UV-resistance locus 8 and UV-B specific signaling in Arabidopsis thaliana

UV-B is a natural component of the sunlight spectrum. As a result of the potentially harmful effects of this radiation, plants have evolved a highly effective suit of protective and repair mechanisms. However, the signalling pathways that control such responses are not yet well known. For example while the photoreceptors responsible for red and blue light responses are well characterised, no such UV-B photoreceptor has yet been identified. Despite this particularly large gap in our knowledge, previous work identified the first UV- B specific signalling component which, unlike the more general stress-associated pathways often seen at high doses, specifically regulates expression of genes in response to even very low fluence rates of UV-B. This protein, UV-RESISTANCE LOCUS 8 (UVR8) regulates the induction of a number of photoprotective genes mostly via the transcription factors ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOGUE (HYH). The end result of this pathway is the production of photoprotective compounds such as the flavonoids which enhance a plants ability to withstand UV-B stress. Thus UVR8 promotes plant fitness under these conditions. While we know that UVR8 binds to chromatin in the promoter region of HY5 and that it accumulates in the nucleus under UV-B, many other questions about this particular protein remain unanswered. For example, we do not yet know if UVR8-mediated UV-B signalling involves other factors which interact with UVR8 nor do we understand the mechanism by which UVR8 localisation is mediated. In addition, although we are aware of the importance of UVR8 in UV-B acclimation, it is unclear what roles might be played by other genes and proteins acting independently of this pathway. Therefore, the aims of this study were to investigate low fluence UV-B pathways that may act independently of UVR8 and to further examine the UVR8 protein itself both in terms of its interactions with other proteins and also in the role of the N-terminal region in regulation of its localisation. To achieve the first of these aims, RNA samples derived from plants treated with low fluence UV-B were submitted for microarray analysis. It was initially determined that the total number of genes induced was roughly equal in both low fluence treated samples and also to that found in the previous microarray performed by Brown et al. (2005) at a comparatively higher fluence. Thus, as only 72 genes have currently been linked to UVR8, there do appear to be many low-fluence UV-B induced pathways besides that regulated by UVR8. Several genes were analysed further using RT-PCR and qPCR methods in order to confirm their independence from the UVR8 signalling pathway components as well as assess their dependence on other hypothesised UV-B sensory mechanisms. It was found that while some genes did seem to be expressed independently of known photoreceptors, DNA damage signals as well as UVR8, HY5, HYH and COP1; one gene was expressed in a COP1-dependent but UVR8 independent manner. It therefore appears that at least four classes of genes are induced by UV-B; low fluence UVR8/HY5/HYH independent COP1 dependent, low fluence UVR8/HY5/HYH/COP1 dependent, low fluence UVR8/HY5/HYH/COP1 independent and finally high fluence non- specific signalling. The second portion of this thesis examined the structure and function of UVR8 in greater detail. To assist in this analysis, the BLAST sequence homology tool was used to probe both the Arabidopsis genome and available green plant sequences. It was found that 23 UVR8-like sequences exist in Arabidopsis but none of these appear to have similar N or C-terminal sequences to UVR8. As these two regions have previously been shown to be of vital importance in UVR8 function (Kaiserli and Jenkins, 2008; Kaiserli unpublished data) it is unlikely that any are acting in a redundant fashion to UVR8. A number of similar proteins to UVR8 can be found in other plant species. These potential homologues however fall into two categories based on their closer similarity with either UVR8 or its close homologue in humans REGULATOR OF CHROMATIN CONDENSATION 1 (RCC1). The wide variety of plant species that did show UVR8-like proteins suggests that this particular means of UV-B acclimation may have arisen relatively early with the colonisation of land plants. Interestingly, many of these likely homologues had a conserved N terminal. The N-terminal of UVR8 has previously been show to have a role in UV-B dependent nuclear accumulation (Kaiserli and Jenkins, 2008). This was examined further in Chapter 4 through the generation of a number of deletion and addition constructs in both a stable Arabidopsis uvr8-1 background as well as transiently in tobacco. From analysis of localisation of these constructs via confocal microscopy it was determined that the first 12 amino acids are sufficient but not necessary for nuclear accumulation, while the first 20 appear to be both necessary and sufficient. Indeed, it was shown that the initial 32 amino acids also confer constitutive localisation of a GFP tag in the nucleus regardless of light condition and despite the presence of a nuclear exclusion signal (NES). It therefore appears that this region, which shows strong conservation with UVR8-like proteins in other plant species, is of vital importance to the nuclear accumulation seen under UV-B. Finally, in Chapter 5, the possibility that UVR8 may be acting as part of a complex was explored. This involved use of size exclusion chromatography to provide approximate sizes of the UVR8 protein complex. It was found that native UVR8 appears to exist in a complex of about 70-90 kDa in size. This suggests that at least one other protein interacts stably with UVR8. Other fusion constructs were also analysed in this way, however the results were more difficult to interpret due the apparent artificial dimerisation of the GFP tag. In summary, the work presented here has shown that although UVR8 dependent pathways are predominant, a variety of low fluence UV-B induced genes and pathways may exist. Homology searches and mutational analyses suggest that the N-terminal region of UVR8 plays a critical role in its function and localisation. Finally, size exclusion chromatography suggests that UVR8 forms a complex in vivo with as yet uncharacterised partner proteins. In total these results provide further insight into the mechanisms UVR8 action and highlight new avenues for both UVR8 dependent and independent UV-B signalling

