The attenuation of radiation in forest canopies has been studied in depth within the photosynthetically active radiation (PAR, 400-700 nm), but we are still lacking knowledge on how the spectral composition of ultraviolet radiation (UV-B 280-315 nm, UV-A 315-400 nm) varies. Advances in knowledge on the effects of UV radiation has led to growing interest in its study as a trigger of regulatory responses in plants, rather than as a stressor, which is now considered to be rare in plants growing under natural conditions. Furthermore, a growing number of studies suggest that there are complex interactions in perception, signalling and responses of plants to solar spectral irradiance. My dissertation research is focussed on the acclimation and adaptation of leaf flavonoids to solar radiation in plant taxa from contrasting environments assessed through the study of optically measured leaf traits. These responses were studied in forest understorey taxa growing in a seasonally dynamic, but mostly low UV radiation environment, and in taxa growing at high elevation experiencing a high UV radiation environment in a "common garden" setting. More precisely, we examined: 1) how understorey spectral irradiance changes across shade, leaf-shade and sunflecks through the spring and among different forest stands, 2) how optically measured leaf traits and leaf flavonoids in understorey plants change through the growing season, 3) how these patterns relate to seasonal changes in spectral irradiance especially those in UV region, 4) do the patterns of optically measured leaf traits from a large set of taxa, mainly growing at high elevation, follow their patterns of phylogenetic relatedness and 5) how do their leaf traits relate to climatic conditions at their original collection sites. To study these questions, we adopted relatively new monitoring approaches, enabling us both to measure simultaneously the in situ spectral irradiance from the UV to near infra-red (NIR) regions, and leaf flavonols/flavones in vivo repeatedly with a leaf-clip in the field. Our analysis revealed a hierarchy among those factors affecting spectral composition of solar radiation in forest understoreys; most importantly understorey position (sunflecks, shade or radiation transmitted through the canopy of leaves), then stand composition, and date during spring. We found the optically measured leaf flavonol/flavone index (Iflav) in forest understorey species to be plastic and to adjust to changes in climatic conditions. Furthermore, species' leaf retention strategy (e.g., summer green, overwintering leaves) and new leaf production were found to affect the Iflav of plants. All these factors are reflected in the seasonal trends we describe in leaf flavonoids, measured optically (Iflav) and via leaf extracts, across understorey plant communities. For mountain environments, our objectives were to determine factors that underpin leaf flavonoid accumulation of high elevation taxa and whether patterns in optically measured leaf traits followed their phylogenetic relatedness or climatic conditions at their origin. Both these patterns could potentially constrain plant responses. To see if either pattern was present, we tested for a phylogenetic signal particularly in Iflav from a large set of taxa growing in a high-elevation environment and the relationship of mean Iflav of plants to climatic variables. The tests for a phylogenetic signal (Pagel's λ, range from 0 to 1) gave intermediate fitted λ values with significant results for Iflav and anthocyanin index (Iant), while for the smaller set of taxa growing in Kumpula Botanical Garden (southern Finland) only chlorophyll index (Ichl) showed significant results. Despite the relatively low signal for Iflav, we identified certain genera with mainly positive local autocorrelations (local Moran's I) meaning they contained species showing either with mostly high or mostly low leaf trait values. This suggests potential limitations in their leaf flavonol/flavone accumulation responses. Hence, some of these genera may be less well prepared against higher maximum UV radiation and may encounter constraints in migrating upwards, if other compensatory photoprotection mechanisms fail. We did not find a relationship between Iflav and climate at the plants' origin, while our results suggested UV irradiance in the plants' current microhabitat to be important, albeit not the only driver for flavonoid accumulation. In most taxa, we did not find a clear indication of constraints on leaf flavonoid accumulation, thus no evidence that high UV radiation is a detrimental factor in their environment. The values of these optically measured leaf traits represent the outcome of complex interactions between the evolutionary and biogeographical history, and acclimation to the current growing conditions of the plants. In general across the three studies, these results provide evidence that optically measured leaf traits related to flavonoid accumulation are largely flexible and acclimate to local changes in the environment, as well as adjusting over the growing season.

