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Jan 12, 2011 - Mitsuru Osaki & Susan S. Miller & Carroll P. Vance. Received: 4 .... Numerous gene transcripts have enhanced accumulation in cluster roots ...
Plant Soil (2011) 342:481–493 DOI 10.1007/s11104-010-0711-9

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Real-time RT-PCR profiling of transcription factors including 34 MYBs and signaling components in white lupin reveals their P status dependent and organ-specific expression Masumi Yamagishi & Keqin Zhou & Mitsuru Osaki & Susan S. Miller & Carroll P. Vance

Received: 4 September 2010 / Accepted: 28 December 2010 / Published online: 12 January 2011 # Springer Science+Business Media B.V. 2011

Abstract Phosphorus (P) is often a limiting macronutrient because of its low availability in soils. White lupin (Lupinus albus L.) plants are well Responsible Editor: Hans Lambers. Electronic supplementary material The online version of this article (doi:10.1007/s11104-010-0711-9) contains supplementary material, which is available to authorized users. M. Yamagishi (*) : M. Osaki Research Faculty of Agriculture, Hokkaido University, N9W9, Kita-ku, Sapporo 060-8589, Japan e-mail: [email protected] K. Zhou Graduate School of Agriculture, Hokkaido University, N9W9, Kita-ku, Sapporo 060-8589, Japan S. S. Miller : C. P. Vance USDA-ARS, Plant Science Research Unit, 1991 Upper Buford Circle, St. Paul, Minnesota 55108, USA C. P. Vance Department of Agronomy and Plant Genetics, University of Minnesota, 1991 Upper Buford Circle, St. Paul, Minnesota 55108, USA Present Address: K. Zhou Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150081, China

adapted to growth under low-P conditions. White lupin acclimation to low-P conditions includes changes in root architecture and enhanced expression of numerous genes encoding for secreted acid phosphatases and phosphate transporters. However, information about transcription factors and signaling proteins that coordinate the P-starvation responses is limited in white lupin. In this study, cDNAs and ESTs encoding for transcription factors and signaling proteins were isolated and their transcription profiles were clarified to facilitate the identification of key signal transduction genes necessary to improve P acquisition, allocation, and use. 34 cDNA fragments of MYB-coiled coil (MYB-CC) and R2R3-MYB, and 26 ESTs encoding for transcription factors and signaling proteins were isolated. Four MYB-CC cDNAs showed high similarity to the transcription factor Phosphate starvation response 1 in Arabidopsis, which has been implicated in regulation of many P-starvation response genes. In addition, deduced amino acid sequences of 29 R2R3-MYB cDNAs showed similarities to Arabidopsis R2R3-MYB proteins. Transcription of the 60 genes, as measured by real-time reverse transcription-PCR, in normal roots, cluster roots, leaves, and shoot tips under P sufficient and low-P conditions revealed that six (10%) and two (3.3%) sequences were either induced or suppressed, respectively, by low-P condition. In addition, 36 genes (60%) showed an organ specific expression.

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Keywords Cluster roots . Lupinus albus L. . MYB-CC . Phosphate starvation response 1 (PHR1) . R2R3-MYB . Signal transduction Abbreviation 3′RACE Rapid amplification of cDNA 3′-end CC coiled coil DAE days after emergence EST expressed sequence tag LaMATE Lupinus albus multidrug and toxin efflux protein LaPT1 Lupinus albus phosphate transporter 1 LaSAP Lupinus albus secreted acid phosphatase P Phosphorus PHR phosphate starvation response RT-PCR reverse transcription-PCR TF transcription factor

Introduction Phosphorus (P) is an essential element required for plant growth. However, because P in soil often forms insoluble complexes with cations, particularly with aluminum and iron, it is frequently unavailable. Thus, P is often a limiting element for plant growth (Vance et al. 2003). White lupin (Lupinus albus L.) plants are well acclimated for growth in low-P environments (Vance et al. 2003). The most well-recognized adaptation is the formation of specialized roots that have prolific, closely spaced lateral roots (rootlets) of determinate growth, called cluster (or proteoid) roots (Watt and Evans 1999b). Cluster roots increase P availability from soil by expanding the root surface area, increasing the release of proton, acid phosphatase, carboxylates, and phenolic compounds (Johnson et al. 1994, 1996a, b; Keerthisinghe et al. 1998; Neumann et al. 1999; Watt and Evans 1999a, b; Yan et al. 2002; Cesco et al. 2010). Acid phosphatases and carboxylates released from cluster roots solubilize P compounds in the rhizosphere into an absorbable form. The P-deficiency status of white lupin is the predominant signal for induction of cluster roots to release carboxylates and acid phosphatase (Johnson et al. 1994, 1996a; Keerthisinghe et al. 1998; Neumann et al. 1999). Numerous gene transcripts have enhanced accumulation in cluster roots, such as Lupinus albus secreted acid phosphatase (LaSAP) (Wasaki et al. 1999; Miller et al. 2001), phosphate transporter 1 (LaPT1) (Liu et al.

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2001), multidrug and toxin efflux protein (LaMATE) (Uhde-Stone et al. 2005) and phosphoenolpyruvate carboxylase (Uhde-Stone et al. 2003a; Peñaloza et al. 2005). P-starvation responses in white lupin are systemic, that is, the P-starvation condition in shoots leads to cluster root formation and to the exudation of carboxylates and acid phosphatase in roots (Keerthisinghe et al. 1998; Shane et al. 2003; Shen et al. 2005). Thus, signaling cascades may transport the message of P deficiency from the shoots to the roots. Long-distance signals such as sugars and microRNA399 have been identified in Arabidopsis (Lin et al. 2009; Vance 2010; Yang and Finnegan 2010), and a sugar signal has been postulated as involved in P-starvation responses in white lupin (Liu et al. 2005; Zhou et al. 2008b). Transcription factors (TFs) are global regulators of many plant developmental and stress responses by regulating the expression of sets of target genes. Phosphate starvation response (PHR) genes in Arabidopsis (AtPHR1) and rice (OsPHR1 and OsPHR2) encode a MYB-coiled coil (CC) transcription factor that regulates P-starvation responses (Rubio et al. 2001; Zhou et al. 2008a). The identification of possible cisregulatory elements among P starvation-induced genes in Arabidopsis revealed that 47.3% (80 of 169) contain the PHR1-responsive element in the 5′ upstream region (Müller et al. 2007). This observation suggests that PHR1 orthologues in other plant species may be a common regulation system for P-starvation response in higher plants. Müller et al. (2007) also found other TF motifs in P-starvation induced genes, indicating that transcription factors other than PHR1 may also participate in the regulation of gene expression induced by P deficiency. The global transcriptional analyses in common bean reveals that 4 and 13 of 372 TFs are either induced or repressed, respectively, in P-deficient roots. Three of the four induced genes are MYB family TFs, one of which is a PHR1 homologue and another is an R2R3-MYB TF (Hernández et al. 2007). In the global transcriptional examination in Arabidopsis, 11 TFs and 11 signaling proteins are upregulated by Pstarvation, and 3 of the 11 TFs are MYB family TFs (Müller et al. 2007). Similarly, in Arabidopsis, 6 of 16 TFs induced by P-starvation belong to the MYB family (Morcuende et al. 2007). These studies indicate that analyses of TFs, especially the MYB family TFs, are necessary for the understanding of gene regulation and signaling cascades of P-starvation responses. However, research on TFs related to P acclimation in species

