This study was the first report that analyze the difference of the hairy root architectures among wild type- and individual rol gene deletion-induced ones. The results showed that comparing to HR WT, HR ΔrolC were the most affected one, which showed significant defects in all parameters analyzed. HR ΔrolB showed significant decrease in branch root number and total branch root length. HR ΔrolD showed significant retard main root length. On the other hand, only HR ΔrolB and HR ΔrolC showed significant decrease in hairy root initiation ability test. These results suggest that rolB and rolC may play a more crucial role in hairy root formation and growing.
We also noted that HR WT performance was among the best in all parameter tested indicate that rol genes play a positive role in hairy root growth, despite their biological function remain to be elucidated.
Microarray and qRT-PCR analyses results indicated that rolB might positively regulate lipid transport, cellular amino acid derivative biosynthesis process, processes regarding to wound response, and response to ethylene stimulus. Meanwhile, the biological processes that may be positively regulated by rolC are response to wounding, response to chemical stimulus, carbohydrate metabolic process, and lipid transport. This information gives us a glance of how the biological functions were manipulated by rolB and rolC in hairy root.
Tables
Table 2-1. Primers used for genotype confirmation. Reverse primer GGATTCCGGCAGCTTCCATT CCAATCTCTTTGATTAGCTCTTCATA AAGTGAGGATACGCACG GTGTTGATCCTGCTGCTGAA GCGCGATTGTCCTGTTTTAT GTGGGCCAGTGCATATAGGT TCAGCCACAGGATCAGAGTG These primers were designed by Jung-Hao Wang (Unpublished data) Forward primer CCTGAGGTCCTTTTCCAACCA TACAAAGCGTGAGAGAGCA ATCAAGGCTGCATCGAAC GGCCATTAATTGGCACCTAC TGAATGCAGCAATCTTCACG GCAGGGCGAATAGCAGTTAG CCGCCCACTACAATGAATTT
Construction Tobacco ACTIN-9 VirA G3PDH pK18HRrolA check pK18HRrolB check pK18HRrolC check pK18HRrolD check
Table 2-2. Primers used for qRT-PCR test Reverse Primer
CCCT A G A CCA CCT T G T G CG CT T G G G A CCCCA A CA G G T CA A G T
TGACTACCAGCATAACGATTCACTA G CA G CCT CA A G G T CA G CA A CA
ATTGGGCTTTACGATCCTG GCTCGCTGGAACTGAATTGTT G CG G CA T T CG G A CCT A CG C CCA G CCCCG CG A T CA A G CT G
ACCAACTTATAAATGGAACAGAG GCACTCATTAGACATCAGTTGGAA ACACATCTCCACCATCAAT TGGCATCGTTGGAGGACTCT G G G A CCCCA A CA G CT CA A G T
Forward primerG G T T CCA T CT G G CT T CCA G T G T G C CG A G CCA T A CA T CA G CA CG CCA
CCTCAAGGTGGAAATAATGGCAATCCCA T CCA A A G G CA A G T G CCCA
AGCCGAAGTACCCGATTA GTCCGCAATCTTCAAACCCATAG G A CT CCG T CCG CCG CT G A T G G CA A CG G A G G T G G T T CG G G
ATACAGAAATGCAGAAATCCC TCAGGTGGTGTGGCGTAA TTGGCTACTCTGTTGCTAA GCTCTGAAGTTGGGTGTATGTGA G G T CCT T T G CT CA G CA CG CA
NCBI accession BQ842876 DR752062 FG637828 EH618856 AB035125 EF051128 X51426 D86629 FG638725 EB444675 EB445883 AF043554 BP526812
