Ferritin gene organization: Differences between plants and animals suggest possible kingdom-specific selective constraints

Ferritin, a protein widespread in nature, concentrates iron ∼10¹¹–10¹²-fold above the solubility within a spherical shell of 24 subunits; it derives in plants and animals from a common ancestor (based on sequence) but displays a cytoplasmic location in animals compared to the plastid in contemporary plants. Ferritin gene regulation in plants and animals is altered by development, hormones, and excess iron; iron signals target DNA in plants but mRNA in animals. Evolution has thus conserved the two end points of ferritin gene expression, the physiological signals and the protein structure, while allowing some divergence of the genetic mechanisms. Comparison of ferritin gene organization in plants and animals, made possible by the cloning of a dicot (soybean) ferritin gene presented here and the recent cloning of two monocot (maize) ferritin genes, shows evolutionary divergence in ferritin gene organization between plants and animals but conservation among plants or among animals; divergence in the genetic mechanism for iron regulation is reflected by the absence in all three plant genes of the IRE, a highly conserved, noncoding sequence in vertebrate animal ferritin mRNA. In plant ferritin genes, the number of introns (n= 7) is higher than in animals (n= 3). Second, no intron positions are conserved when ferritin genes of plants and animals are compared, although all ferritin gene introns are in the coding region; within kingdoms, the intron positions in ferritin genes are conserved. Finally, secondary protein structure has no apparent relationship to intron/exon boundaries in plant ferritin genes, whereas in animal ferritin genes the correspondence is high. The structural differences in introns/exons among phylogenetically related ferritin coding sequences and the high conservation of the gene structure within plant or animal kingdoms suggest that kingdom-specific functional constraints may exist to maintain a particular intron/exon pattern within ferritin genes. In the case of plants, where ferritin gene intron placement is unrelated to triplet codons or protein structure, and where ferritin is targeted to the plastid, the selection pressure on gene organization may relate to RNA function and plastid/nuclear signaling. Received: 25 July 1995 / Accepted: 3 October 1995     

Molecular evolution of nitrate reductase genes

To understand the evolutionary mechanisms and relationships of nitrate reductases (NRs), the nucleotide sequences encoding 19 nitrate reductase (NR) genes from 16 species of fungi, algae, and higher plants were analyzed. The NR genes examined show substantial sequence similarity, particularly within functional domains, and large variations in GC content at the third codon position and intron number. The intron positions were different between the fungi and plants, but conserved within these groups. The overall and nonsynonymous substitution rates among fungi, algae, and higher plants were estimated to be 4.33 × 10⁻¹⁰ and 3.29 × 10⁻¹⁰ substitutions per site per year. The three functional domains of NR genes evolved at about one-third of the rate of the N-terminal and the two hinge regions connecting the functional domains. Relative rate tests suggested that the nonsynonymous substitution rates were constant among different lineages, while the overall nucleotide substitution rates varied between some lineages. The phylogenetic trees based on NR genes correspond well with the phylogeny of the organisms determined from systematics and other molecular studies. Based on the nonsynonymous substitution rate, the divergence time of monocots and dicots was estimated to be about 340 Myr when the fungi–plant or algae–higher plant divergence times were used as reference points and 191 Myr when the rice–barley divergence time was used as a reference point. These two estimates are consistent with other estimates of divergence times based on these reference points. The lack of consistency between these two values appears to be due to the uncertainty of the reference times. Received: 10 April 1995 / Accepted: 10 September 1995     

The Presence of GSI-Like Genes in Higher Plants: Support for the Paralogous Evolution of GSI and GSII Genes

Glutamine synthetase type I (GSI) genes have previously been described only in prokaryotes except that the fungus Emericella nidulans contains a gene (fluG) which encodes a protein with a large N-terminal domain linked to a C-terminal GSI-like domain. Eukaryotes generally contain the type II (GSII) genes which have been shown to occur also in some prokaryotes. The question of whether GSI and GSII genes are orthologues or paralogues remains a point of controversy. In this article we show that GSI-like genes are widespread in higher plants and have characterized one of the genes from the legume Medicago truncatula. This gene is part of a small gene family and is expressed in many organs of the plant. It encodes a protein similar in size and with between 36 and 46% amino acid sequence similarity to prokaryotic GS proteins used in the analyses, whereas it is larger and with less than 25% similarity to GSII proteins, including those from the same plant species. Phylogenetic analyses suggest that this protein is most similar to putative proteins encoded by expressed sequence tags of other higher plant species (including dicots and a monocot) and forms a cluster with FluG as the most divergent of the GSI sequences. The discovery of GSI-like genes in higher plants supports the paralogous evolution of GSI and GSII genes, which has implications for the use of GS in molecular studies on evolution. Received: 4 May 1999 / Accepted: 17 September 1999     

ABA regulates apoplastic sugar transport and is a potential signal for cold-induced pollen sterility in rice

Cold temperatures cause pollen sterility and large reductions in grain yield in temperate rice growing regions of the world. Induction of pollen sterility by cold involves a disruption of sugar transport in anthers, caused by the cold-induced repression of the apoplastic sugar transport pathway in the tapetum. Here we demonstrate that the phytohormone ABA is a potential signal for cold-induced pollen sterility (CIPS). Cold treatment of the cold-sensitive cultivar Doongara resulted in increased anther ABA levels. Exogenous ABA treatment at the young microspore stage induced pollen sterility and affected cell wall invertase and monosaccharide transporter gene expression in a way similar to cold treatment. In the cold-tolerant cultivar R31, ABA levels were significantly lower under normal circumstances and remained low after cold treatment. The differences in endogenous ABA levels in Doongara and R31 correlated with differences in expression of the ABA biosynthetic genes encoding zeaxanthin epoxidase (OSZEP1) and 9-cis-epoxycarotenoid dioxygenase (OSNCED2, OSNCED3) in anthers. The expression of three ABA-8-hydroxylase genes (ABA8OX1, 2 and 3) in R31 anthers was higher under control conditions and was regulated differently by cold compared with Doongara. Our results indicate that the cold tolerance phenotype of R31 is correlated with lower endogenous ABA levels and a different regulation of ABA metabolism.     

