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www.lasbioscic.co Coicaiv & Igaiv Biology 457

Coicaiv & Igaiv Biology 4:4, 457-458; Jly/Ags 2011; 2011 Las Bioscic

ArtICLe Addendum

Addendum to: Arredondo-Peter R, Hargrove MS,

Sarath G, Moran JF, Lohrman J, Olson JS, et al.

Rice hemoglobins: gene cloning, analysis and

oxygen-binding kinetics of a recombinant pro-

tein synthesized in Escherichia coli. Plant Physiol

1997; 115:125966; PMID: 9390447;

DOI: 10.1104/pp.115.3.125

Key words: embryonic organs, geneexpression, hemoglobin, non-symbiotic,Oryza, rice

Abbreviations: Hb, hemoglobin; nsHb,non-symbiotic hemoglobin; RT-PCR,reverse transcriptase-polymerase chainreaction

Submitted: 03/13/11

Accepted: 03/13/11

DOI: 10.4161/cib.4.4.15468

*Correspondence to: Ral Arredondo-Peter;

Email: ra@uaem.mx

Rice (Oryza sativa) contains fve cop-ies o the non-symbiotic hemoglobin(hb) gene, namelyhb1 to hb5. Previousanalysis by RT-PCR revealed that rice

hb1 expresses in roots and leaves andhb2 expresses in leaves. However, it isnot known whether or nothb1 and hb2express in rice embryonic organs. Here,

we report the expression ohb1 and hb2genes in rice embryonic organs usingRT-PCR and specifc oligos or Hb1 andHb2. Our results indicate thathb1 andhb2 genes express in embryonic organsin rice growing under normal condi-tions. Specifcally, hb1 expresses in riceembryos and seminal roots, and hb2

expresses in embryos, coleoptiles andseminal roots. These observations sug-gest that Hb1 and Hb2 coexist and unc-tion in rice embryonic organs.

Non-symbiotic hemoglobins (nsHbs) arewidespread in land plants. Nucleotidesequences coding or these proteinshave been identied in plants rangingrom primitive bryophytes to evolvedangiosperms, including monocots anddicots.1-3 In monocots, rice (Oryzasativa) contains ve copies o the nshb

gene, namelyhb1 to hb5.4,5

Western blotanalysis and immunolocalization byconocal microscopy using polyclonalanti-rice Hb1 antibodies revealed thatnsHbs are localized in specic tissues orice embryonic organs, such as the seedaleurone and scutellum, and o vegetativeorgans, such as the lea schlerenchymaand root cap.6,7 However, these analysesdid not dierentiate among individualnsHbs because detected signals could

Expression o non-symbiotic hemoglobin 1 and 2 genesin rice (Oryza sativa) embryonic organs

Vernica Lira-Ruan,1,2 Mariel Ruiz-Kubli1 and Ral Arredondo-Peter1,*1Laboratorio de Biosica y Biologa Molecular; 2Laboratorio de Fisiologa y Desarrollo Vegetal; Departamento de Bioqumica y Biologa Molecular; Facultad de

Ciencias; Universidad Autnoma del Estado de Morelos; Morelos, Mxico

result rom any one o the rice nsHbs(i.e., Hb1, Hb2, Hb3, Hb4 or Hb5), orrom a combination o them. Previousanalysis by RT-PCR using specic oli-

gos revealed that rice hb1 expresses inroots and leaves, hb2expresses in leaves,and hb5 expresses in embryonic andvegetative organs.5,8 Also, the analysiso an OsNSHB2 promoter used to thegus reporter gene revealed tissue-specicexpression o the rice hb2gene in roots,vasculature o young leaves, fowers andthe pedicel/stem junction o transgenicArabidopsis.9 These observations sug-gest that Hb1, Hb2 and Hb5 coexist inrice leaves and roots. However, it is still

not known whether or not hb1 and hb2express in rice embryonic organs.We analyzed the expression o hb1

and hb2 genes in rice embryonic organsusing RT-PCR and specic oligos or Hb1and Hb2. Seed germination, isolation opoly(A+) RNA, reverse transcription andPCR amplication were perormed asdescribed by Arredondo-Peter et al.8 TheHb1 and Hb2 transcripts are each approx-imately 550 bp in length. Figure 1 showsthat PCR ragments o the expected sizeor Hb1 were amplied rom embryos and

seminal roots, and or Hb2 were ampliedrom embryos, coleoptiles and seminalroots. These PCR ragments were clonedand sequenced, and the resulting sequenceswere identical to those o the rice Hb1and Hb2 transcripts (Genbank acces-sion number U76030.1 and U76031.1,respectively). This result indicates thathb1 and hb2genes express in embryonicorgans in rice growing under normal con-ditions. It was previously reported that

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458 Coicaiv & Igaiv Biology Vol 4 Iss 4

Acknowledgements

Authors wish to express their gratitudeto Miss Gillian Klucas and Dr. GautamSarath or English corrections and useulcomments to improve the contents o thisaddendum.

