Identification and characterization of the GH3 gene family in maize

Journal of Integrative Agriculture 2016, 15(2): 249–261 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Identification and characterization of the GH3 gene family in maize ZHANG Dong-feng, ZHANG Nan, ZHONG Tao, WANG Chao, XU Ming-liang, YE Jian-rong National Maize Improvement Center/Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing 100193, P.R.China

Abstract The phytohormone auxin plays a central role in coordinating plant growth and development. GH3 is one of the three gene families that respond rapidly during auxin stimulation. Here, we report the identification and characterization of the GH3 gene family in maize. A total of 12 GH3 genes were identified, which are not evenly distributed over the 10 maize chromosomes. Maize GH3 protein sequences share a conserved domain which occupies nearly the entire protein. Diversified cis-elements were found in promoters of maize GH3 genes. In this study, the 12 maize GH3 proteins were primarily classified into two phylogenetic groups, similar to the 13 rice GH3 proteins, while 9 of the 19 Arabidopsis GH3 proteins were observed in the third phylogenetic group. Microarray analysis showed that expression of maize GH3 genes is temporally and spatially modulated. Additionally, maize GH3 genes displayed variable changes at transcript level upon pathogen infection. Results presented here provide insight into the diversification and evolution of GH3 proteins, and lay a foundation for the functional characterization of these GH3 genes in future, especially for elucidating the mechanisms of GH3-mediated pathogenesis. Keywords: auxin, GH3 family, pathogen infection, maize (Zea mays L.)

1. Introduction Phytohormone auxin plays a pivotal role in regulating many aspects of plant growth and development, such as light signal transduction, gravitropism, lateral root growth and development, and vascular canalization as well as cell division differentiation (Ulmasov et al. 1997; Woodward and Bartel 2005; Benjamins and Scheres 2008; Rosquete et al. 2012). A large number of genes, called early auxin response genes, are involved in these physiological processes. These early

Received 16 January, 2015 Accepted 7 May, 2015 Correspondence YE Jian-rong, Tel: +86-10-62731135, Fax: +8610-62733808, E-mail: [email protected] © 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61076-0

auxin response genes are divided into three major families, including SMALL AUXIN-UP RNAs (SAURs), GH3 and AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) (Hagen and Guilfoyle 2002; Woodward and Bartel 2005). Most SAUR, GH3 and Aux/IAA genes contain more than one auxin response elements (AuxREs) in their promoter regions (Woodward and Bartel 2005; Fu et al. 2011). In auxin signaling transduction cascade, auxin binds to its receptors, which are members of the TIR1/AFB family of F-box proteins, and triggers the degradation of Aux/IAA transcriptional repressors, thus allowing auxin-response factor (ARF) transcriptional factors that bind specifically to the canonical AuxREs TGTCTC or its variants found in the auxin-responsive promoter elements (some ARFs act as transcriptional activator, while others show transcriptional repressor activity), to regulate the expression of auxin-responsive genes (Li et al. 1994; Ulmasov et al. 1995; Ulmasov et al. 1997). The primary auxin-responsive GH3 family is also called the firefly luciferase family, which is a member of the broad

250

ZHANG Dong-feng et al. Journal of Integrative Agriculture 2016, 15(2): 249–261

acyl-adenylate/thioester-forming enzyme superfamily (Staswick et al. 2002). This superfamily consists of enzymes that catalyze various reactions with a common first step, in which the transfer of adenosine monophosphate (AMP) (from adenosine triphosphate (ATP)) to the carboxylic acid group of an acyl substrate. GH3 family proteins effectively trigger the adenylation or conjugation of amino acid to phytohormones indole-3-acetic acid (IAA) and/or jasmonic acid (JA) (Staswick 2002; Staswick and Tiryaki 2004; Nobuta et al. 2007; Ludwig et al. 2009). During the reaction, GH3 proteins conjugate amino acids to diverse acyl acid-containing substrates through a two-step reaction involving adenylation and transferase activities (Chen et al. 2009, 2010). The GH3 proteins use various amino acids as substrates to form IAA-amido conjugates. Among these conjugates, the IAA-alanine and leucine, serve to temporarily store IAA and can be hydrolyzed to release free IAA when needed, whereas IAA-aspartic acid and IAA-glutamate sequester IAA for degradation (Staswick et al. 2005). GH3 family members are reported to function in plant defense signaling through the maintenance of auxin homeostasis. By the GH3 family proteins controlled auxin conjugation, auxin signaling is suppressed, thus giving evidence for a role of GH3 proteins in plant disease resistance, as the balance exsisted between plant growth and defense during the process of plant development. Arabidopsis AtGH3.2, AtGH3.3, AtGH3.4, AtGH3.6, AtGH3.9, and AtGH3.17 and grapeberry VvGH3.1 have adenylation activity that enables conjugating amino acids to IAA, and AtGH3.5 shows adenylation activity for both IAA and SA (Staswick et al. 2002; Staswick et al. 2005; Chen et al. 2009; Bottcher et al. 2010; Chen et al. 2010). Rice OsGH3.2, OsGH3.8 and OsGH3.13 all have IAA-amido synthetase function (Ding et al. 2008; Zhang et al. 2009; Fu et al. 2011; Westfall et al. 2012). The over-expressings of some GH3-protein encoding genes, AtGH3.5, OsGH3.1, OsGH3.2, or OsGH3.8, induce the transgenic plants display auxin-deficient phenotypes, such as shortened plant height and enhanced plant disease resistance. These suggest an association of enhanced plant disease resistance with suppression of auxin signaling (Nakazawa et al. 2001; Takase et al. 2004; Park et al. 2007; Zhang et al. 2007; Domingo et al. 2009). Another rice GH3 protein OsGH3-8 regulates both auxin and defense signaling in a pathogen-nonspecific manner (Ding et al. 2008). Although the role of GH3 in disease resistance is unclear, a relation between disease susceptibility and GH3-induced IAA-Asp accumulation has been reported (Gonzalez-Lamothe et al. 2012). In fact, the transcription of GH3 genes is not only triggered by endogenous phytohormones auxin, abscisic acid (ABA), ethylene (ER), brassinosteroids (BRs), gibberellins (GAs),

