Hormone regulation of rhizome development in tall fescue

(Festuca arundinacea) associated with proteomic changes controlling

respiratory and amino acid metabolism

Xiqing Ma13 Qian Xu2 William A Meyer3 and Bingru Huang31College of Agro-grassland Science Nanjing Agricultural University Nanjing 210095 PR China 2National Engineering

Laboratory for Tree Breeding Beijing Forestry University Beijing 100083 PR China and 3Department of PlantBiology and Pathology Rutgers the State University of New Jersey New Brunswick NJ 08901 USA

For correspondence E-mail huangaesoprutgersedu

Received 1 February 2016 Returned for revision 17 March 2016 Accepted 18 April 2016 Published electronically 21 July 2016

Background and Aims Rhizomes are underground stems with meristematic tissues capable of generating shootsand roots However mechanisms controlling rhizome formation and growth are yet to be completely understoodThe objectives of this study were to investigate whether rhizome development could be regulated by cytokinins(CKs) and gibberellic acids (GAs) and determine underlying mechanisms of regulation of rhizome formation andgrowth of tall fescue (Festuca arundinacea) by a CK or GA through proteomic and transcript analysis Methods A rhizomatous genotype of tall fescue (lsquoBRrsquo) plants were treated with 6-benzylaminopurine (BAP asynthetic cytokinin) or GA3 in hydroponic culture in growth chambers Furthermore comparative proteomic analy-sis of two-dimensional electrophoresis and mass spectrometry were performed to investigate proteins and associatedmetabolic pathways imparting increased rhizome number by BAP and rhizome elongation by GA3 Key Results BAP stimulated rhizome formation while GA3 promoted rhizome elongation Proteomic analysisidentified 76 differentially expressed proteins (DEPs) due to BAP treatment and 37 DEPs due to GA3 treatmentCytokinin-related genes and cell division-related genes were upregulated in the rhizome node by BAP andgibberellin-related and cell growth-related genes in the rhizome by GA3 Conclusions Most of the BAP- or GA-responsive DEPs were involved in respiratory metabolism and amino acidmetabolism Transcription analysis demonstrated that genes involved in hormone metabolism signalling pathwayscell division and cell-wall loosening were upregulated by BAP or GA3 The CK and GA promoted rhizome forma-tion and growth respectively by activating metabolic pathways that supply energy and amino acids to support celldivision and expansion during rhizome initiation and elongation in tall fescue

Key words Rhizome tall fescue hormone protein transcription

INTRODUCTION

Rhizomes are underground stems that are composed of nodesinternodes and scale leaves which grow horizontally from therhizome nodes in crowns in grasses Lateral and apical buds ofthe rhizome have meristematic tissues capable of generating ad-ventitious roots and new shoots (Jernstedt and Bouton 1985 Liand Beuselinck 1996) Rhizomes serve as storage organs forcarbohydrates nutrients and water Therefore plants with ex-tensive rhizomes are able to establish quickly and are competi-tive in acquiring water and nutrients as well as being persistentthrough prolonged periods of stress (Tao et al 2001 Sackset al 2006 Zhou et al 2014) furthermore rhizomatousplants especially perennial crops have a great potential to pre-serve ecosystems in mountainous areas that are fragile due toserious soil erosion (Cox et al 2002) Rhizomes are also desir-able traits for breeding of forage and turfgrass as they benefitplants by promoting rapid establishment and increasing theirdensity thus helping them to withstand traffic and tolerate bi-otic and abiotic stresses (De Battista and Bouton 1990)Understanding mechanisms of promoting rhizome production

and growth are of great significance for developing rhizoma-tous perennial grass species

Recent research has begun to uncover the molecular mecha-nisms of rhizome development For instance 48 essential tran-scription factors including the YABBY NAM TCP TALEAP2 and bHLH families were identified that are specificallyexpressed or highly enriched in the rhizome tips and elongationzones of wild rice (Oryza longistaminata) (Hu et al 2011 Heet al 2014 Zhang et al 2014) Five candidate transcriptionalprotein complex factors (zinc finger family protein ribosomalprotein S11 nuclear RNA-binding protein mitogen-activatedprotein kinase and 26S proteasome regulator) were detectedand upregulated in the rhizome tips of wild sorghum (Sorghumhalepense and Sorghum propinquum) (Jang et al 2006)REVOLUTA CLAVATA1 and SINAT5 were highly ex-pressed in the rhizome buds of bamboo (Phyllostachys praecox)and contributed to rhizome bud formation and procambial de-velopment (Wang et al 2009) In addition energy metabo-lism-related genes and proteins were also in higher abundancein the rhizomes including monosaccharide transporter and oli-gosaccharyl transferase in S halepense (Jang et al 2006)

VC The Author 2016 Published by Oxford University Press on behalf of the Annals of Botany CompanyAll rights reserved For Permissions please email journalspermissionsoupcom

Annals of Botany 118 481ndash494 2016

doi101093aobmcw120 available online at wwwaoboxfordjournalsorg

b-glucosidase starch branching enzyme and trehalose 6-phos-phate synthase in bamboo (Wang et al 2009) and sucrose syn-thase glyceraldehyde-3-phosphate dehydrogenase (GAPDH)and alcohol dehydrogenase in perennial wild rice (Hu et al2011 He et al 2014) horsetail (Equisetum hyemale)(Balbuena et al 2012) and lotus (Nelumbo nucifera) roots(Cheng et al 2013) Metabolite profiling showed that fructosesucrose glucose-1-phosphate and amino acids including aspar-agine glutamine and methionine accumulated in the rhizometissues (He et al 2014) Some hormone-related genes andproteins were also identified especially gibberellic acid (GA)-responsive cis-element motifs including a pyrimidine boxTATCCA box and CAREs box which were enriched in rhi-zome tips of S halepense (Jang et al 2006) downregulation ofauxin response factor 8 and auxin efflux carrier 3 and upregula-tion of GA 2-b-dioxygenase and GA-regulated proteins weredetected in rhizome tips of O longistaminata (Hu et al 2011)a cytokinin responsive cis element (TATTAG) was also presentin higher abundance in S propinquum (Zhang et al 2014) Heet al (2014) also reported that 24 unigenes of cytokinins (CKs)were enriched in wild rice rhizomes and a higher CK contentwas detected in bamboo rhizomes before transfer to the bambooshoot (Hu et al 1995)

Cytokinins are important regulators and mediate many as-pects of plant growth and development including cell divisionaxillary initiation and outgrowth (Choi and Hwang 2007)Cytokinin derivatives (isopentenyladenine trans-zeatin andcis-zeatin) are primarily synthesized in roots by isopentenyl-transferases (IPTs) riboside 50-monophosphate phosphoribohy-dralase (lonely guy LOG) and cytochrome P450monooxygenases (CYP735A1A2) and degraded by cytokinindehydrogenases (CKXs) The CK signalling pathway is in-volved in the two-component phosphorelay pathway includingtransmembrane CK receptor histidine kinases (HKs) histidinephosphotransfer proteins (HPs) and type-B response regulators(RRs) which switch on a subset of primary CK-response genes(Ha et al 2012) Rice mutants with impaired expression oroverexpression of CK metabolism or signalling pathway genesshowed an abnormal shoot apical meristem inflorescence meri-stem and root system (Ashikari et al 2005 Kurakawa et al2007 Gao et al 2014) loss of apical dominance and adventi-tious shoot formation in transgenic tobacco (Nicotiana taba-cum) (Li et al 1992) and more axillary stems in Arabidopsis(Kuroha et al 2009) further research indicated that cell divi-sion genes such as the gene encoding histone H4 were affectedby CK (Kurakawa et al 2007) Gibberellic acid is another im-portant hormone that mainly regulates cell elongation stimulat-ing elongation of plant organs (Hedden 1997) GAs aresynthesized by gibberellin 20-oxidase (GA20-OX) and 3-oxi-dase and degraded by gibberellin 2-dioxygenase (Yamaguchi2008) For the GA signalling pathway GID1 (GA insensitivedwarf1) which is the GA signal receptor and controls the bind-ing of activity GA interacts with the DELLA proteins whichnegatively regulate stem growth and switch on the downstreamgibberellin-response genes (Sun 2010) In addition the expan-sin and xyloglucan endotransglucosylasehydrolase (XET) genefamilies are involved in cell expansion cell separation and cellwall disassembly and are induced by GA (Jan et al 2004Claeys et al 2014) Despite the widely recognized roles andmechanisms of CK and GA in regulating plant growth

development whether the development of rhizomes is directlyregulated by hormones is not well documented and the mecha-nisms underlying the regulation of rhizome formation andgrowth by CK or GA remain unclear

