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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°C vs. 16°C) affect growth of the sensitive
Petunia (
Previous research covered cold and freezing reactions of
The chilling response of the plant phenotype reflects changes in the habitus like a reduced growth performance, which are the consequences of a disturbed plant metabolism and of the acclimation to the changed environment at transcriptional or post-transcriptional level.
Some phytohormones such as auxins, abscisic acid (ABA) and jasmonic acid (JA) are known to be involved in plant reactions to abiotic stress resulting from low temperatures. Auxin is a key regulatory phytohormone in plant growth and development, while indole-3-acetic acid (IAA) constitutes the most important physiologically active fraction. It plays an important role in plant reactions to environmental changes. Changed plant growth and development in response to cold may be linked to an altered intracellular homeostasis of auxins, which is regulated by local auxin gradients (reviewed in
Among the diverse mechanisms, providing plant plasticity toward a changing environment, the regulation of gene expression plays an important role in the genetic potential of a plant to cope with environmental stresses. In this context, the MYC-like transcription factor (TF) ICE1 from the bHLH family has a crucial control function in cold tolerance in
The molecular and physiological response of
All experiments were carried out with the chilling-sensitive
Detailed growth performance analysis was conducted under greenhouse conditions (control temperature, day/night average: 16.8°C/15.2°C; sub-optimal temperature, day/night average: 13.1°C/11.1°C; 12 h photoperiod with ±130 μmol m-2 s-1 photo-synthetic photon flux density). All other experiments were conducted under climate chamber conditions. Therefore, immediately after potting, rooted cuttings were transferred to a climate chamber to acclimate to control temperatures (day/night: 17°C/15°C) and to develop a good root system. Day length was fixed to a 12 h photoperiod, (photo-synthetic photon flux density: during first week 100 μmol m-2 s-1, from second week onward 150 μmol m-2 s-1 (lighting: fluorescent tubes, FQ80W/865 HO Constant, Lumilux Cool Daylight, Osram, Germany), while the relative humidity was 65%. After 2 weeks of acclimation, half of the plants were moved to a climate chamber with identical conditions but under chilling exposure (day/night: 13°C/11°C). The 4-weeks period of cultivation focused on the vegetative growth of plants. To avoid interference with competing sinks, developing flower buds were continuously removed from the plants as soon as being visible during the course of the experiment.
For determination of growth, the main shoots of 12 plants per treatment were marked at the time point of 0 DoT (days after differentiation of temperature). As growth parameters, the number of newly developed shoots, the increase in length of the main shoot, and the number of newly developed leaves on the main shoot were evaluated weekly over a period of 28 days.
Samples were collected from three plant organs for analyses. As sink tissue, the apex of the main shoot was harvested, including adjacent small leaves (total length <2 cm). For sampling of source tissue, leaf disks (50–120 mg fresh weight for carbohydrates and 200–250 mg for phytohormones) were excised with a cork borer from the middle of each leaf half of young, but fully expanded leaves. For phytohormone and gene expression analyses, the uppermost internode (for microarray only at 21 DoT) was additionally collected. The samples were immediately transferred to liquid nitrogen and stored at -80°C until purification and analysis. The samples for carbohydrates, enzymes, and microarray were collected at 6 h, for phytohormone analyses at 8 h after begin of the photoperiod. Samples for carbohydrate, enzyme and phytohormone analyses were collected at 0, 1, 2, 3, 7, 14, 21, and 28 DoT, samples for the microarray analysis at 1, 3, 7, 21 DoT. Since metabolic and hormone data of 2 DoT gave no information in addition to those of 1 and 3 DoT, these were not included in the results.
Extraction, purification, and analysis of ABA, IAA, and JA by GC-MS/MS were performed as described by
To execute statistical analyses, the STATISTICA software package was used
In order to identify genes with altered expressions in response to sub-optimal temperatures, a petunia-specific microarray was hybridized, which carries 24,816 unigene annotated sequences (
The chilling-sensitive
Considering the crucial role of carbohydrates as protective molecules for plant survival at very low and freezing temperatures, we analyzed the concentrations of the most important sugars fructose (Fru), glucose (Glc), and sucrose (Suc) as well as starch in source leaves and in the shoot apex as important utilization sink. Samples were collected at midday, 6 h after the start of the photoperiod, at the expected climax of sugar concentrations (
While carbohydrate concentrations in the tissues at certain time points are snap-shots of current carbohydrate availability, carbohydrate ratios between source and sink and among each other may indicate bottlenecks of partitioning between carbohydrate fractions and between tissues at the two different temperatures. It becomes apparent from
Considering the changed carbohydrate levels and particularly the changed Suc/hexose equilibrium, we investigated the activities of cell wall bound (cwInv), cytoplasmic (cytInv), and vacuolar (vacInv) forms of invertase. In both, source and sink tissues, activities of cytInv (
In order to unravel if the homeostasis of ABA, IAA, and JA in petunia is altered by exposure to sub-optimal temperature, concentrations of these phytohormones were monitored. Since the stem provides an important transport unit for phytohormones and, furthermore, IAA may control stem-elongation, the uppermost internode was analyzed in addition to the source leaves and the shoot apex. The mild chilling treatment had no measureable impact on levels of JA. For all three tissues, JA concentrations were very low. Actually, most values were below the limits of quantification of 1.5 pmol per injection (
Samples were collected on four dates (1, 3, 7, 21 DoT), to examine fast, midterm and long-term reactions of gene expression to mild chilling temperatures. Even the 4 K reduction of temperature caused significant changes in gene expression. Most M values (log2 of fold change ratios) for differentially expressed genes were between -1 and -3 for down-regulated or between 1 and 3 for up-regulated putative genes, respectively. However, a few genes showed even
