Bowman, respectively. Only the sample laxatum_st2 and laxatum_st3 showed a higher correlation to the samples of the first (glume primordium) developmental stage (Figure 22b). However, the overall correlation of all replicates was very high. The introgression of Bowman NIL457 was only restricted to chromosome 5H in a Bowman background. Since expression values were compared to Bowman as wild-type, an overall high amount of similar gene expression can be expected. The samples of the first two developmental stages are very close together in developmental time. Since multiple spike meristems per sample are harvested to have sufficient tissue for RNA isolation, small developmental differences might introduce ‘noise’ into the analysis. However, as expected for an entirely deleted gene no reads of the gene HvLAX-A were detected in all samples derived from BW457. Thus an error in sampling could be excluded and therefore the analysis was conducted with all replicates and analyzed separately for each developmental stage.
Figure 21: Correlation heat map of all samples and replicates of the RNA-seq expression analysis.
The heatmaps show the correlation of gene expression values between samples and are colored by a red (low correlation) to white (high correlation) scale. a) The correlation matrix was plotted by all genes (a) and only for the differentially expressed genes (b).
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In total, 144 high confidence genes (IBSC, 2012) showed a significant (p < 0.05) differential expression between Bowman and BW457 samples in at least one of the addressed tissues.
Among these, 20 down regulated and 19 upregulated genes showed high differential expression (log2-fold change >2) between Bowman and BW457 (Table 22). A summary of all differential gene expression statistics (normalized mean read counts per gene and sample, log 2 fold change, significance level and their functional annotation (IBSC, 2012) is provided in Table 22 (log2 fold > 2) and Table A21 (log2 fold < 2).
Only 33 genes were significantly differentially regulated between Bowman and BW457 among all three developmental stages. This included three ‘phytohormone associated’ genes, six ‘transcription related’ genes with protein-protein interaction domains as well as 11 unknown proteins according to the published annotation information (IBSC, 2012). Since it is not known at which of the three developmental stages HvLAX-A might act for establishing a wild-type barley spike, the three time points of development were inspected separately for putative candidate genes that showed differential regulation between Bowman and BW457.
Three down-regulated homeobox leucine zipper transcription factors (TFs) were identified in the glume primordium stage in BW457. In the ‘stamen primordium stage two’ one ectopically expressed MADS-box TF gene (MLOC_76418.1) was observed, whereas in stage of
‘completely developed immature spikes’ five transcription factor related genes were up-regulated in lax-a samples compared to Bowman.
The paralogous gene HvCUL4 (AK360734) was expressed at similar levels in both mutant and wild-type genotypes. Thus deletion of HvLAX-A did not induce any compensating over-expression of the paralogous gene HvCUL4 in the analyzed tissues (Figure 22).
The genes BOP1/2 in Arabidopsis were in the focus of intensive research over the last 10 years and a number of genes were described to be regulated by both genes. The orthologs of those genes known to be involved in regulating pathways were inspected for conserved gene regulation between barley and Arabidopsis. A summary table of known interaction partners is given in Table 21. The nucleotide sequences of Arabidopsis genes (http://www.arabidopsis.org) were used to identify homologous barley genes (Table 21). For some genes the entire gene family was listed since the sequence conservation did not allow a prediction of the putative orthologs. Among these genes, HvLAX-A remained the only candidate with high difference in expression between Bowman and BW457 samples. A second gene, a LOB domain containing gene which showed similarity to AS2 of Arabidopsis reached the significance level (p<0,05) of the DE test in the last sampled tissue stage of
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completely developed spikes (Table 23). This indicated that the regulation of BOP-like genes may not be well conserved between Arabidopsis and barley in the analyzed tissues. However, some of the described regulations from Arabidopsis are known from studies of morphological changes in leaf development and thus might be not comparable to the analyzed spike meristem tissue in the present study.
Figure 22: Expression analysis of the genes HvLAX-A and HvCUL4.
The expression values obtained from RNAseq compared between Bowman and BW457 of the three developmental stages (glume primordium, stamen primordium, completely developed spike) for HvLAX-A (a) and HvCUL4 (b). No reads were detected for HvLAX-A in samples of the BW457 mutant line whereas the gene was highly expressed in wild-type tissue. The expression of the paralogous gene HvCUL4 was not affected thus no differences in transcript level could be observed between wild-type and mutant plants.