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other than Arabidopsis is limited. An EST study of P responsive genes in white lupin cluster roots by UhdeStone et al. (2003b) revealed several TFs but only the function of a Scarecrow TF has been evaluated (Sbabou et al. 2010). Although microarray and macroarray have been used to evaluate global gene expression, it is likely that the transcripts of many TF genes may be difficult to detect and quantify due to their low abundance. In Arabidopsis, Affymetrix microarray detected less than 55% of the putative TFs, and the putative TFs of which expression is relatively low showed poor accuracy in their transcription level (Czechowski et al. 2004). Thus, real-time reverse transcription (RT)-PCR is currently necessary to analyze expression of TFs (Czechowski et al. 2004; Hernández et al. 2007; Morcuende et al. 2007). This highly sensitive method exhibits greater precision than microarray (Czechowski et al. 2004). In this study, five MYB-CC and 29 R2R3-MYB cDNA sequences were isolated from white lupin by degenerate primer RT-PCR technique. Other TF sequences and sequences for signaling proteins were selected from a cDNA library made with RNA isolated from cluster roots with rootlet primordia. Expression profiles of TFs and signaling proteins were determined by real-time RTPCR technique among normal roots, cluster roots, leaves, and shoot tips including shoot apical meristems grown under low-P and P sufficient conditions.

Materials and methods Plant growth Seeds of white lupin (Lupinus albus L. cultivars ‘Kievskij mutant’ and ‘Ultra’) were surface-sterilized by bleach solution, pre-cultured in sterilized water for 2 days, and then planted in pots filled with coarse quartz sand. About 100 mL of nutrient solution was supplied every 2 days. +P solution consisted of 3 mM KNO3, 2.5 mM Ca(NO3)2, 0.5 mM Ca(H2PO4)2, 1 mM MgSO4, 0.01 mM Fe(III)-EDTA, 0.023 mM H3BO3, 4 μM MnCl2, 0.46 μM ZnSO4, 0.1 μM CuSO4, and 0.1 μM NaMoO4 (pH 6). -P solution contained 0.5 mM CaSO4 instead of 0.5 mM Ca (H2PO4)2 (Johnson et al. 1994). Plants were grown in a growth incubator at 20°C under a 16 h light—8 h dark condition for several days as described below.

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cDNA library construction from very young cluster roots For cDNA library construction, white lupin plants (‘Ultra’) were harvested at 6 days after emergence (DAE), and only lateral roots bearing the primordia of rootlets (very young cluster roots, Supplementary Fig. 1) were collected. Methods for RNA isolation, preparation, and screening of a cDNA library and generation of expressed sequence tags (ESTs) were as described by Uhde-Stone et al. (2003b). A total of 1,114 clones were sequenced. These sequences have been deposited in DDBJ/EMBL/GenBank as FF836314-FF837416 and AB585974-AB585996. Using the BlastX algorithm, EST sequences were translated and searched against non-redundant protein sequences of the GenBank database. A probability threshold for E values lower than 10−6 was used to assign functions. Isolation of MYB-CC and R2R3-MYB fragments 12 DAE white lupin plants were used for MYB cDNA isolation and transcription analysis because the transcription of P-starvation induced genes such as LaMATE begins at 12 DAE and peaks at 14 DAE under P depleted condition (Uhde-Stone et al. 2003b). Total RNA was extracted and purified using the RNeasy Plant Mini Kit in combination with RNasefree DNase (Qiagen, Tokyo, Japan). cDNA was synthesized by Super Script III reverse transcriptase (Invitrogen Japan, Tokyo, Japan) and the oligo dT primer ((T) 20 ) or the poly T-adapter primer (GGCCACGCGTCGACTAGTAC(T)17). DNA fragments that encode MYB-CC cDNA were amplified by PCR using cDNA of white lupin (‘Kievskij mutant’) and the originally designed degenerate primers, the forward primer TGGACNCCNGARYTNCAYGAR (the conserved amino acid sequence used to design the primer was WTPELHE) and the reverse primer YTTYTGNACYTCCATYTG (QMEVQK). cDNA was derived from shoots (including shoot apical meristems) or roots (consisting of both cluster roots and normal roots) of plants grown under low-P or P sufficient conditions. Similarly, R2R3-MYB cDNA fragments were PCR-amplified using the same cDNA and degenerate primers, the forward primers AARWSNTGYMGNYTNMGNTGG (KSCRLRW), and the re-

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verse primer RTTYTTDATYTCRTTRTCNGTNC (RTDNEIKN) (Rabinowicz et al. 1999). Amplified fragments were cloned into pGEM T-easy vector (Promega, Tokyo, Japan) and sequenced using an ABI DNA sequencer and a BigDye Terminator Sequencing system (Applied Biosystems, Tokyo, Japan). Rapid amplification of cDNA 3′-end (3′ RACE) PCR was done using gene-specific forward primers designed from each of sequences amplified by the degenerate primers and the adapter primer (GGCCACGCGTCGACTAGTAC). 3′ end sequences were cloned and sequenced as described above. Phylogenetic analysis The nucleotide sequence data were analyzed using the 4Peaks software (http://mekentosj.com/4peaks/). Deduced amino acid sequences for white lupin gene products and corresponding homolog genes in other plant species were aligned using CLUSTALW version 1.83 (http://clustalw.genome.jp/) at gap open penalty 10 and gap extension penalty 0.05. The genetic distances were calculated using Kimura 2-parameter (Kimura 1980), and phylogenetic trees were constructed using the neighbor-joining method (Saitou and Nei 1987). One hundred bootstrap replicates were analyzed. Real-time RT-PCR To examine transcript abundance, white lupin plants (‘Kievskij mutant’) were divided into cluster roots, normal (non-cluster) lateral roots, leaves, and shoot tips including shoot apical meristems. 2 μg of total RNA isolated from each plant part was used for RT reaction (20 μL), and the resulting cDNA was diluted 1:10 with DNase-free water. Relative quantification of transcripts was achieved by real time RT-PCR using the Chromo4 real time PCR system (Bio-Rad, Tokyo, Japan). PCR reaction (20 μL) contained 10 μL of 2 x SYBR Premix Ex Taq (Takara, Ohtsu, Japan), 2 μL of cDNA solution and 0.2 μM of each primer. LaUbiquitin (CA410752) mRNA was used as a reference gene (Uhde-Stone et al. 2003b). The ratio between the gene of interest (GOI) and Ubiquitin expression was calculated using the equation: ΔCt ð1 þ EGOI ÞΔCt = 1 þ EUbiquitin where ΔCt is calibrated Ct minus sample Ct in each gene, and E corresponds to the efficiency of each