Probe name A_95_P000541 A_95_P003336 A_95_P003626 A_95_P004391 A_95_P005446 A_95_P007251 A_95_P010771 A_95_P015356 A_95_P019246 A_95_P020646 A_95_P021466 A_95_P027676 A_95_P075770
Table 2-2. Primers used for qRT-PCR test (continuous) Reverse Primer
T G A A A CG A G CCCG A A G CT T G A CA G CG G T G G CA CA G CCT T T G T G
AGCAGCCTCAAGGTCAGCAACA GGGCTAAGGACCTCAAAGG CCA G G T G G CCT T G T G A CCG
AACCTCTCACCACCAAAT CGTTGGCATACTTGGTGGAGACA CG A T G G T A A CA T G G T G CA A A G CC
GCGTAAGAAGCATGAGCATGAATT CCACTACCCATATCGTTATACAA CCTCTCCAACACTCTCCACAAG CA A G G CA G G T G CCCG A G A G CCG CCG G A G CT G T CG A G A A T
Forward primer
G CT G CT A CG G T CT T T G A T CA G CCC G T G T CCCA A CA A G T T G T G T T G T A G C
CCCATCCAAAGGCAAGTGCCCA AAGTTGTTACAGAAGAGACCATCCCT G G T A G G CCCCA CCCT C
TTACCGACTTGATAATATGACTT AGAGGCAGGCATTGGAGACAT CG G G CT A A A T T G CCCA G CCA
GCAGATGAGAAGGTATTACAAGCA CTCATCTCATATTTAGTCCCTCAA CCTCTACACTTCTCTCTGCCATACCCG CT A G CCCCG CG A T CA A G G CA G CA G CA A A G G G A T CG G C
NCBI accession EU123522 CV015982 AB041519 CV021588 DV157577 DW001196 EB428960 EB443821 EB684101 EH623698 EH623367 EB450585 T18330
Probe name A_95_P101828 A_95_P104722 A_95_P112757 A_95_P114413 A_95_P115397 A_95_P121177 A_95_P129257 A_95_P139897 A_95_P153957 A_95_P160832 A_95_P164222 A_95_P174692 A_95_P176232
Table 2-2. Primers used for qRT-PCR test (continuous) Reverse Primer
CT T G G CT G T A G G CG CCG A G G CCCT G G T A G G CCCCA CCCT C T G A A CCCG A G CCA T CG A CA CA
CAAGATAAGCCAAGGGAATTACGA GCTGTTCTCCAGACTGTT CAGGTGGCAAGTTGATAGG GCATAAGTTCAATCAGGTAAGGTG CCG T G A G T CCA T CA T CG CCC
ATTCCTAATGCTGCTTCCAAGA GTTCCTCCAGTTACACCAT GTTGGCATACTTGGCGGAGAC CTTGATCTAGGCGGTGTT CTCAACAGATAGCGGACAATG Forward primerA G T G T CG A G G G CA CA G G G A A G CCA G G T G G CCT T G T G A CCG T G CG A T T A G A A G CG CG G T T G G T
GCGACCAAGCAAAGGAAGA GTTCAAGCCTCATATTACACAA TTGGTTGTGGATATTGTGGAA ATGGTCACGGGAGAGATGCA CA CA A CT T G G G T T G G CT G A G T
ACTATACTCCCTGACCTCTGTTT GCTTCAAGTTCCACAAGTC AGAGGCAGGCATTGGAGACA GCGGTTCCAATGACTATTATG GAGGGGTTCAAGTTTTCTAATTCTNCBI accession EB443656 BQ842956 AB034638 FG636940 BP128741 FG137954 EB679225 DW003388 GU994208 FG191218 FG152217 EB424775 AB263747
Probe name A_95_P202652 A_95_P205197 A_95_P218347 A_95_P223717 A_95_P232164 A_95_P233804 A_95_P244112 A_95_P248077 A_95_P258416 A_95_P260586 A_95_P287758 A_95_P291753 A_95_P306688
Table 3-1. Numbers of differentially expressed genes analyzed by microarray.
Transcriptome comparison Up-regulated* Down-regulated*
HR DrolB vs. HR WT** 6 242
HR DrolC vs. HR WT** 42 208
HR DrolA vs. HR WT*** 217 352
HR DrolD vs. HR WT*** 271 297
* Genes were meshed under 2-fold cut off threshold
** Two biological replicates were performed in a dye-swap manner.
***Only one biological sample had been analzied.
Table 3-2. GO term of down regulated genes in HR ΔrolB compared to HR WT.
