Journal of Heredity Advance Access originally published online on April 3, 2007
Journal of Heredity 2007 98(3):272-276; doi:10.1093/jhered/esm014
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Brief Communications |
Two infA Gene Mutations Independently Originated from a Mutator Genotype in Barley
From the Instituto de Genética E. A. Favret, CICVyA, Instituto Nacional de Tecnología Agropecuaria, CC 25, B1712WAA, Castelar, Provincia de Buenos Aires, Argentina
Address correspondence to Alberto R. Prina at the address above, or e-mail: aprina{at}cnia.inta.gov.ar.
Cytoplasmic line 2 (CL2) is a chlorophyll mutant that was selected from a plastid mutator genotype in barley. The dynamics of greening and plastid development of CL2 first-leaf blade contrasts with that of monocots. Previous characterizations of CL2 suggested that this mutant has a delay of plastid gene translation during embryogenesis. We hypothesize that CL2 is a mutant in the infA gene, which encodes translation initiation factor 1 (IF1). Wild-type barley infA gene differs in some nucleotides from that in wheat, but the corresponding IF1 proteins are identical. However, infA from CL2 carries a point mutation, which leads to an amino acid change in IF1 residue 52. One CL2-like seedling selected from a new mutator pool also carries a point mutation in infA gene, this time leading to a change of the universally conserved amino acid residue 32. Both point mutations were T 
C substitutions. We sequenced the complementary DNA of the infA transcripts from the wild type and CL2 and found that the mutation was conserved at the mRNA level. Results strongly suggest that CL2 and CL2-like are infA gene mutants, this being the first time that a mutant phenotype is attributed to infA gene in a higher plant.
A curious cytoplasmically inherited chlorophyll-deficient mutant, codified as cytoplasmic line 2 (CL2), was previously described in barley (Prina 1996). It presented a time-dependent expression mainly localized on the upper part of the first leaf blade, showing a dynamics of greening and plastid development opposite to that usually observed in monocots (Mullet 1988, 1993; Prina et al. 2003). Previous characterization by transmission electron microscopy and pigment analysis in experiments with antibiotics and different temperatures (Prina et al. 2003) showed that CL2 syndrome produces a delay in plastid protein synthesis during embryogenesis, which is accompanied by a delay and sometimes an impairment of normal plastid development and plastid ribosome formation. On this basis, the infA gene, which encodes the translation initiation factor 1 (IF1), is proposed as a candidate gene responsible for CL2 syndrome.
In bacteria, the rate-limiting step for translation initiation is usually the formation of 30S subunit ternary complex with the mRNA (Pon and Gualerzi 1984; Stern et al. 1997) that, to proceed properly, requires the presence of 3 translation factors (Boelens and Gualerzi 2002). IF1 functions in modulating 30S–IF2 interactions, and the association/dissociation of ribosomal subunits are widely recognized (Dahlquist and Puglisi 2000; Boelens and Gualerzi 2002; Croitoru et al. 2004; Laursen et al. 2005). Plastid translation is generally regulated at the initiation phase (Zerges 2000). Among bacterial genes encoding translation initiation factors only infA gene has homologues residing in the plastome of higher plants (Millen et al. 2001). IF1 is a highly conserved protein that has functional counterparts in all living organisms (Kyrpides and Woese 1998; Roll-Mecak et al. 2001) and belongs to the family of oligonucleotide-binding fold proteins, which also includes the ribosomal protein S1 and the cold shock proteins CspA and CspB. IF1 has shown to have not only structural resemblance but also functional similarities with those cold shock proteins (Laursen et al. 2005). Mutants for infA gene have not been reported in higher plants, and they were rare until recently even in bacteria (Croitoru et al. 2004). No phenotypes have been so far attributed to infA mutants in higher plants, and its functions in chloroplasts are inferred from information in bacteria (Kozak 1983; Millen et al. 2001).
Here we show results of infA gene sequencing in CL2 and CL2-like mutants and 2 different controls. We found one different point mutation in each of the 2 independent mutant origins, both of them affecting conserved residues in the deduced IF1 proteins.
 |
Materials and Methods |
|---|
 Top Materials and Methods Results and DiscussionReferences |
|---|
Plant Material
Seeds of 2-rows spring barley (Hordeum vulgare L.) were used as experimental material.
The following chlorophyll-deficient mutants isolated from the barley chloroplast mutator genotype (Prina 1992) were included in the analysis. 1) CL2, which was described in introduction (Prina 1996; Prina et al. 2003). 2) New independently originated CL2-like mutants selected from new mutator pools of plants carrying mutator genotype. In order to guarantee the independence of new mutational events from those preexisting in the plastome of the original mutator pool, these pools were originated by crossing the mutator genotype as male parent and consequently not contributing to the progenies with plastids previously affected by the mutator genotype. Only one seedling was so far selected by this procedure. 3) Newly selected CL2-like mutants selected from the original mutator genotype pool. Fifteen individual seedlings were isolated. 4) Cytoplasmic line 9 (CL9): homogeneous light green chlorophyll–deficient type (Prina AR, unpublished data), selected from the barley chloroplast mutator original pool, was used as a second control of wild-type infA gene.