RNA silencing is a conserved mechanism that occurs in a broad range of eukaryotes, which is regulated by small RNAs (sRNAs). RNA silencing operates to control gene expression and maintain genome integrity. Virus-induced gene silencing (VIGS) in plants is a natural antivirus mechanism that has adapted from the general RNA silencing system. To counter the antivirus RNA silencing, plant viruses have evolved to encode viral suppressors of RNA silencing (VSRs). Nowadays VIGS is usually referred to as the technology that uses recombinant viruses to knock down the expression of plant endogenous genes. Gerbera hybrida (gerbera) is a model species in the family of Asteraceae. As a highly heterozygous species, gerbera lacks efficient functional genetic approaches other than gene transfer. The aim of the present study was to develop a Tobacco rattle virus (TRV, genus Tobravirus) induced gene silencing system for gerbera, and use TRV VIGS to characterize functions of chalcone synthase (CHS) encoding genes in the plant. Preliminary VIGS experiments on the cultivar Terraregina, by syringe infiltration and applying previously developed TRV vectors, did not result in visible VIGS phenotypes due to the absent of TRV RNA2 in the up non-infiltrated leaves. Consequently, I first aimed to study the mechanism of TRV VIGS, and tried to develop new VIGS vectors based on TRV RNA1. I investigated the role of two important TRV proteins of the 16K VSR and the 29K movement protein (MP) on TRV infection and TRV VIGS, and developed TRV RNA1 based VIGS vectors. For accomplishing this, a series of TRV RNA1 mutants have been constructed to disrupt the 16K, or to replace its 29K with Tobacco mosaic virus (TMV, genus Tobamovirus) 30K MP. TRV RNA1 vector, carrying a fragment of the gene encoding Nicotiana benthamiana PDS to replace part of the 16K sequence, induced PDS gene silencing systemically in N. benthamiana. However, this has found to be less efficiently than the original TRV VIGS system when the wild-type RNA1 and RNA2:PDS were used. The infection experiments demonstrated that 16K was required for TRV long distance movement, and helped in maintaining the integrity of the TRV RNA2 genome. In addition, TRV 29K alone did not suppress RNA silencing in the co-infiltration assay, but it could suppress RNA silencing in the context of RNA1 replication. TRV 29K may be the first VSR whose silencing suppression functions are found to be directly linked to viral replication. The original TRV vector system was finally adopted for VIGS in gerbera. TRV VIGS was optimized for gerbera by screening for TRV sensitive cultivars and by improving its inoculation methods. Intensive gene silencing phenotypes were achieved both in green tissues and in floral tissues, demonstrated by knocking down genes involved in isoprenoid biosynthesis (phytoene desaturase: GPDS; H and I subunits of Mg-chelatase: GChl-H and GChl-I), flower pigmentation (chalcone synthase: GCHS1), and flower development (GLOBOSA-like MADS domain transcription factor: GGLO1). Unexpectedly, a gerbera polyketide synthase encoding gene, G2PS1, that has no apparent connections to the carotenoid or chlorophyll biosynthesis, was knocked down by the photo-bleaching that was induced by the silencing of GPDS, GChl-H and GChl-I, or by the herbicide norflurazon. We have demonstrated for the first time that the using of VIGS in an Asteraceaeous species. Our data also suggested that the selection and use of a marker gene for VIGS should be strictly evaluated. A new CHS encoding gene, GCHS4, was characterized in gerbera. Together with the two previously identified GCHS1 and GCHS3, gerbera CHSs are represented by a three-gene family. Each gerbera CHS shows a distinct expression pattern. GCHS3 is particularly expressed in gerbera pappus. In partnership with the concomitantly expressed GCHS1, they are involved in the biosynthesis of colorless flavonoids. GCHS4 is the only CHS that is naturally expressed in the leaf petiole and inflorescence scape, and it is responsible for cyanidin biosynthesis in those tissues. GCHS4 is also the only CHS that was induced by environmental stresses in the leaf blade. Both GCHS1 and GCHS4 are markedly expressed in gerbera petals, and GCHS4 mRNA actually takes the majority of CHS mRNAs in the later stages of petal development. Nonetheless, VIGS experiments, by target silencing GCHS1 or GCHS4 independently, demonstrated that GCHS1 is the predominant functional CHS in gerbera petals. Thus, GCHS4 in gerbera petals seems to be regulated post-transcriptionally. In conclusion, the results of this study shed new light on the mechanism of TRV VIGS. The established TRV VIGS system provides a valuable tool for functional genomics in gerbera.

