Wheat Zinc Finger Proteins Potentially Involved in Drought Adaptation

Mr Wing-hei Kam (2008). Wheat Zinc Finger Proteins Potentially Involved in Drought Adaptation PhD Thesis, School of Integrative Biology, The University of Queensland.

       
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Author Mr Wing-hei Kam
Thesis Title Wheat Zinc Finger Proteins Potentially Involved in Drought Adaptation
School, Centre or Institute School of Integrative Biology
Institution The University of Queensland
Publication date 2008-05
Thesis type PhD Thesis
Supervisor Prof. Peter M. Gresshoff
Dr. Gang-ping Xue
Dr. Ray Shorter
Total pages 164
Total colour pages 20
Total black and white pages 144
Subjects 270000 Biological Sciences
Formatted abstract Water deficit is one of the major factors limiting wheat productivity in Australia. In
response to water deficit, plants have developed various strategies to cope with stress conditions
through a combination of metabolic, physiological and morphological changes. These drought
adaptive changes rely largely on alterations in gene expression. Therefore, transcriptional
regulators play an important role in drought adaptation. In this thesis regulatory genes from two
zinc finger families (RING and Q-type C2H2 zinc finger) from Triticum aestivum (bread wheat)
were investigated through extensive analyses of their expression profiles, sequence characterisation
and DNA-binding specificity with a primary focus on identifying zinc finger genes involved in
wheat drought adaptation.
RING zinc finger proteins are known for their regulatory role in ubiquitin-mediated protein
degradation and participate in interaction with other regulatory proteins. Seven RING zinc finger
genes from T. aestivum (designated TaRZF) were analysed for expression profiles in various
organs (leaf, root, stem, spike, endosperm and embryo), during leaf development and aging, as well
as in wheat response to drought stress. Expression levels of six of these seven genes varied
markedly among the six organs examined. All seven genes changed their expression levels in the
leaf from the growing to senescing stage. Four genes were responsive to drought stress. A RINGH2
zinc finger gene, TaRZF70, showed differential response to water deprivation, namely upregulation
in the leaf and down-regulation in the root. This differential response was also observed
in abscisic acid (ABA)-treated plants. Sequence analysis revealed that TaRZF70 contains four
RING-H2 domains. Bioinformatic analysis showed a transmembrane domain at the N-terminus of
TaRZF70 and its RING domains sharing homology with previously characterised membraneassociated
ubiquitin ligases. These results indicate that some of these RING zinc finger genes are
involved in wheat response to drought and potentially participate in modulating the levels of some
proteins involved in wheat drought adaptation through the ubiquitin-mediated protein degradation
pathway.
Q-type C2H2 zinc finger proteins contain a plant-specific QALGGH motif in the C2H2
domain and form a subfamily of transcription factors. A total of 47 expressed Q-type C2H2 zinc
finger genes in T. aestivum (designated TaZFP) were identified from the current databases. Protein
sequence analysis for the presence of ERF-associated amphiphilic repressor (EAR) motif
sequences from known transcriptional repressors revealed that 26% of the TaZFP subfamily
members contain an EAR motif. Quantitative RT-PCR analysis of the mRNA distribution of 44
TaZFP genes in various organs revealed that 30 genes were predominantly expressed in the roots.
The majority of the TaZFP genes showed significant changes in their mRNA levels during leafdevelopment and aging. Expression of 37 TaZFP genes in the leaves and roots responded to
drought stress in at least one organ with 74% of the drought-responsive TaZFP genes being downregulated
in the drought-stressed roots. In contrast, only six out of the 44 TaZFP genes showed
expression changes in the leaves with sucrose treatment. Expression of 50% of the droughtresponsive
TaZFP genes (16 genes analysed) did not respond to ABA treatment in the leaves,
indicating that some TaZFP genes are involved in ABA-independent signalling pathways. These
results indicate that the Q-type TaZFP subfamily is likely to have an important role in wheat roots
and is enriched with members that are potentially involved in regulating cellular activities during
drought stress.
The DNA-binding specificity of three representative drought-responsive Q-type 2-fingered
ZFP proteins from T. aestivum (TaZFP22, TaZFP34 and TaZFP46h) was investigated. Analysis of
DNA-binding activity revealed that all three TaZFPs were capable of binding to a dimer of an EP1
motif from the promoter of petunia 5-enolpyruvylshikimate-3-phosphate synthase. Systematic base
substitution analysis of the EP1 motif showed that TaZFP46h bound preferably to a dimer of a Grich
motif (5’-GGGAGTGA). Comparative analysis of DNA-binding activity showed a
considerable overlapping in the DNA-binding specificity among TaZFP22, TaZFP34 and
TaZFP46h. Promoter sequence search identified three potential DNA-binding sites, but only with
low or moderate binding affinity for these TaZFP proteins, in the promoter regions of wheat genes
encoding delta-1-pyrroline-5-carboxylate dehydrogenase (TaP5CDH), starch branching enzyme
IIa (TaSbeIIa) and Heading date 1 (Hd1-3). Quantitative RT-PCR analysis showed that these genes
were also drought-responsive. In particular, TaP5CDH, which is known for its role in the removal
of the toxic intermediate metabolite of proline metabolism in plants, showed a clear pattern of
drought up-regulation in both roots and leaves. However, the identification of a suite of genes
targeted by these drought-responsive TaZFP proteins has to await the availability of a substantial
amount of the wheat genome sequence.
In conclusion this study has made the following achievements: (1) characterised drought
adaptation- and growth-related expression profiles of seven members of the RING zinc finger
family and 44 members of the Q-type C2H2 zinc finger family from wheat, (2) identified members
from these Q-type C2H2 zinc finger genes that are involved in drought response and those that are
potentially involved in vegetative growth, and (3) elucidated the DNA-binding specificity of
drought-responsive 2-fingered TaZFP proteins. These results provide fundamental molecular
information towards understanding of the biological roles of these two important zinc finger
families in wheat.
Keyword wheat, zinc finger, drought, developmental, DNA binding
Additional Notes Pages to be printed in colour: 23, 26, 27, 44, 45, 48, 51, 56, 57, 86, 107, 109, 110, 113, 115, 117, 118, 159, 160, 161

 
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Created: Tue, 18 Nov 2008, 16:34:58 EST by Mr Wing-hei Kam on behalf of Library - Information Access Service