MC182 (accession number of the Institute for Genetics E.A. Favret, Instituto Nacional de Tecnología Agropecuaria [INTA]) was used as wild-type control.
Gene Cloning and Sequencing
Total DNA was extracted from leaves following a standard protocol (Fang et al. 1992). The sequences of interest were amplified by polymerase chain reaction (PCR) with primers designed on the basis of published information of chloroplast DNA sequences of wheat (GenBank accession no. NC_002762), rice (GenBank accession no. NC_001320), and maize (GenBank accession no. NC_001666). A fragment of 930 bp was amplified using primer rpl36+ (5'-CCCCTGTCTTTGTTTATGCTTCG-3') located on the rpl36 gene and primer rps8– (5'-CGAGAGGGTTTTATTGAAAGTGTTCGG-3') on rps8 gene and cloned into pCR2.1-TOPO Vector (Invitrogen TOPO TA Cloning kit). Amplification was performed in a final volume of 25 µl using 20 ng of total DNA, 2.5 µl 10x buffer Taq, 2 µl MgCl2 (25 mM), 1.25 µl dNTPs mix (2.5 mM each nucleotide), 0.8 µl primer rpl36+ (10 µM), 0.8 µl primer rps8– (10 µM), and 0.2 µl Taq Highway (5 U/µl). After denaturation at 94°C for 3 min, the reaction mixtures were heated to 94°C for 30 s, 58°C for 1 min, and 72°C for 1 min, in 30 cycles.
In order to detect possible mistakes made by Taq polymerase amplification, 5 or more independent colonies from each amplified sequence were analyzed, and a consensus sequence for each genotype was obtained. For sequencing the newly selected CL2-like mutants, a new pair of primers which only amplify the infA gene were designed: infAF (5'-ATGACAGAAAAAAAAAATAGGAGAGAA-3') and infAR (5'-CTAATCCTTTGAATCTTTGGTATCCTT-3'). A Pfu Taq polymerase was used for amplification, and the fragments were sequenced as PCR products. Amplification was performed in a final volume of 50 µl using 20 ng of total DNA, 5 µl 10x buffer Taq with MgSO4, 1 µl dNTPs mix (2.5 mM each nucleotide), 1 µl primer infAF (10 µM), 1 µl primer infAR (10 µM), and 0.5 µl Pfu Taq Promega (3 U/µl). After denaturation at 94°C for 5 min, the reaction mixtures were heated to 94°C for 30 s, 52°C for 30 s, and 72°C for 30 s, in 30 cycles.
Most of the sequences were obtained at Laboratorio de Servicios en Biologia Molecular—CNIA, INTA, applying the dye terminator technique (DYEnamic TM ET terminator cycle sequencing premix kit, Amersham, Amersham Biosciences, Buenos Aires, Argentina) with the help of a DNA ABI sequencer 373A. The sequences corresponding to the newly selected CL2-like mutants were acquired from a local private service provider. All sequence data alignments were performed using the ClustalW WWW Service at the European Bioinformatics Institute (http://www.ebi.ac.uk/clustalw) (Chenna et al. 2003).
RNA Isolation and Reverse Transcription–PCR
Total RNA was isolated from 7-day old leaves with Trizol (Invitrogen, Buenos Aires, Argentina) according to the manufacturer's procedure and treated with DNAse RQ1 (Promega, Madison, WI). Complementary DNA (cDNA) was synthesized with SuperScript III reverse transcriptase using the antisense primer infAR, according to the manufacturer's instructions (Invitrogen). Products of reverse transcription were amplified by PCR using Pfu Taq polymerase as described above.
|
Results and Discussion |
|---|
|
TopMaterials and MethodsResults and DiscussionReferences |
|---|
In Figure 1, it can be observed that wild-type barley infA gene sequence (GenBank accession no. AY488512) differed in some nucleotides from that previously reported for wheat (GenBank accession no. NC_002762). However, the corresponding IF1 proteins were identical (Figure 2).