response to wounding 2.80E-06 0.00014
hydrolase activity, hydrolyzing O-glycosyl compounds 7.30E-06 0.00028 hydrolase activity, acting on glycosyl bonds 2.60E-05 0.00066
endomembrane system 3.30E-05 0.0018
response to chemical stimulus 0.00018 0.0068
oxidoreductase activity, acting on peroxide as acceptor 0.00025 0.0038
peroxidase activity 0.00025 0.0038
response to stimulus 0.00026 0.008
response to stress 0.00029 0.008
response to external stimulus 0.0004 0.0095
electron carrier activity 0.0005 0.0064
antioxidant activity 0.00089 0.0097
extracellular region 0.00096 0.026
oxidoreductase activity 0.001 0.0097
lyase activity 0.0013 0.011
transport 0.0015 0.031
establishment of localization 0.0016 0.031
cation binding 0.002 0.014
ion binding 0.002 0.014
cellular amino acid derivative biosynthetic process 0.0021 0.034
localization 0.0022 0.034
response to endogenous stimulus 0.0023 0.034
oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor 0.0026 0.017
response to hormone stimulus 0.0034 0.044
response to ethylene stimulus 0.0035 0.044
oxidoreductase activity, acting on CH-OH group of donors 0.0045 0.027 cellular amino acid derivative metabolic process 0.0067 0.08
response to organic substance 0.013 0.14
carbohydrate metabolic process 0.023 0.24
transition metal ion binding 0.03 0.16
metal ion binding 0.031 0.16
Table 3-3. GO analysis in terms of biological process of HR Δ
rol B
down regulated genes compared with those in HR WT. qRT-PCRp-
value Lipid transport 0.0010 0.0010 0.0010 NA 0.0010 NA 0.0010 Fold 0.0623 0.0192 0.0623 NA 0.0623 NA 0.0575Microarray Fold change 0.3653 0.1956 0.3684 0.2298 0.4126 0.2694 0.2984 0.2858 0.3041 0.3024 0.2387 0.3038 0.2813 0.2746
Description lipid binding extensin-like protein (ELP) protease inhibitor/seed storage/lipid transfer protein (LTP) family protein
GO source AT1G12090 1.00E-32 AT1G12090 1.00E-33 AT1G12090 5.00E-10 AT1G12090 6.00E-12 AT1G12090 9.00E-50 AT1G62500 2.00E-72 AT1G62500 5.00E-76
NCBI accession AB041519 D86629 AB041519 EB450585 AB041519 DV157577 BQ842956
Probe name A_95_P112757 A_95_P015356 A_95_P102367 A_95_P174692 A_95_P112022 A_95_P115397 A_95_P205197
Table 3-3. GO analysis in terms of biological process of HR Δ
rol B
down regulated genes compared with those in HR WT (continued) qRT-PCRp-
value Lipid transport NA NA NA 0.0010 0.0010 0.0020 0.0010 Cellular amino acid derivative biosynthetic process Fold NA* NA NA 0.0594 0.0192 0.0268 0.0495Microarray Fold change 0.1966 0.0741 0.3844 0.3523 0.2197 0.3427 0.1052 0.4772 0.3718 0.1805 0.0432 0.3738 0.3510 0.2227 0.2299 0.1074 0.2937 0.3608
Description protein_coding protease inhibitor/seed storage/lipid transfer protein (LTP) family protein structural constituent of cell wall Encodes a Protease inhibitor/seed storage/LTP family protein DIR1 (DEFECTIVE IN INDUCED RESISTANCE 1) EFE (ETHYLENE-FORMING ENZYME); 1-aminocyclopropane-1-carboxylate oxidase (ACO) MYB DOMAIN PROTEIN 58 (MYB58)
GO source AT1G62510 4.00E-32 AT1G62510 4.00E-35 AT1G62510 5.00E-52 AT1G62510 7.00E-50 AT3G22142 6.00E-14 AT5G48485 6.00E-21 AT5G48485 7.00E-22 AT1G05010 8.00E-57 AT1G16490 2.00E-26
NCBI accession AF043554 AB035125 BQ842876 EH618856 D86629 DW003388 EB443656 AB012857 DW002203
Probe name A_95_P027676 A_95_P005446 A_95_P000541 A_95_P004391 A_95_P109177 A_95_P248077 A_95_P202652 A_95_P029796** A_95_P122162
Table 3-3. GO analysis in terms of biological process of HR Δ
rol B
down regulated genes compared with those in HR WT (continued) qRT-PCRp-
value Cellular amino acid derivative biosynthetic process 0.0010 NA 0.0010 Response to wounding 0.0010 0.0010 0.0010 NAFold 0.2120 NA 0.2976 0.2120 0.2183 0.0260 NA
