Maize mitochondrial genes

IN VITROCELLULAR& DEVELOPMENTALBIOLOGY Volume 22, Number 4, April 1986 9 1986 Tissue Culture Association,Inc.

INVITED REVIEW MAIZE MITOCHONDRIAL

GENES 1

V. K. ECKENRODE AND C. S. LEVINGS, III2 North Carolina State University, Raleigh, North Carolina 27695 (Received 2 January 1986; accepted 2 January 19861

Key words: genome organization; structural R N A genes; protein-encoding gnes; cytoplasmic male sterility. MITOCHONDRIAL ORGANIZATION

Our understanding of gene organization and expression has expanded greatly over the past 15 yr. This has been due primarily to the development of rapid and sophisticated molecular techniques. The capacity to isolate genes by molecular cloning and to analyze them at the nucleotide level has been a major contributor to our increased knowledge. Genes can now be analyzed for their informational content as well as for regulatory elements affecting their expression. All eukaryotic cells contain mitochondria, the centers of energy metabolism where organic molecules are oxidized and electron transport is linked to ATP formation. Plant cells are distinguished from other eukaryotic cells because they contain chloroplasts. Chloroplasts convert light energy into chemical energy and use it to reduce carbon dioxide to carbohydrates. Both organelles, the mitochondria and the chloroplasts, have their own genetic systems. Thus, plant ceils differ from other eukaryotic ceils by containing three distinct genomes: the nuclear, the chloroplast, and the mitochondrial. The nuclear gcnome is the largest of the three and is inherited in a Mendelian fashion. In maize, the haploid nuclear genome is approximately 5.8 X 10~ kilobases (kb) in size (43) and is organized into 10 chromosomes [see (85D for a recent map]. In contrast, the chloroplast and mitochondrial genomes are much smaller and are generally inherited in a non-Mendelian fashion through the maternal parent. In maize, the chloroplast genome is approximately 135 kb in size (50,59), whereas the mitochondrial genome is about 570 kb (57). The organelle genomes consist of naked DNA (40), that is D N A which is not tightly complexed with proteins to form chromosomes. Much more is known about nuclear genes than organelle genes, primarily because Mendelian genes and their mutants are more abundant and more easily studied. The development of molecular biology over the past 15 yr has allowed organelle genomes to be studied in greater depth than previously. The following sections describe our current knowledge of the mitochondrial genes in maize and some other higher plants.

Mitochondria are bounded by an outer membrane that surrounds a highly folded inner membrane. An intermembrane space separates the two membranes and an internal matrix is enclosed by the inner membrane. The major structural elements of mitochondHa are proteins and lipids, the relative proportions of which vary depending on the amount of inner membrane present (92). There are 300 to 400 different polypeptides in the protein component. In yeast, approximately 10% of the mitochondrial protein mass is synthesized in the mitochondria, and the remainder is synthesized in the cytoplasm (82). Mitochondrial biogenesis is the consequence of coordinated interactions between the nuclear and mitochondrial genetic systems. This interaction is receiving increased attention by molecular biologists who are now analyzing the specific genes and gene products involved. Mitochondria of all eukaryotic cells contain mitochondrial D N A (mtDNA) that encodes a small number of essential polypeptides and structural RNAs. Although mtDNA from different sources encodes a similar basic set of products (Table I), the genomes from different organisms are organized in a variety of ways. Animal mtDNA genomes are the smallest and range from 14 to 18 kb in size (41). The animal mitochondrial genes are very tightly packed on a small circular molecule. Even though fungal mtDNAs are organized in circles up to nine times larger than mammalian mtDNAs, from 19 to 108 kb in size (41), they encode a similar number of genes. The "extra" m t D N A in the fungal genomes is accounted for primarily by (A + T)-rich spacers and introns. Plant mtDNAs are much larger and more variable than those of other organisms. Plant mitochondrial genomes range in size from 200 to 2500 kb (41). Even within a single family, the cucurbits, there is a dramatic eight fold difference in mtDNA size, with watermelon at 330 kb and muskmelon at 2500 kb (94). The large genome size is not due to repetitive DNA since less than 10% of the cucurbit DNA is repetitive (94). Although plant mtDNA contains a few genes not found in animal or fungal mtDNAs (Table

