Euphytica 139: 187–197, 2004. C 2004 Kluwer Academic Publishers. Printed in the Netherlands.
187
Molecular and cytological characterization of a cytoplasmic-specific mutant in pima cotton (Gossypium barbadense L.) Mehmet Karaca1,3 , Sukumar Saha1,∗ , Franklin E. Callahan1 , Johnie N. Jenkins1 , John J. Read1 & Richard G. Percy1 1
USDA-ARS, Crop Science Research Laboratory, Genetics and Precision Agriculture Research Unit, Mississippi State, MS 39762, U.S.A.; 2 USDA-ARS, Crop Science Research Laboratory, Maricopa, AZ, U.S.A.; 3 Present address: Department of Field Crops, Faculty of Agriculture, Akdeniz University, Antalya 07059, Turkey; (∗ author for correspondence: e-mail:
[email protected])
Received 23 December 2003; accepted 5 September 2004
Key words: cDNA, chloroplast, cotton, cyt-V, EST, virescent mutant
Summary A cytoplasmic mutant of Gossypium barbadense L., cyt-V was characterized at the morphological, cellular, genetic and molecular levels using comparison analysis with v7 v7 , a nuclear virescent mutant to identify molecular effects of the cyt-V mutation. The yellow phenotype was specific only to leaves in the cyt-V mutant (CM-1-90) but the same phenotype was present in both leaves and cotyledons of v7 v7 , a nuclear virescent mutant, suggesting that cyt-V and v7 v7 , had different organ-specific gene actions. Chlorophyll and carotenoid levels of CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v7 v7 true leaves were significantly lower than in the true leaves of PS-7 × CM-1-90, v7 v7 × CM-1-90 and PS-7. Anatomical studies of chloroplast showed that CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v7 v7 lacked grana in the thylakoids of the mesophyll cells. This indicated that chlorophyll and carotenoid levels correlated with chloroplast structure. SDS-PAGE analysis of thylakoid preparations revealed decreases of several granalocalized PSII proteins in CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v7 v7 . cDNA-AFLP differential display studies identified several differentially expressed transcripts in the leaves of reciprocal crosses (PS-7 × CM-1-90, v7 v7 × CM-1-90 and CM-1-90 × PS-7 and CM-1-90 × v7 v7 ), including one possessing a high sequence homology to a psbA gene. Western blot analysis further confirmed the absence of D1 protein encoded by psbA in CM-1-90 × PS-7 CM-1-90 × v7 v7 and CM-1-90 true leaves. Overall, we studied cyt-V and v7 v7 that both are developmental mutants, as all the virescents of cotton mutants, and as such it was difficult to separate cause and effect in the observation; however, we verified that the source of cyt-V mutation was in chloroplast and elucidated that its gene action was different from v7 v7 . Results indicated that cyt-V is inherited as a single gene but it affects several chloroplast and nucleus-encoded genes. We identified several transcripts that associated with the cyt-V mutation. This study also suggested that chloroplast-encoded gene products might affect the expression of nuclear genes, possibly at the transcriptional stage. Abbreviations: SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; cDNA-AFLP: cDNADNA-amplified fragment length polymorphism
Introduction Cotton is the world’s most important natural textile fiber crop. This genus comprises more than 50 diploid and tetraploid species. Two tetraploid species, G. hirsutum L. and G. barbadense L., account for 90 and 5%,
respectively, of the world’s cotton production (Wendel et al., 1992). Increasing numbers of morphological mutants of cotton are used in genetic mapping studies and in studies of plant development, and occasionally have proven to have agronomic value including male sterility, fertility restoration and susceptibility to certain
188 insects, diseases and environmental stresses (Galau & Wilkins, 1989; Chen et al., 1990; Kohel et al., 2002; Herring et al., 2004). The molecular identification of chloroplastassociated genes in many economically important crop species has been facilitated by the analysis of mutants. One class is the virescent mutants, with yellow leaf color, associated with chloroplast development in cotton (Percy & Kohel, 1999). To date, more than 30 virescent and 2 albino mutants have been genetically characterized in the tetraploid cotton species (Kohel, 1967, 1983; Percival and Kohel, 1974, 1976; Turcotte & Feaster, 1978; Turcotte & Percy, 1988; Zhang & Pan, 1986, 1990; Percy, 1999). However, only a few of these plastid-specific virescent mutants have been studied in detail (Katterman & Endrizzi, 1973; Percy, 1999). Kohel & Benedict (1971) studied a variegated cotton mutant producing leaves or sectors of leaves, which were distinctly yellow in appearance with the chloroplasts containing about half of the carotenoid and chlorophyll contents compared to the wild type. Katterman & Endrizzi (1973) studied a maternally inherited cotton mutant showing white and yellow leaves. White sectors of the mutant leaves were found deficient in the 70S ribosomes, while yellow sectors contained normal 70S ribosomes, compared with the normal leaves. Another maternally inherited morphological mutation (cyt-V) was reported in Pima (Gossypium barbadense L. CM-1-90) cotton and called as cyt-V (Percy, 1999). In the cyt-V mutation, the first to third leaves on the plant show a pale yellow to white color, followed by restoration of a normal green phenotype in the fifth to sixth leaves and in all subsequent leaves. Chloroplast-specific mutants (maternally inherited) can be used to discover chloroplast-associated genes in cotton. Chloroplast biosynthesis requires the products of nuclear and chloroplast-encoded genes which must be co-regulated for successful chloroplast development (Archer et al., 1987; Falbel et al., 1996; Roy & Barkan, 1998). The primary regulation of the chloroplast genes lies at the level of post-transcriptional processing, such as RNA editing and mRNA processing, including polyadenylation and degradation (Gruissem & Schuster, 1993; Karpinska et al., 1997; Lisitsky et al., 1996; Meurer et al., 1996; Karcher & Bock, 1998; Hayes et al., 1999). The mutants we studied are developmental mutants, as all the virescents of cotton appear to be, and as such it is difficult to separate cause and effect in the observation. In this study, a cytoplasmic mutant of Gossypium barbadense L., cyt-V, (CM-1-90) was characterized at the morphological,
cellular, genetic and molecular levels using comparison analysis with v7 v7 , a nuclear virescent mutant to identify molecular effects of the cyt-V, mutation. In this study, we studied a new type of chloroplast-specific virescent mutant, cyt-V and used a differential display method, cDNA-AFLP, which could be used to discover chloroplast- and nuclear-specific genes in cotton.
