Ainsley Chan

Ammonia excretion in the freshwater planarian Schmidtea mediterranea

SUMMARY In aquatic invertebrates, metabolic nitrogenous waste is excreted predominately as ammonia. Very little is known, however, of the underlying mechanisms of ammonia excretion, particularly in freshwater species. Our results indicate that in the non-parasitic freshwater planarian Schmidtea mediterranea, ammonia excretion depends on acidification of the apical unstirred layer of the body surface and consequent ammonia trapping. Buffering of the environment to a pH of 7 or higher decreased the excretion rate. Inhibitor experiments suggested further that the excretion mechanism involves the participation of the V-type H+-ATPase and carbonic anhydrase and possibly also the Na+/K+-ATPase and Na+/H+ exchangers. Alkalinization (pH 8.5, 2 days) of the environment led to a 1.9-fold increase in body ammonia levels and to a downregulation of V-ATPase (subunit A) and Rh-protein mRNA. Further, a 2 day exposure to non-lethal ammonia concentrations (1 mmol l−1) caused a doubling of body ammonia levels and led to an increase in Rh-protein and Na+/K+-ATPase (α-subunit) mRNA expression levels. In situ hybridization studies indicated a strong mRNA expression of the Rh-protein in the epidermal epithelium. The ammonia excretion mechanism proposed for S. mediterranea reveals striking similarities to the current model suggested to function in the gills of freshwater fish.

Altered seed oil and glucosinolate levels in transgenic plants overexpressing the Brassica napus SHOOTMERISTEMLESS gene.

SHOOTMERISTEMLESS (STM) is a homeobox gene conserved among plant species which is required for the formation and maintenance of the shoot meristem by suppressing differentiation and maintaining an undetermined cell fate within the apical pole. To assess further the role of this gene during seed storage accumulation, transgenic Brassica napus (Bn) plants overexpressing or down-regulating BnSTM under the control of the 35S promoter were generated. Overexpression of BnSTM increased seed oil content without affecting the protein and sucrose level. These changes were accompanied by the induction of genes encoding several transcription factors promoting fatty acid (FA) synthesis: LEAFY COTYLEDON1 (BnLEC1), BnLEC2, and WRINKLE1 (BnWRI1). In addition, expression of key representative enzymes involved in sucrose metabolism, glycolysis, and FA biosynthesis was up-regulated in developing seeds ectopically expressing BnSTM. These distinctive expression patterns support the view of an increased carbon flux to the FA biosynthetic pathway in developing transformed seeds. The overexpression of BnSTM also resulted in a desirable reduction of seed glucosinolate (GLS) levels ascribed to a transcriptional repression of key enzymes participating in the GLS biosynthetic pathway, and possibly to the differential utilization of common precursors for GLS and indole-3-acetic acid synthesis. No changes in oil and GLS levels were observed in lines down-regulating BnSTM. Taken together, these findings provide evidence for a novel function for BnSTM in promoting desirable changes in seed oil and GLS levels when overexpressed in B. napus plants, and demonstrate that this gene can be used as a target for genetic improvement of oilseed species.

Transcriptome atlas of the Arabidopsis funiculus – a study of maternal seed subregions

The funiculus anchors the structurally complex seed to the maternal plant, and is the only direct route of transport for nutrients and maternal signals to the seed. While our understanding of seed development is becoming clearer, current understanding of the genetics and cellular mechanisms that contribute to funiculus development is limited. Using laser microdissection combined with global RNA-profiling experiments we compared the genetic profiles of all maternal and zygotic regions and subregions during seed development. We found that the funiculus is a dynamic region of the seed that is enriched for mRNAs associated with hormone metabolism, molecular transport, and metabolic activities corresponding to biological processes that have yet to be described in this maternal seed structure. We complemented our genetic data with a complete histological analysis of the funiculus from the earliest stages of development through to seed maturation at the light and electron microscopy levels. The anatomy revealed signs of photosynthesis, the endomembrane system, cellular respiration, and transport within the funiculus, all of which supported data from the transcriptional analysis. Finally, we studied the transcriptional programming of the funiculus compared to other seed subregions throughout seed development. Using newly designed in silico algorithms, we identified a number of transcriptional networks hypothesized to be responsible for biological processes like auxin response and glucosinolate biosynthesis found specifically within the funiculus. Taken together, patterns of gene activity and histological observations reveal putative functions of the understudied funiculus region and identify predictive transcriptional circuits underlying these biological processes in space and time.

Tissue-specific laser microdissection of the Brassica napus funiculus improves gene discovery and spatial identification of biological processes

Highlight Tissue-specific transcriptomic analysis reveals biological processes contributing to the development of the epidermis, cortex, and vasculature, and how these tissues contribute to the development and function of the canola funiculus.

Vitamin C deficiency improves somatic embryo development through distinct gene regulatory networks in Arabidopsis

Summary Depletion of cellular vitamin C improves somatic embryogenesis in Arabidopsis. Improved embryo number and quality is through changes in gene regulatory network activation and cellular architecture.

