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Expression of non-symbiotic hemoglobin 1 and 2 genes in rice ( Oryza sativa ) embryonic organs

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Abstract

Rice (Oryza sativa) contains five copies of the non-symbiotic hemoglobin (hb) gene, namely hb1 to hb5. Previous analysis by RT-PCR revealed that rice hb1 expresses in roots and leaves and hb2 expresses in leaves. However, it is not known whether or not hb1 and hb2 express in rice embryonic organs. Here, we report the expression of hb1 and hb2 genes in rice embryonic organs using RT-PCR and specific oligos for Hb1 and Hb2. Our results indicate that hb1 and hb2 genes express in embryonic organs in rice growing under normal conditions. Specifically, hb1 expresses in rice embryos and seminal roots, and hb2 expresses in embryos, coleoptiles and seminal roots. These observations suggest that Hb1 and Hb2 coexist and function in rice embryonic organs.
www.landesbioscience.com Communicative & Integrative Biology 457
Communicative & Integrative Biology 4:4, 457-458; July/August 2011; ©2011 Landes Bioscience
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; 115:1259–66; PMID: 9390447;
DOI: 10.1104/pp.115.3.125
Key words: embryonic organs, gene
expression, hemoglobin, non-symbiotic,
Oryza, rice
Abbreviations: Hb, hemoglobin; nsHb,
non-symbiotic hemoglobin; RT-PCR,
reverse transcriptase-polymerase chain
reaction
Submitted: 03/13/11
Accepted: 03/13/11
DOI: 10.4161/cib.4.4.15468
*Correspondence to: Raúl Arredondo-Peter;
Email: ra@uaem.mx
Rice (Oryza sativa) contains five cop-
ies of the non-symbiotic hemoglobin
(hb) gene, namely hb1 to hb5. Previous
analysis by RT-PCR revealed that rice
hb1 expresses in roots and leaves and
hb2 expresses in leaves. However, it is
not known whether or not hb1 and hb2
express in rice embryonic organs. Here,
we report the expression of hb1 and hb2
genes in rice embryonic organs using
RT-PCR and specific oligos for Hb1 and
Hb2. Our results indicate that hb1 and
hb2 genes express in embryonic organs
in rice growing under normal condi-
tions. Specifically, hb1 expresses in rice
embryos and seminal roots, and hb2
expresses in embryos, coleoptiles and
seminal roots. These observations sug-
gest that Hb1 and Hb2 coexist and func-
tion in rice embryonic organs.
Non-symbiotic hemoglobins (nsHbs) are
widespread in land plants. Nucleotide
sequences coding for these proteins
have been identified in plants ranging
from primitive bryophytes to evolved
angiosperms, including monocots and
dicots.1-3 In monocots, rice (Oryza
sativa) contains five copies of the nshb
gene, namely hb1 to hb5.4,5 Western blot
analysis and immunolocalization by
confocal microscopy using polyclonal
anti-rice Hb1 antibodies revealed that
nsHbs are localized in specific tissues of
rice embryonic organs, such as the seed
aleurone and scutellum, and of vegetative
organs, such as the leaf schlerenchyma
and root cap.6,7 However, these analyses
did not differentiate among individual
nsHbs because detected signals could
Expression of non-symbiotic hemoglobin 1 and 2 genes
in rice (Oryza sativa) embryonic organs
Verónica Lira-Ruan,1,2 Mariel Ruiz-Kubli1 and Raúl Arredondo-Peter1,*
1Laboratorio de Biofísica y Biología Molecu lar; 2Laboratorio de Fisiología y Desarrollo Vegetal; Departa mento de Bioquímica y Biología Molecular; Facultad de
Ciencias ; Universidad Autónoma del Estado de Morelos; Morelos, México
result from any one of the rice nsHbs
(i.e., Hb1, Hb2, Hb3, Hb4 or Hb5), or
from a combination of them. Previous
analysis by RT-PCR using specific oli-
gos revealed that rice hb1 expresses in
roots and leaves, hb2 expresses in leaves,
and hb5 expresses in embryonic and
vegetative organs.5,8 Also, the analysis
of an OsNSHB2 promoter fused to the
gus reporter gene revealed tissue-specific
expression of the rice hb2 gene in roots,
vasculature of young leaves, flowers and
the pedicel/stem junction of transgenic
Arabidopsis.9 These observations sug-
gest that Hb1, Hb2 and Hb5 coexist in
rice leaves and roots. However, it is still
not known whether or not hb1 and hb2
express in rice embryonic organs.