The impact of light and temperature on chromatin organisation and plant adaptation

Light and temperature shape the developmental trajectory and morphology of plants. Changes in chromatin organisation and nuclear architecture can modulate gene expression and lead to short and long-term plant adaptation to the environment. Here, we review recent reports investigating how changes in chromatin composition, structure and topology modulate gene expression in response to fluctuating light and temperature conditions resulting in developmental and physiological responses. Furthermore, the potential application of novel revolutionary techniques such as RNA and padlock fluorescence in situ hybridization (FISH), and Hi-C to study the impact of environmental stimuli such as light and temperature on nuclear compartmentalisation in plants is discussed

Multigene manipulation of photosynthetic carbon assimilation increases CO2 fixation and biomass yield in tobacco

Over the next 40 years it has been estimated that a 50% increase in the yield of grain crops such as wheat and rice will be required to meet the food and fuel demands of the increasing world population. Transgenic tobacco plants have been generated with altered combinations of sedoheptulose-1,7-bisphosphatase, fructose-1,6-bisphosphate aldolase, and the cyanobacterial putative-inorganic carbon transporter B, ictB, of which have all been identified as targets to improve photosynthesis based on empirical studies. It is shown here that increasing the levels of the three proteins individually significantly increases the rate of photosynthetic carbon assimilation, leaf area, and biomass yield. Furthermore, the daily integrated measurements of photosynthesis showed that mature plants fixed between 12-19% more CO2 than the equivalent wild-type plants. Further enhancement of photosynthesis and yield was observed when sedoheptulose-1,7-bisphosphatase, fructose-1,6-bisphosphate aldolase, and ictB were over-expressed together in the same plant. These results demonstrate the potential for the manipulation of photosynthesis, using multigene-stacking approaches, to increase crop yields

A High-Throughput Method for Illumina RNA-Seq Library Preparation.