Tall fescue (Festuca arundinacea) is a cool-season perennialgrass species widely used as forage and turfgrass whereas mosttall fescue genotypes are non-rhizomatous or have short determi-nate rhizomes (Burns and Chamblee 1979 Jernstedt andBouton 1985 Saha et al 2015) Previous research has paidincreasing attention to enhancing rhizome production in tall fes-cue and other perennial grass species (Cowan 1956 Porter1958) Limited genetic variation exists in rhizome formation intall fescue and the number and length of rhizomes in tall fescueare affected by photoperiod and temperature (Charrier andStewart 2006 St John et al 2009 Saxena et al 2014)Furthermore information is limited about the underlying mecha-nisms of rhizome initiation and development in tall fescue Wehypothesized that CK and GA may stimulate rhizome formationand growth of tall fescue by regulating proteins or genes in-volved in metabolic processes supporting cell division and elon-gation Therefore the objectives of this study were to investigatewhether rhizome formation and growth could be promoted byCK and GA and determine the major metabolic pathways under-lying regulation of rhizome formation and growth of tall fescueby CK or GA through proteomic and gene expression analysis

MATERIALS AND METHODS

Plant materials and growth conditions

A breeding selection from the Rutgers turfgrass breeding pro-gramme lsquoBRrsquo which forms short rhizomes was examined in thisstudy Mature lsquoBRrsquo plants were collected from the turfgrass re-search farm at Adelphia NJ and transplanted to plastic trays (54 27 6 cm) filled with fritted clay in a greenhouse The growthconditions in the greenhouse were set up as a 14-h photoperiodan average temperature of 20 C and 780 lmol m2 s1 of photo-synthetically active radiation (PAR) with natural sunlight andsupplemented with sodium lamps on cloudy days During the es-tablishment phase plants were irrigated three times per week andfertilized weekly with Hoaglandrsquos nutrient solution (Hoaglandand Arnon 1950) Plants were kept at 6ndash7 cm canopy height bymowing weekly

After 2 months of establishment plants with the same num-ber of tillers without rhizomes and roots were transferred to thehydroponic system with plastic boxes (56 54 15 cm) con-taining 20 L of half-strength Hoaglandrsquos solution in growthchambers Each box had 40 individual plants wrapped insponge strips and held in position with foam board and aeratedwith a pump (115 V 60 Hz Tetra Blacksburg VA) Nutrientsolution was changed every 4 d and pH was maintained at 65every day The growth conditions were controlled at a 14 hlight8 h darkness photoperiod and temperature of 2018 C(daynight) and PAR of 680 lmol m2 s1 at the canopy level

Hormone treatments

After plants had acclimated to the hydroponic system for 5 dhormone treatments were imposed Thirty plants in each of fourcontainers were maintained in either half-strength Hoaglandrsquos

482 Ma et al mdash Hormone regulation of rhizome development

solution or with addition of 6-benzylaminopurine (BAP a syn-thetic cytokinin) (0 01 1 3 and 5 mM) or GA3 (0 01 1 10 and50 mM) Each hormone treatment was replicated in four con-tainers which were arranged in a randomized complete blockdesign

Prior to hormone treatment (0 d) and at 3 6 9 and 12 d ofhormone treatment the numbers of tillers rhizomes and rootsper plant were counted and rhizome length of each plant wasmeasured Each trait was measured on 30 individual plantsfrom each replicate of each treatment

Endogenous cytokinin quantification

Isopentenyl adenosine (iPA) in rhizome nodes (crown tissueswhere rhizomes are initiated excluding tillers and roots) andGA4 in the entire rhizomes were extracted at 12 d and purifiedaccording to Zhang et al (2013) About 50 mg of frozen freshtissue was ground with liquid nitrogen and mixed with 18 mL ofNa-phosphate buffer (50 mM pH 70) containing 002 sodiumdiethyldithiocarbamate as an antioxidant then purified twicewith 1 mL of 1 acetic acid and 1 mL of dichloromethane andsamples were dissolved in 210 mL of methanol and diluted to700 mL in deionized H2O with 01 formic acid

Quantification was iPA was carried out by enzyme-linkedimmunosorbent assay (ELISA) as described by Zhang andErvin (2004) Extracted samples were conjugated to bovineserum albumin (110 000 dilution) incubated overnight at4 C and washed three times with phosphate-buffered saline(50 mM pH 72) Fifty microlitres of the iPA antibody (1200dilution) and 50 mL of the iPA extract were mixed and incu-bated at 37 C for 1 h 100 mL of alkaline phosphatase-labelled goat anti-mouse IgG (11000 dilution Sigma StLouis MO) was added and incubation was continued for an-other 1 h at 37 C One hundred microlitres of substrate solu-tion (3 mg mL1 p-nitrophenyl phosphate in 10 diethanolamine with 05 mM MgCl2 pH 98) was added andincubation was continued for 30 min at 37 C The colour re-action of each sample was determined by measuring absor-bance at 405 nm and iPA concentration was calculated basedon the standard curve

We analysed GA4 according to Zhang et al (2013) using anAgilent liquid chromatographyndashtandem mass spectrometry(LCndashMSMS) system with an ESI sample introduction interface(Agilent Santa Clara CA) consisting of 1290 UPLC (ultra-performance liquid chromatography) and 6490 QQQ (triplequadrupole) The HPLC separation was performed on AgilentZorbax Extend-C18 analytical (46 50 mm 5 mm) andguard (46 12 mm 5 mm) columns The analytes were elutedwith water (mobile phase A) and methanol (B) in 01 for-mic acid in a gradient of 0ndash45 min B increasing from 30 to80 45ndash5 min B increasing to 100 5ndash7 min B at 100 and B decreasing to 30 at 75 min The injection volumewas 10 mL and flow rate was 05 mL min1 The column tem-perature was 40 C The internal standard was C13-labelledindole-3-acetic acid The source parameters were as followsnebulizer pressure 310 kPa dry gas temperature 250 C sheathgas temperature 200 C and gas flow 8 mL min1 We deter-mined GA4 based on retention times ion products and GA4

standards

Protein extraction and two-dimensional electrophoretic separation

Proteins were extracted from rhizome nodes or entire rhi-zomes (the same as those used for the hormone samples) usingthe acetonetrichloroacetic acid (TCA) protein extractionmethod (Xu et al 2010) with modifications Tissues werewashed three times with deionized water and 15 g of frozenfresh tissue was ground using liquid nitrogen Six millilitres ofice-cold precipitation solution (10 TCA 007 2-mercap-toethanol in acetone) was added and the sample was homogen-ized and left overnight Precipitated proteins were pelleted bycentrifugation at 10414 g for 15 min at 4 C and washed threetimes with rinse solution (007 2-mercaptoethanol inacetone) to remove pigments and lipids The pellets were vac-uum-dried and suspended in resolubilization solution (8 M

urea 1 CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) 1 IPG (immobilized pH gradient) buffer[GE Healthcare] 2 M thiourea 1 dithiothreitol [DTT]) andsonicated for 1 h at 4 C The supernatants were obtained aftercentrifugation for 15 min at 4 C Protein concentration wasquantified according to Bradford (1976)