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primer amplification (Pfaffl 2001). Gene expression levels were normalized to that of the organ sample with the lowest expression in order to obtain a calibrated Ct for each gene. E value was estimated using data obtained from the exponential phase of each individual amplification plot (Ramakers et al. 2003). PCR primer sequences and the annealing temperature at PCR amplification are listed in Supplementary Table 1. We verified that all primer pairs amplified a single fragment of the expected size on agarose gels and by melting curve analysis. The expression data were analyzed using a Tukey’s compromise test (P≤0.05) with three biological replications (i.e., the plants from distinct culture pots).

Results cDNA library from very young cluster roots In the effort to isolate genes involved in the cluster root development and in early responses to P deficiency, a cDNA library was constructed from the very young cluster roots (Supplementary Fig. 1). In total, 1,114 clones were sequenced, which were assembled into 241 unigenes. Based on similarity to already known or predicted genes and gene products, the 1,114 ESTs were grouped into four main categories: metabolism (35%), cell cycle and plant development (9%), interaction with the environment (28%), and unknown function (28%). Compared with the published white lupin EST library developed from early stage cluster roots (7 and 10 DAE) and later stage cluster roots (12 and 14 DAE) (Uhde-Stone et al. 2003b), the library in this study showed lower percentage of ESTs involved in carbon and energy metabolism (5% vs. 11%) and, instead, higher percentage of ESTs involved in amino acid/protein metabolism (23% vs. 12%), indicating that physiological condition in very young cluster roots is different from that in early and later stage cluster roots. That the cDNA library in this study included relatively high percentage of clones involved in amino acid/protein metabolism may be because cell division and elongation to develop cluster rootlets are major event in the very young cluster roots. While, carboxylic acid metabolism becomes active in mature cluster roots at 12 and 14 DAE to secrete carboxylates into soil (Uhde-Stone et al. 2003a), and therefore, the

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published cDNA library should include high percentage of EST clones involved in carbon and energy metabolism (Uhde-Stone et al. 2003b). ESTs of 11 transcription factors and 15 signal transduction proteins including protein kinases, protein phosphatases, and calmodulin were selected (Table 1) and used for transcriptional analysis. MYB-CC and R2R3-MYB genes should have important roles in P acquisition as described in the introduction, but only a few MYB sequences were included in the cDNA libraries in this study and by Uhde-Stone et al. (2003b); no sequences for MYBCC TFs and one R2R3-MYB sequence, LaMYB1, in the library by Uhde-Stone et al. (2003b). Thus, we isolated MYB-CC and R2R3-MYB sequences using degenerate primers in the following experiments. Isolation of MYB-CC sequences in white lupin AtPHR1 and its related sequences of AT3G13040, AT2G20400, AT3G04450, AT5G29000, AT2G01060 and AT5G06800 in Arabidopsis and OsPHR1 and OsPHR2 in rice have two conserved domains, single MYB and CC (Rubio et al. 2001; Zhou et al. 2008a). In the effort to isolate PHR1 homologues in white lupin, degenerate primers were designed from these sequences: forward to reverse primers could hybridize with the sequences of MYB and CC domains, respectively. In total, 28 amplified fragments (285 bp) were sequenced, and 7 unique sequences were obtained. Five of the seven sequences showed high similarity to AtPHR1 sequence and were used for further analysis. Because the regions including MYB and CC domains were highly conserved among the five sequences, 3′RACE PCR was carried out to obtain the unique sequences followed by designing genespecific primers for RT-PCR. Amino acid alignment of the five sequences together with AtPHR1 sequence showed both the MYB and CC domains were well conserved. However, in LaMYB-CC2, a portion of the CC domain and a region downstream of it was truncated due to a nonsense mutation (Fig. 1). Because a CC domain is necessary for sequence-specific DNA binding (Rubio et al. 2001), LaMYB-CC2 may not be functional. The Arabidopsis genome contains 15 MYB-CC proteins. They are divided into subgroups I and II according to their similarities. AtPHR1 belongs to subgroup I (Rubio et al. 2001). The neighborjoining tree (Fig. 2) showed that LaMYB-CC1,

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LaMYB-CC3, LaMYB-CC4, and LaMYB-CC5 in white lupin together with AtPHR1, OsPHR1, and OsPHR2 made a single clade (subgroup I). Isolation of R2R3-MYB cDNAs in white lupin R2R3-MYB proteins have two incomplete MYB repeats at an N-terminus, and the degenerate primers amplified a part of the two repeats. In total, 152 amplified fragments (168 bp) were sequenced and classified into 28 unique sequences. Because the MYB repeat region is too conserved to design gene specific primers, 3′RACE PCR was carried out to obtain unique sequences. During the 3′RACE PCR of the LaMYB5, one additional sequence LaMYB6 was obtained. Thus, 29 R2R3-MYB sequences, in total, were isolated from white lupin. Alignment of the deduced amino acid sequences of the 29 R2R3-MYBs and the related sequences in other plant species showed that the MYB repeats were well conserved (Supplementary Fig. 2), and sequences downstream of the repeats were variable (data not shown), indicating that these 29 R2R3-MYBs encode for typical R2R3-MYB sequences. The deduced R2R3-MYB protein sequences in white lupin were compared among themselves and with the MYB sequences from other plant, especially from Arabidopsis (Fig. 3). Arabidopsis R2R3-MYB proteins are categorized into 24 subgroups (Stracke et al. 2001), and 25 of 29 white lupin sequences were assigned to 12 of the 24 subgroups (Fig. 3). Four white lupin sequences, LaMYB27, LaMYB28, LaMYB30 and LaMYB29, clustered with AtMYB85, AtMYB59, AtMYB5 and AtMYB89, respectively, that do not fit into a defined subgroup (Stracke et al. 2001). Two to five white lupin sequences respectively clustered closely in subgroups 9, 1, 11, 7, 4, 16 and 18. Some motifs appeared in R2R3 DNA binding domains of LaMYB sequences (Supplementary Fig. 2). LaMYB8, LaMYB9, LaMYB10 (subgroup 4), and LaMYB30 harbored the bHLH-interaction motif ([D/E]Lx2[K/R]x3Lx6Lx3R, Zimmermann et al. 2004). LaMYB24 (subgroup 20) contained the motif, RWLNYLRPDVRRGNITLE, necessary for interaction with calmodulin (Yoo et al. 2005). Transcriptional analysis Expression of transcripts was evaluated by real time RTPCR in normal roots, cluster roots, leaves, and shoot tips