Presented at the Session-in-Depth on Plant Molecular Genetics at the Thirty-Sixth Annual Meeting of the Tissue Culture Association, New Orleans, LA, 2-6 June 1985. 2 To whom requests for reprints shouM be addressed at Genetics Department, Box 7614, North Carolina State University, Raleigh, NC 27695-7614. 169

170

ECKENRODE AND LEVINGS

1), the difference in gene number is not enough to account for the larger plant genome size. Animal and fungal mitochondrial genomes are arranged in a single circular molecule, whereas plant mtI)NAs often consist of a complex assortment of circular molecules of different sizes and numbers. Restriction endonuclease mapping of the mitochondrial genomes of Brassica campestris (Chinese cabbage, turnip) (73) and maize (57) have provided insight into their organization. The mitochondrial genome of Brassica consists of three major circular molecules. The largest circle is 218 kb in length, and contains the entire sequence complexity of the Brassica genome. This master chromosome contains two direct repeats of about 2 kb that are spaced around the circle, 135 and 83 kb from each other. Recombination between the two repetitive elements produces two circular molecules of 135 and 83 kb. This accounts for the two smaller circles in the Brassica mitocbondrial genome. Similarly, recombination between the two smaller circles can reproduce the largest circle. An analogous model has been proposed for the organization of the maize mitochondrial genome (57), although the maize organization is more complex than Brassica. The maize mitochondrial genome is more than twice as large (570 kb) and contains six pairs of repetitive elements (57). A larger variety of circular DNA molecules can therefore be produced in maize mitochondria than in Brassica. The circular molecules of various sizes observed by electron microscopy (56) are accounted for by Lonsdale's model (57L The relative proportions of the different circles apparently depend on the recombination frequencies among the different pairs of repetitive elements.

whereas those taking part in phosphate metabolism are situated in the intermembrane space. Finally, those involved in glycolysis, phospholipid metabolism, and protein import are found in the outer membrane. To date, 12 genes have been identified and characterized from the maize mitochondrial genome (Table 1). These include genes for six structural RNAs and six polypeptides. Plant mitoribosomes differ from those of animals and fungi in that they contain a unique 5S rRNA species and their large {26S) and small (18S) rRNAs are distinctly larger than the animal and fungal counterparts (51). In comparison, animal mitoribosomes contain 16S and 12S rRNAs whereas fungal mitoribosomes have 21S and 15S rRNAs. The maize mitochondrial 18S rRNA is even larger than its cytoplasmic counterpart; the mitochondrial subunit contains 1968 nucleotides (nt) (17) and the cytoplasmic 1905 nt (61). A 5S RNA species is associated with the large ribosomal subunit of plant mitochondria, chlorplasts, bacteria, and the cytoplasm of eukaryotic cells (29). All 5S rRNA species are related in primary and secondary structures, but those from plant mitochondria differ sufficiently from the others to be considered in a group of their own (29). Coding regions for the mitochondrial rRNAs are arranged in a way unique to plant mitochondria. The 18S and 5S genes are closely linked in a 5' to 3' direction [108 bases apart in maize (16)] and

TABLE 1 MITOCHONDRIAL GENES AND THEIR PRODUCTS

MITOCHONDRIAL GENES Mitochondria possess their own distinct protein synthesizing apparatus. Inasmuch as there is no evidence that any mRNAs are imported into the mitochondria, it is presumed that all the mRNAs translated in mitochondria are products of mitochondrial transcription. Despite the large differences in the size and organization of mitochondrial genomes, mitochondria from the different kingdoms code for many similar protein and structural RNA products (28,41). All mitocbondrial genomes encode ribosomal RNAs, a complement of tRNAs (22-25), and a small number (15-30} of mRNAs {Table IL A few polypeptides belonging to the electron transport chain are always specified by mitochondrial mRNAs. In some fungal systems, mRNAs may specify a ribosomal protein or RNA processing proteins or both (Table 1 ). The remainder of the mitochondrial polypeptides are encoded by nuclear genes, synthesized on cytosolic ribosomes and posttranslationally imported into the mitochondria. These polypeptides are targeted for different destinations within the mitochondria (77). Those participating in nucleic acid replication, transcription and translation, the tricarboxylic acid cycle, fatty acid metabolism, amino acid and nitrogen metabolism, and porphyrin and heme synthesis are found in the matrix. Those involved in electron transport, pbosphorylation reactions, and steroid metabolism are located in the inner membrane,

