Sodium butyrate induces genotoxic stress in function of photoperiod variations and differentially modulates the expression of genes involved in chromatin modification and DNA repair in Petunia hybrida seedlings.

MAIN CONCLUSION: Sodium butyrate applied to Petunia hybrida seeds under a long-day photoperiod has a negative impact (reduced seedling length, decreased production of photosynthetic pigments, and accumulation of DNA damage) on early seedling development, whereas its administration under dark/light conditions (complete dark conditions for 5 days followed by exposure to long-day photoperiod for 5 days) bypasses some of the adverse effects. Genotoxic stress impairs plant development. To circumvent DNA damage, plants activate DNA repair pathways in concert with chromatin dynamics. These are essential during seed germination and seedling establishment, and may be influenced by photoperiod variations. To assess this interplay, an experimental design was developed in Petunia hybrida, a relevant horticultural crop and model species. Seeds were treated with different doses of sodium butyrate (NaB, 1 mM and 5 mM) as a stress agent applied under different light/dark conditions throughout a time period of 10 days. Phenotypic (germination percentage and speed, seedling length, and photosynthetic pigments) and molecular (DNA damage and gene expression profiles) analyses were performed to monitor the response to the imposed conditions. Seed germination was not affected by the treatments. Seedling development was hampered by increasing NaB concentrations applied under a long-day photoperiod (L) as reflected by the decreased seedling length accompanied by increased DNA damage. When seedlings were grown under dark conditions for 5 days and then exposed to long-day photoperiod for the remaining 5 days (D/L), the damaging effects of NaB were circumvented. NaB exposure under L conditions resulted in enhanced expression of HAT/HDAC (HISTONE ACETYLTRANSFERASES/HISTONE DEACTEYLASES) genes along with repression of genes involved in DNA repair. Differently, under D/L conditions, the expression of DNA repair genes was increased by NaB treatment and this was associated with lower levels of DNA damage. The observed DNA damage and gene expression profiles suggest the involvement of chromatin modification- and DNA repair-associated pathways in response to NaB and dark/light exposure during seedling development.

Regulation of biosynthesis and emission of volatile phenylpropanoids/benzenoids in petunia× hybrida flowers by multi-factors of circadian clock, light, and temperature.

Floral volatile phenylpropanoids and benzenoids (VPBs) play important ecological functions and have potential economic applications. Little is known about how multi-factors in integration regulate the formation and emission of floral VPBs. In the present study, we investigated effects of multi factors including endogenous circadian clock, light, and temperature on the formation and emission of VPBs, which are major volatiles in flowers of Petunia× hybrida cv. 'Mitchell Diploid'. Endogenous circadian clock was proposed as the most important factor regulating rhythmic emission of VPBs and expressions of structural genes involved in the upstream biosynthetic pathway of VPBs, but did not affect expression levels of structural genes involved in the downstream pathway and VPBs-related regulators. In contrast to light, temperature was a more constant factor affecting emission of VPBs. VPBs emission could be inhibited within a short time by increasing temperature. The information will contribute to our understanding of emission mechanism of floral volatiles.

Flavonols drive plant microevolution.

The idea that pollinators are in large part responsible for the diversity of flowering plants dates back more than 150 years to Darwin's work, but even modern scientists have struggled to identify specific 'speciation genes' and determine how they influenced flower-pollinator interactions. A new study proposes that a series of mutations in a single gene controlling floral chemicals influenced pollinator preferences, likely resulting in speciation, bringing us closer to finding a speciation gene.

MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation.

Adaptations to new pollinators involve multiple floral traits, each requiring coordinated changes in multiple genes. Despite this genetic complexity, shifts in pollination syndromes have happened frequently during angiosperm evolution. Here we study the genetic basis of floral UV absorbance, a key trait for attracting nocturnal pollinators. In Petunia, mutations in a single gene, MYB-FL, explain two transitions in UV absorbance. A gain of UV absorbance in the transition from bee to moth pollination was determined by a cis-regulatory mutation, whereas a frameshift mutation caused subsequent loss of UV absorbance during the transition from moth to hummingbird pollination. The functional differences in MYB-FL provide insight into the process of speciation and clarify phylogenetic relationships between nascent species.

Mathematical Modeling of the Dynamics of Shoot-Root Interactions and Resource Partitioning in Plant Growth.