Interactions between Arabidopsis DNA repair genes UVH6, DDB1A, and DDB2 during abiotic stress tolerance and floral development

Plants must protect themselves from a spectrum of abiotic stresses. For example, the sun is a source of heat, intense light, and DNA-damaging ultraviolet (UV) rays. Damaged DNA binding protein 1A (DDB1A), DDB2, and UV hypersensitive 6 (UVH6)/XPD are all involved in the repair of UV-damaged DNA - DDB1A and DDB2 in the initial damage recognition stage, while the UVH6/XPD helicase unwinds the damaged strand. We find that, as predicted, Arabidopsis ddb1a and ddb2 mutants do not affect uvh6/xpd UV tolerance. In addition, uvh6 is heat sensitive, and ddb1a and ddb2 weakly enhance this trait. The uvh6 ddb1a and uvh6 ddb2 double mutants also exhibit sensitivity to oxidative stress, suggesting a role for DDB1 complexes in heat and oxidative stress tolerance. Finally, we describe a new uvh6 phenotype, the low penetrance production of flowers with five petals and five sepals. ddb1a and ddb2 suppress this phenotype in uvh6 mutants. Interestingly, heat treatment also induces five-petalled flowers in the ddb1a and ddb2 single mutants. Thus UVH6, DDB1A, and DDB2 all contribute to UV tolerance, heat tolerance and floral patterning.

Gene expression analysis in microdissected shoot meristems of Brassica napus microspore-derived embryos with altered SHOOTMERISTEMLESS levels.

Altered expression of Brassica napus (Bn) SHOOTMERISTEMLESS (STM) affects the morphology and behaviour of microspore-derived embryos (MDEs). While down-regulation of BnSTM repressed the formation of the shoot meristem (SAM) and reduced the number of Brassica MDEs able to regenerate viable plants at germination, over-expression of BnSTM enhanced the structure of the SAM and improved regeneration frequency. Within dissected SAMs, the induction of BnSTM up-regulated the expression of many transcription factors (TFs) some of which directly involved in the formation of the meristem, i.e. CUP-SHAPED COTYLEDON1 and WUSCHEL, and regulatory components of the antioxidant response, hormone signalling, and cell wall synthesis and modification. Opposite expression patterns for some of these genes were observed in the SAMs of MDEs down-regulating BnSTM. Altered expression of BnSTM affected transcription of cell wall and lignin biosynthetic genes. The expression of PHENYLALANINE AMMONIA LYASE2, CINNAMATE 4-4HYDROXYLASE, and CINNAMYL ALCOHOL DEHYDROGENASE were repressed in SAMs over-expressing BnSTM. Since lignin formation is a feature of irreversible cell differentiation, these results suggest that one way in which BnSTM promotes indeterminate cell fate may be by preventing the expression of components of biochemical pathways involved in the accumulation of lignin in the meristematic cells. Overall, these studies provide evidence for a novel function of BnSTM in enhancing the quality of in vitro produced meristems, and propose that this gene can be used as a potential target to improve regeneration of cultured embryos.

Chalazal seed coat development in Brassica napus

The chalazal seed coat (CZSC) is a maternal subregion adjacent to the funiculus which serves as the first point of entry into the developing seed. This subregion is of particular interest in Brassica napus (canola) because of its location within the seed and its putative contribution to seed filling processes. In this study, the CZSC of canola was characterized at an anatomical and molecular level to (i) describe the cellular and subcellular features of the CZSC throughout seed development, (ii) reveal cellular features of the CZSC that relate to transport processes, (iii) study gene activity of transporters and transcriptional regulators in the CZSC subregion over developmental time, and (iv) briefly investigate the contribution of the A and C constituent genomes to B. napus CZSC gene activity. We found that the CZSC contains terminating ends of xylem and phloem as well as a mosaic of endomembrane and plasmodesmatal connections, suggesting that this subregion is likely involved in the transport of material and information from the maternal tissues of the plant to other regions of the seed. Laser microdissection coupled with quantitative RT-PCR identified the relative abundance of sugar, water, auxin and amino acid transporter homologs inherited from the constituent genomes of this complex polyploid. We also studied the expression of three transcription factors that were shown to co-express with these biological processes providing a preliminary framework for the regulatory networks responsible for seed filling in canola and discuss the relationship of the CZSC to other regions and subregions of the seed and its role in seed development.

Predicting transcriptional circuitry underlying seed coat development.

Filling, protection, and dispersal of angiosperm seeds are largely dependent on the development of the maternally derived seed coat. The development of the seed coat in plants such as Arabidopsis thaliana and Glycine max (soybean) is regulated by a complex network of genes and gene products responsible for the establishment and identity of this multicellular structure. Recent studies support the hypothesis that the structure, development, and function of the seed coat are under the control of transcriptional regulators that are specified in space and time. Furthermore, these transcriptional regulators can act in combination to orchestrate the expression of large gene sets. We discuss the underlying transcriptional circuits of the seed coat sub-regions through the interrogation of large-scale datasets, and also provide some ideas on how the identification and analysis of these datasets can be further improved in these two model oilseed systems.

Tissue-specific localization of polyketide synthase and other associated genes in the lichen, Cladonia rangiferina, using laser microdissection

The biosynthesis of two polyketides, atranorin and fumarprotocetraric acid, produced from a lichen-forming fungus, Cladonia rangiferina (L.) F. H. Wigg. was correlated with the expression of eight fungal genes (CrPKS1, CrPKS3, CrPKS16, Catalase (CAT), Sugar Transporter (MFsug), Dioxygenase (YQE1), C2H2 Transcription factor (C2H2), Transcription Factor PacC (PacC), which are thought to be involved in polyketide biosynthesis, and one algal gene, NAD-dependent deacetylase sirtuin 2 (AsNAD)), using laser microdissection (LMD). The differential gene expression levels within the thallus tissue layers demonstrate that the most active region for potential polyketide biosynthesis within the lichen is the outer apical region proximal to the photobiont but some expression also occurs in reproductive tissue. This is the first study using laser microdissection to explore gene expression of these nine genes and their location of expression; it provides a proof-of-concept for future experiments exploring tissue-specific gene expression within lichens; and it highlights the utility of LMD for use in lichen systems.