Proteins were separated using two-dimensional electrophore-sis according to Burgess and Huang (2014) with minor modifi-cations Briefly Immobiline DryStrips (pH3-10 GEHealthcare Piscataway NJ) were rehydrated with 250 mg ofprotein samples in rehydration solution (8 M urea 1 CHAPS(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate)1 IPG (immobilized pH gradient) buffer [GE Healthcare]2 M thiourea 1 DTT 0002 bromophenol blue) Thevoltage settings for first-dimensional isoelectric focusingwere 30 V for 12 h 100 V for 3 h 200 V for 3 h 500 V for1 h 1000 V for 1 h 5000 V for 1 h and 8000 V to a total of80 kVh The samples were then incubated in equilibration buf-fer (50 mM TrisndashHCl (pH 88) 6 M urea 2 sodium dodecylsulphate [SDS] 0002 bromophenol blue 30 glycerol1 DTT) for 15 min twice and again incubated in the samebuffer with 25 iodoacetamide replacing DTT for 20 minFor the second-dimensional electrophoresis a Hoefer SE 600Ruby electrophoresis unit (GE Healthcare Piscataway NJ)was used with 125 SDSndashpolyacrylamide gel Voltage set-tings were 5 mA per gel for 45 min and 18 mA per gel for 7 hGels were stained with Coomassie brilliant blue (CBB) G-250(17 wv ammonium sulphate 34 vv methanol 3 vvO-phosphoric acid 007 wv CCB) and scanned with aTyphoon FLA 9500 (GE Healthcare Piscataway NJ) Gel foreach treatment was replicated four times and the images wereanalysed using SameSpots software (Nonlinear USADurham NC) Protein spots were normalized to the total vol-ume of spots on the gel and automatically matched Gels withhormone treatment were compared with normal control gelsand differentially expressed protein (DEP) spots were selectedfor further analysis (P 005)

Protein identification and functional analysis

Protein spots were manually excised and washed with 30 acetonitrile in 50 mM ammonium bicarbonate before DTT re-duction and iodoacetamide alkylation according to Xu andHuang (2008) Trypsin was used for digestion at 37C over-night The resulting peptides were extracted with 30 mL of 1

Ma et al mdash Hormone regulation of rhizome development 483

trifluoroacetic acid followed by C18 Ziptip desalting thenmixed with 7 mg ml1 a-cyano-4-hydroxycinnamic acid andsubjected to matrix-assisted laser desorptionionization time-of-flight MS (MALDI-TOF-MS) analysis (Applied BiosystemsFramingham MA) Proteins were identified using the PeptideMass Fingerprinting module of the Mascot search engine(V22 Matrix Science Boston MA USA) against the greenplant NCBI database The following parameters were set tryp-sin methionine oxidation carboxyamidomethylation of cyste-ine two missed cleavages precursor mass tolerance 50 ppmand fragment mass tolerance 06 Da Confidence interval valuesgt95 for at least two peptides were considered as successfulidentification

For the classification and functional analysis of identifiedproteins protein homologues were identified against theArabidopsis database (httpswwwarabidopsisorg) and geneontology (GO) was categorized against the Agrigo database(httpbioinfocaueducnagriGOanalysisphp) for biologicalprocess and cellular component analysis the threshold was setat ndashlog10(P value)gt4 Functional classification and regulationnetwork analysis were performed using MapMan software(httpmapmangabipdorgwebguestjsessionid=D943DDE0DBAF371F1B1D5C4F1A2E1597ajp13_mapman_gabipd_org)and the KEGG pathway database (httpwwwgenomejpkeggpathwayhtml)

Gene expression analysis by quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from the same treatments as thoseused for protein analysis using Trizol reagent (Gibco BRL LifeTechnologies Grand Island NY) according to the manufac-turerrsquos instructions as previously described (Ma et al 2016) ATurbo DNA-Free Kit (Ambion Austin TX) was used to removeDNA contamination A High-Capacity cDNA ReverseTranscription Kit (Life Technologies Grand Island NY) wasused for RNA reverse transcription according to the manufac-turerrsquos manual Power SYBR Green PCR Master Mix(Life Technologies Grand Island NY) was used for cDNAamplification on the StepOnePlus Real-Time PCR System(Applied Biosystems Foster City CA) Correspondinghomologous genes of tall fescue were obtained from the bio-fuel feedstock genomics resource (httpbfgrplantbiologymsueduintegrated_searchesshtml) Details of primer sequences andgene bank IDs are provided in Supplementary Data Table S1

Statistical analysis

All data were subjected to analysis of variance according tothe general linear model of SAS 90 (SAS Institute Cary NC)Treatment means were separated using Fisherrsquos protected leastsignificant difference (LSD) test at P 005

RESULTS

Rhizomatous phenotypes

Rhizomatous phenotypes of 3-month old lsquoBRrsquo plants are illus-trated in Fig 1A Each plant had an average of two rhizomes

and the average rhizome length was 397 6 017 cm includingtwo internodes Three days after transplanting to the hydroponicsystem a new rhizome bud was initiated from the crown node(Fig 1B) and after 6 d each plant had an average of078 6 020 rhizome buds initiated with average length196 6 hx00A0028 cm (Fig 1D) Twelve days after transplan-tation the number of rhizomes had increased to 146 6 024 andthe length of rhizomes had increased to 433 6 044 cm(Fig 1C E)

Effects of BAP on rhizome formation

To investigate the effects of CK on the initiation of rhizomescrowns were treated with BAP in the hydroponic solution Thenumber of rhizomes per plant increased during the 12-d treat-ment period in BAP-untreated control plants and plants treatedwith either 01 or 1 mM BAP (Fig 2A) The number of rhizomesper plant increased significantly from 078 and 145 in theBAP-untreated plants to 175 and 21 at 6 and 12 d of treatmentwith 01 mM BAP respectively corresponding figures were183 and 243 with 1 mM BAP treatment (Fig 2A) Plantstreated with 3 and 5 mM BAP displayed phytotoxicity symptoms(leaf deformation) and no increased rhizome numbers were ob-served (data not shown) In addition during treatment withBAP we investigated the number of tillers and new root forma-tion but there were no significant differences between the BAPtreatment and the control (Supplementary Data Fig S1A B)The 1 mM BAP treatment resulted in a 25-fold increase in en-dogenous iPA content compared with the untreated control(Fig 2B)

Proteins in rhizome nodes induced by BAP

More than 502 protein spots were detected on each gel of therhizome nodes (Fig 3 Supplementary Data Table S2) Throughmass spectrometry and protein database analysis a total of 76DEPs were successfully identified with BAP treatment com-pared with the untreated control (Table S2) including 60upregulated and 16 downregulated proteins GO category en-richment indicated that BAP caused rhizome nodes to expressproteins involved in various biological processes which weremainly metabolic processes ie cellular metabolism primarymetabolism carbohydrate metabolism monosaccharide metab-olism and glucose metabolism responses to stress oxidoreduc-tase activity protein folding and glycolysis These DEPs werelocated in intracellular spaces cytoplasm organellesmembrane-bounded organelles mitochondria and envelopes ac-cording to the cellular component (Fig 4A) Functional classifi-cation showed that they were mainly involved in redoxregulation (1579 ) glycolysis (1184 ) amino acid metabo-lism (1184 ) stress response (1053 ) cell development(789 ) the tricarboxylic acid cycle (658 ) photosynthesis(526 ) protein synthesis (395 ) and transport protein(395 ) (Fig 4B Table S2)

In order to understand how CK could stimulate rhizome for-mation it was of particular interest to identify proteins upregu-lated in rhizome nodes by BAP treatment Among proteinsinvolved in redox regulation seven were upregulated includingperoxidase (c373 c387) catalase (c166 c169 c172) ascorbate