JA, and salicylic acid (SA), but also by exogenous light, biotic and abiotic stresses signals (Nakazawa et al. 2001; Staswick et al. 2005; Terol et al. 2006; Khan and Stone 2007; Kazan and Manners 2009; Hoffmann et al. 2011; Spaepen and Vanderleyden 2011). During ripening period, GH3 proteins establish and maintain low IAA concentrations in fruits (Bottcher et al. 2010). Additionally, Arabidopsis GH3 genes display diverse physiological responses to abiotic stimuli. No information about the physiological responses of the 12 maize GH3s to abiotic or biotic stimuli was reported. The quantity and quality of ambient light may influence the biological function of GH3 proteins. Light is necessary for the biosynthesis of Arabidopsis GH3 protein AtGH3.6 which functions in hypocotyl elongation control (Nakazawa et al. 2011). Under red or blue light conditions, overexpression of AtGH3.12 (DFL2) results in a short hypocotyl phenotype, whereas decreasing its expression induces a long hypocotyl under red light (Spaepen and Vanderleyden 2011). AtGH3-5 (AtGH3a) exhibits light-dependent expression (Tanaka et al. 2002; Tanaka et al. 2003). Moreover, auxin and light signal pathways may be connected through the GH3 family members (Neff et al. 1999). There is an evidence supporting that auxin and light phytochrome A (PHYA) signal pathways could be integrated through the suppression of photomorphogenesis repressor CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) via Arabidopsis GH3 protein AtGH3.11 (FIN219) (Hsieh et al. 2000). To date, a total of 19 Arabidopsis and 13 rice GH3 family members have been identified (Hsieh et al. 2000; Hagen and Guilfoyle 2002; Takase et al. 2004). Recently, 15 auxin-responsive GH3 genes were isolated from tomato (Kumar et al. 2012). Herein, we report the analysis of 12 GH3 genes in maize, such as identification and validation of maize GH3 genes based on the B73 sequencing project resource, survey the structure and phylogeny of these ZmGH3 proteins, investigate the expression patterns of maize GH3 genes in various tissues and developmental stages based on microarray data.

2. Results 2.1. Identification and characterization of maize GH3 genes Arabidopsis and rice GH3 genes were obtained by searching the National Center for Biotechnology Information (NCBI, China) database. Then, non-redundant GH3 sequences were used to query the B73 sequence and MaizeGDB databases using TBLASTN and BLASTp. Finally, 12 GH3 genes were identified in the maize genome and designated as ZmGH3-1 to -12 according to their positions on the 10 maize chromosomes (Table 1).

Only one predicted conserved domain was observed in all these maize GH3 protein sequences. There are 2 (ZmGH3-2 and ZmGH3-7) to 6 (ZmGH3-11) exons in these ZmGH3 genes. The non-coding sequences vary dramatically among ZmGH3 genes, and 10 of the 12 ZmGH3 proteins are acidic except two ZmGH3 are alkaline. The domain position is stated in AA (amino acid). NF, not found. 2) (–) means the reverse orientation of the gene in the corresponded chromosome; (+) means the forward orientation of the gene in the corresponded chromosome.

2 1 3 3 2 2 1 4 3 2 5 4 3 2 4 4 3 3 2 5 4 3 6 5 GRMZM2G068701_T01 GRMZM2G378106_T01 GRMZM2G410567_T01 GRMZM2G061005_T01 GRMZM2G061515_T01 GRMZM2G033359_T01 GRMZM2G053338_T03 GRMZM2G414460_T02 GRMZM2G001421_T04 GRMZM2G366873_T01 GRMZM2G162413_T02 GRMZM2G091276_T02 75 958 491–75 961 166 209 110 460–209 113 041 223 632 037–223 638 458 2 946 152–2 948 661 190 085 375–190 087 817 194 424 615–194 428 239 162 669 667–162 672 151 172 501 854–172 507 237 26 345 438–26 350 246 121 797 016–121 799 823 75 985 675–75 990 565 144 700 082–144 703 942 1(+) 2(–) 2(–) 3(–) 3(–) 3(–) 7(–) 7(–) 8(+) 8(+) 8(+) 8(–) ZmGH3-1 ZmGH3-2 ZmGH3-3 ZmGH3-4 ZmGH3-5 ZmGH3-6 ZmGH3-7 ZmGH3-8 ZmGH3-9 ZmGH3-10 ZmGH3-11 ZmGH3-12

1)

Intron no.

Transcript length (bp) 2 487 2 451 1 953 2 066 2 243 2 249 2 381 2 540 2 228 2 574 2 112 1 950 Transcript ID Transcript location Chr.2) Name

Table 1 Characterization of the ZmGH3 gene family members in maize1)

Protein length (aa) 618 614 651 604 644 604 614 547 639 633 623 583

Molecular weight (D) 68 137.9 67 209.5 71 369.9 65 852.6 70 469.5 66 983.5 67 507.1 60 769.5 69 365.9 68 989 68 958.6 65 282.3

Theoretical pI 6.14 5.75 5.33 6.14 6.31 5.62 5.8 5.36 8.11 7.18 6.02 5.59

Domain position 30–592 35–584 54–620 16–589 23–565 28–582 33–588 1–526 62–624 23–584 55–605 8–567

Exon no.

Oryza sativa Maize Fl homologues cDNA GH3.8 BT054476 BT038815 GH3.13 NF GH3.12 BT069100 GH3.1 NF GH3.2 BT066257 BT062907 GH3.11 NF GH3.3 BT035531 GH3.4 BT055956 GH3.5 BT056093 BT061462

ZHANG Dong-feng et al. Journal of Integrative Agriculture 2016, 15(2): 249–261

251

These 12 ZmGH3 genes are not evenly distributed over the 10 maize chromosomes, which scattered across five maize chromosomes: ZmGH3-1 is on chromosome 1, ZmGH3-2 and -3 are on chromosome 2, ZmGH3-3, -4 and -5 are on chromosome 3, ZmGH3-6 and -7 are on chromosome 7, and others are on chromosome 8 (Table 1). Only one predicted conserved domain, which occupies nearly the entire protein, was observed in all these maize GH3 protein sequences. Transcript length of ZmGH3 genes varied from 1 950 (ZmGH3-12) to 2 540 bp (ZmGH3-8). The sizes of ZmGH3 proteins ranged from 547 (ZmGH3-8) to 651 residues (ZmGH3-3). The transcript length did not perfectly match the predicted amino acid length of the protein (the longest full length ZmGH3-8 translated the shortest 547 residues), suggesting that the UTR (untranslated region) sequences vary dramatically among ZmGH3 genes. The molecular masses varied from 61 (ZmGH3-8) to 71 kDa (ZmGH3-3). The pI value revealed that 10 ZmGH3 proteins are acidic, and the remaining two (ZmGH3-9 and ZmGH310) are alkaline (Table 1).