Materials and methods Plant materials All plants used in this study belong to the species Gossypium barbadense L. A maternally inherited dominant cyt-V mutant (CM-1-90), self-pollinated for two successive generations, was used as a parent in reciprocal crosses to produce F1 hybrids with Pima S-7 (PS-7) and the nuclear genome-encoded recessive virescent mutant (v7 v7 ), respectively. These F1 hybrids (CM-190 ( ) × PS-7 ( ), PS-7 ( ) × CM-1-90 ( ), CM-1-90 ( ) × v7 v7 ( ), and v7 v7 ( ) × CM-1-90 ( )) plants were also self-pollinated to generate four different F2 populations. All the F1 plants and parental lines (PS-7, nuclear recessive virescent mutant, v7 v7 , CM-1-90 and another plant (CM-1-90 WT), herein we called wild type in which cyt-V mutation was discovered, were grown in a greenhouse and field. F2 plants were grown in a field to verify the maternal inheritance of the cyt-V mutations and to compare with v7 v7 . Chloroplast structure Young, healthy first true leaves and cotyledons of the four reciprocal hybrids and parental lines (at the 14day-old seedling stage), and after the restoration of green pigmentation for true leaves (at the sixth leaf stage) were fixed in freshly prepared half-strength Karnovsky’s fixative which contained 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) overnight at 4 ◦ C. Fixed samples were rinsed, post-fixed in 2% osmium tetroxide (OsO4), dehydrated in ascending concentrations of ethanol (20– 100%), and subsequently embedded in Spurr’s lowviscosity embedding medium. Polymerization was allowed to proceed overnight at 70 ◦ C. Thin sections were cut and collected on 200 mesh copper grids and stained with uranyl acetate and lead citrate. Samples were then examined with a transmission electron microscopy (JEOL JEM 100CX II TEM) at 60 kV at Mississippi State University Microscopy Center,
189 Mississippi State, MS. At least four different chloroplasts were examined for each sample.
carotenoid contents (µg/mL) using equations described by Lichtenthaler (1987).
RNA extraction, cDNA-AFLP and capillary electrophoresis
Leaf proteins, SDS-PAGE and Western blot
Bulked samples of leaves from several 14-day-old seedlings of each of the CM-1-90 × PS-7, PS-7 × CM-1-90, CM-1-90 × v7 v7 , and v7 v7 × CM-1-90 hybrids grown in a greenhouse were used for total RNA extraction and cDNAs were synthesized as described in Saha et al. (2003). The cDNA-amplified fragment length polymorphism (cDNA-AFLP) differential display studies were performed using the AFLP selective amplification module and primer pairs for large plant genomes according to the manufacturer’s recommendation (PE Applied Biosystems, Foster City, CA). Capillary electrophoresis of the cDNA-AFLP fragments were analyzed as described by Karaca et al. (2002). Isolation and sequencing of cDNA Based on capillary electrophoresis observations, primers that resulted in polymorphisms were reused to amplify differentially expressed bands. Bands of interest were isolated from the gels, cloned using the TOPO TA Cloning Kit (Invitrogen La Jolla, CA) and sequenced at USDA/ARS, Stoneville, MS, using the M13 forward primer. In order to diagnose sequencing errors, at least six clones per sequence were compared for the final sequences. These sequences were then compared to sequences in GenBank using BLAST (Basic Local Alignment Search Tool; Altschul et al., 1997) to find homology with other genes previously known in GenBank databases. Chlorophyll, carotenoid contents Four leaf punches of young, healthy first true leaves of the four reciprocal hybrids and parental lines (at the 14-day-old seedling stage), and after the restoration of green pigmentation for true leaves (at the sixth leaf stage) were obtained using a cork borer (5.5 mm diameter) and the leaf discs were placed in a vial containing 4 mL dimethyl sulfoxide (DMSO, Fisher Scientific, Fairlawn, NJ). A total of four vials per plant were prepared and incubated at room temperature overnight in the dark. One milliliter of the solution was scanned with a spectrophotometer to measure the absorbances at 664, 648, and 470 nm to calculate chlorophyll and
Thylakoid proteins of leaf and cotyledon tissue were isolated and analyzed by SDS-PAGE and Western blot analysis of immunoblotting (anti-D) as previously described by Callahan et al. (1989). Total leaf proteins and soluble protein fractions were isolated and subjected to SDS-PAGE as described by Callahan et al. (1992).