Plants are highly plastic in their potential to adapt to changing environmental conditions. For example, they can selectively promote the relative growth of the root and the shoot in response to limiting supply of mineral nutrients and light, respectively, a phenomenon that is referred to as balanced growth or functional equilibrium. To gain insight into the regulatory network that controls this phenomenon, we took a systems biology approach that combines experimental work with mathematical modeling. We developed a mathematical model representing the activities of the root (nutrient and water uptake) and the shoot (photosynthesis), and their interactions through the exchange of the substrates sugar and phosphate (Pi). The model has been calibrated and validated with two independent experimental data sets obtained with Petunia hybrida. It involves a realistic environment with a day-and-night cycle, which necessitated the introduction of a transitory carbohydrate storage pool and an endogenous clock for coordination of metabolism with the environment. Our main goal was to grasp the dynamic adaptation of shoot:root ratio as a result of changes in light and Pi supply. The results of our study are in agreement with balanced growth hypothesis, suggesting that plants maintain a functional equilibrium between shoot and root activity based on differential growth of these two compartments. Furthermore, our results indicate that resource partitioning can be understood as the emergent property of many local physiological processes in the shoot and the root without explicit partitioning functions. Based on its encouraging predictive power, the model will be further developed as a tool to analyze resource partitioning in shoot and root crops.

Environmental control of branching in petunia.

Plants alter their development in response to changes in their environment. This responsiveness has proven to be a successful evolutionary trait. Here, we tested the hypothesis that two key environmental factors, light and nutrition, are integrated within the axillary bud to promote or suppress the growth of the bud into a branch. Using petunia (Petunia hybrida) as a model for vegetative branching, we manipulated both light quality (as crowding and the red-to-far-red light ratio) and phosphate availability, such that the axillary bud at node 7 varied from deeply dormant to rapidly growing. In conjunction with the phenotypic characterization, we also monitored the state of the strigolactone (SL) pathway by quantifying SL-related gene transcripts. Mutants in the SL pathway inhibit but do not abolish the branching response to these environmental signals, and neither signal is dominant over the other, suggesting that the regulation of branching in response to the environment is complex. We have isolated three new putatively SL-related TCP (for Teosinte branched1, Cycloidia, and Proliferating cell factor) genes from petunia, and have identified that these TCP-type transcription factors may have roles in the SL signaling pathway both before and after the reception of the SL signal at the bud. We show that the abundance of the receptor transcript is regulated by light quality, such that axillary buds growing in added far-red light have greatly increased receptor transcript abundance. This suggests a mechanism whereby the impact of any SL signal reaching an axillary bud is modulated by the responsiveness of these cells to the signal.

γ irradiation with different dose rates induces different DNA damage responses in Petunia x hybrida cells.

In plants, there is evidence that different dose rate exposures to gamma (γ) rays can cause different biological effects. The dynamics of DNA damage accumulation and molecular mechanisms that regulate recovery from radiation injury as a function of dose rate are poorly explored. To highlight dose-rate dependent differences in DNA damage, single cell gel electrophoresis was carried out on regenerating Petunia x hybrida leaf discs exposed to LDR (total dose 50 Gy, delivered at 0.33 Gy min(-1)) and HDR (total doses 50 and 100 Gy, delivered at 5.15 Gy min(-1)) γ-ray in the 0-24h time period after treatments. Significant fluctuations of double strand breaks and different repair capacities were observed between treatments in the 0-4h time period following irradiation. Dose-rate-dependent changes in the expression of the PhMT2 and PhAPX genes encoding a type 2 metallothionein and the cytosolic isoform of ascorbate peroxidase, respectively, were detected by Quantitative RealTime-Polymerase Chain Reaction. The PhMT2 and PhAPX genes were significantly up-regulated (3.0- and 0.7-fold) in response to HDR. The results are discussed in light of the potential practical applications of LDR-based treatments in mutation breeding.

Overexpression of PhEXPA1 increases cell size, modifies cell wall polymer composition and affects the timing of axillary meristem development in Petunia hybrida.

• Expansins are cell wall proteins required for cell enlargement and cell wall loosening during many developmental processes. The involvement of the Petunia hybrida expansin A1 (PhEXPA1) gene in cell expansion, the control of organ size and cell wall polysaccharide composition was investigated by overexpressing PhEXPA1 in petunia plants. • PhEXPA1 promoter activity was evaluated using a promoter-GUS assay and the protein's subcellular localization was established by expressing a PhEXPA1-GFP fusion protein. PhEXPA1 was overexpressed in transgenic plants using the cauliflower mosaic virus (CaMV) 35S promoter. Fourier transform infrared (FTIR) and chemical analysis were used for the quantitative analysis of cell wall polymers. • The GUS and GFP assays demonstrated that PhEXPA1 is present in the cell walls of expanding tissues. The constitutive overexpression of PhEXPA1 significantly affected expansin activity and organ size, leading to changes in the architecture of petunia plants by initiating premature axillary meristem outgrowth. Moreover, a significant change in cell wall polymer composition in the petal limbs of transgenic plants was observed. • These results support a role for expansins in the determination of organ shape, in lateral branching, and in the variation of cell wall polymer composition, probably reflecting a complex role in cell wall metabolism.