2.2. Motif distribution in maize GH3 proteins Compared with other auxin response gene families ARF and Aux/IAA, which are characterized with conserved domains, no conserved regions have been reported in the GH3 family. To decipher sequence characters of these maize GH3 proteins, all these ZmGH3s were employed to mine the Pfam database. Results revealed that only one putative conserved domain with nearly 550 residues length was found in these ZmGH3s (Table 1). This domain occupied nearly the entire protein and was highly conserved among all the 12 maize GH3 proteins. Sequence variation of the putative domain was observed in maize GH3 proteins. The length of domain in ZmGH3-8 was 525 residues. Domains length was extended to 573 residues in ZmGH3-4 (Table 1). Of note, biological function of the putative domain remains elusive. On the basis of the predicted domain, the sequence identity of the domain among these ZmGH3 proteins was analyzed. Some amino acids were identical among all the 12 ZmGH3 proteins, some amino acids were conserved among most ZmGH3 proteins such as ZmGH3-2, -7, -1, -6, -5, -10, -3, and -8, whereas some amino acids were conserved among a few ZmGH3 proteins, for instance, ZmGH3-4, -9, -11, and -12. No more than 5 continuous amino acids are identical among all the 12 ZmGH3 proteins, and peptides with more than 15 amino acids are identical among certain members of this protein family, such as ZmGH3-1, 2 and 7 or ZmGH39, 11 and 12 (Fig. 1). The online functional annotation of these GH3 proteins revealed no motif information. To find conserved motifs in GH3 proteins, GH3 members from maize, rice, and Arabidopsis were collected and analyzed

252

ZHANG Dong-feng et al. Journal of Integrative Agriculture 2016, 15(2): 249–261

Fig. 1 Multiple sequence alignment of the conserved domain of the 12 maize GH3 proteins. There are more than 10% of amino acids are identical among all the members of this protein family, while much more continuous amino acids are identical among certain members of this protein family, such as ZmGH3-1, -2 and -7 or ZmGH3-9, -11 and -12. Black indicates 100% identity, purple indicates >75% identity, blue indicates >50% identity, and yellow indicates >33% identity. Analyses were conducted using ClustalX and DNAMAN.

253

ZHANG Dong-feng et al. Journal of Integrative Agriculture 2016, 15(2): 249–261

with the multitasking extensible messaging environment (MEME) web server. Six conserved motifs with 50 residues in length were identified in these GH3 proteins (Table 2). Most of these maize, rice and Arabidopsis GH3 proteins contained all the six motifs, while only a few contained 4 or 5 motifs (Appendix A).

2.3. Phylogeny of maize GH3 proteins Based on their amino acid sequence similarity, these 12 maize GH3 proteins could be classified into two phylogenetic groups. One group contained eight maize GH3 proteins (ZmGH3-2, -7, -1, -6, -5, -10, -3, and -8), the left

four (ZmGH3-4, -9, -11, and -12) were located in another phylogenetic group (Fig. 2-A). All the maize GH3 genes were interrupted by introns and there are 2 (ZmGH3-2 and -7) to 6 (ZmGH3-11) exons in these ZmGH3 genes (Fig. 2-B). To further investigate the phylogenetic relationship of GH3 proteins, 12 ZmGH3s, 19 AtGH3s, and 13 OsGH3s were adopted in the phylogenetic analysis. These 44 GH3 proteins could be separated into three groups. Maize and rice GH3 proteins were all found in groups I and II. The group III is only inhabited by nine Arabidopsis GH3 proteins (Fig. 3). Actually, some groups could be further classified into a few subgroups. For example, group I might be divided into three subgroups and group II could

Table 2 Identification of conserved motifs in these ZmGH3 proteins1) Motif 1 2 3 4 5 6 1)

Width (AA) 50 50 50 50 50 50

Best possible match

1 – 0.14 0.15 0.11 0.14 0.11

WEGIITRLWPNTKYIDVIVTGSMAQYIPTLEHYSGGLPMVCTMYASSECW VDLVDVKVGHEYELVITTFAGLYRYRVGDILQVTGFHNNAPQFRFVCRKN PDEAILCPDFHQSMYCQMLCGLCQRHEVLRVGAVFAHGFLRAIKFLQKHW VPVVTYEDIKPYIQRIANGDRSPIICGHPITEFLTSSGTSQGERKLMPTI NDVYRQCRRKDKSIGPLEIRVVRPGTFDKLMDYFISRGASINQYKTPRCV SLLMPVMNKYVPGLDKGKAMYFLFVKSETKTPGGLPARPVLTSYYKSDHF

2 0.14 – 0.15 0.15 0.15 0.16

Similarity 3 4 0.15 0.11 0.15 0.15 – 0.14 0.14 – 0.14 0.14 0.12 0.14

5 0.14 0.15 0.14 0.14 – 0.13

6 0.11 0.16 0.12 0.14 0.13 –

The similarities were calculated with the amino acid sequence conservation of the motif. At the beginning of the software start, the width was defined to be 40–50 amino acid (AA), while the giving analysis result was 50 AA in all cases. These motifs were identified with the web server MEME (http://meme.nbcr.net/meme/cgi-bin/meme.cgi), it is done by pasting the protein sequence in the page, then clicking the link icon “go”.

B

A 100 100

ZmGH3-2 Exon

ZmGH3-7

100

Intron

UTR

ZmGH3-1 ZmGH3-6

97

ZmGH3-5 100

ZmGH3-10 ZmGH3-3

86

100

ZmGH3-8 ZmGH3-4 ZmGH3-9

100 100

ZmGH3-11 ZmGH3-12

3

5 0

1

2

3

4

5

6

7

8 (kb)

Fig. 2 Phylogenetic analysis of the 12 maize GH3 proteins and genomic DNA structure of their encoding genes. A, phylogenetic analysis of the 12 ZmGH3 proteins that were collected from the Pfam database. These 12 maize GH3 proteins could be divided into two phylogenetic groups based on amino acid sequence homology. Analysis of data was conducted using MEGA 5.0 with the Poisson method, and the bootstrap test was carried out with 10 000 iterations. B, genomic DNA structures of different phylogenetic ZmGH3 gene groups. 2 to 6 exons were observed in these ZmGH3 genes. Exons, introns and untranslated regions were indicated.

