Figure 1.
Representation of barley grain tissues, morphology of differentiating ETCs and cryosections after microdissection.
(A) Median cross-section of a barley grain at 7 DAF depicting the main grain tissues. (B–D) ETC region in detail at 3, 5 and 7 DAF. Note that in (B) details of a barley grain between 3 and 4 DAF are represented to illustrate the beginning of the cellularization process. (E–G) Representative examples of cryosections after isolation of the ETC region. ETC: endosperm transfer cells, MVB: main vascular bundles, NP: nucellar projection, P: pericarp, SE: starchy endosperm. Bars represent 100 µm.
Table 1.
Comparison of ETC transcriptome data to general and plant-specific databases.
Figure 2.
Comparison of 454 sequences with public barley sequence information.
Venn diagram displays the number of overlapping sequences between ETC-454 contigs and sequences of the HarvEST assembly 35 (BlastN <1E-20). Commonalities using tBlastX searches with different stringencies are given at the bottom.
Figure 3.
The multilevel process of filtering for functional assignment of ETC transcripts not present in HarvEST35.
Dark shaded regions in pie charts indicate the number of hits in databases whereas light coloured regions display the number of transcripts not fulfilling the respective similarity criteria. Non-hitting sequences were further compared to the next protein database with decreasing stringencies (BlastX cut-offs 1E-20 to 1E-10). InterPro search for functional domains was employed for the sequences without sufficient similarity.
Table 2.
A list of putative TCS components found in the transcriptome of differentiating barley endosperm transfer cells (ETCs) by 454 sequencing.
Figure 4.
Phylogenetic relationship of barley TCS elements and counterparts of Arabidopsis thaliana and Oryza sativa.
Phylogenetic trees are based on inferred amino acid sequences from complemented barley 454 contigs (see information in Table 2) and full-length amino acid sequences adopted from different databases (TAIR release 9, Rice Genome Annotation project and NCBI Protein database). Multiple alignments were performed using ClustalW algorithm with BLOSUM protein weight matrix (DNAstar software). Barley sequences are highlighted by blue boxes. Elements from other species are identified by chromosome loci or GenBank accession number. (A) Histidine kinases, colours indicate different subgroups. Arabidopsis PDK was used as outgroup. (B) HPt elements, protein sequences of Zea mays and Triticum aestivum were additionally included in the alignment. Yeast HPt protein YPD1 was used as outgroup. (C–E) Type-A, -B, -C response regulators, amino acid sequences of selected maize response regulators were included in the alignment. For reasons of simplicity not all Arabidopsis and rice elements were included in the phylogenetic trees.
Figure 5.
Transcript levels of barley TCS elements in ETCs and grain tissues determined by qRT-PCR analyses.
At the top, cross-sections of a barley grain at 3 DAF are given as an example for targets used in qRT-PCR analyses. ETC regions at different stages were isolated by LMPC (encircled in green) and the complete leftovers of the tissue sections, e.g. the whole grain tissues without the ETCs, have been collected separately (encircled in purple). Relative expression levels are illustrated by colour code: white- not detectable (nd), light blue- >0–<0.1/low, blue- 0.1–<1.0/intermediate, pink- 1.0–5.0/high, red- >5.0/very high. Assignment of TCS elements according to Table 2.
Figure 6.
Expression profiles of barley TCS elements in ETCs as determined by qRT-PCR analyses.
Expression profiles of candidate genes were grouped by clusters. Cluster 1, genes highly expressed at 3 DAF (and 5 DAF); cluster 2, genes showing a peak of expression at 5 DAF; cluster 3, genes with highest expression levels at 7 DAF. Expression levels are given in the log10 scale, values and standard deviations were calculated from three replicates.
Table 3.
Conserved cis-regulatory elements putatively implicated in ABA signalling pathways.
Figure 7.
Expression profiles of putative ABA-related transcription factors, -signalling elements and ABA-induced transcripts.
Transcript levels in barley ETCs were determined by qRT-PCR analyses. Relative expression is given in the log10 scale, values and standard deviations were calculated from three replicates. Transcripts are assigned to gene families according to sequence homology and functional domains, numbers indicate the contig identifier in the 454 transcriptome assembly.