Nitrogen remobilisation facilitates adventitious root formation on reversible dark-induced carbohydrate depletion in Petunia hybrida

Supplemental data of Figures 2 to 7. (PDF 31 kb

Transcriptome, carbohydrate, and phytohormone analysis of Petunia hybrida reveals a complex disturbance of plant functional integrity under mild chilling stress

Cultivation of chilling-tolerant ornamental crops at lower temperature could reduce the energy demands of heated greenhouses. To provide a better understanding of how sub-optimal temperatures (12 degrees C vs. 16 degrees C) affect growth of the sensitive Petunia hybrida cultivar 'SweetSunshine Williams', the transcriptome, carbohydrate metabolism, and phytohormone homeostasis were monitored in aerial plant parts over 4 weeks by use of a microarray, enzymatic assays and GC-MS/MS. The data revealed three consecutive phases of chilling response. The first days were marked by a strong accumulation of sugars, particularly in source leaves, preferential up-regulation of genes in the same tissue and down-regulation of several genes in the shoot apex, especially those involved in the abiotic stress response. The midterm phase featured a partial normalization of carbohydrate levels and gene expression. After 3 weeks of chilling exposure, a new stabilized balance was established. Reduced hexose levels in the shoot apex, reduced ratios of sugar levels between the apex and source leaves and a higher apical sucrose/hexose ratio, associated with decreased activity and expression of cell wall invertase, indicate that prolonged chilling induced sugar accumulation in source leaves at the expense of reduced sugar transport to and reduced sucrose utilization in the shoot. This was associated with reduced levels of indole-3-acetic acid and abscisic acid in the apex and high numbers of differentially, particularly up-regulated genes, especially in the source leaves, including those regulating histones, ethylene action, transcription factors, and a jasmonate-ZIM-domain protein. Transcripts of one Jumonji C domain containing protein and one expansin accumulated in source leaves throughout the chilling period. The results reveal a dynamic and complex disturbance of plant function in response to mild chilling, opening new perspectives for the comparative analysis of differently tolerant cultivars.BMBF/AgroCluster/WeGa - Horticulture Research NetworkState of BrandenburgFree State of ThuringiaFederal Republic of German

Comprehensive transcriptome analysis unravels the existence of crucial genes regulating primary metabolism during adventitious root formation in Petunia hybrida

To identify specific genes determining the initiation and formation of adventitious roots (AR), a microarray-based transcriptome analysis in the stem base of the cuttings of Petunia hybrida (line W115) was conducted. A microarray carrying 24,816 unique, non-redundant annotated sequences was hybridized to probes derived from different stages of AR formation. After exclusion of wound-responsive and root-regulated genes, 1,354 of them were identified which were significantly and specifically induced during various phases of AR formation. Based on a recent physiological model distinguishing three metabolic phases in AR formation, the present paper focuses on the response of genes related to particular metabolic pathways. Key genes involved in primary carbohydrate metabolism such as those mediating apoplastic sucrose unloading were induced at the early sink establishment phase of AR formation. Transcriptome changes also pointed to a possible role of trehalose metabolism and SnRK1 (sucrose non-fermenting 1- related protein kinase) in sugar sensing during this early step of AR formation. Symplastic sucrose unloading and nucleotide biosynthesis were the major processes induced during the later recovery and maintenance phases. Moreover, transcripts involved in peroxisomal beta-oxidation were up-regulated during different phases of AR formation. In addition to metabolic pathways, the analysis revealed the activation of cell division at the two later phases and in particular the induction of G1- specific genes in the maintenance phase. Furthermore, results point towards a specific demand for certain mineral nutrients starting in the recovery phase

Adventitious rooting declines with the vegetative to reproductive switch and involves a changed auxin homeostasis

Adventitious rooting, whereby roots form from non-root tissues, is critical to the forestry and horticultural industries that depend on propagating plants from cuttings. A major problem is that age of the tissue affects the ability of the cutting to form adventitious roots. Here, a model system has been developed using Pisum sativum to differentiate between different interpretations of ageing. It is shown that the decline in adventitious rooting is linked to the ontogenetic switch from vegetative to floral and is mainly attributed to the cutting base. Using rms mutants it is demonstrated that the decline is not a result of increased strigolactones inhibiting adventitious root formation. Monitoring endogenous levels of a range of other hormones including a range of cytokinins in the rooting zone revealed that a peak in jasmonic acid is delayed in cuttings from floral plants. Additionally, there is an early peak in indole-3-acetic acid levels 6h post excision in cuttings from vegetative plants, which is absent in cuttings from floral plants. These results were confirmed using DR5:GUS expression. Exogenous supplementation of young cuttings with either jasmonic acid or indole-3-acetic acid promoted adventitious rooting, but neither of these hormones was able to promote adventitious rooting in mature cuttings. DR5:GUS expression was observed to increase in juvenile cuttings with increasing auxin treatment but not in the mature cuttings. Therefore, it seems the vegetative to floral ontogenetic switch involves an alteration in the tissue’s auxin homeostasis that significantly reduces the indole-3-acetic acid pool and ultimately results in a decline in adventitious root formation

Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation.