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Table 1 Annotation of expressed sequences isolated from white lupin roots Clone name, putative

Annotation

Accession # of Blast GenBank hit Evalue

BlastX hit

LaWD40a

WD-40 repeat family protein

PREDICTED: hypothetical protein [Vitis vinifera]

LaDELLA

DELLA protein, involved in GA signaling WRKY family transcription factor WD-40 repeat family protein

XP_002266620 0.E +00 BAF62637 4.E128 ABY84659 4.E111 ACU21423 4.E105 XP_002512411 1.E-96

LaWRKY LaWD40b LaJmjC

DELLA protein [Phaseolus vulgaris] transcription factor [Glycine max] unknown [Glycine max]

LaTLP

transcription factor, jumonji (jmjC) domain-containing protein Tubby-like F-box protein

ACU18200

7.E-91 unknown [Glycine max]

LabHLH2

bHLH transcription factor

CBI27511

2.E-62 unnamed protein product [Vitis vinifera]

LabHLH1

bHLH transcription factor

LaHDZIP

homeodomain leucine zipper protein, HDZip bZIP transcription factor

NP_194639 3.E-53 ethylene-responsive family protein [Arabidopsis thaliana] AT4G29100 XP_002511518 2.E-49 homeobox protein, putative [Ricinus communis]

LabZIP

ABI34673

1.E-41 bZIP transcription factor bZIP41 [Glycine max]

Lazfzinc finger (C-x8-C-x5-C-x3-H XP_002516892 2.E-37 CCCH type) family protein LaAKINβγ AKINβγ, protein kinase AAO61673 0.E +00 LaSYM19 SYM19, symbiosis receptor AAY22390 0.E kinase +00 LaCKL Casein Kinase I like NP_194617 2.EAT4G28880 137 LaPP2A2 LaGRAM

serine/threonine protein phosphatase 2A (PP2A) GRAM

LaC3HC4b RING finger protein (C3HC4 type) La14-3-3 14-3-3 like protein

ACU20069 ACJ85492

transcription factor, putative [Ricinus communis]

nucleic acid binding protein, putative [Ricinus communis] AKIN betagamma [Medicago truncatula] symbiosis receptor-like kinase [Lupinus albus] ckl3 (Casein Kinase I-like 3); ATP binding/kinase/protein kinase/protein serine/threonine kinase [Arabidopsis thaliana]

3.Eunknown [Glycine max] 116 1.E-90 unknown [Medicago truncatula]

XP_002283980 1.E-87 PREDICTED: hypothetical protein [Vitis vinifera] Q96452

7.E-86 RecName: Full = 14-3-3-like protein C; AltName: Full = SGF14C, [Glycine max]

LaSERK

SERK (somatic embryogenesis XP_002262698 1.E-74 PREDICTED: similar to somatic embryogenesis receptorreceptor kinase) like kinase 3 [Vitis vinifera]

LaPTK

protein tyrosine kinase family protein Calmodulin-binding family protein serine/threonine protein phosphatase 2A (PP2A)

LaCaMB LaPP2A1

XP_002277797 4.E-70 PREDICTED: hypothetical protein [Vitis vinifera] XP_002525175 4.E-54 calmodulin binding protein, putative [Ricinus communis] XP_002519463 8.E-49 protein phosphatase 2a, regulatory subunit, putative [Ricinus communis]

LaAux/IAA Aux/IAA protein

ACU19961

2.E-48 unknown [Glycine max]

LaCaM

AAT73620

2.E-41 calmodulin cam-207 [Daucus carota]

Calmodulin

LaC3HC4a RING finger protein (C3HC4 type) LaMAPK mitogen-activated protein kinase, MAPK

XP_002524078 4.E-37 ring finger protein, putative [Ricinus communis] ACJ31803

1.E-30 MAPK [Glycine max]

Plant Soil (2011) 342:481–493 Fig. 1 Amino acid sequence alignment of five white lupin MYB-CC together with Arabidopsis PHR1 (AT4G28610). Black and gray boxes show identical and similar amino acid, respectively. Horizontal bars represent the MYB or CC domains

487 LaMYB-CC1 LaMYB-CC3 LaMYB-CC2 AtPHR1 LaMYB-CC4 LaMYB-CC5

A F V EA VNQ L GG S EK A T P KGV L K LM K V EG L T A F V EA VNQ L GG S EK A T P KGV L K LM K V EG L T A F V EA I NQ L GG S EK A T P KGV L K LM K V EG L T A F V EA VN S L GG S ERA T P KGV L K I M K V EG L T A F V EA VNQ L GG S ERA T P KGV L K LM K V EG L T A F V EA VNQ L GG S ERA T P KGV L K LM K V EG L T MYB

LaMYB-CC1 LaMYB-CC3 LaMYB-CC2 AtPHR1 LaMYB-CC4 LaMYB-CC5

A EV E EM K S L D L K T S KG I P EV E EM K S L D L K T S KG I T EV E EMN S L D L K TN KG I T P L E H I T S L D L KGG I G I S S I DD I S S L D L K T G I G I S S I DD I S S L D L K T G I E I

LaMYB-CC1 LaMYB-CC3 LaMYB-CC2 AtPHR1 LaMYB-CC4 LaMYB-CC5

EK D K SA A L I S N T V A V L P S - - - - - - - - - - - - - - - - - - - P V E S L E TN A K I Q I N SN T P E T L L E EMD K P SA S I S S T A I A L P S - - - - - - - - - - - - - - - - - - - P I DN L D T TN - - E DHD K I R L T L P E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S G L T KG T A S T SD SA A K S E - - - - - - - - - - - - - - - - - - - Q ED K K T A D S K EV P E E ET RK C EE L K PG I E T F K A S S S I I DN P SG L S L D A T KD FN A K SN S EA SQVD CY R SGPDQA DN SMA V E E - G S K PG I E T F K A S S ST P EN P S - - - - - - - - - - - - - - - - - GV S L D CCR SGPDQA GA S I A V E E EG S