Reerences

1. Vinogradov SN, Hoogewijs D, Bailly X, Arredondo-

Peter R, Gough J, Dewilde S, et al. A phylogenomicproile o globins. BMC Evol Biol 2006; 6:31-47.

2. Garrocho-Villegas V, Gopalasubramaniam SK,Arredondo-Peter R. Plant hemoglobins: what weknow six decades ater their discovery. Gene: FunctEvol Genom, 2007; 398:78-85.

3. Vinogradov SN, Fernndez I, Hoogewijs D,Arre dondo-Peter R. Phylogenetic relat ionships oplant 3/3 and 2/2 hemoglobins to bacterial and othereukaryotic hemoglobins. Mol Plant 2011; 4:42-58.

4. Lira-Rua n V, Ross E, Sarath G, Klucas RV,Arredondo-Peter R. Mapping and ana lysis o a hemo-globin gene amily rom rice (Oryza sativa). PlantPhysiol Biochem 2002; 40 :199-202.

hb2 apparently does not express in riceroots,8 however in this work Hb2 tran-scripts were detected in rice seminal roots(Fig. 1). This observation suggests thatexpression ohb2is downregulated duringroot development.

The above observations together withthose reported by Arredondo-Peter et al.8

and Garrocho-Villegas et al.

5

suggest thatHb1, Hb2 and Hb5 coexist in rice embry-onic and vegetative organs. Specically,it is likely that Hb1, Hb2 and Hb5 coex-ist and unction in rice embryos, seminalroots and leaves; Hb2 and Hb5 coexist andunction in coleoptiles; and Hb1 and Hb5coexist and unction in roots (Table 1).Results reported here complement ourknowledge o the expression onshb genesin rice organs.

Figure 1. expssio aalysis ohb1a hb2gs i ic byoic ogas. Aplifcaio o h ic Hb1 a Hb2 ascips was po by rt-PCr sig spcifc oligos o Hb1 a Hb2 a coiios scib by Aoo-P al.8

Table 1. expssio onshb gs i ic ogas

nshb gene

Expression* in rice

ReferencesEmbryonic organs Vegetative organs

Embryos Coleoptiles Seminal roots Leaves Roots

hb1 + nd + + + this wok; Aoo-P al.8

hb2 + + + + nd this wok; Aoo-P al.8

hb3 nA nA nA nA nA

hb4 nA nA nA nA nA

hb5 + + + + + Gaocho-Villgas al.5

*nd, o c; nA, o aalyz.

5. Garrocho-Villegas V, Bustos-Rivera G, Gough J,Vinogradov SN, Arredondo-Peter R. Expression andin-silico structural analysis o a rice (Oryza sativa)hemoglobin 5. Plant Physiol Biochem 2008; 46:855-9.

6. Lira-Ruan V, Sarath G, Klucas RV, Arredondo-PeterR. Synthesis o hemoglobins in rice (Oryza sativavar.

Jackson) plants growing i n normal and stress condi-tions. Plant Sci 2001; 161:279-87.

7. Ross EJH, Shearman L, Mathiesen M, Zhou J,Arredondo-Peter R, Sarath G, Kluca s RV. Non-symbiotic hemoglobins are synthesized duri ng germi-nation and in dierentiating cell types. Protoplasma2001; 218:125-33.

8. Arredondo-Peter R, Hargrove MS, Sarath G, MoranJF, Lohrma n J, Olson JS, et al. Rice hemoglobins:gene cloning, analysis and oxygen-binding kinetics oa recombinant protein synthesized in Escherichia coli.Plant Physiol 1997; 115:1259-66.

9. Ross EJH, Stone JM, Elowsky CG, Arredondo-PeterR, Klucas RV, Sarath G. Activation o the Oryzasativanon-symbiotic haemoglobin-2 promoter by thecytokinin-regulated transcription actor, ARR1. JExp Bot 2004; 55:1721-31.