Results Morphology Parental lines, PS-7, CM-1-90, v7 v7 , and CM-1-90WT grown in a greenhouse and field showed uniform morphological appearances indicating that seeds used in this study were homozygous at least for the leaf trait(s). Morphological observations confirmed that the first to third leaves of the cyt-V mutation in CM-1-90 showed a yellow color, persisting through the fifth to sixth true leaves and in all subsequent leaves (Percy, 1999). The yellowish color turned to green gradually as the leaf matured in plants with five or six true leaves. The normal green phenotype was restored and persisted throughout the remainder of the plant development. The transient change of yellow to green color occurred from the bottom of the plant and from the edges of the leaves to the center. Greenhouse and field observations also revealed that all cotyledons of the cyt-V mutant (CM-1-90) were green in color while cotyledons of v7 v7 plants were yellow in color (Figure 1). This indicated that cyt-V and nuclear v7 v7 genes showed similar phenotype in true leaves but differed in cotyledons. The cyt-V mutation, yellow color first true leaves, persisting through the fifth to sixth true leaf stage and green cotyledon color, was observed in F1 CM-1-90 × PS-7 and CM-1-90 × v7 v7 hybrids. As in the cyt-V mutation (CM-1-90), the yellow color also turned to green gradually as the leaf matured in plants with five or six true leaves of CM-1-90 × PS-7 and CM-1-90 × v7 v7 hybrids. Other PS-7 × CM-1-90 and v7 v7 × CM-1-90 F1 hybrid plants (reciprocal crosses) showed normal morphological appearances in true leaf and cotyledon color. Morphological observations indicated that the virescent expression of cyt-V gene was probably altered by light quality since plants grown in the field showed a very deep yellow color
190
Figure 1. Yellow color cotyledons of nuclear genome specific cotton mutant plants (left v7 v7 ) and green color cotyledons of chloroplast genome specific cotton mutant plants (right, CM-1-90).
as compared to the light yellow color when plants were grown in a growth chamber. The leaves of greenhouse-grown plants were intermediate in color. All the F2 cotyledons in CM-1-90 × v7 v7 and CM1-90 × PS-7 were normal green in color and true leaves were yellow in color due to the dominant maternal inheritance of cyt-V gene action. On the other hand, among the 32 F2 plants from self-pollinated v7 v7 × CM-1-90 hybrid, 23 plants showed normal green cotyledons and 9 plants had cotyledons that exhibited a yellowish phenotype clearly indicating the classical 3:1 ratio (with probability of a greater Chi-square value of 0.75) of a monohybrid cross controlled by a recessive nuclear gene (v7 ). All the F2 cotyledons and true leaves from self-pollinated PS-7 × CM-1-90 hybrid had normal phenotype. Chloroplast anatomy Chloroplast anatomy in the first true leaves and cotyledons of CM-1-90 at the 14-day-old seedling stage differed in grana formation (Figure 2). Cotyledon chloroplasts showed normal grana formation while grana in the chloroplasts of true leaves were impaired. Anatomical studies of CM-1-90 × PS7, and CM-1-90 × v7 v7 leaves at the 14-dayold seedling stage hybrids exhibited undeveloped grana compared with PS-7 × CM-1-90 and v7 v7 × CM-1-90 normal green leaves at the 14-day-old seedling stage (Figure 3). The chloroplast development of CM-1-90 × v7 v7 was more severely af-
Figure 4. Electropherogram of cDNA-AFLP differential display technique showing a cyt-V-specific transcript (arrow at 208 bp) that was differentially expressed in CM-1-90 × PS-7 cotton hybrid at the 14-day-old seedling stage but it was absent in PS-7 × CM-1-90 hybrid at the same age seedling stage. Electropherogram presents amplified products sizing between 202 and 210 bp. Blue line represents cDNAs from leaf sample of PS-7 ( ) × CM-1-90 ( ) and green line represents cDNAs from CM-1-90 ( ) × PS-7 ( ) leaf sample. The x-axis represents the size of the DNA fragments and the y-axis shows the amplified products intensity in arbitrary unit.
fected than CM-1-90 × PS-7 (Table 1). No distinct differences in the grana and overall chloroplast structure were observed when the yellow true leaves of mutant plants recovered normal green
191
Figure 2. Ultrastructure of chloroplasts in mesophyll cells of cotton (G. barbadense L.). (A and B) First true leaves chloroplasts of CM-1-90 at the 14-day-old seedling stage. Note that grana development was impaired. (C and D) Cotyledon chloroplasts of CM-1-90 at the 14-day-old seedling stage showing normal development. (Bar = 4µm (A and C) and 1.5 µm (B and D)).
