7. GESTIÓN DE EQUIPOS Y DISPOSITIVOS
7.4. ÁRBOL DE GRUPOS
To investigate CEP gene regulation in Arabidopsis, publicly available data for CEP expression profiles were searched. Of the 12 highly conserved CEP genes, only 5 (CEP1,
3, 5, 9, 13 and 15) were represented on the Affymetrix Arabidopsis microarray chip. AtCEP3, CEP5 and CEP9 were significantly induced by environmental conditions,
particularly nutrient deficiency and abiotic stress (Table 2). For all three genes, nitrate starvation (relative to untreated seedlings) was one of the top three conditions under which a significant induction in gene expression occurred. Indeed for CEP3, this was the only condition where induction was seen at all. CEP5 was highly induced in
numerous hypoxia studies as well as during germination and shift to low pH. CEP9 was also induced in hypoxia studies and during germination and was extremely highly induced during callus formation. Additionally, AtCEP9 was found to be one of the 31 signature genes up-regulated by elevated field CO2 (Li et al., 2006). CEPs1, 13 and 15
were induced by a range of conditions. Notably, CEP1 was induced by IAA treatment (in many different Arabidopsis ecotypes), but not by nitrate starvation. CEPs13 and 15 were both induced by biotic stress, including Pseudomonas syringae and
Golovinomyces cichoracearum infection.
Table 2. Relative change in expression levels for CEP3, CEP5, CEP9, CEP13 and CEP15 under various conditions. Data obtained from Genevestigator (Hruz et al., 2008) filtered for induction only with minimum expression change of 1.5 fold and p < 0.05. Green highlight indicates the top 3 conditions. Specific details of studies undertaken can be found at www.genevestigator.com.
Stimulus Fold Change
CEP1
cold study 6 (Coi) / 20° C/18° C treated rosette samples (Coi) 1.94
decapitation / axillary bud samples (Col-0) 1.51
IAA + Dex / mock treated iaa1GR seedlings 3.49
IAA study 2 / solvent treated seedlings 1.51
IAA study 3 / solvent treated seedlings 1.78
IAA study 6 / mock treated iaa1GR seedlings 2.40
IAA study 7 (Sha) / untreated seedling samples (Sha) 1.54
IAA study 8 (Bay-0) / untreated seedling samples (Bay-0) 3.49 IAA study 8 (Bl-1) / untreated seedling samples (Bl-1) 2.03 IAA study 8 (Bur-0) / untreated seedling samples (Bur-0) 3.70
IAA study 8 (C24) / untreated seedling samples (C24) 1.74
IAA study 8 (Fei-0) / untreated seedling samples (Fei-0) 1.86
IAA study 8 (Sha) / untreated seedling samples (Sha) 2.13
long day (cs26) / short day study 2 (cs26) 1.51
pollen-pistil interaction ( 8.0 hap) / unpollinated pistil samples 2.01 shift SD to LD (5d) / short day shoot apex samples at 16°C (Col-0) 1.95
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Stimulus Fold Change
CEP3
nitrate starvation / untreated seedlings 1.66
CEP5
GA4 study 4 (3h) / untreated Pcga2ox1 hypocotyl samples (3h) 2.71
germination (48h) / seed desiccation 2.29
germination (48h) / stratification (48h) 2.45
hypoxia study 15 (p35S:HF-RPL18) / mock treated p25S:HF-RPL18 root samples 2.12 hypoxia study 17 (pGL2:HF-RPL18) / mock treated pGL2:HF-RPL18 root samples 2.49 hypoxia study 17 (pPEP2:HF-RPL18) / mock treated pPEP2:HF-RPL18 root samples 2.52 hypoxia study 17 (pRPL16B:HF-RPL18) / mock treated pRPL16B:HF-RPL18 root samples 2.93 hypoxia study 17 (pSHR2:HF-RPL18) / mock treated pSHR2:HF-RPL18 root samples 3.36 hypoxia study 17 (pSUC2:HF-RPL18) / mock treated pSUC2:HF-RPL18 root samples 4.04 hypoxia study 17 (pWOL:HF-RPL18) / mock treated pWOL:HF-RPL18 root samples 2.70 hypoxia study 9 (AtERF73/HRE1-RNAi20) / untreated root samples (AtERF73/H... 5.57 hypoxia study 9 (Col-0) / untreated root samples (Col-0) 3.43 iron deficiency / protoplasting / iron deficiency study 8 (24h) 7.18