254

OsG H3-3

99

99

Zm GH 3-1 10 Os 0 GH 3-9 Os GH 310

OsG H3-8

ZmGH3-7

0

10

99

72

99

98

H3 sG O

79

Os GH 312 Zm GH 3-4 Zm GH 3-9

100 46

H G At

99 0 11 10 3 H39 ZmG 3-5 GH Os 12 3GH 11 Zm 3-

ZmGH3-2

ZHANG Dong-feng et al. Journal of Integrative Agriculture 2016, 15(2): 249–261

3 H3AtG

0

99 100 100

100

1

85 1 00

53

92

3-8

0

00

10

0

1

84

OsGH3 -7

3-17

AtG H3-1

AtGH3

-2

AtGH3-4 AtGH3-5

AtGH

3-6

OsG H3-

Zm GH 3-5 10 Os 0 GH 3Zm 4 G H 310

1

9 3GH 1 At 3-1 GH Os 3-8 GH Zm

AtGH

OsGH3-13

ZmGH3-3

At G H3 -1 At 6 GH 3-1 3 AtG H3 -15

0

83

10

100

77 100

-12 H3 G At -7 H3 G At

10

4

H AtG

00

100

58

22

8 H3.1 AtG

100

OsGH3-6

100 99

6 3GH m Z -1 H3 AtG

10

100

AtGH3-10

9 AtGH3.1

-2

Fig. 3 Phylogeny of GH3 proteins from Arabidopsis, rice and maize. Forty-four GH3 proteins are divided into three groups. Red indicates the phylogenetic group I GH3 family members, green shows phylogenetic group II GH3 family members, and blue indicates the phylogenetic group III GH3 family members. The amino acid sequences of these GH3s were obtained from the TIGR database and Pfam database. The phylogenetic analysis was performed using MRBAYES 3.1.2 by the Bayesian method, and the bootstrap test was carried out with 10 000 iterations. Numbers on the nodes indicate clade credibility values. All the maize and rice GH3 famile members are in group I or II, only nine Arabidopsis GH3 famile members are in group III.

be divided into two subgroups (data not shown).

2.4. cis-elements in promoter regions of maize GH3 genes In this study, four kinds of cis-elements were specially surveyed in the genomic DNA sequence from the transcription initiation (+1) to the 2 kb of the upstream promoter region of the 12 ZmGH3 genes, ARFAT, ASF1MOTIFCAMV, AUXREPSIAA4, and NTBBF1ARROLB, which are the most frequent and functionally important cis-elements that involved in auxin response, although there are more than 11 kinds of potential auxin cis-regulatory elements. The cis-element ASF1MOTIFCAMV (TGACG), with 2–5 copies, was found in promoters of all the 12 ZmGH3s except ZmGH3-3 and -5. In contrast, the cis-element NTBBF1ARROLB (ACTTTA)

was detected only in promoters of four maize GH3 genes ZmGH3-3, -5, -8, and -11 with one or two copies. The most important canonical AuxRE, ARFAT (TGTCTC), was found in promoters of the following five maize GH3 genes ZmGH3-1, -3, -6, -9, and -12 with 1–3 copies, AUXREPSIAA4 (GGTCCCAT) was found only in promoters of ZmGH3-7 and -8 with one copy (Table 3).

2.5. Microarray analysis of expression profiles of maize GH3 genes in diverse tissues To describe the time and space expression profiles of these ZmGH3s during maize growth and development, hoping to detect whether there is functional redundance between or among these ZmGH3 genes, expression levels of these 12 maize GH3 genes in 60 different tissue samples from

255

ZHANG Dong-feng et al. Journal of Integrative Agriculture 2016, 15(2): 249–261

Table 3 Identification of auxin reponse cis-elements in the promoters of these ZmGH3 genes Promotor of the ZmGH3 GH3-1

ARFAT/TGTCTC 849(+)

GH3-2

None

GH3-3 GH3-4

489(+) None

GH3-5

None

GH3-6

1 172(+) 987(–) 1 007(–)

GH3-7

None

GH3-8

None

GH3-9

1 103(+) 1 226(+)

GH3-10

None

GH3-11

None

GH3-12

598(+) 1 497(–)

1)

Binding site ID/Sequence pattern1) ASF1MOTIFCAMV/TGACG AUXREPSIAA4/GGTCCCAT 506(+) None 1 931(–) 99(+) None 1 572(+) 1 748(+) 744(–) 1 272(–) None None 389(–) None 446(–) None None 255(+) 368(+) 151(–) 1 138(–) 141(+) 854(–) 1 290(–) 1 022(–) 1 295(–) 1 403(+) 1 345(–) 1 588(–) 30(–) 1 709(–) 1 799(–) 975(+) 954(–) 1 778(–) 288(+) 586(–) 1 444(–)

NTBBF1ARROLB/ACTTTA None None

790(+) 7(–)

None

254(–) 1 423(–) None

388(+)

None

952(+) None

308(–) 683(–) None

None

None

None

1 099(+) 1 762(+)

None

None

Genomic DNA sequence from the transcription initiation (+1) to the 2 kb of the upstream of the 12 ZmGH3 genes were used as promoter sequence analyzed. Four kinds of cis-elements, which are the most frequent and functionally important cis-elements that involved in auxin response, ARFAT, ASF1MOTIFCAMV, AUXREPSIAA4, and NTBBF1ARROLB. The numbers mean the distance of the elements to the transcription initiation, as it was defined to be 1. (+) indicates the cis-elements located in the forward sequence and (–) indicates the cis-elements located in the reverse sequence. GGTCCCAT is named AuxRR core (core of the auxin response region), and TGTCTC is named AuxRE (auxin response element). None indicates the cis-element is not present in the given 2 kb promoter sequence of the corresponded ZmGH3 genes.

maize inbred line B73 were collected from the NimbleGen microarray database. Results showed that relative expression levels of ZmGH3 genes varied dramatically in different tissues and developmental stages. Some of the ZmGH3 genes were only expressed in certain tissues. For example, ZmGH3-3 and -4 were exclusively expressed in the rapidly growing region near the shoot apical meristem (SAM), whereas ZmGH3-6 and -10 tended to express in anthers. In germinating seeds, the expression levels of ZmGH3-3, -4, -5, -9, -10, and -12 were relatively low (Appendix B). These suggesting that ZmGH3-3 and -4 may play major roles in the rapidly growing tissue, and ZmGH3-6 and -10 may be very important for anther development, although maize GH3 genes displayed complex expression profiles and no uniform

expression model could be constructed. Six kinds of tissue patterns could be identified among 60 tissue samples. Anther (tissue no. 13) was classified as tissue pattern I, which was significantly different from other tissue patterns. Tissue pattern II mainly consisted of the early rapidly growing above-ground parts, especially the SAM. Tissue pattern IV correlated closely with microspore development, and tissue pattern VI was related to root development. Because tissue patterns III and V contained many more tissue samples than other tissue patterns, it was difficult to interpret their roles (Appendix C). The clustering result suggests that the close developmental time points and spatial distance of the developing tissues were not markedly linked, these GH3 expressions were under both

256

ZHANG Dong-feng et al. Journal of Integrative Agriculture 2016, 15(2): 249–261

time and space controlled and could not be temporally and/or physically clustered in the same tissue at different development stages. Twelve maize GH3 genes could be divided into three groups according to their expression profiles in 60 tissue samples: the group I contained three genes (ZmGH3-1, -2 and -7), which were abundant in reproductive organs; the group II had six genes (ZmGH3-3, -4, -5, -6, -8, and -10), which were related to the development of microspore; and the group III had three genes (ZmGH3-9, -11 and -12), which were highly expressed in vegetative tissue (Fig. 4). Most of these members were still in the same group in this clustering analysis based on microarray data, such as ZmGH3-1, -2 and -7 or ZmGH3-9, -11 and -12, similar to the two phylogenetic groups division based on sequence similarity (Fig. 2). This suggests that there is a correlation between protein structure and expression patterns of surveyed maize GH3 genes.