color. Chloroplast ultrastructure development in the area of yellowish green leaves exhibited a mixture of normal and abnormal chloroplast grana formations, suggesting that the green color directly correlated with normal grana development. Reduced membrane integrity of the thylakoids was also observed from ultrastructure visualization of whole chloroplasts. Chloroplast length and width of the true leaves mesophyll cells at the 14-day-old seedling stage of CM-190 × PS-7 (4.5 ± 0.41 and 2.2 ± 0.38) and CM-1-90 × v7 v7 (4.4 ± 0.94 and 1.4 ± 0.28) hybrids were much smaller than PS-7 × CM-1-90 (6.1 ± 0.82 and 2.5 ±
0.42) and v7 v7 × CM-1-90 (6.2 ± 0.68 and 2.3 ± 0.84) at the 14-day-old seedling stage (Table 1). However, when the plants were at the sixth leaf stage, chloroplast size differences were not significant. Chloroplasts of CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v7 v7 at the 14-day-old seedling stage were similar in size. Chlorophylls and carotenoids Total chlorophyll and carotenoid contents of CM-1-90 × PS-7, CM-1-90 × v7 v7 and CM-1-90 were lower than PS-7 × CM-1-90, v7 v7 × CM-1-90, PS-7 and CM-190WT in the similar age of true 14-day-old seedling
192
Figure 3. Ultrastructure of chloroplasts in mesophyll cells of cotton (G. barbadense L.). (A) Chloroplasts of CM-1-90 ( ) × PS-7 ( ) hybrid at the 14-day-old seedling stage. Note that grana development was impaired when CM-1-90 was used as a maternal parent. (B) Chloroplasts of CM-1-90 ( ) × PS-7 ( ) hybrid at the sixth leaf stage showing normal chloroplast with many large starch molecules. (C) Chloroplasts of PS-7 ( ) × CM-1-90 ( ) hybrid at the 14-day-old seedling stage showing normal development. (D) Chloroplasts of PS-7 ( ) × CM-1-90 ( ) at the sixth leaf stage with large starch molecules (Bar = 2 µm).
leaves. However, when the leaves of CM-1-90 × PS-7, CM-1-90 × v7 v7 , CM-1-90 restored the green phenotype at sixth leaf stage, chlorophyll and carotenoid contents differences were not significant (Table 1). cDNA-AFLP differential display The cDNA-AFLP differential display identified four transcripts (Expressed Sequence Tags, ESTs) that were present in the yellow true leaves of the CM-1-90 × PS-7
and CM-1-90 × v7 v7 at the 14-day-old seedling stage, but they were absent in the same age green leaves of PS-7 × CM-1-90 and v7 v7 × CM-1-90. One of the transcript, GBLAP, had homology to a leucine aminopeptidase gene (LAP) encoded by nuclear genomes of several eukaryotic organisms including Arabidopsis, Medicago and Magnolia. GBARF had high homology to an ADP-ribosylation factor gene and the two other transcripts (GBNH1 and GBNH2) showed no homology with the GenBank sequences (Table 2). Further
193 Table 1. Chloroplast size, chlorophyll and carotenoid contents of reciprocal crosses and their parental lines grown in a greenhouse
Source
Total Chls (µg/mL)
Crtnd (µg/mL)
Chloroplast length (µm)
Chloroplast width (µm)
PS-7 × CM-1-90 (green true leaves)1
7.02 (±0.73)
2.13 (±0.28)
6.1 (±0.82)
2.5 (±0.42)
CM-1-90 × PS-7 (green true leaves)2
7.73 (±0.74)
2.07 (±0.15)
6.7 (±0.28)
3.3 (±0.58)
CM-1-90 × PS-7 (yellow true leaves)1
1.79 (±0.91)
1.08 (±1.03)
4.5 (±0.41)
2.2 (±0.38)
v7 v7 × CM-1-90 (green true leaves)1
8.63 (±3.45)
2.32 (±0.35)
6.2 (±0.68)
2.3 (±0.84)
CM-1-90 × v7 v7 (green true
leaves)2
8.72 (±1.53)
2.22 (±1.78)
6.2 (±1.55)
3.1 (±1.83)
CM-1-90 × v7 v7 (yellow true leaves)1
1.51 (±0.74)
0.96 (±0.04)
4.4 (±0.94)
1.4 (±0.28)
CM-1-90 (yellow true leaves)1
0.85 (±0.41)
0.66 (±0.37)
4.3 (±1.28)
1.3 (±1.23)
PS-7 (Green true leaves)1
5.77 (±0.69)
1.84 (±1.12)
7.1 (±1.21)
3.2 (±1.31)
CM-1-90 WT (green true leaves)1
8.24 (±1.31)
2.36 (±0.22)
6.8 (±0.67)
2.8 (±0.81)
Chls: chlorophyll, Crtnd: carotenoid. 1 At the 14-day-old seedling stage. 2 At the sixth leaf stage.