Regulation of cellular homeostasis is crucial for proper development, survival, defense responses, programmed cell death and ultimately survival. Maintaining cellular homeostasis requires tight regulation of multiple highly interactive signaling pathways. The apoplast lies at the frontier between the cell and the environment, where the plant perceives environmental cues. Since the apoplast is also a site for cell-to-cell communication, it has an important role in mediating plant-environment interactions. Reactive oxygen species (ROS) are known as both toxic agents and indispensable signaling molecules in all aerobic organisms. A ROS burst in the apoplast is one of the first measurable events produced in response to different biotic and abiotic stresses, eventually leading to the initiation of signal transduction pathways and altered gene expression. Apoplastic ROS signaling is well known to dynamically coordinate multiple signaling pathways in the activation of defense responses in plants. Dissection of the signaling crosstalk within such a signaling network could therefore reveal the molecular mechanisms underlying defense responses. Treatments with ozone (O3) have been adopted as an efficient tool to study apoplastic ROS signaling. Plants exposed to O3 trigger a ROS burst in the apoplast and induce extensive changes in gene expression and alteration of defense hormones, such as salicylic acid (SA), jasmonic acid (JA), and ethylene. Genetic variation in O3 sensitivity among Arabidopsis thaliana accessions or mutants highlights the complex genetic architecture of plant responses to ROS. To gain insight into the genetic basis of apoplastic ROS signaling, a recombinant inbred line (RIL) population from a reciprocal cross between two Arabidopsis accessions C24 (O3 tolerant) and Tenela (O3 sensitive) was used for quantitative trait loci (QTL) mapping. Through a combination of QTL mapping and transcriptomic analyses in the response to apoplastic-ROS treatment, three QTL regions containing several potential candidate genes were identified in this study. In addition, multiple mutants with varying O3-sensitivities were employed to dissect the signaling components involved in the early apoplastic ROS signaling and O3-triggered cell death. A combination of global and targeted gene expression profiling, genetic analysis, and cell death assays was performed to dissect the contribution of hormone signaling and various transcription factors to the regulation of apoplastic ROS-triggered gene expression and cell death. The contributions of SA, JA and ethylene were assessed through analysis of mutants deficient in these hormones, mutants with constitutively activated hormone signaling and the exogenous application of hormones. Plants with elevated SA levels were found to be associated with an attenuated O3 response, whereas simultaneous elimination of SA-dependent and SA- independent signaling components enhanced the response to apoplastic ROS treatment. JA could act as both a positive and negative modifier of apoplastic ROS signaling, which was enhanced when ethylene signaling was also impaired. However, transcriptome analysis of a triple mutant deficient in SA, JA and ethylene revealed that these hormones signaling only contributed part (about 30%) of early-apoplastic ROS-triggered changes in gene expression, suggesting multiple signaling pathways could be required to regulate the apoplastic ROS response via combinatorial or overlapping mechanisms.