484 Ma et al mdash Hormone regulation of rhizome development

peroxidase (c402) and disulphide isomerase (c147) Amongproteins involved in glycolysis six were upregulated includingphosphoglycerate kinase (PGK) (c283) GAPDH (c305 c318c320) fructose-bisphosphate aldolase (FBP) (c294) and phos-phopyruvate hydratase (c218) Among proteins involved inamino acid metabolism nine were upregulated including me-thionine synthases (c85 c86 c91 c92 c96 and c97) adenosyl-homocysteinase (c185) adenosylmethionine synthase (c256)and alanine aminotransferase (c225) In addition four proteinsinvolved in the tricarboxylic acid cycle were upregulated in-cluding pyruvate dehydrogenase (PDH) (c295) malate dehy-drogenase (c323) succinate dehydrogenase (SDH) (c136)aconitate hydratase (c32) and isocitrate dehydrogenase (CDH)(c266) Furthermore five cell division and growth-related pro-teins were also upregulated with BAP treatment in the rhizomenodes including a-tubulin (c224 c226) b-tubulin (c214) celldivision cycle protein (c35) and actin (c268) (Fig 3Supplementary Table S2)

Expression analysis of cytokinin-related genes in rhizome nodes

While proteomics is a powerful approach for identifying abun-dant proteins proteins of low abundance involved in CK metabo-lism or signalling were not found in the proteomic profiles ofrhizome nodes In order to further determine whether CK regula-tion of rhizome formation involved different components of CKmetabolism and the signalling pathway transcript levels of sev-eral cytokinin-related genes were examined using qRTndashPCRanalysis The CK synthesis gene IPT and the metabolic geneCKX were upregulated with BAP treatment in rhizome nodesThe transcript level of the CK signalling pathway receptor histi-dine kinase HK1 and the response regulator orthologous genesRR1 and RR6 were also increased in BAP-treated rhizome nodescompared with the untreated control but the expression level ofHK2 was not different with or without BAP treatment (Fig 5A)Expression levels of cell-division-related gene were also exam-ined and the results indicated that cyclin D2 (CYCD) histone

2 cm

0middot1

cm

2 cm

A B C

Days of treatment Days of treatment

0 6 120 6 12

a

b

c

5

4

3

2

1

0

c

b

a1middot8

1middot6

1middot4

1middot2

1middot0

0middot8

0middot6

0middot4

0middot2

0

No

of r

hizo

mes

per

pla

nt

Ave

rage

leng

th o

f rhi

zom

es (

cm)

ED

FIG 1 Phenotypes of lsquoBRrsquo tall fescue (A) Rhizomatous phenotypes that had been grown in fritted clay for 3 months in the greenhouse (B) A rhizome bud was ob-served 3 d after transplanting to the hydroponic system (C) Rhizomatous phenotypes in hydroponics after 12 d in the growth chamber (D) Average numbers of rhi-zomes per plant in hydroponics Values are mean 6 se for 30 plants (E) Average length of rhizomes in hydroponics Values are mean 6 se for 30 rhizomes

Columns marked with different letters indicate significant differences among treatments based on the LSD value (P005) FW fresh weight

Ma et al mdash Hormone regulation of rhizome development 485

H4 (His4) proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase B (CDKB) were also increased by BAP treat-ment compared with the control (Fig 5B)

Effects of GA3 on rhizome elongation

To further investigate whether GA3 may enhance rhizomegrowth changes in the length of rhizomes of plants treated with

GA3 were examined Plants treated with GA3 had longer rhi-zomes than untreated control plants at 12 d of treatment(Fig 6A) Compared with the untreated control theaverage length of rhizomes with 10 mM GA3 treatment wasincreased by 2486 at 12 d however GA3 treatment atother concentrations had no significant effects on rhizomelength (Fig 6B) Additionally the content of endogenous GA4

was increased by 16 compared with the untreated control(Fig 6C)

0 6 12

Days of treatment

BRndashBAP BR+BAP

2middot5

2middot0

1middot5

1middot0

0middot5

0

ControlBR+BAP(0middot1mM)BR+BAP(1mM)

No

of r

hizo

mes

per

pla

nt

Ca Ca Ca

BaBa

Bb

Ac

Ab

Aa

0

50

100

150

200

250

300

b

a

iPA

con

tent

(ng

gndash1

FW

)

A B

FIG 2 Effects of BAP on rhizomes of lsquoBRrsquo tall fescue (A) Average number of rhizomes per individual plant treated with different concentrations of BAP in hydro-ponics (B) Endogenous iPA content in rhizome nodes of lsquoBRrsquo tall fescue treated with 1 mM BAP Columns marked with different lowercase letters indicate signifi-cant differences among treatments at a given day of sampling based on the LSD value (P005) Columns marked with different uppercase letters indicate

significant differences among sampling time points for a given treatment based on the LSD value (P005) FW fresh weight

FIG 3 Two-dimensional SDSndashPAGE gels of rhizome nodes with BAP treatment in lsquoBRrsquo tall fescue Differentially expressed protein spots are marked There wereat least four replicated gels for each treatment (P 005)

486 Ma et al mdash Hormone regulation of rhizome development

Metabolic process

Monosaccharide metabolic process

Glucose metabolic process

Cellular metabolic processPrimary metabolic process

Response to stressOxidoreductase activity

Carbohydrate metabolic process

Protein folding

GlycolysisCell

IntracellularCytoplasmOrganelle

Membrane-bounced organelleMitochondrion

Envelope

Biological process

Cellular component

0 5 10 15 20 25 30 35 40

No of proteins

Redox regulationGlycolysisAmino acid metabolismStress responseCell developmentTricarboxylic acid cyclePhotosynthesis Protein synthesisTransport proteinNot assigned Major CHO metaolismSecondary metabolismMitochondrial electron transport Oxidative pentose phosphateC1-metabolismFermentationMetal handlingN-metabolism Nucleotide metabolism Hormone metabolism

5middot26 3middot95

3middot95

3middot95

3middot95

2middot63

2middot63

1middot32 1middot32 1middot32 1middot32 1middot32 1middot32 1middot32

6middot58

7middot89

10middot53

11middot84

11middot84 15middot79

A

B

FIG 4 Cluster analysis and functional distribution of differentially expressed proteins of rhizome nodes with BAP treatment in lsquoBRrsquo tall fescue (A) Biologicalprocess and cellular components were analysed against the Arabidopsis database (httpbioinfocaueducnagriGOanalysisphp) (threshold log10(P value) gt 4)

(B) Functional distribution was grouped using MapMan software (httpmapmangabipdorgwebguesthome)

5

4

4

3

2

21

0 0

6

8

10 Rhizome node without BAPRhizome node+BAP

Rhizome node without BAPRhizome node+BAP

FaCYCD FaHis4 FaPCNA FaCDKB

Rel

ativ

e ex

pres

sion

leve

l

Rel

ativ

e ex

pres

sion

leve

l

FaIPT FaCKX FaHK1 FaHK2 FaRR1 FaRR6

A B

FIG 5 Expression levels of genes in rhizome nodes of lsquoBRrsquo tall fescue with BAP treatment (A) Expression levels of genes responsible for CK metabolism and thesignalling pathway in rhizome nodes with BAP treatment (B) Expression levels of genes involved in cell division in crown nodes with BAP treatment FaIPTtRNA dimethylallyltransferase 2-like FaCKX cytokinin oxidase FaHK1 histidine kinase 1 FaHK2 histidine kinase 2 FaRR1 response regulator 1 FaRR6 re-

sponse regulator 6 FaCYCD cyclin-D2-2 FaHis4 histone h4 FaPCNA proliferating cell nuclear antigen FaCDKB cyclin-dependent kinase B1-1