|
|
On the other hand, infA gene sequences from CL2 mutant carried a mutational change in all the 5 independent colonies analyzed in comparison to 11 independent colonies analyzed from barley wild type. It was a T
C transition at nucleotide 157 (GenBank accession no. AY488513), corresponding to a semi-conserved amino acid change of serine to proline at residue 52 of the IF1 protein, which is a highly conserved residue in Poales (Figure 2). Furthermore, it is located in a segment that is homologous to that comprising residues 36–49 in Escherichia coli, which has been pointed out as probably important for IF1 functions and has given several functional mutants (Croitoru et al. 2004). Other 15 CL2-like seedlings were isolated from advanced progenies of natural self-pollinated plants, corresponding to the original chloroplast mutator pool from which the first CL2 mutant was previously isolated (Prina 1996). All the 15 new isolates presented an identical base substitution to that observed in the original CL2. As the independence of the mutational events that originated each of them cannot be established, these results only allow us to conclude that this mutant allele could be easily selected at the level of individual seedlings. On new pools made by crossing the mutator genotype as a male parent, only one CL2-like mutant was isolated and it showed maternal inheritance. Sequence analysis of the infA gene in this new mutant also showed a point mutation, which was observed in all the 7 independent colonies from CL2-like mutant. This was also a T C substitution, but this time at nucleotide 97 (GenBank accession no. AY743911). It corresponded to a conserved amino acid change, from phenylalanine to leucine at residue 32, which is a universally conserved residue of the IF1 protein (Figure 2). Interestingly, CL2-like seedlings greened faster than CL2 seedlings. We observed this difference on progenies of plants harvested at the field nursery. However, given the marked influence of the temperature during embryogenesis on CL2 phenotype expression (Prina et al 2003), we must consider that a conclusive experiment using seeds developed under controlled environmental conditions should be done. Another mutator-induced mutant, CL9, completely different to that described in CL2, was used as an additional control and, as expected, it showed the wild-type infA sequence. Nine independent colonies from CL9 mutant were sequenced.
Both mutations observed were T C substitutions occurring in a first codon position, making it unlikely that they could be subjected to mRNA editing, which in higher plants chloroplasts is mostly restricted to C U conversions located at the second codon position (Sugiura et al. 1998; Bock 2000). However, to confirm if editing could restore the cytidine to uridine in the mRNA, we sequenced the cDNA of the infA transcripts from the wild-type and CL2 mutant, and we found that the mutation was conserved at the mRNA level.
Previously, with a few exceptions recently reported (Drescher et al. 2000; Ahlert et al. 2003; Kuroda and Maliga 2003; Kode et al. 2005), little importance has been given to the influence of plastid genes on the control of early plastid development (Mache et al. 1997; Leon et al. 1998). Mutants in nuclear genes that affect plastid ribosomes were previously reported in barley (Börner et al. 1976) and maize (Walbot and Coe 1979), but in those cases, plastid ribosomes were absent during all the plant cycle. Those unstable genotypes were conspicuously manifested in F2 seedlings and induced a narrow spectrum of chlorophyll deficiencies. On the other hand, the mutator genotype from which CL2 and CL2-like mutants were isolated induces a wide spectrum of chlorophyll mutants, which includes several viable and normal-vigor types (Prina 1992, 1996). Besides, mutator effects are slightly manifested in the F2 generation, and only subtle mutational changes on the plastome have so far been detected on mutator-induced mutants (Ríos et al. 2003; Colombo et al. 2006).
Results strongly suggest that CL2 and CL2-like are infA gene mutants, this being the first time that a mutant phenotype is attributed to infA gene in a higher plant. The conditional expression of CL2 mutants make them a promising experimental material for addressing the functions of IF1 in higher plants and for a better understanding of plastid protein synthesis and plastid development in monocots.
|
Acknowledgments |
|---|
Authors want to thank Ms Helen Ray and the librarian Raúl D. Bassi for helpful technical assistance during manuscript preparation. This work was supported by INTA and Proyecto de Investigación Científica y Técnica #04841, Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Argentina. A.L. had a postgraduate fellowship of ANPCyT. M.J. had a postgraduate fellowship of INTA-ANPCyT.
|
Footnotes |
|---|
Corresponding Editor: Susan Gabay-Laughnan
Received May 10, 2006
Accepted January 10, 2007
|
References |
|---|
|
TopMaterials and MethodsResults and DiscussionReferences |
|---|
-
Ahlert D, Ruf S, Bock R. Plastid protein synthesis is required for plant development in tobacco. Proc Natl Acad Sci USA (2003) 100:15730–15735.
Bock R. Sense from nonsense: how the genetic information of chloroplasts is altered by RNA editing. Biochimie (2000) 82:549–557.[Medline]
Boelens R, Gualerzi CO. Structure and function of bacterial initiation factors. Curr Protein Pept Sci (2002) 3:107–119.[CrossRef][Web of Science][Medline]
Börner T, Schumann B, Hagemann R. Biochemical studies on a plastid ribosome-deficient mutant of Hordeum vulgare. In: Genetics and biogenesis of chloroplasts and mitochondria—Bücher T, Neupert W, Sebald W, Werner S, eds. (1976) North-Holland (Amsterdam): Biomedical Press, Elsevier. 41–48.
Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res (2003) 31:3497–3500.