Blue and UV radiation are environmental cues or sources of information that can shape the morphology and development of plants. It was hypothesized that: H1) long-term treatments of solar blue (400–500 nm), long-UV (350–400 nm) and short-UV (290–350 nm) radiation (starting before seedling emergence) are perceived as different and can trigger distinct morphological, physiological and molecular responses; H2) parental long-term exposure to short-UV radiation before flowering affects response patterns to blue and UV radiation in the offspring; H3) long-term exposure to solar blue, long-UV and/or short-UV radiation enhances drought tolerance; H4) the responses in H1, H2 and H3 are accession-dependent and related to the environments where the accessions originate. To test these hypotheses, three experiments assessed morphological, physiological and molecular responses of accessions of two legume species, faba bean (Vicia faba L.) (I, II) and barrel medic (Medicago truncatula Gaertn.) (III). To impose radiation treatments by attenuating different wavebands of sunlight, four types of plastic filters were used in experiments I and III outdoors. Through pairwise filter comparisons, three different solar wavebands were assessed: blue, long-UV and short-UV radiation. In experiment I, two accessions of V. faba (Aurora; ILB938) originating from contrasting UV environments (southern Sweden; Andean region of Colombia and Ecuador) were grown under the four filters in sunlight. To study the transgenerational effect of solar short-UV radiation, experiment II was established using seeds produced by plants from experiment I and a factorial experiment design combining the two V. faba accessions, two parental UV treatments (full sunlight and exclusion of short-UV radiation) and four offspring light treatments, from the factorial combination of UVB and blue radiation manipulations in a controlled environment. In experiment III, the effect of long-term exposure to solar blue, long-UV and short-UV radiation during growth on the tolerance of subsequent progressive drought was studied in three M. truncatula accessions using the same filter treatments as in experiment I combined with progressive drought treatments imposed by withholding watering for 2 and 7 days to half the plants starting 40 days after sowing. The three M. truncatula accessions, Jemalong A17, HM006 and HM020, originate from Australia, France and Tunisia, respectively. After long-term natural light treatments (I, III), blue light but not long-UV or short-UV radiation, significantly regulated plant morphology and transcript abundance. In contrast, both solar blue and short-UV radiation, but not long-UV radiation, induced the accumulation of total flavonoids in leaves of V. faba (I) and M. truncatula (III). Moreover, simultaneous exposure to blue and UVB radiation had a synergetic effect on the induction of flavonoid accumulation (II). In V. faba, the variations of flavonoid composition and gene expression between the two accessions were consistent throughout the two successive generations (I, II). In V. faba, the transgenerational effect of short-UV radiation altered the morphological responses of the progenies to blue light, and it also affected flavonoid accumulation of the offspring in response to UVB radiation. Moreover, the transgenerational effects differed in the two accessions (II): in Aurora, the parental exposure to solar short-UV radiation led to a near-doubling of total quercetin concentration in response to UVB radiation in the progeny, while this was not observed in ILB938. The difference of responses to blue and UV radiation in these two accessions are consistent with adaptation to contrasting UV environments. In M. truncatula, long-term exposure to both solar blue and UV radiation pre-acclimated plants to subsequent slowly imposed drought, as observed in the transcriptomic result in accession Jemalong A17 that drought (2 and 7 days without watering) did not regulate differentially expressed genes (DEGs) under the filter transmitting blue and UV radiation. In contrast, drought increased transcript abundance of several previously described stress-inducible genes under all other filters. In the light of transcriptomic and flavonoid responses to filter and drought treatments, two processes potentially contribute to light-driven acclimation to drought: 1) increased flavonoid accumulation under blue and short-UV radiation could enhance the capability to scavenge drought-induced reactive oxygen species (ROS); 2) down-regulation of genes involved in light reactions of photosynthesis by blue light could reduce the generation of ROS when stomata close. In conclusion, under long-term sunlight treatment, blue light modified plants’ morphology and transcript change while both blue and short-UV radiation induced the accumulation of flavonoids; a transgenerational effect of short-UV radiation influenced offspring responses to blue and UVB radiation differently in the two accessions; both blue and UV radiation contributed to pre-acclimation toward subsequent drought by functioning as environmental cues rather than stressors even if the specific responses differed among accessions. Thus, the results support the four hypotheses.

