- Title
- An investigation of transcriptional regulation of wall ingrowth formation in plant transfer cells
- Creator
- Arun Chinnappa, Kiruba Shankari
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2016
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Transfer cells (TCs) form wall ingrowths as an anatomical adaption to increase plasma membrane surface area to achieve enhanced rates of nutrient transport at apoplasmic-symplasmic bottlenecks in nutrient transport pathways in plants. TC formation occurs widely across the plant kingdom and also in algae and fungi. TCs can form as a consequence of normal development processes in plants or can be induced to form by biotic or abiotic stress. Various signaling pathways involving auxin, ethylene and reactive oxygen species are known to induce the formation of wall ingrowths in TCs, and abiotic factors such as cold and high light, the latter acting through jasmonic acid signaling, are reported to influence wall ingrowth deposition. Transcript profiling studies have shown that hundreds of genes are likely involved in regulating the biosynthesis of wall ingrowths, however, despite the importance of this process to nutrient transport in plants, little is known of the genetic regulation of wall ingrowth deposition. Hence, in this thesis, a combination of approaches were used in an attempt to identify transcription factors regulating wall ingrowth formation in TCs. First, a non-targeted strategy employing RNA sequencing (RNA-Seq) of epidermal TCs in cotyledons of Vicia faba was used to identify transcription factors (TFs) and other cohorts of genes that showed extensive transcriptional regulation in epidermal cells undergoing trans-differentiation to become TCs. A role for selected V. faba TFs in regulating wall ingrowth deposition was then investigated by examining T-DNA insertional mutants of orthologous genes in Arabidopsis thaliana. V. faba is a cool season grain-legume crop used extensively for human nutrition and livestock fodder and also as a model to study aspects of plant physiology, including epidermal TC development in cotyledons. Despite its agricultural importance, however, very limited genomic information is available for this species, in part due to its extreme genome size of approximately 13 Gb. Therefore, to provide a reference map for subsequent RNA-Seq, a genome-wide de novo assembly was generated by Next Generation Sequencing. Total RNA pooled from different vegetative and reproductive tissues at different stages of development, along with cultured cotyledons induced to form adaxial epidermal TCs, was subjected to deep sequencing using the Illumina Hi-Seq 2000 platform. Sixty five million reads were generated from 100-bp paired-end sequencing and de novo assembly was undertaken from this read set using CLC Genomics Workbench. An optimum assembly using word size (K-mer) 47 and bubble size 300 was obtained which yielded 21,297 transcripts, of which 80.6% (17,160 contigs) were functionally annotated with GO terms. The assembly was validated by comparing against sequenced V. faba mRNAs from NCBI which also included transcripts known to have role in TC development, as well as sequenced CesA genes. This transcriptome map identified 726 transcription factors representing 31 of the 58 families of known plant TFs. For RNA-Seq analysis of epidermal TC development, total RNA obtained from pooled epidermal and storage parenchyma tissues of cotyledons cultured for 0, 3, 9 and 24 h was subjected to 100-bp paired-end sequencing which yielded 368 million reads in total (42-48 million reads per sample). Log₂ fold-changes in RPKM values were calculated for both epidermal and storage parenchyma samples. Using stringent thresholds of Log₂ fold-change differences in epidermal and storage parenchyma samples, differentially expressed transcripts specific to epidermal TCs were identified. This analysis identified 444 transcripts that were up-regulated specifically in epidermal TCs, of which 22 were annotated as transcription factors. Prominent amongst this list were members of the MYB and ERF families, along with a trihelix GT-3B TF which ranked as the highest fold-change transcript in epidermal cells undergoing trans-differentiation to become epidermal TCs. A similar analysis was carried out to identify 172 epidermal-specific down-regulated transcripts, of which 10 were annotated as transcription factors. This cohort included homeobox leucine zipper proteins, MADS-box and zinc finger CCCH-domain TFs. In addition to the categories of epidermal-specific up- or down-regulated genes, a third class of transcripts defined as “epidermal-enhanced” were identified. In this category, 198 transcripts, 20 of which were transcription factors, showed a ≥25-times higher fold-change in epidermal tissue compared to storage parenchyma across