Animal (6 ) Mitochondrial Gene/Product

1 4 - 1 8 k ba

Ribosomal RNAs Large Subunit Small Subunit 5S Transfer RNAs Cytochrome c oxidase Subunit I Subunit II Subunit III Ubiquinol cytochrome c reductase Apo-cytochrome b F0-ATPase complex Subunit 6 Subunit 8 Subunit 9 F,-ATPase complex Subunit Alpha Ribosomal protein NADH-ubiquinone oxidoreductase Number of subunits RNA processing enzymes Intron 2 CoB maturase Intron 4 CoB maturase 9S RNA Unassigned reading frames ~

size.

Yeast/Other Fungi (6 ) 1 9 - 1 0 8 k ba

Plant 2 0 0 - 2 5 0 0 k b~

16S 12S -22

21S 15S -23-25

26Sa2 18S('7~ +-,~ ~30

+ + +

+ + +

+ (471 + (3,~ ?

"4-

-4-

+ 123

+ + _

+ + +/_

+ (2,~ ? + ~27~

_ --

_ +

+ c9 ?

6(tBi

_/6i4s:

9

--? 1

-44+ ~98%), suggesting strong functional or evolutionary constraints or both. Interestingly, an insertion sequence is found at the same position in the introns of the rice and wheat genes (4,49). All three plant mitochondrial introns can be folded into potential secondary structures that are similar to those predicted for Group I I introns (49) of fungal mitochondria (63). There is no indication that the plant mitochondrial introns encode maturase-like activities. Northern transfer analyses indicate that the transcriptional patterns of the maize mitochondrial protein-coding

genes a r e variable and sometimes complex (Table 3). The genes for coI (47) and atp-alpha (9) have the simplest Northern patterns, with only two apparent R N A species each. The other four genes, coil, cob, atp6, and atpg, hybridize to R N A patterns with three to eight different species. In each of these transcriptional patterns, the smallest R N A is considerably larger than needed to accommodate its protein-coding capacity. For example, the smallest detected transcript for the maize atp9 gene is approximately 400 at, whereas the protein-codiug region is 222 nt (27). The smallest transcripts for each of the other genes, c o l coII, cob, atp6, and atp-alpha are at least 400 bases longer than the minimum required, and in many cases are approximately twice the minimum. The largest detected transcripts are huge relative to their protein-coding regions. The 1950 nt R N A species proposed as the primary transcript for the atp9 gene is almost nine times larger than the 222 nt coding region (27). Little is known about the events responsible for splicing introns and processing the primary messenger R N A in plant mitochondria. The complexity of maize mitochondrial transcriptional patterns is partially explained by chimeric genes, where short regions of D N A homologous to one gene are present within the transcriptional unit of another gene. A region of 128 bp ending at a position eight nucleotides 5' to the probable initiation codon (4,45,49) of the c o i l gene (34) is 97% homologous to a 122 bp region within the protein-coding region of maize atp6 (26).

TABLE 3 CODING REGION AND TRANSCRIPT LENGTHS OF MAIZE MITOCHONDRIAL GENES

Gene

Coding Region Length (n0

Intron Length

Transcript Lengths

(n0

lnt)

Protein Coding Genes col "7) coil (s2)

1584 780

-794

cob '~2~ atp6 (2~

1164 873

---

atp9 (271

222

--

1524

--

atp-alpha (9~

2400,2300 6000,5100, 3900, 3500, 3200, 2450, 1950 9000,4300, 2250 6800,4500, 4100, 3300, 2400, 2200, 1900, 1600 1950,1650, 1000, 850 650, 400 5000,2600

Structural RNA Genes 5S rRNA "6) 18S rRNA "71 26S rRNA {22) tRNAM., (InitiatorPTM tRNAM*' (Elongator) c~'~ tRNAA,p ~75~

126 1968 3546

----

.9 ? ?