Table 2. Differentially expressed transcripts between F1 hybrids (CM-1-90 × PS-7 and CM-1-90 × v7 v7 ) with yellow true leaves and F1 hybrids (PS-7 × CM-1-90 and v7 v7 × CM-1-90) with green true leaves EST
Source
Length (bp)
GenBank homologya
GBPSII
PS-7 × CM-1-90 v7 v7 × CM-1-90
634
gi|5881673| Arabidopsis thaliana, gi|2924257| Nicotiana tabacum, gi|7636084| Spinacia oleracea, gi|343022| Pisum sativum, and gi|17149410| Medicago truncatula chloroplast genomes and photosystem II protein genes
GBPSBA
PS-7 × CM-1-90 v7 v7 × CM-1-90
480
gi|11306| Gossypium hirustum, gi|16755684|, Lactuca sativa, gi|515373| Arabidopsis thaliana, gi|16224268| Prunus leveilleana, gi|2924257| Nicotiana tabacum gi|17149410| Medicago truncatula and gi|4097504| Magnolia pyramidata D1 protein (psbA) and chloroplast genomic DNA
GBLAP
CM-1-90 × PS-7 CM-1-90 × v7 v7
265
gi|14334665| Arabidopsis thaliana putative leucine aminopeptidase (At2g24200) mRNA, gi|17063167| A. thaliana At2g24200/F27D4.11 mRNA, gi|16393| A. thaliana mRNA for leucine aminopeptidase, and gi|1483562| Petroselinum crispum mRNA for leucine aminopeptidase
GBARF1
CM-1-90 × PS-7 CM-1-90 × v7 v7
407
gi|17154684| Gossypium hirsutum (ARF1 gene), gi|15810604| Arabidopsis thaliana putative ADP-ribosylation factor 1 (At1g70490) mRNA, gi|21212358| Zea mays PCO085483 mRNA sequence, gi|1132482| Oryza sativa (japonica cultivar-group) mRNA for ADP-ribosylation factor, and gi|7643793| Capsicum annuum ADP-ribosylation factor mRNAs
GBNH1
CM-1-90 × PS-7 CM-1-90 × v7 v7 CM-1-90 × PS-7 CM-1-90 × v7 v7
384
No homology found
434
No homology found
GBNH2
a Based on GenBank BLAST analysis, not all significant homologies are shown. E values of homologies are at least −20 or over. The nucleotide sequences reported in this table have been submitted to GenBank under accession numbers AY239296, AY239297, AY239298, AY239299, AY239300, and AY239301.
194 studies are required to asses function(s) of these transcripts. The cDNA-AFLP technique also identified two ESTs that were present in PS-7 × CM-1-90 and v7 v7 × CM-1-90 hybrids at 14-day-old seedling stage but they were absent in CM-1-90 × PS-7, CM-1-90 × v7 v7 hybrids at the same age stage (Table 2). We identified two another transcripts that expressed only in the green leaves of PS-7 × CM-1-90 and v7 v7 × CM-1-90, but they were not present in CM-1-90 × PS-7 and CM-1-90 × v7 v7 (Figure 4). One of these transcripts (GBPSBA) showed high homology to psbA gene and the transcript GBPSII showed high level of homology to a photosystem II (PSII)-associated protein (Table 2). SDS-PAGE and Western blot analysis The large and small subunits of RUBISCO (about 50 and 15 kDa, respectively), and several undefined proteins in the stroma, thylakoids and total leaf extracts were found to be less abundant in CM-1-90 × PS-7, CM-1-90 × v7 v7 , CM-1-90 in comparison to that of PS-7 × CM-1-90, v7 v7 × CM-1-90, PS-7 and CM1-90WT. Thylakoid membrane proteins of about 16 and 25 kDa were in abundance in PS-7 × CM-1-90, v7 v7 × CM-1-90, PS-7 and CM-1-90WT leaves compared to the CM-1-90 × PS-7, CM-1-90 × v7 v7 , CM-1-90 true leaves (Figure 5). The banding profiles of several soluble proteins in the leaves of PS-7 × CM-1-90, v7 v7 × CM-1-90, PS-7 and CM-1-90WT were also different from CM-1-90 × PS-7, CM-1-90 × v7 v7 , and CM-1-90. Western blot analysis, using immunoblotting (anti-D) for D1 protein as a target, demonstrated that D1 protein encoded by psbA gene was not present in CM-1-90 × PS-7, CM-1-90 × v7 v7 , CM-1-90 true leaves further confirming the cDNAAFLP differential display results. Cotyledons of CM1-90 and v7 v7 showed differences in protein profile but both contained D1 protein encoded by psbA gene (Figure 5). Discussion A series of nuclear virescent mutants affecting chloroplast development have been found in cotton. Most of them are readily identifiable due to their yellow colored leaves and reported to be controlled by recessive genes (Percival & Kohel, 1976; Kohel, 1983; Percy & Kohel, 1999; Percy, 1999). Our preliminary observa-
Figure 5. SDS-PAGE and Western blot gels showing protein bandings of cotton leaf and cotyledon samples. Upper picture is SDSPAGE analysis. Lane M: protein size marker, lane 1: CM-1-90WT, lane 2: PS-7 ( ) × CM-1-90 ( ), lane 3: CM-1-90 ( ) × PS-7 ( ), lane 4: v7 v7 ( ) × CM-1-90 ( ), lane 5: CM-1-90 ( ) × v7 v7 ( ), lane 6: CM-1-90, (cyt-V), lane 7: cotyledons of CM-1-90 (cyt-V) and lane 8: cotyledons of v7 v7 . Bottom picture is the Western blot analysis of the SDS-PAGE gel using immunoblotting (anti-D) for D1 protein encoded by psbA gene as a target.