2.6. Expression profiles of maize GH3 genes upon pathogen infection To investigate the response of maize GH3 genes to exogenous pathogen attack, a pair of near-isogenic lines (NILs), differed in quantitative trait locus QTL-qRfg1 (Yang et al. 2010), were used, although the locus has nothing to do with the GH3 proteins. The line with resistance to Fusarium graminearum induced stalk rot is named R-NIL and the other one susceptible to stalk rot is S-NIL. The expression profiles of the 12 ZmGH3 genes were investigated using the RNA-seq data with the R- and S-NIL were collected 0, 6, 18, and 48 hours after inoculation. Maize GH3 genes displayed variable expression profiles upon pathogen attack in the two NILs (Fig. 5-A). Expression levels of ZmGH3-4 and -5 were relatively low either before or after pathogen inoculation in the two NILs; the expressions of ZmGH3-1, -2, -7, and -9 were decreased upon infection in both R- and S-NIL. Transcript levels of ZmGH3-3, -4 and -10 were increased in R-NIL but decreased in S-NIL during pathogen infection. The expression levels of ZmGH3-8, -11 and -12 were not significantly different between the two NILs during pathogen infection (Fig. 5-A). The ZmGH3 genes could be clustered into two classes according to their expression profiles during pathogen infection: class I contained ZmGH3-4, -5, -8, -10, and -12, and other ZmGH3 genes were clustered to class II. The expression level of the family members in class I was all very low (reads per kilobase per million mapped reads (RPKM )<2) in maize root. Usually, the family members with similar expression pattern during the whole fungus infection process (such as ZmGH3-1, -2, -7, and -9) or at certain time point (ZmGH3-3, -6 and -11) were clustered in one class (Fig. 5-B). When compared with the two groups derived from

ZmGH3-12 ZmGH3-11

Group III

ZmGH3-9 ZmGH3-4 ZmGH3-3 ZmGH3-6 ZmGH3-10

Group II

ZmGH3-5 ZmGH3-8 ZmGH3-7 ZmGH3-2

Group I

ZmGH3-1

Fig. 4 Clustering of maize GH3 genes based on microarray data. Maize GH3 genes are clustered according to their expression data. Twelve ZmGH3 genes are divided into three groups. Group I contains GH3-1, -2 and -7, group II contains GH3-3, -4, -5, -6, -8, and -10, and group III contains GH3-9, -11 and -12. Most of the members were still in the same group in this clustering analysis based on microarray data, similar to the two phylogenetic groups division based on sequence similarity. Brown indicates the members in phylogenetic group I and blue indicates the members in phylogenetic group II shown in Fig 2.

phylogenetic analysis (Fig. 2-A), the same group members, ZmGH3-2, -7, -1, -6, and -3, displayed similar expression profiles during pathogen infection. However, the expression profiles of ZmGH3-5, -10 and -8, which were in one phylogenic group, were significantly different under pathogen infection condition. This is also true for ZmGH3-9 and -11 in another phylogenic group (Fig. 5-B).

3. Discussion 3.1. Diversity analysis of maize GH3 genes The GH3 gene family is widely distributed in plant kingdom. GH3 genes have been identified and characterized in Arabidopsis, rice, and tomato at genome scale (Hagen and Guilfoyle 2002; Terol et al. 2006; Kumar et al. 2012). In this study, 12 GH3 genes were identified from the maize genome (Fig. 1, Table 1). These 12 maize GH3 genes are distributed over 5 out of the 10 maize chromosomes. We did not observe that ZmGH3 genes are clustered in the maize genome. In contrast, two GH3 genes clusters were found in the Arabidopsis genome; five AtGH3s (AtGH3-12 to -16) are on chromosome 5, and three AtGH3s (AtGH3-18 and -19) are on chromosome 1 in tandem repeats (Hagen and

257

ZHANG Dong-feng et al. Journal of Integrative Agriculture 2016, 15(2): 249–261

A

ZmGH3-1

RPKM (reads per kilobase per million mapped reads) value

15

150

10

100

5

50

0

0

0

6

18

48

ZmGH3-4

0.3

0.1 0

6

18

48

ZmGH3-7

60

5 0

6

18

48

ZmGH3-5

40

2

20

1

0

0

0

6

18

48

ZmGH3-10

2.0

5

0

0

0

6

18

48

6

18

48

ZmGH3-6

40

10 0

6

18

48

ZmGH3-8

0

0

6

18

48

ZmGH3-9

80 60 40 20

0

6

18

48

ZmGH3-11

0

0

6

18

48

ZmGH3-12

4 3

10

0.5

0

20

15

1.0

0

30

3

1.5

B

10

075 0.50 0.25 0

ZmGH3-3

20 15

1.00

0.2

0

R-NIL S-NIL ZmGH3-2

2

0

6 18 Time (h)

48

1 0

0

6

18

48

ZmGH3-9 ZmGH3-7 ZmGH3-2 ZmGH3-3

Class II

ZmGH3-1 ZmGH3-11 ZmGH3-6 ZmGH3-5 ZmGH3-4 ZmGH3-10

Class I

ZmGH3-8 ZmGH3-12

Fig. 5 Expression pattern of maize GH3 genes upon infection with Fusarium graminearum. A, expression profile of ZmGH3 genes during pathogen infection in two NILs (near-isogenic lines). R-NIL, resistant NIL; S-NIL, susceptible NIL. B, clustering analysis of ZmGH3 genes based on RPKM values from RNA-seq. The maize family members with similar expression patterns during the whole fungus infection process (ZmGH3-1, -2, -7, and -9) or at certain time point (ZmGH3-3, -6 and -11) were clustered in one class.