74

--

?

74 74

---

.9 ?

MAIZE MITOCHONDRIAL GENES

Consequently, Northern analysis with an atp6 coding region-specific probe could detect trasnscripts from both genes. A similar situation may exist with a maize coil probe containing its 5' flanking sequences. Moreover in addition to the colI gene itself, DNA fragments containing homology to the 5' exon from the coII gene have been detected by hybridization in the wheat, maize, and rye mitochondrial genomes (4). The effect these additional fragments have on transcriptional patterns is unknown. Regulatory sequences important to transcriptional initiation and termination have received little attention in plant mitochondria. Isaac et al. (47) have determined the 5' end of two mRNAs coding for the maize coI gene by $1 nuclease analysis. Close to the 5' end of the longer mRNA is a nonanucleotide (TCATAAGTA) which is homologous at seven out of nine positions to a conserved sequence thought to be important in transcriptional initiation in yeast (71). Moon et al. (66) have studied the 5' termini of two transcripts for colI from pea. They note that both transcripts originate from a similar position within a repeated sequence containing 12 nucleotides (AAATCACGTAAGL The sequences noted for the maize and pea genes bear little homology to each other, and it has not been rigorously demonstrated that either sequence plays a role in transcription. Transcriptional termination has not been studied in plant mitochondrial genes. Shine and Dalgarno (86) proposed that a specific sequence (CCUCC) at the 3' end of E. coli 16S rRNA is an mRNA recognition site. Steitz and Jakes (91) reported that sequences complementary to the ShineDalgarno sequence are located near the 5' ends of the majority of E. coli mRNAs. Dawson et al. (23) have proposed that plant mitochondrial mRNAs contain potential ribosome binding sites. They suggest a sequence containing from three to five out of eight nucleotides complementary to eight nucleotides near the 3' terminus of maize mt 18S rRNA and located within 25 nucleotides 5' to the initiation codon may be a ribosomal binding site. Additional experimental verification is needed to determine whether this sequence functions as an mRNA recognition site.

UNIDENTIFIED MITOCHONDRIALGENES Animal and fungal mitochondrial DNAs contain 11 protein-coding genes that have not been identified in the maize mitochondrial genome. These include genes for cytochrome oxidase subunit III, Fo-ATPase subunit 8, a ribosomal protein, two cytochrome b maturases, and six subunits of the NADH-ubiquinone oxidoreductase (Table 1}. Recently, the products of six human mitochondrial unassigned reading frames, URF1, URF2, URF3, URF4, URF4L, and URF5 (URFs) have been identified as components of the enzyme complex NADH-Q reductase (18). URF-specific antipeptide antisera and antibiotic sensitivities were utilized in these determinations. This leaves one expressed open reading frame (62) unassigned in mammalian mitochondria.

173

Yeast mitochondrial DNA does not encode sequences homologous to the six NADH-Q reductase reading frames (18). However, Ise et al. (48) have recently established the mitochondrial translation of six subunits of NADH-Q reductase in Neurospora crassa and indicate that others (personal communication) have found Neurospora mitochondrial genes corresponding to three of the human NADH-Q reductase genes. Furthermore, Brown et al. (13) have identified open reading frames in the Aspergillus nidulans mitochondrial genome that are homologous to the mammalian URFI and URF4 genes and Scazzocchio et al. (81) report other Aspergillus open reading frames homologous to URF3 and URF5. Also, the mtDNA of Chlamydomonas reinhardtii contains a gene with amino acid homology to URF2 (76), and DNA sequence homologies to U R F I have been detected in the mitochondrial genome of maize (81). These findings indicate that genes encoding subunits of the NADH-Q reductase may exist in maize mitochondria. Only three tRNA genes have been identified in the maize mitochondrial genome. Inasmuch as there is no evidence that RNAs are imported into the mitochondria, it is expected that the plant mitochondrial genome encodes the entire complement of tRNA genes. Due to the prokaryotic nature of the structural RNA molecules, a full set of 32 tRNA genes may be encoded in maize and other plant mitochondria. Using a combination of genetic and molecular analyses, a structural RNA necessary in processing tRNA transcripts in yeast mitochondria has been identified (64). As yet a similar gene has not been reported in maize mitochondria. In the S type of cytoplasmic male-sterile (cms) maize, two plasmid-like elements, S-1 and S-2, exist in addition to the main mitochondrial genome (80). The nucleotide sequences of S-I (72) and S-2 (55} have been determined. Together, the two molecules contain four large unidentified open reading frames. These open reading frames have not yet been correlated with specific proteins or with the cms trait. It may be that they code for proteins important in the replication and maintainence of the plasmid-like molecules, rather than typical mitochondrial gene products. Finally, approximately 30 polypeptides are synthesized by isolated maize mitochondria (42). Because only six mitochondrially encoded polypeptides h a v e been identified (Table I), approximately two dozen more polypeptide-coding genes probably remain to be identified. Some of these may be similar to those already reported in mitochondria of other organisms. However, the occurrence of "extra" plant mitochondrial genes, the 5S rRNA and the FI-ATPase alpha subunit, and the large size of the plant mitochondrial genome suggests that a few more unique genes may be identified.