Ma et al mdash Hormone regulation of rhizome development 487

Identification and functional classification of proteins with GA3

treatment in rhizomes

In order to identify proteins and associated metabolic pro-cesses that could be altered by GA3 and associated with GAstimulation of rhizome elongation protein profiles wereanalysed and compared in the rhizomes of plants treated withGA3 and untreated control plants Two-dimensional electropho-resis separated more than 530 proteins spots in each rhizome(Fig 7) A total of 37 DEPs were successfully identified in rhi-zomes treated with GA3 compared with those of the untreatedcontrol 30 were upregulated and 7 were downregulated byGA3 treatment (Fig 7 Supplementary Data Table S3) GO an-notation indicated that the biological processes of the rhizomealtered by GA3 treatment mainly included cellular processesmetabolic processes primary metabolic processes response tostress and metabolism of nitrogen compounds cellular ketonesoxoacids and carboxylic acid (Fig 8A) Based on the cellularcomponent analysis those DEPs were mainly located in intra-cellular spaces organelles membrane bounded organelles cy-toplasm plastids chloroplasts mitochondria membranes andenvelopes (Fig 8A) The GA3-responsive DEPs were mainlyclassified into the following functional categories glycolysis(1892 ) amino acid metabolism (1892 ) stress response(135 ) photosynthesis (1081 ) redox regulation (541 )and transport protein (541 ) according to MapMan functionalanalysis (Fig 8B)

The GA3-upregulated proteins in rhizomes could play impor-tant roles in GA stimulation of rhizome elongation Seven pro-teins involved in amino acid metabolism were upregulatedincluding methionine synthases (r113 r114 r117 r118 r120)and s-adenosylmethionine synthases (r285 and r289) ThreeGAPDH (r344 r367 and r375) three FBP (r338 r339 andr340) proteins and alcohol dehydrogenase (r290) involved inglycolysis were also upregulated with GA3 treatment In addi-tion the abundance of UDP-glucose 6-dehydrogenase (r224)involved in cell wall development glutamine synthetase (r307)involved in nitrogen metabolism and glycine-rich RNA bindingprotein (r586) involved in RNA regulation and transcriptionwere increased with GA3 treatment in rhizome tissues (Fig 7Supplementary Data Table S3)

Expression analysis of gibberellin-related and cellgrowth-related genes in rhizomes

Gibberellin biosynthesis and signalling pathway gene expres-sion levels were also examined in order to determine the possi-ble involvement of different components of GA metabolismand signalling pathways regulating rhizome elongation Thetranscript levels of ent-kaurene oxidase (KO) GA20-OX1 andDELLA were increased in GA3-treated rhizomes compared withthe control but the expression levels of GA20-OX2 andSPINDLY were not significantly different from the control(Fig 9) Several cell-elongation-related genes that were avail-able in the tall fescue EST database were also analysed includ-ing expansin (EXP) and endotransglucosylasehydrolases(XTH) family genes The transcript levels of EXPA5EXPANA11 EXPB2 XET1 and XET2 were increased in rhi-zomes treated with GA3 compared with the untreated control

BR-GA3 BR+GA3 2 cm

ControlGA3(0middot1mM)

GA3(1mM)

GA3(50mM)GA3(10mM)

Days of treatment0 3 6 9 12

0

1

2

3

4

5

6

7

6

5

4

3

2

1

0

ndash1

Ave

rage

leng

th o

f rhi

zom

es(c

m)

a

Rhizome-GA3 Rhizome+GA3

GA

4 co

nten

t (ng

g-1

FW

)

A

B

C

FIG 6 Effects of GA3 on rhizomes of lsquoBRrsquo tall fescue (A) Rhizomatous pheno-types with GA3 treatment in hydroponics after 12 d in the growth chamberWhite arrows indicate rhizomes (B) Average length of rhizomes with GA3

treatment in hydroponics Values are mean 6 se of 30 rhizomes Vertical barsare LSD values (P 005) indicating significant differences among treatmentsat a given day (C) Endogenous GA4 content in rhizomes treated with 10 mM

GA3 Columns marked with different letters indicate significant differencesamong treatments based on the LSD value (P 005) FW fresh weight

488 Ma et al mdash Hormone regulation of rhizome development

but EXPB11 with GA3 treatment was not different from thecontrol (Fig 9)

DISCUSSION

While numerous studies reported effects of CK and GA on vari-ous plant growth and development processes to our knowledgethis is the first report that demonstrates the stimulative effectsof CK on rhizome formation and of GA on rhizome elongationin a perennial grass species This finding is of great significancefor the development of rhizomatous grass species which areknown to be more resilient and to have a greater potential forrecuperation from stress damage as described in theIntroduction The proteomic and transcript analyses in thisstudy showed that the underlying mechanisms of regulation ofrhizome formation by CK and of rhizome elongation by GA arecomplex involving multiple metabolic processes and multiplecomponents of CK and GA metabolism and signalling path-ways as discussed in detail below

Proteomic analysis identified 76 proteins either up- ordownregulated by CK treatment which were classified intomultiple functional categories including redox regulation(1579 ) glycolysis (1184 ) amino acid metabolism(1184 ) stress response (1053 ) cell development(789 ) the tricarboxylic acid cycle (658 ) photosynthesis(526 ) protein synthesis (395 ) and transport protein(395 ) The respiratory pathways including glycolysis thetricarboxylic acid cycle and the mitochondrial electron trans-port chain are essential for plant growth and development be-ing involved in energy provision amino acid biosynthesis and a

wide range of other physiological functions (Plaxton 1996Fernie et al 2004) In our study 36 out of 76 (4736 ) pro-teins were involved in the respiratory pathways most of themwere upregulated with BAP treatment compared with theuntreated control (Fig 10) For instance FBP (c294) GAPDH(c318 c305 c320) PGK (c283 and c297) and bifunctional eno-lase 2 (c218) ndash involved in the glycolysis pathway in the cyto-sol ndashshowed greater accumulation and have also been shownto be upregulated in wild rice rhizomes (Hu et al 2011 Heet al 2014) Also showing higher abundance in rhizome nodeswith BAP treatment were PDH (c295) aconitate hydratase(c32) CDH (c266) SDH (c136) malate dehydrogenase (c323)and ATPase complex subunits (c132 c195 c193 c196) in-volved in the tricarboxylic acid cycle and the electron transportchain in mitochondria Along with the process of energy metab-olism the biosynthesis of amino acids including methioninealanine serine and glycine was also enhanced with BAP treat-ment It has been reported that metabolites involved in aminoacid and carbohydrate metabolites and metabolites in the tricar-boxylic acid cycle were increased in ipt-transgenic creepingbentgrass with associated higher endogenous CKs (Merewitzet al 2011 2012) Furthermore higher accumulation of aminoacid and intermediate products of the tricarboxylic acid cycleassociated with increased contents of endogenous iPA trans-zeatin and trans-zeatin riboside were found in the Arabidopsism132 mutant (Yu et al 2012) Energy metabolism-relatedgenes and proteins such as sucrose synthase GAPDH and alco-hol dehydrogenase were identified in rhizomes of perennial rice(He et al 2014) wild sorghum (Jang et al 2006) bamboo(Wang et al 2009) and horsetail (Balbuena et al 2012) Our

FIG 7 Two-dimensional SDSndashPAGE gels of tall fescue rhizomes with GA3 treatment Differentially expressed proteins are marked There were least four repeatgels for each treatment (P005)

Ma et al mdash Hormone regulation of rhizome development 489

results suggest that integrated networks involved in respiratoryenergy and amino acid metabolism could be activated by CKwhich provides energy products to support cell division and thesubsequent initiation of rhizomes in tall fescue