The transcription of genes in the source tissue (
The differentially regulated genes belonged to various functional groups, whose response to mild chilling varied regarding date and plant organ. Numbers of regulated genes in the different groups are illustrated in
Also at the level of individual genes, three phases of chilling response became apparent (
Genes related to different stages of carbohydrate metabolism were regulated under chilling stress. For example, one gene coding for the mitochondrial F1 ATPase subunit, a PS light reaction ATP synthase (GO_drs21P0003D18_R_ab1), was down-regulated in the apex at 1 DoT and in the source at 3 DoT. Three genes coding for AAA-type ATPase were up-regulated at 21 DoT, two in the source leaves (DY395838_1, GO_drs21P0002F21_R_ab1) and one (GO_drs12P0011F24_F_ab1) in the internode. One gene coding for photosystem II subunit R (cn675) was down-regulated in the apex within the early phase (1 and 3 DoT) but in the source and in the internode at 21 DoT. At the same date, down-regulation of four genes coding for RuBisCO activase (cn2051, cn2052, cn2053, cn3274), a type of chaperone that is essential to promote and maintain the catalytic activity of RuBisCO (reviewed in
One gene coding for expansin (GO_drpoolB-CL8367Contig1), which is required for leaf growth, was constitutively up-regulated in the source at all dates (1/3/7/21 DoT,
Exposure of the chilling-sensitive
Our findings indicate that the observed growth depression is the outcome of a functional disturbance of the whole plant resulting from a highly dynamic and complex molecular physiological stress response. This is mirrored by changes in carbohydrate metabolism, phytohormone homeostasis, and gene expression patterns in three aerial parts of the plant, which constitute important functional units for plant growth.
Carbohydrate metabolism provides the backbone of plant growth, which is dependent on the availability of carbon sources in the plant and on the transport from source tissues to growing sinks. Furthermore, growth is dependent on carbohydrate utilization at the site of growth. Up to now, only few studies have investigated the response of carbohydrate metabolism to mild chilling temperatures and those studies did not consider different functional units of the plant.
To cover the dynamic of both sides of the source-sink network of aerial parts of petunia plants, we monitored carbohydrate levels in the uppermost fully developed leaves as carbohydrate source tissue and in the shoot apex as important utilization sink over a period of 28 days. Even though the microarray data revealed a disturbance of the photosynthetic machinery at transcriptional level, the metabolic data does not reflect a carbohydrate shortage at the source side. By contrast, ‘Williams’ reacted to exposure to sub-optimal temperatures with a sudden strong increase of hexoses and sucrose already within the first day of treatment, followed by an accumulation of starch from the second day on. While the accumulation of carbohydrates in source leaves was maintained over the whole chilling period of 4 weeks, the increase of carbohydrates in the apex during the first days was followed by intermediate approximation to concentrations of the control between 7 and 14 DoT, and from 21 DoT onward, turned into a reversed situation with lower hexose and starch levels in chilled plants when compared to the controls (
Soluble sugars are well known to increase in plant cells at low temperatures and to act there as cryoprotectants that shelter plant cells from freezing (reviewed in
Since invertases, which are responsible for the turnover of Suc to Fru and Glc and thereby contribute to sink activity (
Considering the magnitude of carbohydrate responses to the mild chilling (
Phytohormones are known to be involved in reactions of plants to biotic and abiotic stresses. In rice, JA contents were increased and JA biosynthesis and signaling were induced under cold stress (
Considering the protective roles of both, ABA biosynthesis and reduced ethylene-signaling in tolerance of tomato against moderately sub-optimal temperature stress,
Beyond the changes discussed above, the microarray data reflects a comprehensive phase- and organ-specific transcriptional response of the sensitive
The transcriptome data reflects a chilling-induced phase- and organ-specific regulation of diverse genes, which control DNA replication and maintenance as well as TFs involved in stress signaling. In the long-term phase, genes coding for different histones, as well as two genes coding for one chromomethylase, similar to chromomethylase 3 from
Even a mild chilling stress, realized with a temperature reduction of only 4 K, leads to a complex disturbance of plant functional integrity of a chilling-sensitive petunia cultivar, detectable at the levels of carbohydrates, phytohormones, and gene expression. The data as a whole reveals a holistic stress response: under chilling, specific functional units of the plant are readjusted and fine-tuned in different directions, so that the whole plant enhances the chance to survive at the expense of growth. Taking in account all presented data, a response model is proposed for a chilling-sensitive cultivar, which comprises three consecutive phases (summarized in
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Sabine Czekalla, Baerbel Broszies, Klaus Fricke, and Kerstin Fischer (all IGZ) for their excellent technical support in cultivating the petunia plants, sampling, performing the plant hormone analysis and extracting the RNA for the microarray studies. We also thank Dr. Siegfried Zerche and Dr. Rita Zrenner for helpful advices regarding the fertilization of plants and analysis of microarray data. We thank Beate ter Hell and Dr. Dirk Ludolph from the Lehr- und Versuchsanstalt für Gartenbau Ahlem for their support in developing the chilling test conditions and Selecta Klemm GmbH & Co. KG (Stuttgart) for recommending the cultivar and providing rooted cuttings. The work was funded by the BMBF (Federal Ministry of Education and Research of the Federal Republic of Germany) within the BMBF-AgroClustEr: WeGa – Horticulture Research Network – Product- und Production safety in intensive Plant production systems and further supported by ministries of the state of Brandenburg, the Free State of Thuringia and the Federal Republic of Germany.
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