Microarray Fold change 0.4216 0.4881 0.4460 0.4916 0.4216 0.3354 0.2023 0.3944 0.3273 0.3551 0.4198 0.3768 0.3273 0.2679 0.1019 0.0532
Description RNS1 (RIBONUCLEASE 1) AT5MAT; O-malonyltransferase/ transferase At3g29590 (At5MAT) ACS9 (1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 9) ACS6 (1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID (ACC) SYNTHASE 6) RNS1 (RIBONUCLEASE 1) ATHCHIB (ARABIDOPSIS THALIANA BASIC CHITINASE) putative wound-responsive protein
GO source AT2G02990 8.00E-92 AT3G29590 3.00E-33 AT3G49700 1.00E-113 AT4G11280 2.00E-67 AT2G02990 8.00E-92 AT3G12500 4.00E-81 AT3G12500 5.00E-13 AT4G10265 2.00E-12
NCBI accession AB034638 AB176525 EU123523 EU123522 AB034638 X51426 CV015982 CV021588
Probe name A_95_P218347 A_95_P293088 A_95_P283463 A_95_P101828 A_95_P218347 A_95_P010771 A_95_P104722 A_95_P114413
Table 3-3. GO analysis in terms of biological process of HR Δ
rol B
down regulated genes compared with those in HR WT (continued) qRT-PCRp-
value Response to wounding 0.0010 0.0010 0.0010 0.0010 0.0010 Response to ethylene stimulus NAFold 0.0881 0.0592 0.0463 0.0784 0.2976 NA
Microarray Fold change 0.3635 0.3192 0.2231 0.2429 0.4916 0.2019 0.2287 0.1830 0.1291 0.2589 0.3768 0.2168
Description wound-responsive family protein ACS6 (1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID (ACC) SYNTHASE 6) ATPRB1 pathogenesis related protein, encodes a basic PR1-like protein
GO source AT4G10270 1.00E-15 AT4G10270 2.00E-21 AT4G10270 2.00E-22 AT4G10270 3.00E-25 AT4G11280 2.00E-67 AT2G14580 2.00E-53
NCBI accession T18330 EB443821 DR752062 BP526812 EU123522 EB444675
Probe name A_95_P176232 A_95_P139897 A_95_P003336 A_95_P075770 A_95_P101828 A_95_P020646
Table 3-3. GO analysis in terms of biological process of HR Δ
rol B
down regulated genes compared with those in HR WT (continued) qRT-PCRp-
value Response to ethylene stimulus 0.0010 0.0010 0.0010 0.0010 * NA represent no qRT-PCR data available ** Dark background represent probes without appropriate NCBI accession Fold 0.0260 0.2183 0.0260 0.2976Microarray Fold change 0.1929 0.3354 0.2023 0.4916 0.1433 0.2679 0.1019 0.3768
Description PR4 (PATHOGENESIS-RELATED 4) ATHCHIB (ARABIDOPSIS THALIANA BASIC CHITINASE) ACS6 (1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID (ACC) SYNTHASE 6)
GO source AT3G04720 3.00E-12 AT3G12500 4.00E-81 AT3G12500 5.00E-13 AT4G11280 2.00E-67
NCBI accession CV015982 X51426 CV015982 EU123522
Probe name A_95_P110557 A_95_P010771 A_95_P104722 A_95_P101828
Table 3-4. GO results of HR ΔrolC down regulated genes compared to HR WT.
Term
p-value
FDRlipid transport 6.00E-12 6.40E-10
lipid localization 9.10E-12 6.40E-10
macromolecule localization 5.20E-05 0.0024
response to chemical stimulus 0.00019 0.0041
localization 0.00022 0.0041
transport 0.00014 0.0041
establishment of localization 0.00015 0.0041
response to stimulus 0.00023 0.0041
response to wounding 0.00052 0.0081
carbohydrate metabolic process 0.0011 0.015
ion transport 0.0063 0.075
response to metal ion 0.0064 0.075
response to inorganic substance 0.0095 0.1
response to stress 0.012 0.11
response to external stimulus 0.012 0.11
response to hormone stimulus 0.011 0.11
response to endogenous stimulus 0.018 0.15
Table 3-5. GO analysis in terms of biological process of HR Δ
rol C
down regulated genes compared with those in HR WT. qRT-PCRp-
value Lipid transport 0.0010 0.0010 0.0010 0.0010 0.0010 0.0010 0.0010 Fold 0.3602 0.1946 0.3602 0.3451 0.2479 0.1642 0.1254Microarray Fold change 0.2092 0.0756 0.2662 0.1643 0.1774 0.0685 0.0072 0.3996 0.4885 0.4105 0.3838 0.3770 0.3576 0.1461