LaMYB-CC1 LaMYB-CC3 LaMYB-CC2 AtPHR1 LaMYB-CC4 LaMYB-CC5

E S T Q D A S T KQK RDD A KN A S EH E L GGDQ F A A P L S K RM K S L E S T QD A CK KQK RDD A K - - - - H E L GDDQ F SAQ L S K RM K S L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E S PQ P K RP K I DN - - - - - - - - - - - - - - - - - - - - - - - - - - PQMGA KHD S P - - - - - - - - MQH V I GDD V SAQA S K RK R TN E P EMGA KHD S P KGQ P S EN A KQH V I S GDD SVQA S K RK R TN E

Fig. 2 A neighbor-joining phylogenetic tree of MYB-CC proteins in white lupin, Arabidopsis, and rice. The regions shown in Fig. 1 were used for tree construction. Numbers next to the nodes are bootstrap values from 100 replications. The bar indicates a genetic distance of 0.1. The deduced amino acid sequences were retrieved from the DDBJ/EMBL/GenBank databases. AtPHR1 (AT4G28610), AT3G13040, AT2G20400, AT3G04450, AT5G29000, AT2G01060, AT5G06800, AT3G12730, and AT5G45580 in Arabidopsis, and OsPHR1 (Os03g0329900) and OsPHR2 (Os07g0438800) in rice. I and II indicate the subgroups (Rubio et al. 2001)

I I I I I I

YH V K SH L QK Y R T A RY K P E P S E - V T SV K K L YH V K SH L QK Y R T A RY K P E P S E EG S S EK S L YH V K SH L QK Y R T A RY K P E P S E - G I S E K K L YH V K SH L QK Y R T A RY RP E P S E T G S P E RK L YH V K SH L QK Y R T A RY RP E S S E - GV T E RK T YH V K SH L QK Y R T A RY RP E S S E - GG T EK K T

T E T L RMQM E L QK R L H EQ L E I Q R E L Q I Q I E NQGK R L QMM F E KQ I T EA L R L QM E L QK R L H EQ L E I Q R E L Q I Q I E NQGK R L QKM F E KQ I T E T L R L QM E L QK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T EA L R L QM EVQKQ L H EQ L E I Q RN L Q L R I E EQGK Y L QMM F E KQN T EA L Q L QM EVQK R L H EQ L E I Q RN L Q L R I E EQGRC L QMM F E KQC T EA L R L QM EVQK R L H EQ L E I Q RN L Q L R I E EQGRY L QMM F E KQC CC domain

including shoot apical meristems under P sufficient or low-P growth conditions. Among 60 genes, the transcript abundance of LaMYB5, LaMYB6, LaMYB7, LaMYB20, LaWRKY, and LaCaM was stimulated by low-P condition, while that of LaMYB19 and LaPTK was suppressed by low-P condition (Fig. 4). Low-P condition stimulated the transcription of LaMYB5 and LaMYB6 in shoot tips. Induction of LaMYB7 transcription by low-P was significant in leaves. Transcripts of LaMYB20 and LaWRKY in cluster roots were more abundant under low-P condition than under P sufficient condition. Transcription of LaCaM was significantly induced in cluster roots by low-P. Transcripts of LaMYB19 and LaPTK were more abundant in roots than in shoots, and decreased in normal roots and in cluster roots, respectively, under low-P condition. LaMYB17, LaMYB19, LaMYB24, LaMYB27, LaMYB28, LaMYB30, LaWRKY, LabHLH1, LazfCCCH, LaSYM19, and LaPTK showed increased transcript abundance in roots as compared to shoots (Supplementary Fig. 3, LaMYB19, LaWRKY, and LaPTK are shown in Fig. 4). Among them, transcript accumulation of Lazf-CCCH was higher in cluster roots than in other organs. Transcripts for LaMYBCC4, LaMYB3, LaMYB4, LaMYB12, LaMYB26, LaDELLA, LaPP2A2, LaPP2A1, and LaMAPK was higher in leaves and shoot tips than those in roots, and transcript level of LaMYB6, LaMYB9, LaMYB13, LaMYB14, LaMYB15, LaMYB21, LaMYB22,

488

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Fig. 3 A neighbor-joining phylogenetic tree of R2R3-MYB proteins in white lupin and other related proteins. Only R2 (partial) and R3 repeat region was used for tree construction. Numbers next to the nodes are bootstrap values from 100 replications. 25 of the 29 white lupin sequences were assigned to 12 of the 24 subgroups of Stracke et al. (2001), which are shown in the right side of each clade. The bar indicates a genetic distance of 0.02. The deduced amino acid sequences were retrieved from the DDBJ/EMBL/GenBank databases. AtMYB17 (AT3G61250), AtMYB60 (AT1G08810), AtMYB102 (AT4G21440), AtMYB15 (AT3G23250), AtMYB85 (AT4G22680), AtMYB59 (AT5G59780),

AtMYB112 (AT1G48000), AtMYB12 (AT2G47460), AtMYB111 (AT5G49330), AtMYB11 (AT3G62610), AtMYB4 (AT4G38620), AtMYB5 (AT3G13540), AtMYB123/TT2 (AT5G35550), AtMYB83 (AT3G08500), AtMYB45 (AT3G48920), AtMYB36 (AT5G57620), AtMYB33 (AT5G06100), AtMYB1 (AT3G09230), and AtMYB44 (AT5G67300) in Arabidopsis, AmMIXTA (CAA55725) in Antirrhinum majus, NtMYB1 (AAB41101) in Nicotiana tabacum, PhODORANT1 (Q50EX6) in Petunia hybrida, VvMYBPA1 (CAJ90831) in Vitis vinifera, and HvGAMYB (CAA61021) in Hordeum vulgare

L a M Y B 2 3 , L a M Y B 2 5 , L a W D 4 0 b , L a J m jC , LaAKINßγ, LaCKL, La14-3-3, LaCamB, and LaAux/ IAA was higher in shoot tips than in other organs (Supplementary Fig. 3, LaMYB6 is in Fig. 4).

25 white lupin R2R3-MYBs were grouped into 12 subgroups (Fig. 3). The cDNA in the same subgroups often exhibited the similar expression profiles, e.g., LaMYB13, LaMYB14, and LaMYB15 (subgroup 9)

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489

Fig. 4 Quantification of transcript abundance of LaMYB5, LaMYB6, LaMYB7, LaMYB20, LaWRKY, LaCaM, LaMYB19 and LaPTK in normal roots (NR), cluster roots (CR), leaves (Leaf), and shoot tips including shoot apical meristems (ST) of white lupin plants grown under P sufficient (white bars) and low-P (dark gray bars) growth conditions (12 DAE). Vertical bars show standard errors of three biological replicates. Within each figure, different letters above the bars represent significant differences at the 5% level. LaUbiquitin was used to normalize the expression

were predominantly expressed in shoot tips, and the transcription of LaMYB21, LaMYB22, and LaMYB23 (subgroup 18) was higher in shoot tips (Supplementary Fig. 3), suggesting that genes in the same subgroups have a similar functions. R2R3-MYB proteins in the same subgroups often show functional redundancy in Arabidopsis (Stracke et al. 2001, 2007). The interesting discrepancy was LaMYB19 and LaMYB20 in the subgroup 16. The expression of LaMYB19 was suppressed by low-P in normal roots while that of LaMYB20 was stimulated by low-P in cluster roots (Fig. 4). That is, transcription of the two genes was affected by low-P condition but in an opposite fashion.