light study 4 (cli186) / dark grown cli186 seedlings 1.53
KNO3 study 2 (root) / mock treated root samples (Col-0) 1.96
KNO3 study 5 (1.5h) / N depletion study 2 2.13
N depletion (Col-0) / Seedlings grown under N-replete condition (Col-0) 3.36
nitrate starvation / untreated seedlings 6.38
P deficiency (late) / high Pi treated whole plant samples (late) 1.65 P deficiency study 4 (root) / mock treated root samples (Col-0) 1.66
shift NPA to naxillin (2h) / NPA study 3 3.11
shift to pH 4.6 (24h) / mock treated root samples (24h) 1.56
shift to pH 4.6 (6h) / mock treated root samples (6h) 1.55
sucrose study 3 (Col-7) / untreated seedlings (Col-7) 1.54
sulfur deficiency study 2 (3h) / mock treated root samples 2.45 CEP9
2,4-D + kinetin (ckh1-1) / 2,4-D study 2 (ckh1-1) 1.98
2,4-D + kinetin study 2 (ckh1-1) / 2,4-D study 2 (ckh1-1) 4.83
blue study 2 / low light grown seedlings (Col-0) 1.54
callus formation study 3 (25d + 1d) / untreated hypocotyl samples (35d) 34.97 callus formation study 3 (7d + 1d) / untreated hypocotyl samples (7d) 11.66 GA4 study 4 (3h) / untreated Pcga2ox1 hypocotyl samples (3h) 1.76
germination (48h) / seed desiccation 1.69
germination (48h) / stratification (48h) 2.99
glucose (4h) / untreated seedlings 1.59
hypoxia study 17 (p35S:HF-RPL18) / mock treated p35S:HF-RPL18 root samples 1.90 hypoxia study 17 (pSCR:HF-RPL18) / mock treated pSCR:HF-RPL18 root samples 1.72 hypoxia study 17 (pSUC2:HF-RPL18) / mock treated pSUC2:HF-RPL18 root samples 5.31 hypoxia study 17 (pWOL:HF-RPL18) / mock treated pWOL:HF-RPL18 root samples 2.16 lincomycin+R+B (0.5umol m-2 s-1) / R+B (0.5umol m-2 s-1) 1.52
KCl / KCl (48h) / KNO3 / KNO3 (48h) 1.52
N depletion (Col-0) / Seedlings grown under N-replete condition (Col-0) 4.07
nitrate starvation / untreated seedlings 5.83
primisulfuron-methyl (24h) / mock treated leaf samples (24h) 1.87
selenate / untreated root samples 2.14
sulfometuron methyl (24h) / mock treated leaf samples (24h) 5.18
CEP13
P. syringae pv. syringae study 2 (OE7a-1) / non-infected leaf samples (OE7a-1) 1.78 P. syringae pv. syringae study 2 (OE7a-1) / P. syringae pv. syringae (OE7a-1) 2.13 P. syringae pv. tomato study 17 (lht1-1) / untreated leaf samples (lht1-1) 1.60
16° C (det3) / untreated etiolated seedlings (det3) 1.71
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Stimulus Fold Change
low nitrogen / high nitrogen treated rosette samples 1.53
long day (cs26) / short day study 2 (cs26) 1.54
CEP15
2,4-D + kinetin study 2 (ckh2-1) / 2,4-D study 2 (ckh2-1) 1.68
BL/H3BO3 (10d) / untreated cell culture samples 4.72
BL/H3BO3 (2d) / untreated cell culture samples 7.91
BL/H3BO3 (4d) / untreated cell culture samples 5.51
BL/H3BO3 (6d) / untreated cell culture samples 7.07
BL/H3BO3 (8d) / untreated cell culture samples 5.73
callus formation (12h) / untreated root samples 3.35
callus formation (24h) / untreated root samples 5.05
callus formation (48h) / untreated root samples 7.94
callus formation (96h) / untreated root samples 6.21
cold / cordycepin (24h+1h) / cordycepin (1hr) 1.52
cold study 2 (late) / untreated root samples (late) 2.45