Guilfoyle 2002). Multiple members of a given gene family reflect a succession of genomic rearrangements and expansions owing to extensive duplication and diversification that

frequently occur during evolution (Danilevskaya et al. 2008). Although exon numbers of these maize GH3 genes were variable (Fig. 2-B), their coding sequences were relatively

258

ZHANG Dong-feng et al. Journal of Integrative Agriculture 2016, 15(2): 249–261

conserved. Conserved domain of the GH3 protein may be necessary for their key roles in plant life. Proper auxin quantity and gradient are critical for normal plant growth and development. Too much auxin would activate the GH3 genes, and their activation convert free auxin to amino acid conjugates to maintain auxin homeostasis (Fu et al. 2011; Rosquete et al. 2012). In promoters of the maize GH3 genes, many cis-elements, such as ARFAT, ASF1MOTIFCAMV, AUXREPSIAA4, and NTBBF1ARROLB, were identified (Table 3). Diversified cis-elements in promoters of the maize GH3 genes are required for the fine-tuning of auxin responses. Phylogenetic analysis indicated that all rice and maize GH3 family members fall into phylogenetic group I or II, but group III contained only nine Arabidopsis GH3 genes (Fig. 3). This is consistent with the results of a previous research, which showed that all 15 tomato GH3 family members are also clustered into phylogenetic groups I and II (Kumar et al. 2012). The function of the group I members is reported to adenylate JA, the group II is reported to adenylate IAA and the function of the group III members is unknown until now. More GH3 genes need to be identified from sequenced species, especially from the Brassicaceaes, to test whether the phylogenetic group III is Brassicaceae-specific. Undoubtedly, origin, distribution and function of GH3 members in the phylogenetic group III need to be further investigated.

3.2. Expression profile analysis of maize GH3 genes Originally, the microarray data were collected to describe the time and space expression profiles of these ZmGH3s during maize growth and development, hoping to find some relation about the functional redundance between or among these ZmGH3 genes. However, the results of the microarray analysis presented here showed that the relative expression levels of these ZmGH3 genes varied dramatically in different tissues and developmental stages, the spatiotemporal transcription of maize GH3 genes was very complex (Appendix B). The result did not give any useful information about their functional redundance. Maybe this collection only provides an easy reference for anybody who is interested in the surveying the time and space expression profiles of these 12 ZmGH3s. Based on their expression profiles, 12 maize GH3s are divided into three groups, which is in inconsistent with the phylogenetic results from protein sequence information (Fig. 2). Inconsistency of the clustering analysis based on expression patterns and the protein sequence may be due to the fact that it is the upstream promoter sequence, especially the cis-elements that determine the spatiotemporal expression profile of the gene. And the dramatically different spatiotemporal transcription patterns of these ZmGH3 genes were consistent with the

different auxin responsive cis-regulatory elements presented in the promoter region of these maize GH3 genes (Table 3).

3.3. Response of maize GH3 genes to pathogen infection There are innumerable evidences supporting that plant hormone auxin plays a fundamental role in defense against pathogen infection. An intact auxin signaling pathway promotes susceptibility to Pseudomonas syringae is demonstrated in the auxin signaling mutant axr2 (auxin resistant 2) that showed increased resistance to the pathogen (Ding et al. 2008). A report shows that down regulates the expression of the auxin receptor gene TIR1, leads to the suppression of auxin-responsive gene expression and reduced disease development, while the tir1 mutant shows increased resistance to P. syringae (Navarro et al. 2006). The host auxin signal plays a role in pathogen-associated-molecular-pattern-triggered immunity and effector-triggered susceptibility (Jones and Dangl 2006). Exogenous auxin application enhances host susceptibility to bacterial pathogens and promotes symptoms development (Navarro et al. 2006). Many bacterial pathogens produce IAA themselves; this property may be part of the strategy used by the pathogen to circumvent the plant defense system (Khalid et al. 2004; Spaepen et al. 2007). We investigated the transcription responses of maize GH3 genes. Results showed that responses of the maize GH3 members to pathogen infection are significantly different (Fig. 5-A). The expression level of the family members in class I was all very low (RPKM<2) in maize root, they might don’t have biological function or effect on root growth or physiological process. Accordingly, there was no continuous response (increase of decrease) for the maize GH3 members in class I. While some maize GH3 members (such as ZmGH3-2, -7, -1, -9, and -3) from class II displayed similar expression profiles (increase or decrease) during pathogen infection, the expression profiles of other ZmGH3 members in class II were not continuous under pathogen infection condition (Fig. 5-A and B). Mechanisms of various responses of maize GH3 genes to pathogen attack remains unclear.

4. Conclusion We identified 12 GH3 genes over the 10 maize chromosomes, analyzed the diversified cis-elements in promoters of these ZmGH3 and found a conserved domain which occupies nearly the entire protein. These 12 ZmGH3 proteins were primarily classified into two phylogenetic groups. These ZmGH3 were temporally and spatially modulated during maize growth and development and they displayed variable changes at transcript level upon pathogen infection.

ZHANG Dong-feng et al. Journal of Integrative Agriculture 2016, 15(2): 249–261

5. Materials and methods 5.1. Identification of GH3 genes in maize genome The GH3 genes from Arabidopsis and rice, including 48 genomic DNA sequences, 1 EST, and 25 amino acid sequences deposited in the NCBI database (http://www.ncbi. nlm.nih.gov/) were firstly collected to construct an initial sequence library. The redundant sequences were excluded. The partial assembled maize GH3 sequences from our RNAseq data were first used to blast the databank, all the found sequences were compared and assembled to find out the complete full length sequences. All the non-redundant GH3 sequences were then used to mine the publicly available B73 maize sequencing (http://www.maizesequence.org/index. html), TIGR (http://maize.jcvi.org/) and MAGI (MAGI_4 http:// magi.plantgenomics.iastate.edu/) databases. All potential hits to the GH3 family were assembled, and then additional mining rounds were conducted to achieve nearly complete coverage of genomic and/or transcript sequences.

5.2. Structural analysis of maize GH3 genes Information about maize GH3 genes, including accession number, chromosomal location, open reading frame length, exon and intron numbers was retrieved from the B73 maize sequencing database. Full-length cDNA sequences of maize GH3 genes were obtained from the maize full-length cDNA project website (http://www.maizecdna.org/) (Soderlund et al. 2009). The exon/intron boundaries of maize GH3 genes were analyzed using the gene structure displayer (http://gsds.cbi.pku.edu.cn/) (Guo et al. 2007). Physical and biochemical parameters of maize GH3 proteins were estimated using the ExPASy web server (http://expasy.org/ tools/protparam.html). Characterized regions of GH3 proteins were identified through the Pfam database (http://pfam.sanger.ac.uk/). Additionally, the MEME web server (http://meme.nbcr.net/ meme4_1/cgibin/meme.cgi) was adopted to display motif distribution of GH3 proteins from maize, rice and Arabidopsis. Parameters were set as follows: the occurrence of a single motif was 0 or 1 per sequence, motif length ranged from 40 to 50 amino acid residues, the maximum number of motifs to find was 6, and other parameters were default. To survey cis-elements in promoter regions of maize GH3 genes, 2 kb of genomic sequences upstream of the start codon were retrieved from the B73 sequencing database. Four kinds of cis-elements, including binding site ID ARFAT (sequence pattern: TGTCTC), binding site ID ASF1MOTIFCAMV (sequence pattern: TGACG), binding site ID AUXREPSIAA4 (sequence pattern: GGTCCAT),

259

and binding site ID NTBBF1ARROLB (sequence pattern: ACTTTA) (http://bioinfo.cau.edu.cn/ProFITS/BS_anno.php source=PLACE&BS=ASF1MOTIFCAMV) were chosen to query the PLACE database (http://www.dna.affrc.go.jp/ PLACE/).