Discussion AtPHR1 homologous sequences in white lupin AtPHR1 is involved in many P-starvation responses in Arabidopsis, and sequences related to the PHR1responsive element (GNATATNC) are often found in the promoter region of P-starvation responsive genes (Rubio et al. 2001; Müller et al. 2007). The PHR1-

responsive element also appears in promoter regions of white lupin P-starvation responsive genes, LaSAP, LaPT1, and LaMATE (Liu et al. 2005; Uhde-Stone et al. 2005; Zinn et al. 2009). Thus, it is not unexpected that genes homologous to AtPHR1 should act in important roles in P-starvation responses in white lupin. Four MYB-CC cDNA sequences in white lupin showed high similarity to AtPHR1 (Figs. 1 and 2). Transcript accumulation of the white lupin MYB-CC cDNAs was detected in all plant parts tested and was not stimulated by low-P condition (Supplementary Fig. 3). Transcription of AtPHR1 in Arabidopsis and OsPHR1 and OsPHR2 in rice is also detected in roots and shoots and is not affected by P-starvation condition (Nilsson et al. 2007; Morcuende et al. 2007; Zhou et al. 2008a). Determining the functions of these white lupin homologues of AtPHR1 will contribute to our understanding of the common signaling pathway involving PHR1 among higher plants. Putative R2R3-MYBs involved in stress responses R2R3-MYB proteins form one of the largest families of TFs: 126 in Arabidopsis and 109 in rice, and act in

490

important roles in higher plants such as the regulation of plant secondary metabolism and the determination of cell identity and fate (Riechmann et al. 2000; Stracke et al. 2001; Yanhui et al. 2006). 29 R2R3-MYB cDNAs were isolated from white lupin in this study while more than 100 R2R3-MYB genes are in Arabidopsis and rice genomes. This small number may not reflect the entire complement of R2R3-MYB gene complement in white lupin because the expression of R2R3-MYB genes is often induced by hormone or stresses such as drought and cold and is often specific to organs such as flowers and fruits (Kranz et al. 1998; Yanhui et al. 2006; Yamagishi et al. 2010). Among the four R2R3-MYB sequences that showed enhanced expression under low-P condition (Fig. 4), LaMYB5, LaMYB6, and LaMYB7 were in subgroups 1 and 2 (Fig. 3). Among subgroup 1 R2R3-MYBs in Arabidopsis, AtMYB30 and AtMYB60 are involved in disease resistance (Vailleau et al. 2002) and in drought response (Cominelli et al. 2005), respectively. LaMYB7 (subgroup 2) in white lupin showed high similarity to AtMYB15/Y19 and AtMYB13 in Arabidopsis (subgroup 2) and NtMYB1 in tobacco. Transcription of AtMYB15 and AtMYB13 is responsive to cold stress (Agarwal et al. 2006) and to dehydration, ABA, light, and wounding (Kirik et al. 1998), respectively. While that of NtMYB1 is stimulated by virus infection (Yang and Klessig 1996). These observations suggest that white lupin R2R3-MYBs in subgroups 1 and 2 may have functions involved in response to biotic and abiotic stresses including the stresses caused by P depletion. Putative R2R3-MYBs involved in phenylpropanoid biosynthesis LaMYB11 and LaMYB12 have similarity to AtMYB11, AtMYB12, and AtMYB111 (subgroup 7), by comparison sequences of LaMYB8, LaMYB9, and LaMYB10 resembled those of AtMYB4 (subgroup 4), and LaMYB30 showed similarity to VvMYBPA1 and AtMYB123/TT2 (Fig. 3). Because AtMYB4, AtMYB11, AtMYB12, AtMYB111, AtMYB123, and VvMYBPA1 are implicated in regulation of phenylpropanoid biosynthesis (Nesi et al. 2001; Bogs et al. 2007; Stracke et al. 2007), white lupin R2R3-MYB, LaMYB8, LaMYB9, LaMYB10, LaMYB11, LaMYB12, and LaMYB30, may have similar functions in regulating phenylpropanoid path-

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way. White lupin roots accumulate and exude high amount of flavonoids, especially isoflavone derivatives, in order to control the growth of microorganisms in the rhizosphere to prevent the microbial degradation of secreted carboxylates (Weisskopf et al. 2006a, b; Cesco et al. 2010). It will be of interest to know whether these R2R3-MYB genes are involved in the regulation of flavonoid biosynthesis in roots. Calcium signaling The transcriptional profile of 11 TFs and 15 signaling proteins showed that transcription of one calmodulin gene LaCaM was enhanced by low-P condition in cluster roots (Fig. 4). Calmodulin is known to play multiple regulatory roles in signal transduction in eukaryotes (Kim et al. 2009). Global gene expression analysis in common bean shows that the calmodulin RTS_137_H03 (CV543649) was upregulated by P stress (Hernández et al. 2007). RTS_137_H03 and LaCaM have sequence similarity (data not shown). These results suggest that calmodulin may be involved in P-starvation responses. The calmodulinbinding motif appeared in the R2R3 domain of LaMYB24, subgroup 20 R2R3-MYB (Supplementary Fig. 2), transcripts of which were abundant in roots (Supplementary Fig. 3). Arabidopsis MYB2 (subgroup 20), which possesses the same calmodulinbinding motif, interacts with calmodulin resulting in increased salt tolerance (Yoo et al. 2005). Anther subgroup 20 R2R3-MYB protein in Arabidopsis, AtMYB62, is shown to be involved in the regulation of the P deficiency responses (Devaiah et al. 2009) although interaction of AtMYB62 with calmodulin is not investigated. Thus, it may be intriguing to evaluate the interaction between LaMYB24 and LaCaM and the correlation of LaMYB24 with P acquisition. WRKY transcription factors Transcripts of LaWRKY accumulated mainly in normal roots and cluster roots, and its transcription was induced by low-P condition in cluster roots (Fig. 4). The WRKY family of TFs is unique to plants and is involved in the wide variety of stress responses (Eulgem et al. 2000). In Arabidopsis, AtWRKY75 (Devaiah et al. 2007), AtWRKY6 and AtWRKY42

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(Chen et al. 2009) are involved in the P-starvation response. LaWRKY had low sequence similarity to AtWRKY6, AtWRKY42, and AtWRKY75 (data not shown). However, many WRKY genes are reported to be responsive to P deficient stress in common bean and their functions concerning to the P deficient responses are discussed (Tesfaye et al. 2007).