tions suggested that like the majority of the virescent mutants, the cyt-V mutant was expressed predominantly in the young stage of growth, but expression varied widely with light intensity and duration, and was environmentally sensitive (Johnston & Taliaferro, 1975; Fabel et al., 1996; Chen et al., 1999). Chloroplast size and grana formation in the true leaves of CM-1-90 × v7 v7 at early development stage was more severely affected than that of CM-1-90 × PS-7. Reduced levels of chloroplast size, chlorophyll and carotenoid contents suggested that both cyt-V and v7 v7 mutations affected chlorophyll and carotenoid biogenesis. It was not surprising to note that both chlorophylls and carotenoids were less abundant during the mutant expression in the leaves since carotenoids located in photosynthetic membranes are present in
195 the form of chlorophyll–carotenoid–protein complexes and are involved in light harvesting, chlorophyll photoprotection and stabilization of the plant light-harvesting complexes (Vishnevetsky et al., 1999). Morphological observations indicated that the gene action of cyt-V was initiated at the time of first true leaf development but not in cotyledons, whereas gene activity of the nuclear gene v7 v7 occurred in both cotyledons and true leaves of the mutant plants. Anatomical chloroplast structures showed that chloroplast structures in the first true leaves of CM-1-90 were different from cotyledons but this difference was not observed between the cotyledons and true leaves of v7 v7 mutation. This indicated that the mode of gene action controlled by cytoplasmic (cyt-V) and nuclear gene (v7 v7 ) differed, yet produced a similar visual phenotype in young seedlings. Physiologically, cotyledons perform special functions, serving as storage organs of carbohydrates and other food products for germination and therefore develop differently than normal leaves of young seedlings. Results indicated that two transcripts (GBARF1 and GBLAP in Table 2) were more abundant in the true leaves of CM-1-90 × PS-7 and CM-1-90 × v7 v7 in comparison to PS-7 × CM-1-90 and v7 v7 × CM-1-90. One of these transcripts, GBLAP, had significant homology to leucine aminopeptidase and other one, GBARF1, was homology to ADP-ribosylation factor genes. These two genes are normally nuclear genome-encoded and specifically express when the plants are under salt, wounding, drought stresses and bacterial pathogen infection (Guo et al., 1996; Chao et al., 1999; Powner et al., 2002). Increased activity of these genes in mutant phenotype was possibly activates stress-signaling pathways to escape the damaged chloroplast integrity resulting in either increasing (inducing) or declining (inhibiting) the transcripts for several nuclear-encoded chloroplast-localized proteins or chloroplast-encoded gene products including leucine aminopeptidase and ADP-ribosylation factor or ARF1 genes. Based on the cDNA-AFLP differential display and Western blot studies we found that psbA gene expression was absent in the true leaves of CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v7 v7 at the 14day-old seedling stage but it was present in PS-7 × CM-1-90, v7 v7 × CM-1-90 and CM-1-90WT at the same old stage along with the cotyledons of CM1-90 and v7 v7 parental lines (GBPSBA in Table 2). Although experimental studies have focused on the light-dependent translation of psbA mRNA, which
encodes the D1 protein, the relationship of psbA gene to proper functioning of photosystem II has not yet been established (Rochaix, 2001). Allakhverdiev et al. (2002) reported that psbA gene was differentially expressed in salt or drought stress. Our results indicated that cyt-V mutation was probably affected translation of psbA and several other nuclear and chloroplast genes including PSII-associated protein genes. There are several reports indicating cytoplasmic gene products influencing nuclear gene expression (Kircher et al., 1999; Galston, 2001). Chloroplast-encoded genes have been suggested to play many important roles in plant development, not only in chloroplast-specific genes but also for other nuclear-encoded chloroplast-related genes and cytoplasmic male sterility (Kirti et al., 1998; Kircher et al., 1999; Galston, 2001; Guan et al., 2004). Inhibition of the transcription and translation of the psbA gene in CM-1-90, CM-1-90 × PS-7 and CM-190 × v7 v7 true leaves at early stages and recovery of the transcription and translation of this gene in leaves at later development stages suggested that the cyt-V mutation was not present in the cis-acting site with the psbA gene but was probably associated with regulatory element(s) of psbA gene. Shen et al. (2001) reported that the 5 untranslated region (5 UTR) of the psbA mRNA was a key site for RNA–protein interactions in the post-transcriptional regulation of gene expression. Tyystjarvi et al. (2002) reported that the