Table 1.
Features of the response of tobacco to drought stress mRNA, metabolite, and promoter levels
| Tissue | Observations | Comments | |
|---|---|---|---|
| UP-REGULATED GENES | |||
| NtERF187 | Leaves and roots | 295-fold induced after 40 minutes in root. 23-fold in leaf after one hour. | Similar to Arabidopsis drought-inducible AtERF53, that regulates drought-responsive gene expression by binding to the GCC box and/or dehydration-responsive element (DRE) in the promoter of downstream genes. Overexpression of AtERF53 driven by the CaMV35S promoter resulted in an unstable drought-tolerant phenotype. |
| NtERF114 and NtERF202 | Leaves | Rapid and transient up-regulation in leaves with a maximum of 37-55-fold induction after one hour. | Similar to Arabidopsis AtDREB1a/AtCBF3 which is involved in response to low temperature, drought, and abscisic acid. |
| NtERF228 | Leaves and roots | Rapid and transient up-regulation in roots with a maximum of 127-fold induction at the first time point. Similar rapid and transient up-regulation in leaves. | Similar to Arabidopsis AtDREB1a/AtCBF3 which is involved in response to low temperature, drought, and abscisic acid. |
| NtMYB149 | Roots only | Rapid up-regulation reaching 131-fold after 40 minutes. Not inducible in leaves. | Similar to AtMYB15. AtMYB15 is involved in ABA-, ethylene-, and JA-mediated signaling pathways, the response to salt stress, and the response to water deprivation |
| NtERF218 | Leaves and roots | Rapid and transient up-regulation in both tissues. | Similar to Arabidopsis AtDREB1a/AtCBF3 which is involved in response to low temperature, drought, and abscisic acid. |
| NtWRKY1 | Leaves and roots | Rapid and transient induction in leaves (28-fold). Low level induction in roots. | The apparent ortholog of AtWRKY33, which had been shown to play major roles in the response to stress including abiotic stress. |
| NtERF75 | Leaves and roots | Strong (130-fold) late induction in leaves. Late and lower level induction in roots. | Similar to a member of the DREB subfamily A-6 in Arabidopsis. There are 8 members in this subfamily including RAP2.4. |
| Ninja-family protein AFP3/ABI five-binding protein 3 | Leaves only | 25-fold induced in leaves. Not induced in roots. (CHO_OF648xm02r1) | The Arabidopsis ortholog acts as a negative regulator of abscisic acid (ABA) responses and stress responses. Also called ABI five-binding protein 3. |
| Glutathione peroxidases | 75-fold induced in leaves and 24-fold in roots. (CHO_OF6818xm12r1 and FG645026) | Control of H2O2 homeostasis, and linking ABA and H2O2 signaling in stomatal closure. | |
| Protein phosphatase 2C genes | Leaves and roots | Several genes up-regulated 20–70 fold in leaves and roots. (CHO_OF4760xf16r1 and EST EB442706) | Protein phosphatase PP2Cs acts as constitutive negative regulators of SnRK2 kinases whose autophosphorylation is required for kinase activity towards downstream targets in the ABA signaling network. |
| NtUPLL1 and NtUPLL2 | Leaves and roots | NtUPLL1 is the most strongly up-regulated gene in leaves (291-fold) and both genes are strongly induced in both leaves and roots. (CHO_OF4952xo16r1 and CHO_OF569xh04r1) | Similar to the Arabidopsis U-Box E3 ubiquitin ligases AtPUB18 and AtPUB19 that negatively regulate ABA-mediated stomatal closure and drought stress responses. |
| ABA 8'-hydroxylase CYP707A1 | Leaves and roots | Transiently up-regulated in the leaf (28-fold after 40 minutes). 6-fold in roots. (EST TC18468) | Play a major regulatory role in controlling the level of ABA in plants. Catabolizes ABA. |
| 5-Epiaristolochene 1,3-Dihydroxylase | Leaves and roots | 33-fold transiently induced in leaves. Not induced in roots. (EST AM821089) | Capsidiol is produced by Solanaceae plants in response to stresses such as pathogen or elicitor challenge. |
| Cytochrome P450 CYP94C1 | Leaves and roots | Transiently up-regulated with a peak of 115-fold after 40 minutes in roots. Up-regulated later and less in leaves. (CHO_OF3036xp15r1, CHO_OF4654xf08r1 and CHO_OF3295xn18r1) | Arabidopsis cytochrome P450, CYP94C1 is involved in JA-Ile oxidation. The enzyme catalyzes catabolic turnover of JA-Ile. CYP94C1 and CYP94B3 catalyze successive oxidation steps in JA-Ile turnover. |
| Cytochrome P450 CYP94B3 | Leaves and roots | Transiently up-regulated with a peak of 101-fold after 40 minutes in roots. Up-regulated later and less in leaves. (EST TC39596 and CHO_OF646xl21r1) | Arabidopsis cytochrome P450, CYP94C1 is involved in JA-Ile oxidation. The enzyme catalyzes catabolic turnover of JA-Ile. CYP94C1 and CYP94B3 catalyze successive oxidation steps in JA-Ile turnover. |
| Anthocyanidin synthase | Roots | 61-fold induced after one hour of drought. (CHO_OF559xd02r1) | Catalyzes the penultimate step in the biosynthesis of anthocyanins |
| UDP-glycosyltransferase 74B1 | Roots | 54-fold induced after four hours of drought. (CHO_OF354xn10f1) | Involved in glucosinolate biosynthesis. |
| Inositol polyphosphate 5-phosphatase | Roots | 35-fold after four hours of drought. (EST AM835516) | Predicted to modulate the phosphoinositide pathway, ABA levels and drought responses. |