Conclusions TFs act as master regulators that coordinate the expression of suites of genes in several developmental processes. Thus, TFs and signaling protein genes control many aspects of plant development and stress tolerance. Understanding the function of TFs and signaling proteins involved in P acquisition in white lupin will have broad applications for understanding and engineering efficient soil nutrient uptake in other plants. In this study, 60 cDNAs/ESTs encoding for TFs and signaling proteins were isolated from white lupin. Transcription estimated by real-time RT-PCR revealed 6 (10%) of the 60 corresponding genes were induced and 2 (3.3%) were suppressed by low-P condition, and 36 (60%) showed organ-specific expression. This information of the sequences and the expression profiles will be a valuable starting point for further research on these genes.

Financial source This research was supported by a GrantsIn-Aid for Scientific Research (No. 17658032), the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References Agarwal M, Hao Y, Kapoor A, Dong C-H, Fujii H, Zheng X, Zhu J-K (2006) A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance. J Biol Chem 281:37636–37645 Bogs J, Jaffe FW, Takos AM, Walker AR, Robinson SP (2007) The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol 143:1347–1361 Cesco S, Neumann G, Tomasi N, Pinton R, Weisskopf L (2010) Release of plant-borne flavonoids into the rhizosphere and their role in plant nutrition. Plant Soil 329:1–25 Chen YF, Li LQ, Xu Q, Kong YH, Wang H, Wu WH (2009) The WRKY6 transcription factor modulates PHOSPHATE1 expression in response to low Pi stress in Arabidopsis. Plant Cell 21:3554–3566

491 Cominelli E, Galbiati M, Vavasseur A, Conti L, Sala T, Vuylsteke M, Leonhardt N, Dellaporta SL, Tonelli C (2005) A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance. Curr Biol 15:1196–1200 Czechowski T, Bari RP, Stitt M, Scheible WR, Udvardi MK (2004) Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J 38:366–379 Devaiah BN, Karthikeyan AS, Raghothama KG (2007) WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiol 143:1789–1801 Devaiah BN, Madhuvanthi R, Karthikeyan AS, Raghothama KG (2009) Phosphate starvation responses and gibberellic acid biosynthesis are regulated by the MYB62 transcription factor in Arabidopsis. Mol Plant 2:43–58 Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trend Plant Sci 5:199–206 Hernández G, Ramírez M, Valdés-López O, Tesfaye M, Graham MA, Czechowski T, Schlereth A, Wandrey M, Erban A, Cheung F, Wu HC, Lara M, Town CD, Kopka J, Udvardi MK, Vance CP (2007) Phosphorus stress in common bean: root transcript and metabolic responses. Plant Physiol 144:752–767 Johnson JF, Allan DL, Vance CP (1994) Phosphorus stressinduced proteoid roots show altered metabolism in Lupinus albus. Plant Physiol 104:657–665 Johnson JF, Allan DL, Vance CP (1996a) Phosphorus deficiency in Lupinus albus. Altered lateral root development and enhanced expression of phosphoenolpyruvate carboxylase. Plant Physiol 112:31–41 Johnson JF, Allan DL, Vance CP, Weiblen G (1996b) Root carbon dioxide fixation by phosphorus-deficient Lupinus albus. Contribution to organic acid exudation by proteoid roots. Plant Physiol 112:19–30 Keerthisinghe G, Hocking PJ, Ryan PR, Delhaize E (1998) Effect of phosphorus supply on the formation and function of proteoid roots of white lupin (Lupinus albus L.). Plant Cell Environ 21:467–478 Kim MC, Chung WS, Yun D-J, Cho MJ (2009) Calcium and calmodulin-mediated regulation of gene expression in plants. Mol Plant 2:13–21 Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120 Kirik V, Kölle K, Wohlfarth T, Miséra S, Bäumlein H (1998) Ectopic expression of a novel MYB gene modifies the architecture of the Arabidopsis inflorescence. Plant J 13:729–742 Kranz HD, Denekamp M, Greco R, Jin H, Leyva A, Meissner RC, Petroni K, Urzainqui A, Bevan M, Martin C, Smeekens S, Tonelli C, Paz-Ares J, Weisshaar B (1998) Towards functional characterization of the members of the R2R3-MYB gene family from Arabidopsis thaliana. Plant J 16:263–276 Lin W-Y, Lin S-I, Chiou T-J (2009) Molecular regulators of phosphate homeostasis in plants. J Exp Bot 60:1427–1438 Liu J, Uhde-Stone C, Li A, Vance C, Allan D (2001) A phosphate transporter with enhanced expression in pro-

492 teoid roots of white lupin (Lupinus albus L.). Plant Soil 237:257–266 Liu J, Samac DA, Bucciarelli B, Allan DL, Vance CP (2005) Signaling of phosphorus deficiency-induced gene expression in white lupin requires sugar and phloem transport. Plant J 41:257–268 Miller SS, Liu J, Allan DL, Menzhuber CJ, Fedorova M, Vance CP (2001) Molecular control of acid phosphatase secretion into the rhizosphere of proteoid roots from phosphorusstressed white lupin. Plant Physiol 127:594–606 Morcuende R, Bari R, Gibon Y, Zheng W, Pant BD, Bläsing O, Usadel B, Czechowski T, Udvardi MK, Sttitt M, Scheible W-R (2007) Genome-wide reprogramming of metabolism and regulatory networks of Arabidopsis in response to phosphorus. Plant Cell Environ 30:85–112 Müller R, Morant M, Jarmer H, Nilsson L, Nielsen TH (2007) Genome-wide analysis of the Arabidopsis leaf transcriptome reveals interaction of phosphate and sugar metabolism. Plant Physiol 143:156–171 Nesi N, Jond C, Debeaujon I, Caboche M, Lepiniec L (2001) The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed. Plant Cell 13:2099–2114 Neumann G, Massonneau A, Martinoia E, Römheld V (1999) Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin. Planta 208:373– 382 Nilsson L, Müller R, Nielsen TH (2007) Increased expression of the MYB-related transcription factor, PHR1, leads to enhanced phosphate uptake in Arabidopsis thaliana. Plant Cell Environ 30:1499–1512 Peñaloza E, Muñoz G, Salvo-Garrido H, Silva H, Corcuera LJ (2005) Phosphate deficiency regulates phosphoenolpyruvate carboxylase expression in proteoid root clusters of white lupin. J Exp Bot 56:145–153 Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45 Rabinowicz PD, Braun EL, Wolfe AD, Bowen B, Grotewold E (1999) Maize R2R3 Myb genes: sequence analysis reveals amplification in the higher plants. Genetics 153:427–444 Ramakers C, Ruijter JM, Lekanne Deprez RH, Moorman FM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66 Riechmann JL, Heard J, Martin G, Reuber L, Jiang C-Z, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, Creelman R, Pilgrim M, Broun P, Zhang JZ, Ghandehari D, Sherman BK, Yu G-L (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290:2105–2110 Rubio V, Linhares F, Solano R, Martin AC, Iglesias J, Leyva A, Paz-Ares J (2001) A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes Dev 15:2122–2133 Saitou N, Nei N (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Sbabou L, Bucciarelli B, Miller S, Liu J, Berhada F, Filali-Maltouf A, Allan D, Vance C (2010) Molecular analysis of