5.3. Multiple alignment and phylogenetic analysis of GH3 proteins Multiple alignment of maize GH3 proteins was performed using the ClustalX utility with default parameters (Thompson et al. 1997). To survey the phylogeny of GH3 proteins, Arabidopsis and rice GH3 genes were obtained from the TAIR (The Arabidopsis Information Resource, http://www. arabidopsis.org/) and Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/index.shtml) databases. Phylogenetic analysis of GH3 proteins was performed with the neighbor-joining method and 1 000 bootstrap trials using the molecular evolutionary genetics analysis (MEGA) software (Larkin et al. 2007).

5.4. RNA sequencing (RNA-seq) data generation A quantitative trait locus-qRfg1 (QTL-qRfg1) that confers resistance to Gibberella stalk rot was identified in our lab (Yang et al. 2010). A pair of near-isogenic lines (NILs), an R-NIL line with the resistant QTL-qRfg1 and a susceptible S-NIL line lacking the resistant QTL-qRfg1, were developed. The introgression size was estimated to be ~170 kb in R-NIL, and more than 99% of the genetic background was recovered to the recurrent inbred line Y331. Although this locus has nothing to do with the GH3 protins, these NILs were used to analysis expression profiles of these maize GH3 genes during maize disease resistance or disease development response. Both R-NIL and S-NIL were inoculated with Fusarium graminearum Schwabe conidia, and roots of NILs were collected 0, 6, 18, and 48 hours after inoculation (hai) for RNA sequencing. Expression patterns of maize GH3 genes were surveyed using RNA-seq data (Ye et al. 2013). The protocols for inoculating and testing resistance to F. graminearum and RNA sequencing, calculation and normalization of gene expression, defining differentially expressed genes (DEGs) were directly described in the reference (Ye et al. 2013) in detail.

5.5. Microarray data analysis A total of 60 samples were collected at various developmental stages from 11 tissues of maize inbred line B73 (germinating seed, root, seedling, shoot apical meristem, internodes, cob, tassel/anthers, silk, leaf, husk, and seed). Three biological replicates were collected for each tissue

260

ZHANG Dong-feng et al. Journal of Integrative Agriculture 2016, 15(2): 249–261

type and three technical replications were used for microarray analysis. The NimbleGen genechip containing 330 788 probes was used for microarray analysis according to the procedure described by Sekhon et al. (2011). The relative expression data were collected from the maize database of the BioArray Resource for Plant Biology (http://bar.utoronto. ca/efp_maize/cgibin/efpWeb.cgi). All relative expression values were adjusted by the log2ratio, which is equal to the expression level of a given GH3 gene vs. the expression level of the control. Therefore, a positive (or negative) value means that the expression level of a given GH3 gene was higher (or lower) than the control. The control was a normalization of the data using a robust multi-chip average (RMA) algorithm performed by Roche NimbleGen with NimbleScan software.

Acknowledgements We acknowledge special thanks to Prof. Wang Yijun from Yangzhou University for valuable discussion and critical reading of the manuscript. This work was financially supported by the National Natural Science Foundation of China (31371625). Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

References Benjamins R, Scheres B. 2008. Auxin: The looping star in plant development. Annual Review of Plant Biology, 59, 443–465. Bottcher C, Keyzers R A, Boss P K, Davies C. 2010. Sequestration of auxin by the indole-3-acetic acid-amido synthetase GH3-1 in grape berry (Vitis vinifera L.) and the proposed role of auxin conjugation during ripening. Journal of Experimental Botany, 61, 3615–3625. Chen Q, Zhang B, Hicks LM, Wang S, Jez J M. 2009. A liquid chromatography-tandem mass spectrometry-based assay for indole-3-acetic acid-amido synthetase. Analytica Chimica Acta, 390, 149–154. Chen Q, Westfall C S, Hicks L M, Wang S, Jez J M. 2010. Kinetic basis for the conjugation of auxin by a GH3 family indole-acetic acid-amido synthetase. Journal of Biological Chemistry, 285, 29780–29786. Danilevskaya O N, Meng X, Hou Z, Ananiev E V, Simmons C R. 2008. A genomic and expression compendium of the expanded PEBP gene family from maize. Plant Physiology, 146, 250–264. Ding X, Cao Y, Huang L, Zhao J, Xu C, Li X, Wang S. 2008. Activation of the indole-3-acetic acid-amido synthetase GH3-8 suppresses expansin expression and promotes salicylate- and jasmonate-independent basal immunity in rice. The Plant Cell, 20, 228–240. Domingo C, Andrés F, Tharreau D, Iglesias D J, Talón M. 2009.

Constitutive expression of OsGH3.1 reduces auxin content and enhances defense response and resistance to a fungal pathogen in rice. Molecular Plant-microbe Interactions, 22, 201–210. Fu J, Yu H, Li X, Xiao J, Wang S. 2011. Rice GH3 gene family: Rregulators of growth and development. Plant Signaling & Behavior, 6, 570–574. Gonzalez-Lamothe R, El Oirdi M, Brisson N, Bouarab K. 2012. The conjugated auxin indole-3-acetic acid-aspartic acid promotes plant disease development. The Plant Cell, 24, 762–777. Guo A Y, Zhu Q H, Chen X, Luo J C. 2007. GSDS: A gene structure display server. Genetics, 29, 1023–1026. (in Chinese) Hagen G, Guilfoyle T. 2002. Auxin-responsive gene expression: Genes, promoters and regulatory factors. Plant Molecular Biology, 49, 373–385. Hoffmann M, Hentrich M, Pollmann S. 2011. Auxin-oxylipin crosstalk: relationship of antagonists. Journal of Integrative Plant Biology, 53, 429–445. Hsieh H L, Okamoto H, Wang M, Ang L H, Matsui M, Goodman H, Deng X W. 2000. FIN219, an auxin-regulated gene, defines a link between phytochrome A and the downstream regulator COP1 in light control of Arabidopsis development. Genes and Development, 14, 1958–1970. Jones J D G, Dangl J L. 2006. The plant immune system. Nature, 444, 323–329. Khalid A, Tahir S, Arshad M, Zahir Z A. 2004. Relative efficiency of rhizobacteria for auxin biosynthesis in rhizosphere and non-rhizosphere soils. Australian Journal of Soil Research, 42, 921–926. Kazan K, Manners J M. 2009. Linking development to defense: Auxin in plant-pathogen interactions. Trends in Plant Science, 14, 373–382. Khan S, Stone J. 2007. Arabidopsis thaliana GH3. 9 in auxin and jasmonate cross talk. Plant Signaling & Behavior, 2, 483–485. Kumar R, Agarwal P, Tyagi A K, Sharma A K. 2012. Genomewide investigation and expression analysis suggest diverse roles of auxin-responsive GH3 genes during development and response to different stimuli in tomato (Solanum lycopersicum). Molecular Genetics & Genomics, 287, 221–235. Larkin M A, Blackshields G, Brown N P, Chenna R, McGettigan P A, McWilliam H, Valentin F, Wallace I M, Wilm A, Lopez R, Thompson J D, Gibson T J, Higgins D G. 2007. Clustal W and clustal X version 2.0. Bioinformatics, 23, 2947–2948. Li Y, Liu Z B, Shi X, Hagen G, Guilfoyle T J. 1994. An auxininducible element in soybean SAUR promoters. Plant Physiology, 106, 37–43. Ludwig M, Ulmasov T, Shi X, Und N M, Decker E L, Reski R. 2009. Moss (Physcomitrella patens) GH3 proteins act in auxin homeostasis. New Phytologist, 181, 323–338. Nakazawa M, Yabe N, Ichikawa T, Yamamoto Y Y, Yoshizumi T, Hasunuma K, Matsui M. 2001. DFL1, an auxin-responsive GH3 gene homologue, negatively regulates shoot cell