dark / 21°C (640 and 1280 min) / moderate light / 21° C (640 and 1280 min) 1.92 G.cichoracearum study 2 (18h) / non-infected whole rosette sample (Col-0) 1.74 G.cichoracearum study 2 (36h) / non-infected whole rosette sample (Col-0) 2.01 P. syringae pv. Tomato study 11 (Ler) / untreated leaf disc samples (Ler) 1.74 iron deficiency / protoplasting / iron deficiency study 8 (24 h) 1.91 salicylic acid study 7 (npr1-1 sni1 ssn2-1) / solvent treated whole plant samples 1.60
On the basis of the in silico expression analysis, together with preliminary data obtained within the lab (Radzman, Imin and Djordjevic, 2012, personal
communication) it was hypothesised that CEPs were responding primarily to
environmental stimuli. To investigate this further, the expression of six Group I CEPs and three Group II CEPs were examined under various growth conditions. Plants were grown for six days on standard ½ MS medium before being transferred to various treatments for 24 h (Table 3). The expression of all CEP genes was perturbed by the environmental stimuli tested in the root or shoot tissues or both, except for CEP15. As the environmental stimuli tested were by no means comprehensive, it is possible that
CEP15 expression is responsive to other environmental factors. These results suggest
that CEP1 and CEP2 are not detectable in the root under the conditions tested using Taqman probes.
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Table 3. CEPs are induced by environmental cues. Plants were grown on standard ½ MS medium for 6 days before being transferred to specified treatments or to ½ MS medium (control). Root and shoot tissue was harvest 24 h after transfer. qRT-PCR was performed using Taqman probes and data was analysed using the ΔΔCT method. Expression shown is relative to a control treatment (transfer to standard ½ MS medium for 24 hours). n.d. indicates no reproducible data could be obtained, suggesting genes are not expressed. n.t. indicates not tested. Fold change ± standard error is shown. *P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Green highlight indicates significant results.
Tissue Treatment CEP1 CEP2 CEP3 CEP4 CEP5 CEP9 CEP13 CEP14 CEP15
root 0 mM nitrogen n.d. n.d. 10.15 ± 0.58*** 1.60 ± 0.23* 2.13 ± 0.21** 1.30 ± 0.14 1.61 ± 0.80 1.04 ± 0.14 1.05 ± 0.08 0.25 mM nitrate n.d. n.d. 1.31 ± 0.08 1.17 ± 0.12 1.79 ± 0.08*** 1.29 ± 0.35 2.29 ± 0.08 1.09 ± 0.02 0.82 ± 0.06 0.25 mM NH4Cl n.d. n.d. 1.36 ± 0.03 1.26 ± 0.05* 0.71 ± 0.02*** 0.48 ± 0.02*** 2.31 ± 0.26 1.21 ± 0.03 0.84 ± 0.03 100 mM mannitol n.d. n.d. 0.57 ± 0.13 2.35 ± 0.49* 1.08 ± 0.21 0.34 ± 0.03** 2.67 ± 0.66 0.87 ± 0.06 1.25 ± 0.16 100 mM NaCl n.d. n.d. 2.00 ± 0.13* 1.68 ± 0.17** 1.08 ± 0.08 1.70 ± 0.41 1.00 ± 0.15 1.48 ± 0.29 1.04 ± 0.04 1000 ppm CO2 n.d. n.d. 0.18 ± 0.03*** 1.02 ± 0.1 1.11 ± 0.13 0.90 ± 0.41 0.58 ± 0.44 1.15 ± 0.04* 1.23 ± 0.06 shoot 0 mM nitrogen 4.40 ± 0.97** 0.46 ± 0.06** 1.16 ± 0.16 0.70 ± 0.09 0.65 ± 0.10 1.39 ± 0.18 0.62 ± 0.01* 0.96 ± 0.07 0.89 ± 0.04 0.25 mM nitrate 3.72 ± 0.11** 4.94 ± 3.18* 5.89 ± 0.37*** 4.34 ± 4.91 3.62 ± 2.08 n.t. n.t. n.t. n.t. 0.25 mM NH4Cl 0.80 ± 0.03 0.63 ± 0.08* 1.14 ± 0.11 1.01 ± 0.91 1.7 ± 0.51 n.t. n.t. n.t. n.t. 100 mM mannitol 4.79 ± 0.56*** 1.28 ± 0.08* 2.49 ± 0.34** 1.86 ± 0.16* 1.83 ± 0.16* 1.50 ± 0.04 1.63 ± 0.23 3.82 ± 0.46** 1.04 ± 0.08 100 mM NaCl 2.85 ± 0.26** 0.77 ± 0.26 1.75 ± 0.98 1.53 ± 0.93 1.05 ± 0.65 1.75 ± 0.63 0.77 ± 0.41 1.26 ± 0.01 0.85 ± 0.15 1000 ppm CO2 1.29 ± 0.09 0.94 ± 0.14 1.45 ± 0.12 1.65 ± 0.46 1.04 ± 0.19 1.06 ± 0.06 1.06 ± 0.06 1.06 ± 0.06 1.06 ± 0.06