Description lipid binding extensin-like protein (ELP) EXTENSIN-LIKE PROTEIN (ELP) protease inhibitor/seed storage/lipid transfer protein (LTP) family protein protease inhibitor/seed storage/lipid transfer protein (LTP) family protein
GO source AT1G12090 1.00E-32 AT1G12090 1.00E-33 AT1G12090 9.00E-50 AT1G62500 2.00E-72 AT1G62500 5.00E-76 AT1G62510 4.00E-32 AT1G62510 4.00E-35
NCBI accession AB041519 D86629 AB041519 DV157577 BQ842956 AF043554 AB035125
Probe name A_95_P112757 A_95_P015356 A_95_P112022 A_95_P115397 A_95_P205197 A_95_P027676 A_95_P005446
Table 3-5. GO analysis in terms of biological process of HR Δ
rol C
down regulated genes compared with those in HR WT (continued) qRT-PCRp-
value Lipid transport 0.0010 0.0010 0.0010 0.0010 Carbohydrate metabolic process 0.0010 NA 0.0010 0.0450 Fold 0.5521 1.0033 0.1946 0.1077 0.2927 NA* 0.3002 0.4036Microarray Fold change 0.3719 0.4030 0.0865 0.3192 0.2151 0.4876 0.1914 0.4358 0.0943 0.4515 0.4378 0.3384 0.3560 0.2664 0.4150 0.3404 0.4459 0.3582
Description protease inhibitor/seed storage/lipid transfer protein (LTP) family protein constituent of cell wall Encodes a Protease inhibitor/seed storage/LTP family protein DIR1 (DEFECTIVE IN INDUCED RESISTANCE 1); lipid binding polygalacturonase polygalacturonase glycoside hydrolase family 28 protein / polygalacturonase (pectinase) family protein polygalacturonase, putative / pectinase, putative polygalacturonase glycoside hydrolase family 28 protein / polygalacturonase (pectinase) family protein xyloglucan:xyloglucosyl transferase, putative
GO source AT2G10940 2.00E-54 AT3G22142 3.00E-66 AT3G22142 6.00E-14 AT5G48485 6.00E-21 AT1G10640 2.00E-80 AT1G48100 8.00E-88 AT1G60590 4.00E-77 AT3G61490 1.00E-148 AT4G37800 1.00E-88
NCBI accession FG191218 FG137954 D86629 DW003388 EB428960 DW001196 FG152217 EB679225
Probe name A_95_P260586 A_95_P233804 A_95_P109177 A_95_P248077 A_95_P129257 A_95_P121177 A_95_P287758 A_95_P244112 A_95_P290538**
Table 3-5. GO analysis in terms of biological process of HR Δ
rol C
down regulated genes compared with those in HR WT (continued) qRT-PCRp-
value Carbohydrate metabolic process 0.0679 NA Response to chemical stimulus NA 0.1568Fold
0.9363
NA NA0.898
Microarray Fold change 0.4273 0.142 0.1557 0.3779 0.3989 0.3861 0.432 0.1085 0.1921
0.4277 0.3906 0.4249 0.4091 0.4723 0.4281 0.2947 0.2515 0.2724
Description acidic endochitinase (CHIB1) acidic endochitinase (CHIB1 glycosyl hydrolase family 17 protein XYL4; hydrolase, hydrolyzing O-glycosyl compounds / xylan 1,4-beta-xylosidase 17.8 kDa class I heat shock protein (HSP17.8-CI) putative auxin-responsive protein PETE1 (PLASTOCYANIN 1); copper ion binding / electron carrier One of two Arabidopsis plastocyanin genes
GO source AT5G24090 1.00E-105 AT5G24090 1.00E-66 AT5G24090 4.00E-93 AT5G55180 1.00E-17 AT5G64570 1.00E-101 AT1G07400 3.00E-59 AT1G29450 8.00E-31 AT1G76100 1.00E-59 AT1G76100 3.00E-42
NCBI accession EB684101 EB424775 GU994208 FG636940
Probe name A_95_P030516 A_95_P159427 A_95_P013991 A_95_P153957 A_95_P291753 A_95_P258416 A_95_P223717 A_95_P105042 A_95_P111392
Table 3-5. GO analysis in terms of biological process of HR Δ
rol C
down regulated genes compared with those in HR WT (continued) qRT-PCRp-
value Response to chemical stimulus NA NA 0.001 0.001 0.001 NAFold NA NA 0.1797 0.161 0.161 NA
Microarray Fold change 0.1151 0.2264 0.1121 0.1091 0.0405 0.1023 0.0937 0.4368 0.2695 0.4193 0.301 0.301 0.444 0.473 0.3992 0.3879
Description PETE1 (PLASTOCYANIN 1); copper ion binding / electron carrier One of two Arabidopsis plastocyanin genes ADH1 (ALCOHOL DEHYDROGENASE 1) ATPRB1 pathogenesis related protein, encodes a basic PR1-like protein PR4 (PATHOGENESIS-RELATED 4) ATHCHIB (ARABIDOPSIS THALIANA BASIC CHITINASE) CCH (COPPER CHAPERONE)