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10% of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentosephosphate pathwa

Jasmonate promotes auxin-induced adventitious rooting in dark-grown Arabidopsis thaliana seedlings and stem thin cell layers by a cross-talk with ethylene signalling and a modulation of xylogenesis

Background: Adventitious roots (ARs) are often necessary for plant survival, and essential for successful micropropagation. In Arabidopsis thaliana dark-grown seedlings AR-formation occurs from the hypocotyl and is enhanced by application of indole-3-butyric acid (IBA) combined with kinetin (Kin). The same IBA + Kin-treatment induces AR-formation in thin cell layers (TCLs). Auxin is the main inducer of AR-formation and xylogenesis in numerous species and experimental systems. Xylogenesis is competitive to AR-formation in Arabidopsis hypocotyls and TCLs. Jasmonates (JAs) negatively affect AR-formation in de-etiolated Arabidopsis seedlings, but positively affect both AR-formation and xylogenesis in tobacco dark-grown IBA + Kin TCLs. In Arabidopsis the interplay between JAs and auxin in AR-formation vs xylogenesis needs investigation. In de-etiolated Arabidopsis seedlings, the Auxin Response Factors ARF6 and ARF8 positively regulate AR-formation and ARF17 negatively affects the process, but their role in xylogenesis is unknown. The cross-talk between auxin and ethylene (ET) is also important for AR-formation and xylogenesis, occurring through EIN3/EIL1 signalling pathway. EIN3/EIL1 is the direct link for JA and ET-signalling. The research investigated JA role on AR-formation and xylogenesis in Arabidopsis dark-grown seedlings and TCLs, and the relationship with ET and auxin. The JA-donor methyl-jasmonate (MeJA), and/or the ET precursor 1-aminocyclopropane-1-carboxylic acid were applied, and the response of mutants in JA-synthesis and -signalling, and ET-signalling investigated. Endogenous levels of auxin, JA and JA-related compounds, and ARF6, ARF8 and ARF17 expression were monitored. Results: MeJA, at 0.01 μM, enhances AR-formation, when combined with IBA + Kin, and the response of the early-JA-biosynthesis mutant dde2–2 and the JA-signalling mutant coi1–16 confirmed this result. JA levels early change during TCL-culture, and JA/JA-Ile is immunolocalized in AR-tips and xylogenic cells. The high AR-response of the late JA-biosynthesis mutant opr3 suggests a positive action also of 12-oxophytodienoic acid on AR-formation. The crosstalk between JA and ET-signalling by EIN3/EIL1 is critical for AR-formation, and involves a competitive modulation of xylogenesis. Xylogenesis is enhanced by a MeJA concentration repressing AR-formation, and is positively related to ARF17 expression. Conclusions: The JA concentration-dependent role on AR-formation and xylogenesis, and the interaction with ET opens the way to applications in the micropropagation of recalcitrant species

Sebacinales Everywhere: Previously Overlooked Ubiquitous Fungal Endophytes

Inconspicuous basidiomycetes from the order Sebacinales are known to be involved in a puzzling variety of mutualistic plant-fungal symbioses (mycorrhizae), which presumably involve transport of mineral nutrients. Recently a few members of this fungal order not fitting this definition and commonly referred to as ‘endophytes’ have raised considerable interest by their ability to enhance plant growth and to increase resistance of their host plants against abiotic stress factors and fungal pathogens. Using DNA-based detection and electron microscopy, we show that Sebacinales are not only extremely versatile in their mycorrhizal associations, but are also almost universally present as symptomless endophytes. They occurred in field specimens of bryophytes, pteridophytes and all families of herbaceous angiosperms we investigated, including liverworts, wheat, maize, and the non-mycorrhizal model plant Arabidopsis thaliana. They were present in all habitats we studied on four continents. We even detected these fungi in herbarium specimens originating from pioneering field trips to North Africa in the 1830s/40s. No geographical or host patterns were detected. Our data suggest that the multitude of mycorrhizal interactions in Sebacinales may have arisen from an ancestral endophytic habit by specialization. Considering their proven beneficial influence on plant growth and their ubiquity, endophytic Sebacinales may be a previously unrecognized universal hidden force in plant ecosystems

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The current assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n=14) containing 32,928 and 36,697 protein-coding genes, respectively. The Petunia lineage has experienced at least two rounds of paleohexaploidization, the older gamma hexaploidy event, which is shared with other Eudicots, and the more recent Solanaceae paleohexaploidy event that is shared with tomato and other Solanaceae species. Transcription factors that were targets of selection during the shift from bee- to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral color patterns and pollination systems. The high quality genome sequences will enhance the value of Petunia as a model system for basic and applied research on a variety of unique biological phenomena