With the introduction of cost effective, rapid, and superior quality next generation sequencing techniques, gene expression analysis has become viable for labs conducting small projects as well as large-scale gene expression analysis experiments. However, the available protocols for construction of RNA-sequencing (RNA-Seq) libraries are expensive and/or difficult to scale for high-throughput applications. Also, most protocols require isolated total RNA as a starting point. We provide a cost-effective RNA-Seq library synthesis protocol that is fast, starts with tissue, and is high-throughput from tissue to synthesized library. We have also designed and report a set of 96 unique barcodes for library adapters that are amenable to high-throughput sequencing by a large combination of multiplexing strategies. Our developed protocol has more power to detect differentially expressed genes when compared to the standard Illumina protocol, probably owing to less technical variation amongst replicates. We also address the problem of gene-length biases affecting differential gene expression calls and demonstrate that such biases can be efficiently minimized during mRNA isolation for library preparation

Let it bloom: crosstalk between light and flowering signalling in Arabidopsis

The terrestrial environment is complex, with many parameters fluctuating on daily and seasonal basis. Plants in particular, have developed complex sensory and signalling networks to extract and integrate information about their surroundings, in order to maximise their fitness and mitigate some of the detrimental effects of their sessile lifestyles. Light and temperature each provide crucial insight on the surrounding environment and in combination allow plants to appropriately develop, grow and adapt. Crosstalk between light and temperature signalling cascades allow plants to time key developmental decisions accordingly to ensure they are “in sync” with their environment. In this review, we discuss the major players that regulate light and temperature signalling, and the cross‐talk between them, in reference to a crucial developmental decision faced by plants: to bloom or not to bloom

A New Advanced Backcross Tomato Population Enables High Resolution Leaf QTL Mapping and Gene Identification.

Quantitative Trait Loci (QTL) mapping is a powerful technique for dissecting the genetic basis of traits and species differences. Established tomato mapping populations between domesticated tomato (Solanum lycopersicum) and its more distant interfertile relatives typically follow a near isogenic line (NIL) design, such as the S. pennellii Introgression Line (IL) population, with a single wild introgression per line in an otherwise domesticated genetic background. Here, we report on a new advanced backcross QTL mapping resource for tomato, derived from a cross between the M82 tomato cultivar and S. pennellii This so-called Backcrossed Inbred Line (BIL) population is comprised of a mix of BC2 and BC3 lines, with domesticated tomato as the recurrent parent. The BIL population is complementary to the existing S. pennellii IL population, with which it shares parents. Using the BILs, we mapped traits for leaf complexity, leaflet shape, and flowering time. We demonstrate the utility of the BILs for fine-mapping QTL, particularly QTL initially mapped in the ILs, by fine-mapping several QTL to single or few candidate genes. Moreover, we confirm the value of a backcrossed population with multiple introgressions per line, such as the BILs, for epistatic QTL mapping. Our work was further enabled by the development of our own statistical inference and visualization tools, namely a heterogeneous hidden Markov model for genotyping the lines, and by using state-of-the-art sparse regression techniques for QTL mapping

Regulation of Arabidopsis gene expression by low fluence rate UV-B independently of UVR8 and stress signaling

UV-B exposure of plants regulates expression of numerous genes concerned with various responses. Sudden exposure of non-acclimated plants to high fluence rate, short wavelength UV-B induces expression via stress-related signaling pathways that are not specific to the UV-B stimulus, whereas low fluence rates of UV-B can regulate expression via the UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8). However, there is little information about whether non-stressful, low fluence rate UV-B treatments can activate gene expression independently of UVR8. Here, transcriptomic analysis of wild-type and uvr8 mutant Arabidopsis exposed to low fluence rate UV-B showed that numerous genes were regulated independently of UVR8. Moreover, nearly all of these genes were distinct to those induced by stress treatments. A small number of genes were expressed at all UV-B fluence rates employed and may be concerned with activation of eustress responses that facilitate acclimation to changing conditions. Expression of the gene encoding the transcription factor ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 13 (ANAC13) was studied to characterise a low fluence rate, UVR8-independent response. ANAC13 is induced by as little as 0.1 μmol m−2 s−1 UV-B and its regulation is independent of components of the canonical UVR8 signaling pathway COP1 and HY5/HYH. Furthermore, UV-B induced expression of ANAC13 is independent of the photoreceptors CRY1, CRY2, PHOT1 and PHOT2 and phytochromes A, B, D and E. ANAC13 expression is induced over a range of UV-B wavelengths at low doses, with maximum response at 310 nm. This study provides a basis for further investigation of UVR8 and stress independent, low fluence rate UV-B signaling pathway(s)

