BACKGROUND: Identification of transgene insertion sites in plant genomes has practical implications for crop breeding and is a stepping stone to analyze transgene function. However, single copy sequences are not always easy to localize in large plant genomes by standard approaches. RESULTS: We employed flow cytometric chromosome sorting to determine chromosomal location of barley sucrose transporter construct in three transgenic lines of common wheat. Flow-sorted chromosomes were used as template for PCR and fluorescence in situ hybridization to identify chromosomes with transgenes. The chromosomes carrying the transgenes were then confirmed by PCR using DNA amplified from single flow-sorted chromosomes as template. CONCLUSIONS: Insertion sites of the transgene were unambiguously localized to chromosomes 4A, 7A and 5D in three wheat transgenic lines. The procedure presented in this study is applicable for localization of any single-copy sequence not only in wheat, but in any plant species where suspension of intact mitotic chromosomes suitable for flow cytometric sorting can be prepared.
- Klíčová slova
- Flow cytometric sorting, Hordeum vulgare, HvSUT1, Single chromosome amplification, Transgene localization, Triticum aestivum,
- Publikační typ
- časopisecké články MeSH
The shape of the maternal pericarp affects cereal grain mass and yield. Pericarp growth was analysed by magnetic resonance imaging (MRI), revealing topological maps of mobile water in developing pericarp of barley (Hordeum vulgare) and displaying tissue regions actively elongating in specific temporal-spatial patterns. Correlation analysis of MRI signals and growth rates reveals that growth in length is mediated by dorsal and also lateral rather than ventral regions. Growth in thickness is related to ventral regions. Switching from dorsal to ventral growth is associated with differential expression of axial regulators of the HD-ZipIII and Kanadi/Ettin types, and NPH3 photoreceptors, suggesting light-mediated auxin re-distribution. Auxin increases with the highest levels in the basal pericarp at 6 days after fertilization (DAF), together with transcriptionally up-regulated auxin transport and signalling. Gibberellin biosynthesis is transcriptionally up-regulated only later, and levels of bioactive gibberellins increase from 7 to 13 DAF, with higher levels in ventral than dorsal regions. Differential gene expression related to cell expansion indicates genes related to apoplast acidification, wall relaxation, sugar cleavage, water transport, and cell wall biosynthesis. Candidate genes potentially involved in pericarp extension are distinguished by their temporal expression, representing potential isoforms responsible for dorsal-mediated early growth in length or ventral-mediated late growth in thickness.
- Klíčová slova
- Auxin, barley pericarp, cell expansion, directed growth, gibberellic acid, grain length, growth dynamics, magnetic resonance imaging, transcript profiling.,
- MeSH
- gibereliny metabolismus MeSH
- ječmen (rod) genetika růst a vývoj metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- magnetická rezonanční tomografie MeSH
- regulace genové exprese u rostlin * MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- semena rostlinná genetika růst a vývoj metabolismus MeSH
- stanovení celkové genové exprese MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- gibereliny MeSH
- kyseliny indoloctové MeSH
- regulátory růstu rostlin MeSH
- rostlinné proteiny MeSH
In cereal grains, the maternal nucellar projection (NP) constitutes the link to the filial organs, forming a transfer path for assimilates and signals towards the endosperm. At transition to the storage phase, the NP of barley (Hordeum vulgare) undergoes dynamic and regulated differentiation forming a characteristic pattern of proliferating, elongating, and disintegrating cells. Immunolocalization revealed that abscisic acid (ABA) is abundant in early non-elongated but not in differentiated NP cells. In the maternally affected shrunken-endosperm mutant seg8, NP cells did not elongate and ABA remained abundant. The amounts of the bioactive forms of gibberellins (GAs) as well as their biosynthetic precursors were strongly and transiently increased in wild-type caryopses during the transition and early storage phases. In seg8, this increase was delayed and less pronounced together with deregulated gene expression of specific ABA and GA biosynthetic genes. We concluded that differentiation of the barley NP is driven by a distinct and specific shift from lower to higher GA:ABA ratios and that the spatial-temporal change of GA:ABA balances is required to form the differentiation gradient, which is a prerequisite for ordered transfer processes through the NP. Deregulated ABA:GA balances in seg8 impair the differentiation of the NP and potentially compromise transfer of signals and assimilates, resulting in aberrant endosperm growth. These results highlight the impact of hormonal balances on the proper release of assimilates from maternal to filial organs and provide new insights into maternal effects on endosperm differentiation and growth of barley grains.
- Klíčová slova
- Assimilate transfer, barley endosperm, gibberellin-to-abscisic acid balances, maternal–filial communication, nucellar projection, seg8 barley mutant.,
- MeSH
- buněčná diferenciace fyziologie MeSH
- gibereliny metabolismus MeSH
- ječmen (rod) metabolismus MeSH
- kyselina abscisová metabolismus MeSH
- regulace genové exprese u rostlin fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- gibereliny MeSH
- kyselina abscisová MeSH