Cytokinins may regulate rhizome formation by the direct ef-fects of increasing endogenous cytokinin content and regulatoryeffects through CK responses or signalling In this study theendogenous iPA content increased along with increased tran-script levels of the CK synthesis gene IPT the CK signallingpathway receptor histidine kinase HK1 and the response regu-lator orthologous genes RR1 and RR6 These results suggestthat exogenous application of CK regulates expression levels ofrelated genes such as JcRRA9 and JcRRA17 in Jatropha (Panet al 2014) and SIHK4 SIRRAs and SICKX in tomato(Lycopersicon esculentum) roots and leaves (Gupta et al 2013Shi et al 2013) or are affected in the CK-deficient ipt mutantcompared with the wild type in Arabidopsis (Nishiyama et al2012) In addition cell-division-related gene expression levelsof cyclin D2 (CYCD) histone H4 (His4) proliferating cell nu-clear antigen (PCNA) and cyclin-dependent kinase B (CDKB)were also upregulated by BAP treatment CYCD His4 andPCNA which are involved in RNA and DNA replication at G1

Metabolic processCellular process

Primary metabolic processResponse to stress

Nitrogen compound metabolic processCellular ketone metabolic process

Oxoacid metabolic processCarboxylic acid metabolic process

CellIntracellular

OrganelleMembrane-bounded organelle

CytoplasmPlastid

ChloroplastMitochondrion

MembraneEnvelope

Biological process

Cellular component

0 5 10 15 20No of proteins

A

B Glycolysis5middot41 5middot41

2middot7 2middot7

2middot7

2middot7

2middot7

8middot11

18middot92

18middot92

13middot51

10middot81

5middot41

Amino acid metabolism

Stress response

Photosynthesis

Redox regulation

Transport Protein

Tricarboxylic acid cycle

Major CHO metabolism

Mitochondrial electron transport

Cell wall

N-metabolism

RNA regulation of transcription

Others

FIG 8 Cluster analysis and functional distribution of differentially expressed proteins of rhizomes with GA3 treatment in lsquoBRrsquo tall fescue (A) Biological processesand cellular components were analysed against the Arabidopsis database (httpbioinfocaueducnagriGOanalysisphp) (threshold log10(P value)gt 4) (B)

Functional distribution was grouped using MapMan software (httpmapmangabipdorgwebguesthome)

6Rhizome without GA3

Rel

ativ

e ex

pres

sion

leve

l

Rhizome +GA35

4

3

2

1

0

FaKO

Fa20O

X1

Fa20O

X2

FaDELL

A

FaSPIN

DLY

FaEXPA5

FaEXPA11

FaEXPB2

FaEXPB11

FaXET1

FaXET2

FIG 9 Expression level of genes in rhizomes of lsquoBRrsquo tall fescue with GA3 treat-ment FaKO ent-kaurene FaGA20OX1 gibberellin 20-oxidase1 FaGA20OX2gibberellin 20-oxidase2 FaEXPA5 EXPANSIN A5 FaEXPA11 EXPANSINA11 FaEXPB2 EXPANSIN B2 FaEXPB11 EXPANSIN B11 FaXET1 xylo-

glucan endotransglycosylase1 FaXET2 xyloglucan endotransglycosylase 2

490 Ma et al mdash Hormone regulation of rhizome development

and S phase and CDKB which is involved in the G2-M phaseof cell cycle were found to be involved in bud dormancy devel-opment in pea (Pisum sativum) and sorghum (Stafstrom andSussex 1992 Kebrom et al 2010a b) which suggested thatcell-division-related genes involved in the CK signalling path-way were upregulated and responsible for shoot apical meri-stem and axillary meristem development (Muller and Leyser2011 Schaller et al 2014) Our results suggest that CK stimu-lation of rhizome formation in tall fescue involves both directand regulatory effects which could lead to the upregulation ofexpression levels of downstream genes controlling cell divisionand cycles and thereby subsequently increase the numbers ofrhizomes in tall fescue

It is widely known that GA plays key roles in controlling cellelongation In this study rhizome elongation was indeed pro-moted by GA3 treatment Proteomic analysis of rhizomes for

proteins regulated by GA that could contribute to enhanced rhi-zome elongation found that 21 out of 37 (5675 ) GA-responsive proteins in the rhizome of tall fescue were involvedin energy metabolism and amino acid biosynthesis Most ofthem were also confirmed to be abundant in rhizomes of otherplant species without GA treatment including wild rice (Huet al 2011 He et al 2014) and sacred lotus (Nelumbo nuci-fera) (Kim et al 2013) or showed greater accumulation in thestolons of strawberry (Fragaria ananassa) (Fang et al 2011)For instance GAPDH (r344 r367 and r375) FBP (r338 r339and r340) and phosphoglycerate mutase (r185) which are in-volved in glycolysis were increased with GA3 treatment in rhi-zomes compared with the untreated control and methioninesynthase (r113 r114 r117 r118 and r120) and adenosylmethio-nine synthase which are involved in methionine metabolismwere increased with GA3 treatment in rhizomes In addition

Glycolysis

Glucose

Glucose-6P

Fructose-6P

c291

c418c422

c291

Glyceraldehyde-3P

Glycerate-13P2

Glycerate-3P

Glycerate-2P

Phosphoenolpyruvate

PyruvateEthanolMethionine

Acetyl-CoA

Oxaloacetate

TCA cycleCitrate

Isocitrate

SuccinateFumarate

ATP

ComplexV

ComplexIV

ComplexIIIComplex

I

ComplexII

Co Q

Cyt

c

Malate

Electron transport chain

α-Ketoglutarate

Serine

glycine

Alanine

Fructose-16P2Dihydroxyacetone-3P

Gluconolactone-6P

c229

c166 c169 c172c117

c117

c294c294

c305

c283 c297

c218

c225c262

c85 c86 c91c97c96c92

c185 c254 c256

c323 c32

c266

c136c132 c195

c196c193

c295

c211

c318 c320

Xylucose-5P

Gluconate-6P

Ribulose-5P

Glycolate

FIG 10 Differentially expressed proteins in rhizome nodes involved in the respiratory metabolism pathway according to the KEGG pathway in lsquoBRrsquo tall fescue Redcolour indicates proteins that were upregulated with BAP treatment compared with the control and black colour indicates proteins that were downregulated with

BAP treatment compared with the control

Ma et al mdash Hormone regulation of rhizome development 491

proteins responsive to protein synthesis were identified in rhi-zomes of tall fescue with GA3 treatment in this study includingprotein disulphide isomerase-like (r192) heat shock protein(HSP) 90 protein (r77) mitochondrial HSP (r146 and r153) andHSP 70 (r128) These data suggest that GA stimulation of rhi-zome elongation could result mainly from the activation of re-spiratory and amino acid metabolism although multiplemetabolic processes were altered due to GA3 treatment in therhizome of tall fescue Several GA-associated genes were iden-tified in the rhizomes of sorghum (Jang et al 2006) wild rice(He et al 2014) bamboo (Wang et al 2009) and lotus (Chenget al 2013) without GA treatment In our study the endoge-nous GA4 content increased along with increased transcript lev-els of ent-kaurene (KO) and GA 20 oxidase (GA20-OX) inrhizomes treated with GA3 and the expression level of DELLAwas also increased with GA3 treatment which might be relatedto the rapid degradation of DELLA protein during rhizomeelongation Our results indicate that GA synthesis and signal-ling pathway genes were increased with the application of GA3which might enhance the expression of downstream genessuch as cell wall growth genes Indeed the transcript levels ofexpansins and XET genes were higher in rhizomes treated withGA3 compared with the untreated control A previous studyshowed that internode elongation was possibly promoted byupregulation of XET and expansins associated with higher lev-els of endogenous GA4 in wheat (Triticum aestivum) (Zhanget al 2007) and higher XET activity was detected with exoge-nous application of GA3 in elongation leaves of barley(Hordeum vulgare) (Smith et al 1996) The combination of re-sults from proteomic profiling and transcription analysis sug-gested that application of GA3 enhanced the expression ofgenes involved in energy and protein synthesis and upregulatedcell wall loosening genes expression and subsequently pro-moted rhizome elongation