cotyledon culture. Of the 20 transcription factors defined as “epidermal-enhanced”, almost half were members of the WRKY family, consistent with the induction of epidermal TCs in V. faba cotyledons representing a stress response. A manuscript describing this RNA-Seq study has been submitted to Frontiers in Plant Science and Chapter 3 of this thesis comprises the submitted manuscript plus additional unpublished material. Since no transformation system is available for V. faba, to test the role of transcription factors identified from the RNA-Seq study, a strategy involving analysis of orthologous genes in Arabidopsis thaliana (Arabidopsis) was adopted. Phloem parenchyma (PP) cells in minor veins of Arabidopsis leaves trans-differentiate to become PP TCs, and wall ingrowth deposition in these TCs can be visualised by confocal microscopy following pseudo-Schiff staining using propidium iodide. This development enabled phenotypic screening of wall ingrowth deposition in PP TCs in T-DNA insertional mutants of Arabidopsis genes orthologous to those identified in V. faba. Arabidopsis orthologs of MYB (AtMYB20), WRKY (AtWRKY33, AtWRKY41, ATWRKY48) and trihelix transcription factors (AtGT-3B) were identified along with each paralog (AtMYB43, AtWRKY44, AtWRKY53, AtWRKY57 and AtGT-3A), respectively. Double mutants were created for all pairs except the trihelix transcription factors and then scored for wall ingrowth deposition in PP TCs by confocal microscopy of propidium iodide-stained leaves. However, no detectable change in the extent of wall ingrowth deposition was seen in any of the four double mutants analysed by this approach. Similarly, no change was seen in any of the WRKY double mutants exposed to low temperature, a growth condition reported to induce wall ingrowth deposition in PP TCs of minor veins. Single T-DNA insertional mutants in either AtGT-3B or AtGT-3A also failed to show any phenotype associated with wall ingrowth deposition in PP TCs as revealed by confocal microscopy. AtMYB20, and its closest paralog, AtMYB43, are known components of the transcriptional cascade required for secondary wall formation in Arabidopsis. Therefore the atmyb20/atmyb43 double mutant generated in this study was also examined for general effects on secondary wall deposition in Arabidopsis. While Fourier transform infrared (FTIR) spectroscopy revealed quantitative differences in carbohydrate profiles of stem and leaf extracts from wild type and the double mutant, and phloroglucinol staining of stem sections revealed somewhat reduced levels of secondary wall deposition in the double mutant, surprisingly, no visible phenotype was detected in tracheary elements adjacent to PP TCs in leaf minor veins. This preliminary analysis of the atmyb20/atmyb43 double mutant therefore suggests differences in secondary wall biogenesis in vascular tissue between stem and minor veins. The phenotypic analysis in Arabidopsis of orthologous transcription factors discovered by the RNA-Seq study, as well as the more detailed analysis of the atmyb20/atmyb43 double mutant is reported in Chapter 4 of this thesis. In summary, this study generated a genome-wide de novo transcriptome map of V. faba which not only enriches the genetic resources available for this important grain legume, but also served as a template for subsequent transcript profiling by RNA-Seq. Analysis of epidermal TC development in cotyledons of V. faba by RNA-Seq identified cohorts of transcription factors, amongst other genes, that showed strong epidermal-specific or epidermal-enhanced transcriptional regulation across epidermal TC development, thus suggesting regulatory roles for these transcription factors in this process. However, a genetic analysis of orthologous genes in Arabidopsis, including genes known to regulate secondary wall deposition, failed to establish a role for these transcription factors in regulating TC development. A discussion of the strengths and limitations of the approach used in this study to identify putative transcriptional regulators of TC development is presented in Chapter 4. While this approach was ultimately unsuccessful, the study nonetheless identified numerous transcription factors in V. faba that warrant further investigation as putative regulators of the transcriptional cascades required for wall ingrowth building in TCs. Development of a robust transformation protocol for V. faba or disruption of gene activity by amiRNA constructs delivered by biolistic bombardment into cotyledons may offer a way forward to achieve this goal.
- Subject
- transfer cells; vicia faba; arabidopsis; RNA-Seq; transcriptional regulation
- Identifier
- http://hdl.handle.net/1959.13/1314410
- Identifier
- uon:22763
- Rights
- Copyright 2016 Kiruba Shankari Arun Chinnappa
- Language
- eng
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