DDRT-PCR法で単離された58種類のストレス応答遺伝子のうち37種類に
ついて、水耕栽培実生に「材料と方法」2-1-3記載の方法により各種pHおよび Al処理を行った後、根と地上部における転写を解析した（Fig. 15）。転写解析
は半定量RT-PCRにより行い、遺伝子の発現量の差はPCR産物の電気泳動像
から、内部標準GAPDH遺伝子の発現量を考慮しつつ、目視にて判断した。
転写解析の結果、解析を行った全ての遺伝子は根において発現していること が認められた。これらのうち、19遺伝子は低 pHとAl処理の両方またはいず れか一方の処理に応答して発現上昇した。19遺伝子のうち、7遺伝子（MATE ファミリートランスポーター（AmMATE1 [E12c-2])、PDR (pleiotropic drug resistance) タ イ プ ABC ト ラ ン ス ポ ー タ ー （PDR2 [Al52] ）、CYP94A1 (A07c-2)、serine acetyltransferase (SAT [Al21])、4-hydroxycinnamoyl-CoA
処理の両方で発現上昇したが、3遺伝子（neomenthol dehydrogenase (NMD [D04c-1]) 、purple acid phosphatase (PAP [Al02’]) 、serine/threonine protein kinase (STK3 [Al19])）は1時間処理のみ、5遺伝子（Aサブファミ リー（ABCA [A08c-1]） およびPDRタイプ（PDR1 [Al46]) ABCトランス ポーター、aspagagine synthetase (AS [B04c-1])、methyltransferase-like (MT [C02c-1])、SHOOT2 (E24c-1)）は24時間処理のみ、coatmer subunit β’ (β’-COP [C03c-1])は低pH・1時間処理と低pH/Al・24時間処理に応答し ていた。Cation/calcium exchanger (CAX [A09c-1])と細胞膜型 H+-ATPase (HA [Al09’])の2遺伝子は、低pH・24時間処理のみ、β-D-xylosidase (β-XYL [D03c-1])は5.0 mM Al・24時間処理にのみ応答して発現上昇した。
上述の19 遺伝子のうち5遺伝子（AmMATE1、PDR2、GT1、SHOOT2、
β’-COP）は、地上部でも高濃度のAl処理（2.0および5.0、または5.0 mMの み）に応答して発現が上昇し、4-CLも低pH・1時間処理と低pH/Al・24時間 処理で地上部において発現上昇したが、他の遺伝子は地上部でのストレス応答 は認められなかった。
一方、上述の19遺伝子を除く他の18遺伝子では、class I chitinase (CHIT [Al35])が地上部でのみ低pH/Al・24時間処理に応答して発現上昇したが、そ の他の遺伝子は、根と地上部のいずれにおいてもストレス処理に応答した明確 な転写レベルの変化が認められなかった (Fig. 15)。
Acacia mangium in this study h* mM** 1 242 5 Transporter 1518AB839199LAL+ +MATE family transporterAmMATE1Eucalyptus camaldulensis0.0 1713AB839182PH− +Cation/calcium exchangerCAXMedicago truncatula0.0 2838AB839181LpA+ −ABC transporter A subfamilyABCAVitis vinifera0.0 1362AB839202LpA+ +Nitrate and chloride transporterNiClTM. truncatula0.0 1461AB839203LpA+ +Amino acid permeaseAAPPopulus trichocarpa0.0FS584846 4302AB839212LH+ +− +Pleiotropic drug resistance proteinPDR1Glycine max0.0 4446AB839214LH+ −+ +Pleiotropic drug resistance proteinPDR2M. truncatula0.0 2865AB839220LH+ ++ +Plasma membrane H+ -ATPaseHASesbania rostrata0.0 2328AB839218HAL+ +Potassium transporterKTGossypium hirsutum0.0 1575AB839219HAL− +Hexose transporterHTM. truncatula0.0 Metabolic enzyme 2292AB839196PH− +β-D-Xylosidaseβ-XYLG. max0.0FS590492 GR480913 1767AB839184LpA− +Asparagine synthetaseASG. max0.0 732AB839189LpA+ +Methyltransferase-likeMTG. max3.0e-117 858AB839194LpA+ −Phosphoglycerate mutase-likePGMG. max2.0e-162 918AB839197LpA+ +Neomenthol dehydrogenaseNMDG. max9.0e-133 2778AB839200LpA+ −LipoxygenaseLOXS. rostrata0.0GR480838 1524AB839180LH+ −+ +Cytochrome P450 94A1CYP94A1V. vinifera0.0FS585227 2079AB839187LH+ ++ +Rhamnogalacturonate lyaseRGLM. truncatula0.0 964* HAL+ +1-Aminocyclopropane-1- carboxylate oxidase-likeACCOG. max9.0e-170 OrganismE-valueIdentity to ESTd)AbbreviationLengtha) (bp)Accession numberDetection conditionb)Expressionc) Functional category and homology