CONCLUSIONS

Based on phenotypic responses to hormones and proteomic pro-filing as well as gene expression related to the regulation of

rhizome formation and elongation by CK or GA we have pro-posed a potential regulatory mechanism of rhizome initiationand elongation in tall fescue (Fig 11) The increased number ofrhizomes with CK treatment and the increased length of rhi-zomes resulting from GA3 treatment could be associated withthe activation of respiratory metabolism amino acid metabo-lism redox regulation the stress response major carbohydratemetabolism and upregulation of hormone-responsive gene ex-pression thereby through their integrated actions providingenergy products to support cell division and subsequent rhi-zome development in tall fescue The biological functions andassociated molecular factors of CK- or GA-regulated proteinsimparting enhanced rhizome initiation and elongation deservefurther investigation which will provide new insights into thedevelopment of rhizomatous perennial grass species

SUPPLEMENTARY DATA

Supplementary data are available online at wwwaoboxfordjournalsorg and consist of the following Figure S1 effects of 6-benzylaminopurine (BAP) on tillers and new roots of lsquoBRrsquo tallfescue (A) Average numbers of tillers per plant in hydroponicsconditions (B) Average numbers of new roots per plant in hy-droponic conditions Values are mean 6 se of 30 rhizomesColumns marked with different letters indicate significant dif-ferences among treatments based on the LSD value (P005)Table S1 sequences of tall fescue primers used for qRTndashPCRanalysis Table S2 differentially expressed proteins in rhizomenodes treated with BAP Table S3 differentially expressed pro-teins in rhizomes treated with GA3

ACKNOWLEDGEMENTS

This research was supported by the China PostdoctoralScience Foundation (Grant No 2014M561672) the NationalNatural Science Foundation of China (Grant No 31572153)and Rutgers Center of Turfgrass Science

Respiratory metabolism

Amino acid metabolismCell division andprotein synthesis

(CYCDHis4PCNACDKBExpansins XETs celldivision cycle proteinelongation factors 2tubulins and elF4A-1)

Redox regulationHormone(cytokininGA3)

Stress response

Major CHO metabolism

Hormone regulated genesFestuca arundinacea

FIG 11 A model for hormone-regulated rhizome development in tall fescue lsquoBRrsquo

492 Ma et al mdash Hormone regulation of rhizome development

LITERATURE CITED

Ashikari M Sakakibara H Lin S et al 2005 Cytokinin oxidase regulates ricegrain production Science 309 741ndash745

Balbuena TS He R Salvato F Gang DR Thelen JJ 2012 Large-scale prote-ome comparative analysis of developing rhizomes of the ancient vascularplant Equisetum hyemale Frontiers in Plant Science 3 131

De Battista J Bouton J 1990 Greenhouse evaluation of tall fescue genotypesfor rhizome production Crop Science 30 536ndash541

Bradford MM 1976 A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry 72 248ndash254

Burgess P Huang B 2014 Root protein metabolism in association with im-proved root growth and drought tolerance by elevated carbon dioxide increeping bentgrass Field Crops Research 165 80ndash91

Burns JC Chamblee DS 1979 Adaptation In Buckner R Bush L eds Tallfescue Agronomy Monograph 20 9ndash30

Charrier S Stewart A 2006 Breeding of rhizomatous turf tall fescue InMercer CF ed Breeding for success diversity in action In Proceedings ofthe 13th Australasian Plant Breeding Conference Christchurch NewZealand 18ndash21 April 2006 383ndash387

Cheng L Li S Yin J Li L Chen X 2013 Genome-wide analysis of differen-tially expressed genes relevant to rhizome formation in lotus root (Nelumbonucifera Gaertn) PLoS One 8 e67116

Choi J Hwang I 2007 Cytokinin perception signal transduction and role inplant growth and development Journal of Plant Biology 50 98ndash108

Claeys H De Bodt S Inze D 2014 Gibberellins and DELLAs central nodes ingrowth regulatory networks Trends in Plant Science 19 231ndash239

Cowan JR 1956 Tall fescue Advances in Agronomy 8 283ndash318Cox T Bender M Picone C et al 2002 Breeding perennial grain crops

Critical Reviews in Plant Sciences 21 59ndash91Fang XP Ma HS Lu DZ Yu H Lai W Ruan S 2011 Comparative proteo-

mics analysis of proteins expressed in the I-1 and I-2 internodes of straw-berry stolons Proteome Science 9 26

Fernie AR Carrari F Sweetlove LJ 2004 Respiratory metabolism glycoly-sis the TCA cycle and mitochondrial electron transport Current Opinion inPlant Biology 7 254ndash261

Gao S Fang J Xu F et al 2014 CYTOKININ OXIDASEDEHYDROGENASE4integrates cytokinin and auxin signaling to control rice crown root formationPlant Physiology 165 1035ndash1046

Gupta S Shi X Lindquist IE Devitt N Mudge J Rashotte AM 2013

Transcriptome profiling of cytokinin and auxin regulation in tomato rootJournal of Experimental Botany 64 695ndash704

Ha S Vankova R Yamaguchi-Shinozaki K Shinozaki K Tran L-SP 2012

Cytokinins metabolism and function in plant adaptation to environmentalstresses Trends in Plant Science 17 172ndash179

He R Salvato F Park J-J et al 2014 A systems-wide comparison of red rice(Oryza longistaminata) tissues identifies rhizome specific genes and pro-teins that are targets for cultivated rice improvement BMC Plant Biology14 46

Hedden P 1997 Gibberellin biosynthesis eLS doi1010029780470015902a0023720

Hoagland DR Arnon DI 1950 The water-culture method for growing plantswithout soil Circular California Agricultural Experiment Station 3471ndash32

Hu C Jin A Zhang Z 1995 Change of endohormone in mixed bud on Leibamboo rhizome during differentiation Journal of Zhejiang ForestryCollege 13 1ndash4

Hu F Wang D Zhao X et al 2011 Identification of rhizome-specific genes bygenome-wide differential expression analysis in Oryza longistaminataBMC Plant Biology 11 18

Jan A Yang G Nakamura H et al 2004 Characterization of a xyloglucanendotransglucosylase gene that is up-regulated by gibberellin in rice PlantPhysiology 136 3670ndash3681

Jang CS Kamps TL Skinner DN Schulze SR Vencill WK Paterson AH

2006 Functional classification genomic organization putatively cis-actingregulatory elements and relationship to quantitative trait loci ofsorghum genes with rhizome-enriched expression Plant Physiology 1421148ndash1159

Jernstedt J Bouton J 1985 Anatomy morphology and growth of tall fescuerhizomes Crop Science 25 539ndash542

Kebrom TH Brutnell TP Finlayson SA 2010a Suppression of sorghum axil-lary bud outgrowth by shade phyB and defoliation signalling pathwaysPlant Cell amp Environment 33 48ndash58

Kebrom TH Brutnell TP Hays DB Finlayson SA 2010b Vegetative axillarybud dormancy induced by shade and defoliation signals in the grasses PlantSignaling amp Behavior 5 317ndash319

Kim M-J Nelson W Soderlund CA Gang DR 2013 Next-generationsequencing-based transcriptional profiling of sacred lotus ldquoChina antiquerdquoTropical Plant Biology 6 161ndash179

Kurakawa T Ueda N Maekawa M et al 2007 Direct control of shoot meri-stem activity by a cytokinin-activating enzyme Nature 445 652ndash655

Kuroha T Tokunaga H Kojima M et al 2009 Functional analyses ofLONELY GUY cytokinin-activating enzymes reveal the importance of thedirect activation pathway in Arabidopsis The Plant Cell 21 3152ndash3169

Li B Beuselinck P 1996 Rhizomatous Lotus corniculatus L II Morphologyand anatomy of rhizomes Crop Science 36 407ndash411