GO source AT1G76100 7.00E-37 AT1G76100 7.00E-44 AT1G77120 1.00E-106 AT1G77120 3.00E-46 AT2G14580 2.00E-53 AT3G04720 3.00E-12 AT3G12500 5.00E-13 AT3G56240 7.00E-30
NCBI accession EH623367 EB444675 CV015982 CV015982 BP128741
Probe name A_95_P113472 A_95_P109467 A_95_P164222 A_95_P144057 A_95_P020646 A_95_P110557 A_95_P104722 A_95_P232164
Table 3-5. GO analysis in terms of biological process of HR Δ
rol C
down regulated genes compared with those in HR WT (continued) qRT-PCRp-
value Response to chemicall stimulus 0.001 0.001 0.001 0.001 0.001 NA 0.001 0.001 Fold 0.3695 0.4959 0.4959 0.1847 0.8341 NA 0.3161 0.0924Microarray Fold change 0.4895 0.182 0.2865 0.2957 0.2218 0.2717 0.3272 0.3272 0.2793 0.4854 0.3277 0.3632 0.2812 0.4 0.3634 0.4321 0.3765 0.4726
Description ACS6 (1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID (ACC) SYNTHASE 6); putative L-lactate dehydrogenase putative peroxidase YSL2 (YELLOW STRIPE LIKE 2) auxin-responsive family protein PDC2 (pyruvate decarboxylase-2) gibberellin-regulated family protein gibberellin-regulated family protein xyloglucan:xyloglucosyl transferase, putative
GO source AT4G11280 2.00E-67 AT4G17260 1.00E-104 AT4G17260 2.00E-41 AT5G06730 5.00E-34 AT5G24380 3.00E-51 AT5G53590 4.00E-22 AT5G54960 0 AT5G59845 1.00E-27 AT5G65730 2.00E-93
NCBI accession EU123522 EB445883 FG637828 AB263747 EH623698 EF051128 FG638725
Probe name A_95_P101828 A_95_P021466 A_95_P158992 A_95_P003626 A_95_P306688 A_95_P160832 A_95_P007251 A_95_P019246 A_95_P293048
Table 3-5. GO analysis in terms of biological process of HR Δ
rol C
down regulated genes compared with those in HR WT (continued) qRT-PCRp-
value Response to wounding 0.001 0.001 0.001 0.001 0.001 0.001 * NA represent no qRT-PCR data available ** Dark background represent probes without appropriate NCBI accessionFold 0.4414 0.3695 0.2237 0.2953 0.0583 0.3185
Microarray Fold change 0.0937 0.4895 0.2474 0.1598 0.1413 0.2476
0.3992 0.4854 0.2978 0.2796 0.1692 0.2516
Description ATHCHIB (ARABIDOPSIS THALIANA BASIC CHITINASE) ACS6 (1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID (ACC) SYNTHASE 6) wound-responsive family protein wound-responsive family protein
GO source AT3G12500 5.00E-13 AT4G11280 2.00E-67 AT4G10270 1.00E-15 AT4G10270 3.00E-25 AT4G10270 2.00E-22 AT4G10270 2.00E-21
NCBI accession X51426 EU123522 T18330 BP526812 DR752062 EB443821
Probe name A_95_P104722 A_95_P101828 A_95_P176232 A_95_P075770 A_95_P003336 A_95_P139897
Figures
Figure 1-1. Hairy root.
After A. rhizogenes infecting healthy 8-week-old tobacco leaf discs (a), hairy roots start to emerge from the infecting site (b). Each hairy root can be cultured independently in solid (c) or liquid (d) phytohormone-free medium. Images provided by Jung-Hao Wang, unpublished data.
(a) (b)
(c) (d)
Figure 1-2. A. rhizogenes under microscope.
A. rhizogenes is a rod-shape, gram-negative soil bacterium. It can infect plant cell and transforme it into hair root. Image retrieved from:
http://commtechlab.msu.edu/sites/dlc-me/zoo/microbes/agrobacterium.html
Figure 1-3. Molecular mechanisms of how A. rhizogenes infects plant cell.
Phenolic compound (open circle) will be released as a plant signals after plant injury.
This signal can be sensed by the VirA/G two component systems (yellow circle) of A.
rhizogenes, and resulted in T-DNA (green bar) transform and insert into plant nucleus.
The expression of the inserted T-DNA will transformed the plant cell into hairy root morphology. Image provided by Jung-Hao Wang, unpublished.
Figure 1-4. The TL-DNA region of A. rhizogenes strain A4.