1158AB839205HAL+ +NADH dehydrogenase subunit 4NADSilene latifolia0.0FS587598 975AB839208HAL+ +Serine acetyltransferaseSATNicotiana tabacum2.0e-125 1542AB839209HAL+ +4-Hydroxycinnamoyl-CoA ligase4-CLCoffea arabica0.0 1620AB839210HAL+ +DihydropyrimidinaseDHPM. truncatula0.0FS588531 972AB839211HAL+ +Class I chitinaseCHITAcacia koa0.0 1410AB839213HAL+ +GlycosyltransferaseGT1Pueraria montana0.0 1869AB839216HAL+ +Purple acid phosphatasePAPG. max0.0 1512AB839221HAL+ +Cytochrome P450 71DCYP71DG. max5.0e-175 1407AB839223HAL+ +UDP-glycosyltransferaseGT2G. max0.0 Transcription factor 2562AB839188LpA+ −Auxin response factor-likeARFG. max0.0FS592678 1362AB839201LpA+ −SHOOT2 proteinSHOOT2G. max7.0e-158 1353AB839217HAL+ +Basic leucine zipper proteinbZIPPhaseolus vulgaris0.0FS586684 Signal transduction 2400AB839183LpA+ −Serine/threonine protein kinase- likeSTK1G. max0.0 1221* LpA+ −Calcium-dependent protein kinase-likeCDPKG. max2.0e-11 1935AB839191LpA+ −Cysteine-rich receptor-like protein kinaseCRKG. max0.0 624AB839192LpA+ −Protein MKS1-likeMKSG. max8.0e-71 1926AB839198LpA+ −Receptor-like protein kinaseRLKG. max0.0 1423*HAL+ +Serine/threonine protein kinaseSTK2G. max1.0e-145 1149AB839207HAL+ +Serine/threonine protein kinaseSTK3G. max0.0 538*HAL− +GTP-binding proteinGBPV. vinifera2.0e-30 558AB839222HAL+ +Protein kinasePKG. max1.0e-115
Other 682* LpA+ +DNA-directed RNA polymeraseDDRPM. truncatula2.0e-10 3048AB839185LpA+ −Putative E3 ubiquitin ligaseE3ULP. trichocarpa0.0 363AB839186LpA+ −Heavy metal associated domain containing proteinHMAG. max2.0e-29 744AB839224LpA− +Expansin-likeEXPG. max8.00E-93 1986AB839195LpA+ −Armadillo repeat-containing proteinARPM. truncatula0.0 297* LpA+ −Proton pump-interactor 1-likePPIG. max2.0e-11 939AB839190LH− +− +Coatomer subunit β'-2-likeβ'-COPG. max2.0e-179 1098AB839193LH+ ++ +MYND-type zinc finger proteinMYNDArabidopsis thaliana4.0e-149FS587256 1257AB839204HAL+ +Extensin-like proteinEXTG. max0.0 788** HAL+ +60S Ribosomal protein-like60SG. max1.0e-46 Unknown 1230AB839206HAL+ +Uncharacterized proteinG. max3.0e-155 366AB839215HAL+ +Uncharacterized proteinG. max2.0e-42 1290*LpA+ −No hits 1131** LpA+ −No hits 811*LpA+ −No hits 1241** HAL+ +No hits 932*HAL+ +No hits 1377*HAL− +No hits : partial sequence and not cloned. ** : estimated pseudogene and not cloned. databases (FS: Suzuki et al., 2012; GR: Yong et al., 2011).
nt. LpA: low-pH and low- : time for low-pH and/or low-pH/Al treatments. ** : Al concentration of high-Al treatment. + and -: up-regulated and similar level compared with control, .
Al-resistance gene homologs detected in Acacia mangium in this study 1 h24 h0.1251 h24 h1 h24 h 1443N.D.N.D.N.D.N.D.N.D.N.D.N.D.N.D.Aluminum-activated malate transporterGlycine max NP_00123798967At1g0843042 1545−−−−−−−−−Aluminum-activated citrate transporterGlycine max NP_00123872283At1g5134069 1278−−−−−−−−−Sensitive to proton rhizotoxicity 1-likeGlycine max XP_00355620674At1g3437058FS585053 873−−+++−−++ABC transporter I family member 17Glycine max XP_00658491079At1g6794070FS588102 1890−−−−−−−−+ABC transporter B family member 25-likeCicer arietinum XP_00450865886At5g3904074 840−−+++N.D.−++Aluminum sensitive 3-likeGlycine max XP_00353697284At2g3733076 . At the amino acid level. databases (Suzuki et al., 2012).