photosystem II (PSII) reaction center protein D1 undergoes rapid light-dependent turnover, which is caused by photoinhibition. Another transcript (GBPSII in Table 2), showing high homology to PSII-associated protein was present in the true leaves of PS-7 × CM-1-90 and v7 v7 × CM-1-90, but it was missing in CM-1-90 × PS-7, CM1-90 × v7 v7 at the 14-day-old seedling stage. This gene expression was probably co-regulated with the expression of the psbA gene which was also reduced in CM-190 × PS-7, CM-1-90 × v7 v7 at the 14-day-old seedling stage. In Arabidopsis, a nuclear-encoded arc (accumulation and replication of chloroplasts) gene mutant has been identified which exhibited altered chloroplast numbers in mesophyll cells, but the thylakoid ultrastructure was unaffected (Pyke, 1997). However, the cyt-V mutation was associated with defective thylakoid membrane development as evidenced by the altered protein fingerprint profiles and anatomical levels. Overall, we studied cyt-V and v7 v7 both are developmental mutants, as all the virescents of cotton appear to be, and as such it was difficult to separate cause and effect in the observation. However, we verified that the
196 source of cyt-V mutation was in chloroplast and elucidated that its gene action was different from v7 v7 . Results indicated that cyt-V is inherited as a single gene but it affects several chloroplast and nucleus-encoded genes. This study suggested that chloroplast-encoded gene products might affect the expression of nuclear genes, possibly at the transcriptional stage. Acknowledgments We would like to thank Dr. Allan Zipf, Plant and Soil Science Department of Alabama A&M University and for his help in reviewing the manuscript. This paper was funded by the Scientific Research Projects Administration unit of Akdeniz University. References Allakhverdiev, S.I., Y. Nishiyama, S. Miyairi, H. Yamamoto, N. Inagaki, Y. Kanesaki & N. Murata, 2002. Salt stress inhibits the repair of photodamaged photosystem II by suppressing the transcription and translation of psbA genes in Synechocystis. Plant Cell Physiol 130: 1443–1453. Altschul, S.F., T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller & J. Lipman, 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402. Archer, E., H. Kathleen & T. Bonnett, 1987. Characterization of a virescent chloroplast mutant of tobacco. Plant Physiol 83: 920– 8255. Callahan, F.E., W.P. Wergin, N. Nelson, M. Edelman & A.K. Matoo AK., 1989. Distribution of thylakoid proteins between stromal and granal lamellae in Spirodela. Plant Physiol 91: 629–635. Callahan, F.E., F.M. Davis & W.P. Williums, 1992. Steady-state polypeptide profiles of whorl tissue from Lepidoptera-resistant and susceptible corn inbred lines. Crop Sci 32: 1203–1207. Chao, W.S., Y.O. Gu, V. Pautot, E.A. Bray & L.L. Walling, 1999. Leucine aminopeptidase RNAs, proteins, and activities increase in response to water deficit, salinity, and the wound signals systemin, methyl jasmonate, and abscisic acid. Plant Physiol 120: 979–992. Chen, Z., G.H. Liang, S. Muthukrishnan & K.D. Kofoid, 1990. Chloroplast DNA polymorphism in fertile and male sterile cytoplasms of sorghum (Sorghum bicolor (L.) Moench). Theor Appl Genet 80: 727–731. Chen, M., M. Jensen & S. Rodermel, 1999. The yellow variegated mutant of Arabidopsis is plastid autonomous and delay in chloroplast biogenesis. Heredity 90: 207–214. Falbel, T.G., J.B. Meehl & L.A. Staehelin, 1996. Severity of mutant phenotype in a series of chlorophyll-deficient wheat mutants depends on light intensity and the severity of the block in chlorophyll synthesis. Plant Physiol 112: 821–823. Galau, G.A. & T.A. Wilkins, 1989. Alloplasmic male sterility in AD allotetraploid Gossypium hirsutum upon replacement of its resident A cytoplasm with that of D species G. harknessii. Theor Appl Genet 78: 23–30. Galston, A.W., 2001. A tale of two pigments. Plant Physiol 126: 32–34.
Gruissem, W. & G. Schuster, 1993. In: J.G. Belasco & G. Brawerman (Eds.), Control of mRNA Stability, Academic Press, pp. 329–365. Guan, X.Q., S.J. Zhao, D.Q. Li & H.R. Shu, 2004. Photoprotective function of photorespiration in several grapevine cultivars under drought stress. Photosynthetica, 42: 31–36. Guo, Y.Q.,W.S. Chao & L. L. Walling, 1996. Localization and posttranslational processing of the wound-induced leucine aminopeptidase proteins of tomato. J Biol Chem 271: 25880–25887. Hayes, R., J. Kuda & W. Gruissem, 1999. Degrading chloroplast mRNA. The role of polyadenylation. TIBS 24: 199–202. Herring, A.D., D.L. Auld, M.D. Ethridge, E.F. Hequet, E. Bechere, C.J. Green & R.G. Cantrell, 2004. Inheritance of fiber quality and lint yield in a chemically mutated population of cotton, Euphytica 136: 333–339. Johnston, R.A. & C.M. Taliaferro, 1975. Effects of temperature and light intensity on the expression of a variegated leaf pattern in bermudagrass. Crop Sci 15: 