We analyzed the expression of hb1
and hb2 genes in rice embryonic organs
using RT-PCR and specific oligos for Hb1
and Hb2. Seed germination, isolation of
poly(A+) RNA, reverse transcription and
PCR amplification were performed as
described by Arredondo-Peter et al.8 The
Hb1 and Hb2 transcripts are each approx-
imately 550 bp in length. Figure 1 shows
that PCR fragments of the expected size
for Hb1 were amplified from embryos and
seminal roots, and for Hb2 were amplified
from embryos, coleoptiles and seminal
roots. These PCR fragments were cloned
and sequenced, a nd the resulting sequences
were identical to those of the rice Hb1
and Hb2 transcripts (Genbank acces-
sion number U76030.1 and U76031.1,
respectively). This result indicates that
hb1 and hb2 genes express in embryonic
organs in rice growing under normal con-
ditions. It was previously reported that
458 Communicative & Integrative Biology Volume 4 Issue 4
Acknowledgements
Authors wish to express their gratitude
to Miss Gillian Klucas and Dr. Gautam
Sarath for English corrections and useful
comments to improve the contents of this
addendum.
References
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hb2 apparently does not express in rice
roots,8 however in this work Hb2 tran-
scripts were detected in rice seminal roots
(Fig. 1). This observation suggests that
expression of hb2 is downregulated during
root development.
The above observations together with
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and Garrocho-Villegas et al.5 suggest that
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onic and vegetative organs. Specifically,
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ist and function in rice embryos, seminal
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function in coleoptiles; and Hb1 and Hb5
coexist and function in roots (Table 1).
Results reported here complement our
knowledge of the expression of nshb genes
in rice organs.
Figure 1. Expression analysis of h b1 and h b2 genes in rice embryonic organs. Amplication of the rice Hb1 and Hb2 transcripts was per formed by RT-
PCR using specic oligos for Hb1 and Hb2 and conditions described by Arredondo-Peter et al.8
Tab le 1. Expression of nsh b genes in rice organs
nshb gene
Expression* in rice
ReferencesEmbryonic organs Vegetative organs
Embryos Coleoptiles Seminal roots Leaves Roots
hb1 +ND + + + This work; Arredondo-Peter et al.8
hb2 + + + + ND This work; Arredondo-Peter et al.8
hb3 NA NA NA NA NA
hb4 NA NA NA NA NA
hb5 + + + + + Garrocho-Villegas et al.5
*ND, not detected; NA, not analyzed.
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Vinogradov SN, Arredondo-Peter R. Expression and
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a recombin ant protein s ynthesized in Escherichia coli.
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R, Kluc as RV, Sarath G. Activation of the Oryza
sativa non-symbiotic haemoglobin-2 promoter by t he
cytok inin-regulated tran scription factor, AR R1. J
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A hemoglobin (hb) gene family was identified in rice, consisting of four functional hb copies located in BAC inserts OSM11719 and OSM11676, which contain the gene clusters hb1/hb3/hb4 and hb2, respectively. Sequence comparison with databases revealed that a number of potential promoters exist upstream from the hb genes suggesting that these genes are differentially expressed in rice.
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Land plants and algae form a supergroup, the Archaeplastida, believed to be monophyletic. We report the results of an analysis of the phylogeny of putative globins in the currently available genomes to bacterial and other eukaryote hemoglobins (Hbs). Archaeplastida genomes have 3/3 and 2/2 Hbs, with the land plant genomes having group 2 2/2 Hbs, except for the unexpected occurrence of two group 1 2/2 Hbs in Ricinus communis. Bayesian analysis shows that plant 3/3 Hbs are related to vertebrate neuroglobins and bacterial flavohemoglobins (FHbs). We sought to define the bacterial groups, whose ancestors shared the precursors of Archaeplastida Hbs, via Bayesian and neighbor-joining analyses based on COBALT alignment of representative sets of bacterial 3/3 FHb-like globins and group 1 and 2 2/2 Hbs with the corresponding Archaeplastida Hbs. The results suggest that the Archaeplastida 3/3 and group 1 2/2 Hbs could have originated from the horizontal gene transfers (HGTs) that accompanied the two generally accepted endosymbioses of a proteobacterium and a cyanobacterium with a eukaryote ancestor. In contrast, the origin of the group 2 2/2 Hbs unexpectedly appears to involve HGT from a bacterium ancestral to Chloroflexi, Deinococcales, Bacilli, and Actinomycetes. Furthermore, although intron positions and phases are mostly conserved among the land plant 3/3 and 2/2 globin genes, introns are absent in the algal 3/3 genes and intron positions and phases are highly variable in their 2/2 genes. Thus, introns are irrelevant to globin evolution in Archaeplastida.