Identity to ESTd)CellRoot Low-pH24h Al (mM) GeneIdentity (%)c)
Result of Blast search Low-pH5 mM Al HomologyOrganism and accession no.Identity (%)b)
Length (bp)
Gene expressiona) Identy to Arabidopsis Al resistance gene
F
Fig. 1. A. mangium seedlings at 10 days after sowing on an agar medium showing various growth rates.
F
Fig. 2. Effect of pH on the root growth of A. mangium seedlings. Bars indicate SE (n = 9). Same alphabets show no significant difference at p = 0.05 by Tukey test.
pH Relative root length (% of control) 0
20 40 60 80 100 120 140
6.0 4.3 3.5 3.0 ab
b a
ab
F
Fig. 3. Effect of Al concentration on root growth of A. mangium seedlings.
Bars indicate SE (n = 44 -66). Same alphabets show no significant difference at p = 0.05 by Tukey test.
AlCl3 concntration (mM) 0
20 40 60 80 100 120 140
0 0.05 0.1 0.5 1 2 5
a a a a a a
b
Relative root length (% of control)
F
Fig. 4. Hematoxylin staining of Al-treated A. mangium roots. A, control (0.0 mM AlCl3); B, 0.05 mM AlCl3; C, 1.0 mM AlCl3; D, 5.0 mM AlCl3.
200 μm A
B
C
D
F
Fig. 5. Morin staining of Al-treated A. mangium roots. A and F, control (0.0 mM AlCl3); B and G, 0.05 mM AlCl3; C and H, 0.1 mM AlCl3; D and I, 0.5 mM AlCl3; E and J, 5.0 mM AlCl3. A – E, light microscope. F – J, fluorescence microscope.
A
B
C
D
E
F
G
H
I
J
F
Fig. 6. Cross section of Al-treated A. mangium roots with morin staining. A and C, 0.1 mM AlCl3; B and D, 5.0 mM AlCl3; A and B, light microscope; C and D, fluorescence microscope.
100 μm A
B
C
D
F
Fig. 7. Evans blue staining of Al-treated A. mangium roots. A, control (0.0 mM AlCl3, pH 4.3); B, 0.5 mM AlCl3; C, 1.0 mM AlCl3; D, 5.0 mM AlCl3.
A
B
C
D
200 μm
F
Fig. 8. Suspension-cultured A. mangium cells in low-pH and high Al media.
A, pH 5.8; B, pH 2.5; C, 3.0 mM AlCl3 (pH 3.0).
F
Fig. 9. Effect of Al on cell growth of A. mangium cultures . Bars indicate SE (n = 16). Same alphabets show no significant difference at p = 0.05 by Tukey test.
0 20 40 60 80 100 120
0 0.05 0.1 0.5 1 2 5 AlCl3 concntration (mM)
Relative cell growth (% of control)
a
b c
c
a a
d
F
Fig. 10. Numbers of stress-responsive genes detected in two analyses using cultured A. mangium cells. Analysis I, low-pH and low-pH/Al conditions;
analysis II, high-Al conditions.
2 31 1
Low-pH Low-pH/Al
Analysis I
0 25 6
2 mM Al 5 mM Al
Analysis II
27 7 24
Analysis I Analysis II
F
Fig. 11. Expression analysis of genes involved in stress resistance in A.
mangium detected through degenerate PCR.
Fig. 12. Al-induced efflux of organic acids from cultured A. mangium cells determined by GC analysis. A, secretions from control cells; B, secretions from cells treated with 2.0 mM AlCl3; C, standard samples. Cit, citric acid;
B
C A
F
Fig. 13. Al-induced efflux of citrate from cultured A. mangium cells measured by UPLC analysis.
AlCl3 concentration (mM)
Excretion of citrate (µg/g cells)
0.0 5.0 10.0 15.0 20.0
0.0 0.1 2.0 5.0
F
Fig. 14. Low-pH-induced efflux of proton from cultured A. mangium cells.
A and B, control (pH 5.8); C and D, pH 3.0. A and C, cells; B and D, wash solution.
A
B
C
D
Fig. 15. Organ-specific expression of selected genes in Acacia mangium seedlings.
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