The rol genes are shown in orange arrows while the other open reading frames (ORFs) are shown in green. The left and right borders are shown in blue lines. NCBI accession number K03313.
strain A4 TL-DNA
21126 bp
rolB rolC
rolD
rolA orf2
orf3
orf8 orf4
orf17n orf18
Right border
Left border Convolvulus arvensis DNA
Figure 2-1. Target region of the pK18HRrol check primers.
Blue line indicated the individual rol genes, and the red arrow indicated the forward and reverse of pK18HRrol check primer. Therefore, PCR fragment of deleted rol gene should have PCR product smaller than that of the wild type.
1"""2""3"""4""5"""6 1"""2""3"""4""5"""6 1"""2""3"""4""5"""6
Figure 2-2. Measuring hairy root architecture.
The hairy root elongate from the sub-cultured 1.5 cm hairy root tip was defined as the main root (dash line). Branch root was defined as the roots directly protrude from the main root (asterisk). Total branch root length was defined as the total length of branch root. Branch root density was defined as the branch number per cm of main root (cm-1).
Figure 2-3. Explanation of Gene Ontology diagram.
The left column demonstrate the ten level of significant differences expressed by color in the block. The right column demonstrates the types of arrows used in the diagram.
Retrieved from: http://bioinfo.cau.edu.cn/agriGO/graphresult.php
Figure 3-1. Genotype confirmation of A. rhizogenes strain A4 and its derivatives.
Ten ng of genomic DNA of A. rhizogenes strain A4 and its derivatives were used as the templates for amplification using the following primers: G3PDH, VirA, pK18HRrolA check, pK18HRrolB check, pK18HRrolC check, and pK18HRrolD check. The primer designation is shown in (B) where blue line indicated the gene to be deleted, and the red arrow indicated the forward and reverse of pK18HRrol check primer. Therefore, PCR fragment of deleted rol gene should have PCR product smaller than that of the wild type, as shown in (A). For example, ΔrolA has corresponding pK18rolA product of smaller size than that of wild type. g3pdh and virA served as a bacterium control and Root
Figure 3-2. Growth curve of wild type and the rol gene-deficient A. rhizogenes strain A4.
OD600 were recorded every 3 hours after inoculation. Three biological replicates were performed in each group. The results showed that wild type A. rhizogenes strain A4 grew slower than its rol gene-deficient derivatives during 12-24 h after inoculation.
However, there was no significant differences between all groups when incubated longer than 24 h. All genotypes reached stationary phase at about 48 h.
Figure 3-3. Confirmation of hairy root genotype.
Hairy root genotype was confirmed using chromosome DNA by pK18HRrol primers. In each genotype of hairy root, four independent clones are shown here as a representative data. A. rhizogenes A4 served as a positive control of full length rol genes, while W38 served as a negative control. No template, on the other hand, served as a negative reagent control. The numbers of each primer were 1: tobacco ACTIN-9, 2: VirA, 3:
pK18HRrolA, 4: pK18HRrolB , 5: pK18HRrolC , 6: pK18HRrolD.
1""2"""3""4"""5""6
M HR WT114 1""2"""3""4"""5""6 1""2"""3""4"""5""6 1""2"""3""4"""5""6 HR WT118 HR WT228 HR WT230
1""2"""3""4"""5""6
M HR ΔrolA102 1""2"""3""4"""5""6 1""2"""3""4"""5""6 1""2"""3""4"""5""6 HR ΔrolA103 HR ΔrolA10 HR ΔrolA13 1 2 3 4 5 6
M HR ΔrolC214 1""2"""3""4"""5""6 1""2"""3""4"""5""6 1""2"""3""4"""5""6 HR ΔrolC216 HR ΔrolC101 HR ΔrolC5
1""2"""3""4"""5""6
M HR ΔrolD101 1""2"""3""4"""5""6 1""2"""3""4"""5""6 1""2"""3""4"""5""6 HR ΔrolD114 HR ΔrolD117 HR ΔrolD118 1""2"""3""4"""5""6
M HR ΔrolB116 1""2"""3""4"""5""6 1""2"""3""4"""5""6 1""2"""3""4"""5""6 HR ΔrolB117 HR ΔrolB214 HR ΔrolB217
500
Figure 3-4. Phenotypes of A. rhizogenes A4 and its derivatives-infected tobacco leaf discs at 21 dpi.
Thirty independent leaf discs were infected in each group. Systematic sampling chosen-leaf discs are shown. Hairy root numbers were less in ΔrolB- and ΔrolC-induced leaf discs than WTA4-induced ones. Moreover, hairy root length in ΔrolB- and ΔrolC-induced leaf discs were also shorter, indicated that ΔrolB and ΔrolC were less virulent.
Figure 3-5. Days to the first root emergence post induction (DREPI).