Glyceraldehyde-3-phosphate dehydrogenase subunits A and B are essential to maintain photosynthetic efficiency

In plants, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) reversibly converts 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate coupled with the reduction of NADPH to NADP+. The GAPDH enzyme that functions in the Calvin Benson Cycle is assembled either from four glyceraldehyde-3-phosphate dehydrogenase A subunits (GAPA) proteins forming a homotetramer (A4) or from two GAPA and two glyceraldehyde-3-phosphate dehydrogenase B subunit (GAPB) proteins forming a heterotetramer (A2B2). The relative importance of these two forms of GAPDH in determining the rate of photosynthesis is unknown. To address this question, we measured the photosynthetic rates of Arabidopsis (Arabidopsis thaliana) plants containing reduced amounts of the GAPDH A and B subunits individually and jointly, using T-DNA insertion lines of GAPA and GAPB and transgenic GAPA and GAPB plants with reduced levels of these proteins. Here we show that decreasing the levels of either the A or B subunits decreased the maximum efficiency of CO2 fixation, plant growth, and final biomass. Finally, these data showed that the reduction in GAPA protein to 9% wild-type levels resulted in a 73% decrease in carbon assimilation rates. In contrast, eliminating GAPB protein resulted in a 40% reduction in assimilation rates. This work demonstrates that the GAPA homotetramer can compensate for the loss of GAPB, whereas GAPB alone cannot compensate fully for the loss of the GAPA subunit

Native Environment Modulates Leaf Size and Response to Simulated Foliar Shade across Wild Tomato Species

The laminae of leaves optimize photosynthetic rates by serving as a platform for both light capture and gas exchange, while minimizing water losses associated with thermoregulation and transpiration. Many have speculated that plants maximize photosynthetic output and minimize associated costs through leaf size, complexity, and shape, but a unifying theory linking the plethora of observed leaf forms with the environment remains elusive. Additionally, the leaf itself is a plastic structure, responsive to its surroundings, further complicating the relationship. Despite extensive knowledge of the genetic mechanisms underlying angiosperm leaf development, little is known about how phenotypic plasticity and selective pressures converge to create the diversity of leaf shapes and sizes across lineages. Here, we use wild tomato accessions, collected from locales with diverse levels of foliar shade, temperature, and precipitation, as a model to assay the extent of shade avoidance in leaf traits and the degree to which these leaf traits correlate with environmental factors. We find that leaf size is correlated with measures of foliar shade across the wild tomato species sampled and that leaf size and serration correlate in a species-dependent fashion with temperature and precipitation. We use far-red induced changes in leaf length as a proxy measure of the shade avoidance response, and find that shade avoidance in leaves negatively correlates with the level of foliar shade recorded at the point of origin of an accession. The direction and magnitude of these correlations varies across the leaf series, suggesting that heterochronic and/or ontogenic programs are a mechanism by which selective pressures can alter leaf size and form. This study highlights the value of wild tomato accessions for studies of both morphological and light-regulated development of compound leaves, and promises to be useful in the future identification of genes regulating potentially adaptive plastic leaf traits

Let it bloom: cross‐talk between light and flowering signaling in Arabidopsis

The terrestrial environment is complex, with many parameters fluctuating on daily and seasonal basis. Plants in particular, have developed complex sensory and signalling networks to extract and integrate information about their surroundings, in order to maximise their fitness and mitigate some of the detrimental effects of their sessile lifestyles. Light and temperature each provide crucial insight on the surrounding environment and in combination allow plants to appropriately develop, grow and adapt. Crosstalk between light and temperature signalling cascades allow plants to time key developmental decisions accordingly to ensure they are “in sync” with their environment. In this review, we discuss the major players that regulate light and temperature signalling, and the cross‐talk between them, in reference to a crucial developmental decision faced by plants: to bloom or not to bloom