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In rice (Oryza sativa var. Jackson) at least three copies of hemoglobin (hb) gene exist. Rice hb1 and hb2 genes are differentially expressed in roots and leaves from mature plants. We used polyclonal antibodies raised to recombinant rice Hb1 and Western blotting to analyze the synthesis of Hbs in rice plants growing under normal or stress conditions. Results showed that rice Hbs are synthesized in coleoptiles, seminal roots and embryos from seeds germinated for 6 days, and also in leaves and roots from plants 2-14 weeks after germination. Analysis of Hb synthesis in stressed rice showed that: (i) level of Hbs was higher in etiolated than control plants, (ii) level of Hbs increased in roots from flooded rice, and (iii) level of Hbs did not change under oxidative (H(2)O(2)), nitrosative (SNP) and hormonal (2,4-D) stresses. These results suggest that the effect of light withdrawal in etiolated leaves and O(2)-limiting conditions in flooded roots, but not oxidative, nitrosative and hormonal stresses, modulate the synthesis of rice Hbs.
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Nonsymbiotic hemoglobins (ns-Hbs) previously have been found in monocots and dicots; however, very little is known about the tissue and cell type localization as well as the physiological function(s) of these oxygen-binding proteins. We report the immunodetection and immunolocalization of ns-Hbs in rice (Oryza sativa L.) by Western blotting and in situ confocal laser scanning techniques. Ns-Hbs were detected in soluble extracts of different tissues from the developing rice seedling by immunoblotting. Levels of ns-Hbs increased in the germinating seed for the first six days following imbibition and remained relatively constant thereafter. In contrast, ns-Hb levels decreased during leaf maturation. Roots and mesocotyls contained detectable, but low levels of ns-Hbs. Split-seed experiments revealed that ns-Hbs are synthesized de novo during seed germination and are expressed in the absence of any signal originating from the embryo. Immunolocalization of ns-Hbs by confocal microscopy indicated the presence of ns-Hbs primarily in differentiated and differentiating cell types of the developing seedling, such as the aleurone, scutellum, root cap cells, sclerenchyma, and tracheary elements. To our knowledge, this is the first report of the specific cellular localization of these proteins during seedling development.
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This review describes contributions to the study of plant hemoglobins (Hbs) from a historical perspective with emphasis on non-symbiotic Hbs (nsHbs). Plant Hbs were first identified in soybean root nodules, are known as leghemoglobins (Lbs) and have been characterized in detail. It is widely accepted that a function of Lbs in nodules is to facilitate the diffusion of O(2) to bacteroids. For many years Hbs could not be identified in plants other than N(2)-fixing legumes, however in the 1980s a Hb was isolated from the nodules of the non-legume dicot plant Parasponia, a hb gene was cloned from the non-nodulating Trema, and Hbs were detected in nodules of actinorhizal plants. Gene expression analysis showed that Trema Hb transcripts exist in non-symbiotic roots. In the 1990s nsHb sequences were also identified in monocot and primitive (bryophyte) plants. In addition to Lbs and nsHbs, Hb sequences that are similar to microbial truncated (2/2) Hbs were also detected in plants. Plant nsHbs have been characterized in detail. These proteins have very high O(2)-affinities because of an extremely low O(2)-dissociation constant. Analysis of rice Hb1 showed that distal His coordinates heme Fe and stabilizes bound O(2); this means that O(2) is not released easily from oxygenated nsHbs. Non-symbiotic hb genes are expressed in specific plant tissues, and overexpress in organs of stressed plants. These observations suggest that nsHbs have functions additional to O(2)-transport, such as to modulate levels of ATP and NO.
Article
This work reports the analysis of an additional hemoglobin (hb) gene copy, hb5, in the genome of rice. The amino acid sequence of Hb5 differs from the previously determined rice Hbs 1-4 in missing 11 residues in helix E. Transcripts of hb5 were found to be ubiquitous in rice organs, and hormone- and stress-response promoters exist upstream of the rice hb5 gene. Furthermore, the modeled structure of Hb5 based on the known crystal structure of rice Hb1 is unusual in that the putative distal His is distant from the heme Fe. This observation suggests that Hb5 binds and releases O(2) easily and thus that it functions as an O(2)-carrier or in some aspects of the O(2) metabolism.
Synthesis of hemoglobins in rice
  • V Lira-Ruan
  • G Sarath
  • Rv Klucas
  • R Arredondo-Peter
Lira-Ruan V, Sarath G, Klucas RV, Arredondo-Peter R. Synthesis of hemoglobins in rice (Oryza sativa var.