| NtWRKY69, NtWRKY3, NtWRKY10, and NtWRKY12 | Leaves (NtWRKY3 and 69) and roots (others) | All show early induction (20–40 minutes) | Apparent Solanaceae-specific induction of genes in Group IId. Tomato SlWRKY10 is also induced by drought in leaves. Potential genes for improvement of Solanaceae drought responses. |
| DOWN-REGULATED GENES | |||
| Heat shock proteins HSF25 and HSP40/DnaJ | Roots | HSF25-like gene down-regulated 39-fold in roots only. HSP40/DnaJ-like gene 16-fold down-regulated in roots only. (CHO_OF623xn12f1 and EST AM780669) | Function in unfolded protein binding, heat shock protein binding. |
| bZIP102 | Leaves | mRNA level goes down 11-fold in leaves | Closest Arabidopsis proteins are AtbZIP34 and AtbZIP61. Function unclear. |
| METABOLITES | |||
| 4-hydroxy-2-oxoglutaric acid (KHG) | Roots | Rapid early increase and 70-fold increase by 4 hours. | Possible novel mechanism to restart respiration upon water availability after drought. Appears specific to tobacco/Solanaceae as there is no increase in level during drought in soybean. |
| Mannitol and trehalose | Roots | Later time points were marked by a sharp increase in mannitol and trehalose. | Act as an osmoprotectants (compatible solute). |
| Galactinol and Raffinose | Leaves | In the leaf, galactinol and raffinose were undetectable until the final 240 min time point, suggesting an activation of the pathway due to the stress. | The raffinose pathway can provide osmolytes in situations of low water potential. |
| Oxidized glutathione (GSSG) and dehydroascorbate | Roots and leaves | GSSG levels increase 12-fold in roots. Dehydroascorbate levels double in leaves. | The glutathione-ascorbate cycle detoxifies hydrogen peroxide which is a reactive oxygen species and the cycle is activated in tobacco as a response to drought. |
| γ-aminobutyrate (GABA) | Roots | GABA levels increase 7.8-fold in roots. | The GABA shunt is a stress response pathway, the functions of which include controlling cytoplasmic pH, maintaining C/N balance by converting glutamate in the cytosol to succinate in the TCA cycle, and aiding in oxidative stress protection by generating NADH and succinate. |
| Glycine and serine | Leaves | Dramatic reduction of glycine and serine levels in leaves to 2-4% of initial values. | Tobacco tissues down-regulate photorespiration during drought as a mechanism to reduce the accumulation of toxic ammonia. |
| Inosine | Roots | Increases nearly 50-fold. | Probable nucleotide salvage pathway to recycle nucleosides. Inosine is formed by the deamination of adenosine. |
| HORMONES | |||
| ABA (abscisate/abscisic acid) | Roots and leaves | The ABA concentration increased 8-fold after four hours in root tissue. ABA 8'-hydroxylase CYP707A1 genes are strongly and transiently up-regulated in the leaf. Many ABA responsive genes are up-regulated in both tissues. Components of ABA signaling such as protein phosphatase 2C genes are up-regulated. | ABA clearly plays a central role in regulating drought responses in tobacco. |
| JA (Jasmonate) | Roots | All of the biosynthetic enzyme genes in the JA biosynthetic pathway are rapidly and coordinately up-regulated in roots. At the metabolite level, there was a biphasic increase in N-delta-acetylornithine, which rises in response to JA. Many JA signaling components such as JAZ repressors are differentially regulated. | JA clearly plays an important role in the response to drought in tobacco, especially in the roots. |
| Ethylene (Ethene) | Roots and leaves | The biosynthetic enzyme genes in the ethylene biosynthetic pathway show up-regulation with strong tissue-specific up-regulation of ACC synthase genes and, to a lesser extent, ACC oxidase genes. | Ethylene plays a role in the regulation of drought responses. |
| PROMOTERS | |||
| NtWRKY69 | Leaves | Inducible by drought. Expression progresses upwards from the root and is initially in the vascular tissue before expression in all of the leaf. Also inducible by cold and possibly wounding. | Drought inducible promoter for leaf-inducible expression. ABA independent. Expression initially follows the vascular tissue upwards from the roots before spreading into all leaf cells. Contains three potential bHLH binding sites (CANNTG), one W box (TTGACT), one MYB binding site (CGGTCA). |
| One of the Group IId genes that our data suggest may be part of a Solanaceae-specific response to drought. | |||
| NtWRKY3 | Leaves | Inducible by drought. Also inducible by cold and possibly wounding | Drought and cold inducible promoter. One of the Group IId genes that our data suggest may be part of a Solanaceae-specific response to drought. |
| NtWRKY70 | Leaves | Inducible by drought. Also inducible by cold and wounding | Drought, wound, and cold inducible promoter. |
| NtUPLL2 | Leaves | Inducible by drought. Also inducible by cold and possibly wounding | Drought and cold inducible promoter. |
| NtGolS | Leaves | Inducible by drought. Also inducible by cold and wounding | Drought, wound, and cold inducible promoter. |
The first column contains up- and down-regulated genes at the mRNA level, metabolites, hormones, and promoters that are prominent features of drought stress responses in tobacco. The second column describes which tissue(s) are involved. The third column details our observations in this report. The fourth column discusses the observations in a wider context. Transcription factor names are taken from the TOBFAC database.