Colombo N, Ríos RD, Prina AR. Plastome analysis of barley chroloplast mutator-induced mutants. J Basic App Genet (2006) 17:5–9.
Croitoru V, Bucheli-Witschel M, Hägg P, Abdulkarim F, Isaksson LA. Generation and characterization of functional mutants in the translation initiation factor IF1 of Escherichia coli. Eur J Biochem (2004) 271:534–544.[Web of Science][Medline]
Dahlquist KD, Puglisi JD. Interaction of translation initiation factor IF1 with the E. coli ribosomal A site. J Mol Biol (2000) 299:1–15.[CrossRef][Web of Science][Medline]
Drescher A, Ruf S, Calsa T Jr, Carrer H, Bock R. The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes. Plant J (2000) 22:97–104.[CrossRef][Web of Science][Medline]
Fang G, Hammar S, Grumet R. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biotechniques (1992) 56:52–54.
Kode V, Mudd EA, Iamtham S, Day A. The tobacco plastid accD gene is essential and is required for leaf development. Plant J (2005) 44:237–244.[CrossRef][Web of Science][Medline]
Kozak M. Comparison of initiation of protein synthesis in Procaryotes, Eucaryotes, and Organelles. Microbiol Rev (1983) 47:1–45.
Kuroda H, Maliga P. The plastid clpP1 protease gene is essential for plant development. Nature (2003) 425:86–89.[CrossRef][Medline]
Kyrpides NC, Woese CR. Universally conserved translation initiation factors. Proc Natl Acad Sci USA (1998) 95:224–228.
Laursen BS, Sorensen HP, Mortensen KK, Sperling-Petersen HU. Initiation of protein synthesis in bacteria. Microbiol Mol Biol Rev (2005) 69:101–123.
Leon P, Arroyo A, Mackenzie S. Nuclear control of plastid and mitochondrial development in higher plants. Annu Rev Plant Physiol Plant Mol Biol (1998) 49:453–480.[CrossRef][Web of Science][Medline]
Mache R, Zhan DX, Lerbs-Mache S, Harra KH, Villain P, Gauvincs S. Nuclear control of early plastid differentiation. Plant Physiol Biochem (1997) 35:199–203.[Web of Science]
Millen RS, Olmstead RG, Adams KL, Palmer JD, Lao NT, Heggie L, Kavanagh TA, Hibberd JM, Gray JC, Morden CW, et al. Many parallel losses of infA from chloroplast DNA during angiosperm evolution with multiple independent transfers to the nucleus. Plant Cell (2001) 13:645–658.
Mullet J. Chloroplast development and gene expression. Annu Rev Plant Physiol Plant Mol Biol (1988) 39:475–502.[CrossRef][Web of Science]
Mullet J. Dynamic regulation of chloroplast transcription. Plant Physiol (1993) 103:309–313.[CrossRef][Web of Science][Medline]
Pon CL, Gualerzi CO. Mechanism of protein biosynthesis in prokaryotic cells. Effect of initiation factor IF1 on the initial rate of 30S initiation complex formation. FEBS Lett (1984) 175:203–207.[CrossRef][Web of Science][Medline]
Prina AR. A mutator nuclear gene inducing a wide spectrum of cytoplasmically inherited chlorophyll deficiencies in barley. Theor Appl Genet (1992) 85:245–251.[Web of Science]
Prina AR. Mutator-induced cytoplasmic mutants in barley: genetic evidence of activation of a putative chloroplast transposon. J Hered (1996) 87:385–389.
Prina AR, Arias MC, Lainez V, Landau A, Maldonado S. A cytoplasmically inherited mutant controlling early chloroplast development in barley seedlings. Theor Appl Genet (2003) 107:1410–1418.[CrossRef][Web of Science][Medline]
Ríos RD, Saione H, Robredo C, Acevedo A, Colombo N, Prina AR. Isolation and molecular characterization of atrazine tolerant barley mutants. Theor Appl Genet (2003) 106:696–702.[Web of Science][Medline]
Roll-Mecak A, Shin BS, Dever TE, Burley SK. Engaging the ribosome: universal IFs of translation. Trends Biochem Sci (2001) 26:705–709.[CrossRef][Web of Science][Medline]
Stern DB, Higgs DC, Yang J. Transcription and translation in chloroplasts. Trends Plant Sci (1997) 2:308–315.[CrossRef][Web of Science]
Sugiura M, Hirose T, Sugita M. Evolution and mechanism of translation in chloroplasts. Annu Rev Genet (1998) 32:437–459.[CrossRef][Web of Science][Medline]
Walbot V, Jr Coe EH. Nuclear gene iojap conditions a programmed change to ribosome-less plastids in Zea mays. Proc Natl Acad Sci USA (1979) 76:2760–2764.
Zerges W. Translation in chloroplasts. Biochimie (2000) 82:583–601.[Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||