The hairy root inducing rate of ΔrolB and ΔrolC were significant lower than that of the WT A4 (p =1.1x10-3, and 8.4x10-6, respectively). On the other hand, although ΔrolD has lower root emergence rate before 16 dpi, the emergence rate at 21 dpi shows no significant differences than that of WT A4 (p = 1.0). Significant differences test were performed using Mann-Whiyney U test adjusted with Bonferroni correction. The number of replications in each group was 60, 60, 60, 45, and 52, respectively.
Figure 3-6. Primary root number per leaf disc (RL ratio) at 21 dpi.
Hairy root number induced by A. rhizogenes strain A4 and its derivatives were recorded.
The bar represents the mean and the error bar represents the standard deviation.
Significant differences test using Mann-Whiyney U test adjusted with Bonferroni correction revealed significant differences existed between WTA4 and ΔrolB and between WTA4 and ΔrolC. The number of replications in each group was 60, 60, 60, 45, and 52, respectively. (** p < 0.01, *** p < 0.001).
Figure 3-7. Selected hairy root appearances after 18 days post sub-culturing.
The hairy roots were selected systematically according to their main root length. Each row represents a specific genotype. The population of HR ΔrolB and ΔrolC groups seems to show shorter main root length and retard branch root system. The independent clones observed in HR WT, HR DrolA, HR DrolB, HR DrolC, and HR DrolD, were 84,93,65,82, and 58.
HR#WT
HR#ΔrolA
HR#ΔrolB
HR#ΔrolC
HR#ΔrolD
Figure 3-8. Main root length of A. rhizogenes A4 and its rol gene-deficient derivatives.
Population distribution of each groups are shown in (A). Main root length is shown in x-axis while percentage of the population shown in y-axis. Box plot of each group are shown in (B) where the dots represent the outliers. Significant differences were analyzed using permutation by ANOVA with 1000 replications against HR WT. (*** p
< 0.001).
Figure 3-9. Branch root number of A. rhizogenes A4 and its rol gene-deficient derivatives.
Population distribution of each groups are shown in (A). Branch root number is shown in x-axis while percentage of the population shown in y-axis. The population range is small in HR ΔrolC comparing with the other groups. Moreover, branch root number of HR ΔrolC also decreased drastically. Box plot of each group are shown in (B) where the dots represent the outliers. Significant differences were analyzed using permutation by ANOVA with 1000 replications against HR WT. (** p < 0.01, *** p < 0.001).
Figure 3-10. Total branch root length of A. rhizogenes A4 and its rol gene-deficient derivatives.
Population distribution of each groups are shown in (A). Total branch root length is shown in x-axis while percentage of the population shown in y-axis. Box plot of each group are shown in (B) where the dots represent the outliers. Significant differences were analyzed using permutation by ANOVA with 1000 replications against HR WT. (*
p < 0.05, *** p < 0.001).
Figure 3-11. Branch root density of A. rhizogenes A4 and its rol gene-deficient derivatives.
Population distribution of each groups are shown in (A). Total branch root length is shown in x-axis while percentage of the population shown in y-axis. Box plot of each group are shown in (B) where the dots represent the outliers. Significant differences were analyzed using permutation by ANOVA with 1000 replications against HR WT.
(*** p < 0.001).
Figure 3-12. The constitutive expressed gene under 2-fold cut off.
The red circle represents genes extracted in first biological replicate, the blue circle represents the second. The purple one represents the constitutive expressed genes.
Numbers within the area represent gene numbers.
HR#ΔrolB#vs.#HR#WT
Up+#regulate
784 6 57352 242 107
Down+#regulate
345 42 92
HR#ΔrolC#vs.#HR#WT
304 208 301
Figure 3-13. Gene ontology of HR ΔrolB down-regulated genes.
Gene ontology was performed by AgriGO under default conditions (p-value 0.05). Each box represents a gene group according to the molecular function with brief description in the box, where the p-value was also shown. The color of the box turns from white to yellow to red when the significant difference increases. Lipid binding was the most enriched group, indicating that lipid binding may be regulated by rolB. Hydrolyzing O-glycosyl compounds and peroxidase activity were also affected.
Figure 3-14. Gene ontology of HR ΔrolC down-regulated genes.
Gene ontology was performed by AgriGO under default conditions (p-value 0.05). Each box represents a gene group according to the molecular function with brief description in the box, where the p-value is also shown. The color of the box turns from white to yellow to red when the significant different increases. Lipid binding was the most drastic affected group. Besides, carbohydrate metabolic process and respond to wounding are also extracted.
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