Li Y Hagen G Guilfoyle TJ 1992 Altered morphology in transgenic tobaccoplants that overproduce cytokinins in specific tissues and organsDevelopmental Biology 153 386ndash395

Ma X Zhang J Huang B 2016 Cytokinin-mitigation of salt-induced leaf se-nescence in perennial ryegrass involving the activation of antioxidant sys-tems and ionic balance Environmental and Experimental Botany 1251ndash11

Merewitz EB Gianfagna T Huang B 2011 Photosynthesis water use androot viability under water stress as affected by expression of SAG12-ipt con-trolling cytokinin synthesis in Agrostis stolonifera Journal of ExperimentalBotany 62 383ndash395

Merewitz EB Du H Yu W Liu Y Gianfagna T Huang B 2012 Elevated cy-tokinin content in ipt transgenic creeping bentgrass promotes drought toler-ance through regulating metabolite accumulation Journal of ExperimentalBotany 63 1315ndash1328

Muller D Leyser O 2011 Auxin cytokinin and the control of shoot branchingAnnals of Botany 107 1203ndash1212

Nishiyama R Le DT Watanabe Y et al 2012 Transcriptome analysesof a salt-tolerant cytokinin-deficient mutant reveal differential regula-tion of salt stress response by cytokinin deficiency PLoS One 7e32124

Pan B-Z Chen M-S Ni J Xu Z-F 2014 Transcriptome of the inflorescencemeristems of the biofuel plant Jatropha curcas treated with cytokinin BMCGenomics 15 974

Plaxton WC 1996 The organization and regulation of plant glycolysis AnnualReview of Plant Biology 47 185ndash214

Porter HL 1958 Rhizomes in tall fescue Agronomy Journal 50 493ndash494Sacks E Dhanapala M Tao D Cruz MS Sallan R 2006 Breeding for peren-

nial growth and fertility in an Oryza sativaO longistaminata populationField Crops Research 95 39ndash48

Saha MC Talukder S Azhaguvel P Mukhergee S Chekhovskiy K

2015 Deciphering drought tolerance in tall fescue [Lolium arundina-ceum (Schreb) Darbysh] In Budak H Spangenberg G edsMolecular breeding of forage and turf Cham Switzerland Springer1ndash7

Saxena P Huang B Bonos SA Meyer WA 2014 Photoperiod and temperatureeffects on rhizome production and tillering rate in tall fescue [(Schreb)Darby] Crop Science 54 1205ndash1210

Schaller GE Street IH Kieber JJ 2014 Cytokinin and the cell cycle CurrentOpinion in Plant Biology 21 7ndash15

Shi X Gupta S Lindquist IE Cameron CT Mudge J Rashotte AM 2013

Transcriptome analysis of cytokinin response in tomato leaves PLoS One 8e55090

Smith RC Matthews PR Schunmnn PH Chandler PM 1996 The regulationof leaf elongation and xyloglucan endotransglycosylase by gibberellin inlsquoHimalayarsquo barley (Hordeum vulgare L) Journal of Experimental Botany47 1395ndash1404

St John R Fry J Bremer DJ Keeley S 2009 Establishment rate and lateralspread of Festuca arundinacea cultivars International Turfgrass SocietyResearch Journal 11 481ndash487

Stafstrom JP Sussex IM 1992 Expression of a ribosomal proteingene in axillary buds of pea seedlings Plant Physiology 1001494ndash1502

Sun T 2010 Gibberellin-GID1-DELLA a pivotal regulatory module for plantgrowth and development Plant Physiology 154 567ndash570

Ma et al mdash Hormone regulation of rhizome development 493

Tao D Hu F Yang Y et al 2001 Rhizomatous individual was obtained frominterspecific BC2F1 progenies between Oryza sativa and Oryza longistami-nata Rice Genetics Newsletter 18 11ndash13

Wang K Peng H Lin E et al 2009 Identification of genes related to the devel-opment of bamboo rhizome bud Journal of Experimental Botany 61551ndash561

Xu C Huang B 2008 Root proteomic responses to heat stress in two Agrostisgrass species contrasting in heat tolerance Journal of Experimental Botany59 4183ndash4194

Xu C Sibicky T Huang B 2010 Protein profile analysis of salt-responsive pro-teins in leaves and roots in two cultivars of creeping bentgrass differing insalinity tolerance Plant Cell Reports 29 595ndash615

Yamaguchi S 2008 Gibberellin metabolism and its regulation Annual Reviewof Plant Biology 59 225ndash251

Yu H Du X Zhang F et al 2012 A mutation in the E2 subunit of the mitochon-drial pyruvate dehydrogenase complex in Arabidopsis reduces plant organ

size and enhances the accumulation of amino acids and intermediate prod-ucts of the TCA cycle Planta 236 387ndash399

Zhang T Zhao X Wang W et al 2014 Deep transcriptome sequencing of rhi-zome and aerial-shoot in Sorghum propinquum Plant Molecular Biology84 315ndash327

Zhang X Ervin E 2004 Cytokinin-containing seaweed and humic acid extractsassociated with creeping bentgrass leaf cytokinins and drought resistanceCrop Science 44 1737ndash1745

Zhang X Ervin EH Evanylo GK Li J Harich K 2013 Corn and soybeanhormone and antioxidant metabolism responses to biosolids under two crop-ping systems Crop Science 53 2079ndash2089

Zhang Y Ni Z Yao Y Nie X Sun Q 2007 Gibberellins and heterosis of plantheight in wheat (Triticum aestivum L) BMC Genetics 8 40

Zhou Y Lambrides CJ Fukai S 2014 Drought resistance and soil water ex-traction of a perennial C4 grass contributions of root and rhizome traitsFunctional Plant Biology 41 505ndash519

494 Ma et al mdash Hormone regulation of rhizome development

Tao D Hu F Yang Y et al 2001 Rhizomatous individual was obtained frominterspecific BC2F1 progenies between Oryza sativa and Oryza longistami-nata Rice Genetics Newsletter 18 11ndash13

Wang K Peng H Lin E et al 2009 Identification of genes related to the devel-opment of bamboo rhizome bud Journal of Experimental Botany 61551ndash561

Xu C Huang B 2008 Root proteomic responses to heat stress in two Agrostisgrass species contrasting in heat tolerance Journal of Experimental Botany59 4183ndash4194

Xu C Sibicky T Huang B 2010 Protein profile analysis of salt-responsive pro-teins in leaves and roots in two cultivars of creeping bentgrass differing insalinity tolerance Plant Cell Reports 29 595ndash615

Yamaguchi S 2008 Gibberellin metabolism and its regulation Annual Reviewof Plant Biology 59 225ndash251

Yu H Du X Zhang F et al 2012 A mutation in the E2 subunit of the mitochon-drial pyruvate dehydrogenase complex in Arabidopsis reduces plant organ

size and enhances the accumulation of amino acids and intermediate prod-ucts of the TCA cycle Planta 236 387ndash399

Zhang T Zhao X Wang W et al 2014 Deep transcriptome sequencing of rhi-zome and aerial-shoot in Sorghum propinquum Plant Molecular Biology84 315ndash327

Zhang X Ervin E 2004 Cytokinin-containing seaweed and humic acid extractsassociated with creeping bentgrass leaf cytokinins and drought resistanceCrop Science 44 1737ndash1745

Zhang X Ervin EH Evanylo GK Li J Harich K 2013 Corn and soybeanhormone and antioxidant metabolism responses to biosolids under two crop-ping systems Crop Science 53 2079ndash2089

Zhang Y Ni Z Yao Y Nie X Sun Q 2007 Gibberellins and heterosis of plantheight in wheat (Triticum aestivum L) BMC Genetics 8 40

Zhou Y Lambrides CJ Fukai S 2014 Drought resistance and soil water ex-traction of a perennial C4 grass contributions of root and rhizome traitsFunctional Plant Biology 41 505ndash519

494 Ma et al mdash Hormone regulation of rhizome development