Molecular Analysis of Bud Outgrowth in Monocotyledonous Plants

Chuong Ngo (2011). Molecular Analysis of Bud Outgrowth in Monocotyledonous Plants PhD Thesis, School of Biological Sciences, The University of Queensland.

       
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Author Chuong Ngo
Thesis Title Molecular Analysis of Bud Outgrowth in Monocotyledonous Plants
School, Centre or Institute School of Biological Sciences
Institution The University of Queensland
Publication date 2011-11
Thesis type PhD Thesis
Supervisor A/Prof Christine Beveridge
Dr Prakash Lakshmanan
Dr Scott Hermann
Total pages 181
Total colour pages 15
Total black and white pages 166
Language eng
Subjects 060702 Plant Cell and Molecular Biology
060703 Plant Developmental and Reproductive Biology
060705 Plant Physiology
Abstract/Summary Shoot architecture in plants is largely shaped through shoot branching processes. Considerable progress has been made in elucidating the regulatory mechanisms of branching in dicot species, however comparatively little has been done in monocot species. In dicots CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) encodes an enzyme important for the synthesis of the carotenoid-derived shoot branching inhibition signal, strigolactone. Dicot CCD8s involved in branching include Arabidopsis MAX4, pea RMS1 and petunia DAD1. Unlike dicots, which have only one CCD8 locus, two CCD8 homologs have been identified from the monocot plants rice, sugarcane and sorghum. The rice CCD8 paralogs D10 and D10like share 72.3% similarity to each other at the nucleotide level; at the protein level similarity is 72.6% positives and 61% identity. D10 was found to be the functional equivalent of MAX4/RMS1/DAD1 in rice through branching mutant analysis (Arite et al., 2007). In this thesis, the involvement of D10 and D10like in monocot shoot branching was examined through a different approach. Using rice as the experimental system, D10 and D10like specific hairpin RNA (hpRNA) constructs were used to knock-down these genes. Full-length D10 and D10like coding sequences were also used in over-expression lines. Expression of key genes known to be involved in branching regulation were studied in D10 and D10like knock-down lines to ascertain the impact of D10 or D10like expression on their activity. To test if a dicot-like branching regulatory network is operational in rice, the response of D10 and D10like to decapitation and their interaction with auxin was observed. In addition, the functions of D10 and D10like were tested in a dicot species, by overexpressing in Arabidopsis wild-type and max4 mutant backgrounds. Analysis of D10 and D10like over-expression and knock-down lines of rice confirmed that D10 is the ortholog of MAX4/RMS1/DAD1; however the role of D10like is unclear. Multiple sequence alignments showed D10 clustered with MAX4/RMS1/DAD1 and maize, sorghum and sugarcane CCD8s. Rice D10like clustered with D10like homologs from sugarcane and sorghum. Altering D10 function in rice using hpRNA technology increased tiller number and decreased plant height. No changes were found for tiller number, plant height, leaf morphology or biomass in the D10like hpRNA line. The D10-D10like-double hpRNA line showed no additive phenotype when compared to the D10 hpRNA line. In wild-type rice expression of D10 was highest in root tips and in the root-shoot transition area. D10like expression was found to be highest in the root-shoot area. Expression of both genes was undetectable in leaf tissue. A decrease in expression of D10 was found after the decapitation of the apical meristem and also when auxin transport was reduced in intact plants using the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA). Contrary to the results in pea, exogenous application of auxin after decapitation could not restore D10 expression levels in rice. However, root-fed auxin has been shown to up-regulate D10 expression in intact rice plants (Arite et al., 2007). The inability of exogenous auxin to restore D10 transcript level in a decapitated plant may be due to an over-riding effect of wounding. Transcript levels of D10like also decreased in response to decapitation and could not be restored by exogenous auxin application. However, D10like transcript increased when auxin transport was reduced in intact plants through the use of NPA. Interestingly, D3/OsMAX2 and OsTB1 which are both involved in branching regulation showed increased transcript levels after decapitation. D10 and D10like could not restore the phenotype of a highly branched max4 Arabidopsis mutant back to that of a non-branching WT, indicating that they may not be functional in dicots. This was unexpected as rice HTD1, also involved in strigolactone synthesis, rescued the Arabidopsis max3 mutant (Zou et al., 2006). Both D10 and D10like had no effect on branching when over-expressed in an Arabidopsis WT background. In previous studies over-expression of native MAX4 did not reduce rosette branch numbers (Bainbridge et al., 2005) and may be due to MAX4 acting post-transcriptionally and increased RNA transcript levels having no effect on branching inhibitor levels. Rice has two CCD8 genes, of which D10 was found to function in branching control. The role of D10like is not known, however it is not involved in vegetative branching control. The study presented here shows that molecular processes involved in controlling shoot branching present in dicot species are also functioning in a monocot plant, rice.
Keyword Rice
Arabidopsis
Shoot Branching
D10
D10like
Auxin
Feedback regulation
Rnai
Additional Notes 28, 53-54, 57-58, 60-61, 68, 71, 94, 98, 105, 112-114

 
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Created: Mon, 18 Jun 2012, 22:04:03 EST by Chuong Ngo on behalf of Library - Information Access Service