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Table 21: Arabidopsis genes involved in regulatory pathways of BOP1/2
Name Short GeneID comments
BLADE ON PETIOLE 1 BOP1 AT3G57130.1 BLADE ON PETIOLE 2 BOP2 AT2G41370.1
ASYMMETRIC LEAVES 1 AS1 AT2G37630.1 MYB-domain protein
ASYMMETRIC LEAVES 2 AS2 AT1G65620.1 Lateral organ boundaries (LOB) JAGGED JAG AT1G68480.1 zinc finger transcription factor NUBBIN NUB AT1G13400.1 zinc finger transcription factor
LEAFY LFY AT5G61850.1
PUCHI PUCHI AT5G18560.1 no homolog in barley PERIANTHIA PAN AT1G68640.1 TGA8 TF
APETALA1 AP1 AT1G69120.1 MADS-box protein AGAMOUS-LIKE 24 AGL24 AT4G24540.1 MADS-box protein KNOTTED1-LIKE -
HOMEOBOX GENE 6
KNAT6 AT1G23380.1
class I knotted1-like homeobox BREVIPEDICELLUS BP AT4G08150.1 class I knotted1-like homeobox PENNYWISE PNY AT5G02030.1 BEL1-LIKE Homeodomain HOMEOBOX GENE 1 ATH1 AT4G32980.1
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Table 22: Differentially expressed (log2_fold >2) genes between Bowman and BW457
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Table 23: Differences in expression values (log2-fold change) between Bowman and BW457 for homologous barley genes of known AtBOP1/2 regulated genes.
Class1 Gene2 log2FC_gl log2FC_st log2FC_cd
BOP1 AK360734.1 -0.03 0.04 0.26
BOP2 MLOC_61451.6 -12.09* -10.53* -13.24*
KNOX1-like MLOC_57232.2 0.11 -0.05 0.37
MLOC_11678.1 0.28 0.07 0.11
AK369536 -0.98 0.97 0.95
AK376780 0.09 -0.01 0.01
AK373181 -0.04 0.14 0.13
AK358211 -0.16 -0.01 -0.19
Ap1-like
AK360697 -0.04 0.10 0.30
MLOC_61901.1 -0.34 -0.14 0.11
AK361227 -0.07 0.28 0.20
LFY MLOC_14305.1 -0.23 0.19 0.16
AS1 –like AK372839 -0.61 0.55 0.23
AK372889 -0.44 -0.03 0.33
LOB- like genes AK373607 -0.32 -0.13 -0.49 MLOC_61156.1 -0.94 -0.29 -0.57*
MLOC_55239.1 -0.67 0.43 -0.43
MLOC_52276.7 0.28 -0.05 -0.11
MLOC_51325.1 0.37 1.60 -0.38
MLOC_58304.1 -0.77 0.03 0.21
AK373051 -1.09 0.34 0.20
MLOC_11838.1 -0.20 1.26 0.35
yabby like MLOC_60897.1 -0.21 0.03 0.42
MLOC_18466.1 -0.40 0.02 -0.05
MLOC_70653.1 -0.19 -0.27 -0.16 MLOC_66260.1 -1.42 -0.44 -0.32
MLOC_70676.2 -0.12 0.16 0.02
JAG/NUB MLOC_4391.1 -0.56 0.21 0.00
AGL24-like
AK374424 -0.70 -0.04 0.44
AK370732 -0.31 0.11 -0.02
AK250344.1 -0.47 -0.34 -0.13
AK374272 -0.38 -0.15 0.09
AK360697 -0.04 0.10 0.30
PNY-like MLOC_66099.1 -0.13 0.42 0.01
MLOC_3638.2 -0.03 -0.06 0.25
ATH1-like(bel1- like)
MLOC_53364.1 0.13 -0.11 0.07
MLOC_5414.1 0.00 0.09 0.27
MLOC_68213.2 -0.13 0.35 0.97
AK367579 1.56 3.42** -1.11
AK358615 -0.30 -0.08 -0.21
AK360144 -0.22 -0.39 -0.34
MLOC_3638.2 -0.03 -0.06 0.25
PAN-like
AK362993 -0.12 -0.29 -0.08
AK357239 -0.06 0.14 -0.26
AK359391 -0.01 0.23 0.13
MLOC_64008.1 -0.03 0.11 0.00
AK363931 0.27 0.15 0.10
MLOC_14596.3 -0.22 0.15 0.15
1 Genes and gene classes reported to be regulated by BOP1/2 in Arabidopsis
2 Homologous barley gene models (IBSC, 2012) with changes in expression levels for the 3 different analyzed tissues between Bowman and BW457. Genes with less than 10 reads/gene were excluded and considered at not expressed
* significant differentially expressed (p<0,05); ** 19 reads mapped in single sample. Most likely artifact
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