Plant vascular tissues are supporting and conductive tissues composed of two major components, xylem and phloem. These tissues transport water, food, hormones and minerals within the plant. In my thesis work, I used the Arabidopsis root as a model system to study vascular tissue formation. The first part of my thesis work is focused on the formation of xylem, the water transporting tissue. In the Arabidopsis root, the xylem is organized as an axis of cell files with two distinct cell fates: the central metaxylem and the peripheral protoxylem. It has been previously reported that high and low expression levels of the class III HD-ZIP transcription factors promote metaxylem and protoxylem identities, respectively. In this work, we provide evidence that auxin biosynthesis promotes HD-ZIP III expression and metaxylem formation. We observed that plants with mutations in auxin biosynthesis genes, such as trp2-12, wei8 tar2, or the quintuple yucca mutant, as well as plants treated with a pharmacological inhibitor of auxin biosynthesis, show reduced expression of the HD-ZIP III genes accompanied by specific defects in metaxylem formation. We were able to induce a partial rescue of the metaxylem defects by introducing an endogenous auxin supply. In addition, some of the patterning defects can be suppressed by synthetically elevating HD-ZIP III expression in the stele of the Arabidopsis root. The second part of my thesis work is focused on phloem tissue formation. Phloem is the tissue responsible for long-distance molecular transport and signaling. The conductive components of the phloem, the sieve elements, rely on specific junctions between the conducting cells in the form of highly perforated sieve areas. We identified mutations in the CHER1 (CHOLINE TRANSPORTER LIKE 1) locus of Arabidopsis which result in altered phloem conductivity, reduced sieve pore density, and defects in sieve pore formation. CHER1 encodes a member of a poorly characterized choline transporter-like protein family in plants and animals. We provide data showing that CHER1 facilitates choline transport, localizes to the trans-Golgi network, and is associated with the late stage of phragmoplast formation during cytokinesis. Interestingly, CHER1 has a sustained, polar localization in forming sieve plates, which is consistent with its function in the elaboration of the sieve areas.

To face the constant challenges from numerous pathogens in the environment, sophisticated defense systems have evolved in plants. Reactive oxygen species (ROS) and phytohormones are important cellular compounds that regulate plant defense systems to overcome biotic stresses from different pathogens. Against biotrophic pathogens, which require living host cells, hypersensitive cell death response (HR), a type of programed cell death mediated by ROS and salicylic acid (SA), is effective for immunity. However, to necrotrophic pathogens, which take host cell death as a hallmark of a successful colonization, the roles of ROS and phytohormones in the manipulation of cell death during plant defense are more complex. In this work, we utilized the model necrotrophic pathogen Botrytis cinerea (Botrytis; grey mold) and the model plant Arabidopsis thaliana (Arabidopsis), using mutants in reverse genetic screens, especially radical-induced cell death1 (rcd1) and botrytis susceptible1 (bos1), were used to study the functions of ROS and phytohormones in plant-Botrytis interactions. It was found that Botrytis-triggered signaling in Arabidopsis mostly overlapped with the signaling triggered by apoplastic ROS but not intracellular ROS. However, rcd1 and bos1 exhibited opposite symptoms in response to Botrytis and apoplastic ROS. This suggested that the resistance signaling regulated by RCD1 or BOS1 were distinct from a more common signaling programs induced by Botrytis and apoplastic ROS. Further study revealed that RCD1 negatively regulated Botrytis resistance independent of stress-hormones. RCD1 positively regulated Botrytis-toxin sensitivity and brassinosteroid (BR) signaling, which was demonstrated to negatively regulate plant resistance to Botrytis. In the BOS1 study, suppression of abscisic acid (ABA)-elicited cell death and control of cell death spread were identified as pivotal functions of BOS1 in its regulation of host resistance to Botrytis. This work emphasized the negative roles of both BR and ABA in response to Botrytis infection. Considering the established facts that: 1) ABA promoted plant cell death, 2) BR deficiency leads to delayed senescence, and 3) the ROS burst causes damage to both host and Botrytis; this work supports the view that cell death control plays a pivotal role in plant-Botrytis interactions, where defense combined with less cell death confers plants with an advantage in the battle against Botrytis.