Plant Soil (2011) 342:481–493 SCARECROW genes expressed in white lupin cluster roots. J Exp Bot 61:1351–1363 Shane MW, De Vos M, De Roock S, Lambers H (2003) Shoot P status regulates cluster-root growth and citrate exudation in Lupinus albus grown with a divided root system. Plant Cell Environ 26:265–273 Shen J, Li H, Neumann G, Zhang F (2005) Nutrient uptake, cluster root formation and exudation of protons and citrates in Lupinus albus as affected by localized supply of phosphorus in a split-root system. Plant Sci 168:837– 845 Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4:447–456 Stracke R, Ishihara H, Huep G, Barsch A, Mehrtens F, Niehaus K, Weisshaar B (2007) Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J 50:660–677 Tesfaye M, Liu J, Allan DL, Vance CP (2007) Genomic and genetic control of phosphate stress in legumes. Plant Physiol 144:594–603 Uhde-Stone C, Gilbert G, Johnson JMF, Litjens R, Zinn KE, Temple SJ, Vance CP, Allan DL (2003a) Acclimation of white lupin to phosphorus deficiency involves enhanced expression of genes related to organic acid metabolism. Plant Soil 248:99–116 Uhde-Stone C, Zinn KE, Ramírez M, Li A, Vance CP, Allan DL (2003b) Nylon filter arrays reveal differential gene expression in proteoid roots of white lupin in response to phosphorus deficiency. Plant Physiol 131:1064–1079 Uhde-Stone C, Liu J, Zinn KE, Allan DL, Vance CP (2005) Transgenic proteoid roots of white lupin: a vehicle for characterizing and silencing root genes involved in adaptation to P stress. Plant J 44:840–853 Vailleau F, Daniel X, Tronchet M, Montillet JL, Triantaphylidès C, Roby D (2002) A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack. Proc Natl Acad Sci USA 99:10179–10184 Vance CP (2010) Quantitative trait loci, epigenetics, sugars, and microRNAs: quaternaries in phosphate acquisition and use. Plant Physiol 154:582–588 Vance CP, Uhde-Stone C, Allan DL (2003) Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol 157:423– 447 Wasaki J, Omura M, Osaki M, Ito H, Matsui H, Shinano T, Tadano T (1999) Structure of a cDNA for an acid phosphatase from phosphate-deficient lupin (Lupinus albus L.) roots. Soil Sci Plant Nutr 45:439–449 Watt M, Evans JR (1999a) Linking development and determinacy with organic acid efflux from proteoid roots of white lupin grown with low phosphorus and ambient or elevated atmospheric CO2 concentration. Plant Physiol 120:705– 716 Watt M, Evans JR (1999b) Proteoid roots. Physiology and development. Plant Physiol 121:317–323 Weisskopf L, Abou-Mansour E, Fromin N, Tomasi N, Santelia D, Edelkott I, Neumann G, Aragno M, Tabacchi R, Martinoia E (2006a) White lupin has developed a complex

Plant Soil (2011) 342:481–493 strategy to limit microbial degradation of secreted citrate required for phosphate acquisition. Plant Cell Environ 29:919–927 Weisskopf L, Tomasi N, Santelia D, Martinoia E, Langlade NB, Tabacchi R, Abou-Mansour E (2006b) Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type to cluster-root stage. New Phytol 171:657–668 Yamagishi M, Shimoyamada Y, Nakatsuka T, Masuda K (2010) Two R2R3-MYB genes, homologues of petunia AN2, regulate anthocyanin biosyntheses in flower tepals, tepal spots and leaves of Asiatic hybrid lily. Plant Cell Physiol 51:463–474 Yan F, Zhu Y, Muller C, Zorb C, Schubert S (2002) Adaptation of H+-pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency. Plant Physiol 129:50–63 Yang XJ, Finnegan PM (2010) Regulation of phosphate starvation responses in higher plants. Ann Bot 105:513– 526 Yang Y, Klessig DF (1996) Isolation and characterization of a tobacco mosaic virus-inducible myb oncogene homolog from tobacco. Proc Natl Acad Sci USA 93:14972–14977 Yanhui C, Yang X, He K, Liu M, Li J, Gao Z, Lin Z, Zhang Y, Wang X, Qiu X, Shen Y, Zhang L, Deng X, Luo J, Deng

493 XW, Chen Z, Gu QuLJ (2006) The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant Mol Biol 60:107–124 Yoo JH, Park CY, Kim JC, Heo WD, Cheong MS, Park HC, Kim MC, Moon BC, Choi MS, Kang YH, Lee JH, Kim HS, Lee SM, Yoon HW, Lim CO, Yun DJ, Lee SY, Chung WS, Cho MJ (2005) Direct interaction of a divergent CaM isoform and the transcription factor, MYB2, enhance salt tolerance in Arabidopsis. J Biol Chem 280:3697–3706 Zhou J, Jiao F, Wu Z, Li Y, Wang X, He X, Zhong W, Wu P (2008a) OsPHR2 is involved in phosphate-starvation signaling and excessive phosphate accumulation in shoots of plants. Plant Physiol 146:1673–1686 Zhou K, Yamagishi M, Osaki M, Masuda K (2008b) Sugar signaling mediates cluster root formation and phosphorus starvation-induced gene expression in white lupin. J Exp Bot 59:2749–2756 Zimmermann IM, Heim MA, Weisshaar B, Uhrig JF (2004) Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins. Plant J 40:22–34 Zinn KE, Liu J, Allan DL, Vance CP (2009) White lupin (Lupinus albus) response to phosphorus stress: evidence for complex regulation of LaSAP1. Plant Soil 322:1–15