ZHANG Dong-feng et al. Journal of Integrative Agriculture 2016, 15(2): 249–261

elongation and lateral root formation, and positively regulates the light response of hypocotyl length. The Plant Journal, 25, 213–221. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, Voinnet O, Jones J D G. 2006. A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science, 312, 436–439. Neff M M, Nguyen S M, Malancharuvil E J, Fujioka S, Noguchi T, Seto H, Tsubuki M, Honda T, Takatsuto S, Yoshida S. 1999. BAS1: A gene regulating brassinosteroid levels and light responsiveness in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 96, 15316–15323. Nobuta K, Okrent R A, Stoutemyer M, Rodibaugh N, Kempema L, Wildermuth M C, Innes R W. 2007. The GH3 acyl adenylase family member PBS3 regulates salicylic aciddependent defense responses in Arabidopsis. Plant Physiology, 144, 1144–1156. Park J E, Seo P J, Lee A K, Jung J H, Kim Y S, Park C M. 2007. An Arabidopsis GH3 gene, encoding an auxin-conjugating enzyme, mediates phytochrome B-regulated light signals in hypocotyl growth. Plant Cell Physiology, 48, 1236–1241. Rosquete M R, Barbez E, Kleine-Vehn J. 2012. Cellular auxin homeostasis: Gatekeeping is housekeeping. Molecular Plant, 5, 772–786. Sekhon R S, Lin H, Childs K L, Hansey C N, Buell C R, de Leon N, Kaeppler S M. 2011. Genome-wide atlas of transcription during maize development. The Plant Journal, 66, 553–563. Soderlund C, Descour A, Kudrna D, Bomhoff M, Boyd L, Currie J, Angelova A, Collura K, Wissotski M, Ashley E. 2009. Sequencing, mapping, and analysis of 27,455 maize fulllength cDNAs. PLoS Genetics, 5, e1000740. Spaepen S, Vanderleyden J. 2011. Auxin and plant-microbe interactions. Cold Spring Harbor Perspectives in Biology, 3, 1–13. Spaepen S, Vanderleyden J, Remans R. 2007. Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiology Reviews, 31, 425–448. Staswick P E. 2002. Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic, salicylic, and indole-3-acetic acids in an assay for adenylation. The Plant Cell, 14, 1405–1415. Staswick P E, Serban B, Rowe M, Tiryaki I, Maldonado M T, Maldonado M C, Suza W. 2005. Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. The Plant Cell, 17, 616–627. Staswick P E, Tiryaki I. 2004. The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis. The Plant Cell, 16, 2117–2127.

261

Takase T, Nakazawa M, Ishikawa A, Manabe K, Ichikawa T, Takahashi N, Shimada H, Manabe K, Matsui M. 2004. ydk1-D, an auxin-responsive GH3 mutant that is involved in hypocotyl and root elongation. The Plant Journal, 37, 471–483. Takase T, Nakazawa M, Ishikawa A, Manabe K, Matsui M. 2003. DFL2, a new member of the Arabidopsis GH3 gene family, is involved in red light-specific hypocotyl elongation. Plant and Cell Physiology, 44, 1071–1080. Tanaka S, Mochizuki N, Nagatani A. 2002. Expression of the AtGH3a gene, an Arabidopsis homologue of the soybean GH3 gene, is regulated by phytochrome B. Plant and Cell Physiology, 43, 281–289. Terol J, Domingo C, Talon M. 2006. The GH3 family in plants: Genome wide analysis in rice and evolutionary history based on EST analysis. Gene, 371, 279–290. Thompson J D, Gibson T J, Plewniak F, Jeanmougin F, Higgins D G. 1997. The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876–4882. Ulmasov T, Liu Z B, Hagen G, Guilfoyle T J. 1995. Composite structure of auxin response elements. The Plant Cell, 7, 1611–1623. Ulmasov T, Murfett J, Hagen G, Guilfoyle T J. 1997. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. The Plant Cell, 9, 1963–1971. Woodward A W, Bartel B. 2005. Auxin: Regulation, action, and interaction. Annals of Botany, 95, 707–735. Westfall C S, Herrmann J, Chen Q, Wang S, Jez J M. 2010. Modulating plant hormones by enzyme action: The GH3 family of acyl acid amido synthetases. Plant Signaling & Behavior, 5, 1607–1612. Yang Q, Yin G M, Guo Y L, Zhang D F, Chen S J, Xu M L. 2010. A major QTL for resistance to Gibberella stalk rot in maize. Theoretical and Applied Genetics, 121, 673–687. Ye J R, Guo Y L, Zhang D F, Zhang N, Wang C, Xu M L. 2013. Cytological and molecular characterization of QTL-qRfg1 which confers resistance to Gibberella stalk rot in maize. Molecular Plant-Microbe Interactions, 26, 1417–1428. Zhang S W, Li C H, Cao J, Zhang Y C, Zhang S Q, Xia Y F, Sun D Y, Sun Y. 2009. Altered architecture and enhanced drought tolerance in rice via the down-regulation of indole-3acetic acid by TLD1/OsGH3.13 activation. Plant Physiology, 151, 1889–1901. Zhang Z, Li Q, Li Z, Staswick P E, Wang M, Zhu Y, He Z. 2007. Dual regulation role of GH3.5 in salicylic acid and auxin signaling during Arabidopsis-Pseudomonas syringae interaction. Plant Physiology, 145, 450–464. (Managing editor WANG Ning)