445–447. Karaca, M., S. Saha, J.N. Jenkins, A. Zipf, R. Kohel & D.M. Stelly, 2002. Simple sequence repeat (SSR) markers linked to the Ligon Lintless (Li1 ) mutant in cotton. J Hered 93: 221–224. Karcher, D. & R. Bock, 1998. Site-selective inhibition of plastid RNA editing by heat shock and antibiotics: A role for plastid translation in RNA editing. Nucl Acids Res 26: 1185–1190. Karpinska, B., S. Karpinski & J.E. Hallgren, 1997. The chlB gene encoding a subunit of lighting-dependent protochlorophyllide reductase is edited in chloroplasts of conifer. Curr Genet 31: 343– 347. Katterman, F.H. & J.E. Endrizzi, 1973. Studies on the 70S ribosomal content of a plastid mutant in Gossypium hirsutum. Plant Physiol 51: 1138–1139. Kircher, S., L. Kozma-Bognar, L. Kim, E. Adam, H. Harter, E. Schafer & F. Nagy, 1999. Light quality-dependent nuclear import of the plant photoreceptors phytochrome A and B. Plant Cell 11: 1445–1456. Kirti, P.B., S. Prakash & K. Gaikwad, 1998. Chloroplast substitution overcomes leaf chlorosis in a Morichandia arvensis-based cytoplasmic male sterile Brassica juncea. Theor Appl Genet 97: 1179–1182. Kohel, R.J., 1967. Variegated mutants in cotton, Gossypium hirsutum L. Crop Sci 490–492. Kohel R.G., 1974. Genetic analysis of a new virescent mutant in cotton. Crop Sci 14: 525–527. Kohel, R.J., 1983. Genetic analysis of virescent mutants and the identification of virescent v12 , v13 , v14 , v15 and v16 v17 in upland cotton. Crop Sci 23: 289–291. Kohel, R.J. & C.R. Benedict, 1971. Description and CO2 metabolism of aberrant and normal chloroplasts in variegated cotton Gossypium hirsutum L. Crop Sci 11: 486–488. Kohel, R.J., M.D. Stelly & J. Yu, 2004. Tests of six cotton (Gossypium hirsutum L.) mutants for association with aneuploids. Heredity, 93: 130–132. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: Pigments of photosynthesis. Methods Enzymol 148: 350–352. Lisitsky, I., P. Klaff & G. Schuster, 1996. Addition of destabilizing poly(A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA. Proc Natl Acad Sci USA 93: 13398–13403. Meurer, J., A. Berger & P. Westhoff, 1996. A nuclear mutant of Arabidopsis with impaired stability on distinct transcripts of the plastid psbB, psbD/C, ndhH and ndhC operons. Plant Cell 8: 1193–1207.
197 Percival, A.E. & R.J. Kohel, 1974. Genetic analysis of virescent mutants in cotton. Crop Sci 14: 439–440. Percival, A.E. & R.J. Kohel, 1976. New virescent cotton mutant linked with the marker gene yellow petal. Crop Sci 16: 503–504. Percy, R.G., 1999. Inheritance of cytoplasmic-virescent cyt-V and dense-glanding dg mutants in American pima cotton. Crop Sci 39: 372–374. Percy, R.G. & Kohel, R.J., 1999. Qualitative genetics. In: C.W. Smith & J.T. Cothren (Eds.), Cotton Origin, History, Technology and Production, Wiley, NY, USA, pp. 319–360. Powner, D.J., M.N. Hodgkin & M.J. Wakelam, 2002. Antigenstimulated activation of phospholipase D1b by Rac1, ARF6, and PKCalpha in RBL-2H3 cells. Mol Biol Cell 13: 1252–1562. Pyke, K.A., 1997. The genetic control of plastid division in higher plant. Am J Bot 84: 1017–1027. Rochaix, J.D., 2001. Posttranscriptional control of chloroplast gene expression. From RNA to photosynthetic complex. Plant Physiol 125: 142–144. Roy, L.M. & A. Barkan, 1998. A Sec Y homologue is required for the elaboration of the chloroplast thylakoids membrane and for normal chloroplast gene expression. J Cell Biol 141: 385–395. Saha, S., M. Karaca, J.N. Jenkins, A.E. Zipf, O.U.K. Reddy, A.E. Pepper & R. Kantety, 2003. Simple sequence repeats as useful resources to study transcribed genes of cotton. Euphytica 130: 355–364.
Shen, Y., A. Danon & D.A. Christopher, 2001. RNA binding-proteins interact specifically with the Arabidopsis chloroplast psbA mRNA 5 untranslated region in a redox-dependent manner. Plant Cell Physiol 42: 1071–1078. Turcotte, E.L. & C.V. Feaster, 1978. Inheritance of three genes for plant color in American pima cotton. Crop Sci 18: 149– 150. Turcotte, E.L. & R.G. Percy, 1988. Inheritance of a second virescent mutant in American pima cotton. Crop Sci 28: 1018– 1019. Tyystjarvi, T., I. Tuominen, M. Herranen, E.M. Aro & E. Tyystjarvi, 2002. Action spectrum of psbA gene transcription is similar to that of photoinhibition in Synechocystis sp. PCC 6803. FEBS LETT 516: 167–171. Vishnevetsky, M., M. Ovadis & A. Vainstein, 1999. Carotenoid sequestration in plants: The role of carotenoid associated proteins. Trends Plant Sci 4: 232–235. Wendel, J.F., C.I. Brubakel & A.E. Percival, 1992. Genetic diversity in Gossypium–Hirsutum and the origin of upland cotton. Am J Bot 79: 1291–1310. Zhang, T.Z. & J.J. Pan, 1986. Genetic identification of 12 virescent mutants of upland cotton. Acta Gossypii Sinica 2: 78–90. Zhang, T.Z. & J.J. Pan, 1990. Allelic tests of 11 virescent mutants and genetic identification of virescent v22 in upland cotton. Jiangsu Agric Sci 6: 24–24.