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The most notable perturbation was a ten-fold increase in CEP3 expression in the roots under nitrogen depletion. This induction was not seen in the shoots, or under nitrogen limiting conditions in the roots. However, significant induction in the shoots was seen under low nitrate (i.e. 0.25 mM), but not limitation of ammonium. These data indicate that the response of CEP3 to low nitrogen is both tissue and nitrogen source specific. These results are further corroborated by Tabata et al. (2014), who found that CEP3 was induced significantly following 6, 24 and 48 h hours of nitrogen starvation. CEP3 was up-regulated two-fold in response to increased salt levels in the roots and increased osmotic strength in the shoots. CEP3 was down-regulated in response to a 24 h exposure to increased CO2 levels in the roots only.
The expression of other CEP genes was also perturbed under the conditions tested.
CEP4 expression was induced in the roots by nitrogen depletion and ammonium
limitation, but not by nitrate limitation. The strongest induction in CEP4 expression was seen by increased osmotic strength in both the roots and shoots. CEP5 was induced in the roots under nitrogen depletion and nitrate limitation, and repressed under ammonium limitation. The only change in CEP13 expression was repression seen in the shoots under nitrogen depletion. CEP14 expression was increased slightly in the roots under increased CO2 levels and more strongly in the shoots under increased
osmotic pressure.
CEP1 was up-regulated in the shoots under nitrogen depletion and nitrate limitation,
but not ammonium limitation. Increased osmotic strength and increased salt levels also induced CEP1 in the shoots. CEP2 expression was down-regulated in the shoots under nitrogen depletion and ammonium limitation, but was induced by nitrate limitation. Using a promoter-GUS fusion, Ohyama et al. (2008) showed that CEP1 was expressed in 14-day-old plants in the shoot apical meristem and in developing lateral root primordia. Using qRT-PCR, it was also shown that CEP2 was expressed in roots (Ohyama et al., 2008). The discrepancy in our results may be due to the fact that 7- day-old plants do not have many lateral root primordia and as we were taking whole root samples the expression may have been diluted. Additionally, the expression of
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CEP9 expression was repressed under ammonium limitation as well as increased
osmotic strength in the roots. The expression of CEP9 was not significantly induced in either roots or shoots. Surprisingly, we did not see a change in CEP9 expression under increased CO2 levels as it has been reported to be a signature gene induced by
elevated field CO2 levels (Li et al., 2006) . There are several differences in experimental
conditions that may account for this discrepancy. Firstly, Li et al. (2006) grew plants in the field, in soil, not on plates. Secondly, above-ground parts of older plants exposed to CO2 for 12 days were analysed, compared to the 24 h exposure in these
experimenets. Thirdly, plants were exposed to CO2 at 550 ppm, whereas they were
exposed to 800 ppm here. These differences in conditions are likely to contribute to the differences in CEP9 expression observed in our study.
The data presented here indicated that CEP expression is perturbed by different environmental stimuli. This implicates CEPs as regulators of plant development in response to environmental stress. Expression changes were specific to roots and shoots and each CEP gene tested had a different expression profile.