Light-induced vegetative anthocyanin pigmentation in Petunia.

The Lc petunia system, which displays enhanced, light-induced vegetative pigmentation, was used to investigate how high light affects anthocyanin biosynthesis, and to assess the effects of anthocyanin pigmentation upon photosynthesis. Lc petunia plants displayed intense purple anthocyanin pigmentation throughout the leaves and stems when grown under high-light conditions, yet remain acyanic when grown under shade conditions. The coloured phenotypes matched with an accumulation of anthocyanins and flavonols, as well as the activation of the early and late flavonoid biosynthetic genes required for flavonol and anthocyanin production. Pigmentation in Lc petunia only occurred under conditions which normally induce a modest amount of anthocyanin to accumulate in wild-type Mitchell petunia [Petunia axillaris x (Petunia axillaris x Petunia hybrida cv. 'Rose of Heaven')]. Anthocyanin pigmentation in Lc petunia leaves appears to screen underlying photosynthetic tissues, increasing light saturation and light compensation points, without reducing the maximal photosynthetic assimilation rate (A(max)). In the Lc petunia system, where the bHLH factor Leaf colour is constitutively expressed, expression of the bHLH (Lc) and WD40 (An11) components of the anthocyanin regulatory system were not limited, suggesting that the high-light-induced anthocyanin pigmentation is regulated by endogenous MYB transcription factors.

Reduction of benzenoid synthesis in petunia flowers reveals multiple pathways to benzoic acid and enhancement in auxin transport.

In plants, benzoic acid (BA) is believed to be synthesized from Phe through shortening of the propyl side chain by two carbons. It is hypothesized that this chain shortening occurs via either a beta-oxidative or non-beta-oxidative pathway. Previous in vivo isotope labeling and metabolic flux analysis of the benzenoid network in petunia (Petunia hybrida) flowers revealed that both pathways yield benzenoid compounds and that benzylbenzoate is an intermediate between L-Phe and BA. To test this hypothesis, we generated transgenic petunia plants in which the expression of BPBT, the gene encoding the enzyme that uses benzoyl-CoA and benzyl alcohol to make benzylbenzoate, was reduced or eliminated. Elimination of benzylbenzoate formation decreased the endogenous pool of BA and methylbenzoate emission but increased emission of benzyl alcohol and benzylaldehyde, confirming the contribution of benzylbenzoate to BA formation. Labeling experiments with 2H5-Phe revealed a dilution of isotopic abundance in most measured compounds in the dark, suggesting an alternative pathway from a precursor other than Phe, possibly phenylpyruvate. Suppression of BPBT activity also affected the overall morphology of petunia plants, resulting in larger flowers and leaves, thicker stems, and longer internodes, which was consistent with the increased auxin transport in transgenic plants. This suggests that BPBT is involved in metabolic processes in vegetative tissues as well.

A physiological and morphological study on the injury caused by exposure to the air pollutant, peroxyacetyl nitrate (PAN), based on the quantitative assessment of the injury.

A method for the numerical assessment of the foliar injury caused by the photochemical oxidant, peroxyacetyl nitrate (PAN), was devised, using three injury indices: fresh weight (FW) loss, decreased photosynthetic pigment content, and increased ion leakage, which can be measured using the same leaves. The injury indices clearly indicated a larger number of PAN-sensitive leaves and a more severe level of injury in the PAN-sensitive variety of Petunia hybrida, White Champion (WHITE), compared to the PAN-tolerant variety, Blue Champion (BLUE). FW and photosynthetic pigment content decreased correlatively in both varieties, but ion leakage increased only in WHITE. Morphological observations revealed that ion leakage started concurrently with the start of plasmolysis-like symptoms at the mesophyll cells of injured WHITE leaves, whereas FW loss corresponded to the shrinkage of cells without loss of their round shape in BLUE leaves. PAN injuries measured by the injury indices were markedly increased in the presence of light, and the morphological changes following PAN exposure were similar to those caused by the superoxide-generating chemical, paraquat. The results suggested that PAN injuries indicated by the three injury indices are all light-dependent, but are caused through several independent mechanisms.