MUTATIONS IN MAIZE MITOCHONDRIALDNA Mitochondrial mutants are rare in higher plants, presumably because they are most often lethal or

174

ECKENRODE AND LEVINGS

undetectable. Several mitochondrial mutants are recognized in maize, cytoplasmic male sterility (cms), disease susceptibility (54), and nonchromsomol stripe mutants (ncs) (69). Two ncs mutations have been described and each correlated with specific changes in their m t D N A restriction fragments. Whether these m t D N A changes are responsible for ncs trait remains to be established. The cytoplasmic male-sterile trait is encountered in many higher plant species. Although the specific mechanisms causing male sterility may differ from species to species, the cms trait is characterized by the inability of the plant to produce viable pollen and by a non-Mendelian pattern of inheritance. Abundant evidence suggests that the cms trait in maize is due to a mitochondrial gene mutation [see { 5 3 ) for review]. Restriction patterns of m t D N A from male-fertile, malesterile (54,78), and revertant plants (35) are readily distinguishable and provide a means of identifying the various maize male-sterile cytoplasms {78). In contrast, restriction patterns of chloroplast D N A are nearly indistinguishable among the various cytoplasms. Restriction patterns of m t D N A from sorghum also differ between fertiles and steriles {79). In vitro translational studies have shown additional differences between fertile and male-sterile maize mitochondria {31,32). For example, in the male-sterile cms-T a unique polypeptide with a molecular weight of 13 000 is synthesized in isolated mitochondria that is absent from fertile maize {30). In the presence of the nuclear restorer genes for cms-T, Rfl, and Rf2, a specific reduction in the amount of the 13 000 M r polypeptide is observed. This result demonstrates that nuclear genes can regulate the expression of a specific mitochondrial polypeptide. Variations in the polypeptide patterns produced by mitochondria from other male-sterile maize cytoplasms {51) and from male-sterile sorghum and faba bean {8) have also been detected. Maize plants containing the T cytoplasm are susceptible to the fungal pathogen Bipolaris mayd/s, race T, and its toxin {T-toxin) whereas fertile and other male-steriles are not (51). When challenged with the T-toxin, mitochondria from cms-T plants undergo rapid changes in the permeability of the inner membrane, and oxidative phosphorylation is uncoupled {39,60). Mitochondria from normal plants, from other male-sterile cytoplasms (65), and from male-fertile cms-T revertants (12,36) are not affected by the toxin. Inasmuch as the cms trait and susceptibility to the T-toxin have not been separated experimentally or in nature, the two traits may be due to a single gene or to linked loci. Electron microscopy studies have also linked the male-sterile trait to the mitochondria. Warmke and Lee (95) have observed a premature degeneration of the mitochondria of the tapetum, the innermost anther cell wall layer, of cms-T plants during pollen development. A similar difference in plastid or other organelle morphology is not detected during pollen development. Finally, although the evidence strongly implicates mitochondrial gene mutations with the cms trait, the particular genes and their roles are not yet established.

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MAIZE MITOCHONDRIAL GENES

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