Expression of exogenous genes under the control of endogenous HSP70 and CAB promoters in the Closterium peracerosum-strigosum-littorale complex

A unicellular charophyte alga, Closterium peracerosum–strigosum–littoralecomplex (C. psl. complex), has been studied in order to obtainbasic information regarding sexual reproduction in plants. Systemsfor gene introduction and transient expression were developedfor endogenous genes using phleomycin resistance (ble) and Chlamydomonasgreen fluorescent protein (cgfp) genes as selection markers.These genes have codon usage similar to that of genes in theC. psl. complex. To drive these genes strongly into C. psl.complex cells, two native promoters of the C. psl. complex genome—CpHSP70and CpCAB1—were linked to a ble::cgfp fusion gene andintroduced into the cells by particle bombardment. Following2 d of incubation, we found 500 cells expressing GFP under thecontrol of the CpHSP70 promoter, which were identified followingheat shock treatment at 42°C, and 100 cells expressing GFPunder the control of the CpCAB1 promoter, which were observedin lit conditions. In contrast, the GFP signal was only detectedin two cells when ble::cgfp under control of the cauliflowermosaic virus 35S promoter was introduced. The ble::cgfp fusionprotein was detected in the nucleus, whereas the single cgfpprotein was detected in the cytoplasm. Our results indicatethat the newly isolated native promoters of CpHSP70 and CpCAB1are useful tools for inducing exogenous gene expression underheat shock and lit conditions, respectively. In addition, thisstrategy can be used for transient assays, such as the intracellularlocalization of unknown gene products in the C. psl. complex.     

Isolation and Characterization of a Carbonic Anhydrase Homologue from the Zebrafish (Danio rerio)

We have isolated a 29,000-Da carbonic anhydrase (CA) protein from the zebrafish, Danio rerio, sequenced two peptide fragments, and tentatively identified it as a high-activity CA by inhibition kinetics. We have also characterized a 1,537-bp message whose deduced sequence of 260 amino acids matches that of the isolated protein. This CA is clearly an α-CA based on the similarity of its sequence to that of other members of the α-CA gene family. A phylogenetic analysis suggested CAH-Z diverged after the branching of the CA-V and CA-VII genes and prior to the duplications that generated the CA-I, CA-II, and CA-III genes of amniotes. This marks the first characterization of the mRNA and its protein product from the CA gene of a teleost. Received: 31 March 1996 / Accepted: 8 September 1996     

Gene Conversion and Evolution of Daphniid Hemoglobins (Crustacea, Cladocera)

The extracellular hemoglobins of cladocerans derive from the aggregation of 12 two-domain globin subunits that are apparently encoded by four genes. This study establishes that at least some of these genes occur as a tandem array in both Daphnia magna and Daphnia exilis. The genes share a uniform structure; a bridge intron separates two globin domains which each include three exons and two introns. Introns are small, averaging just 77 bp, but a longer sequence (2.2–3.2 kb) separates adjacent globin genes. A survey of structural diversity in globin genes from other daphniids revealed three independent cases of intron loss, but exon lengths were identical, excepting a 3-bp insertion in exon 5 of Simocephalus. Heterogeneity in the extent of nucleotide divergence was marked among exons, largely as a result of the pronounced diversification of the terminal exon. This variation reflected, in part, varying exposure to concerted evolution. Conversion events were frequent in exons 1–4 but were absent from exons 5 and 6. Because of this difference, the results of phylogenetic analyses were strongly affected by the sequences employed in this construction. Phylogenies based on total nucleotide divergence in exons 1–4 revealed affinities among all genes isolated from a single species, reflecting the impact of gene conversion events. In contrast, phylogenies based on total nucleotide divergence in exons 5 and 6 revealed affinities among orthologous genes from different taxa. Received: 8 March 1999 / Accepted: 14 July 1999     

Clustering of Tissue-Specific Genes Underlies Much of the Similarity in Rates of Protein Evolution of Linked Genes

Are genes nonrandomly distributed around the genome and might this explain why it was found that, in the mouse genome, proteins of linked genes evolve at similar rates? Anecdotal evidence suggests that the similarity of expression of linked genes might, in part, explain the similarity in their rates of evolution. Immune system genes, for example, are known to evolve at a high rate and sometimes cluster in the genome. Here we develop methods for statistical tests of similarity of expression of linked genes and report that there is a significant tendency for genes of similar expression breadth to be linked. Significantly, when we exclude tissue specific genes from our sample, the similarity in rates of protein evolution of linked genes is greatly diminished, if not abolished. This diminution is not a sampling artifact. In contrast, while half of the immune genes in our sample reside in 1 of 10 immune clusters in the mouse genome, this clustering appears not to affect the extent of local similarity in rates of evolution. The distribution of placentally expressed genes, in contrast, does have an effect.     

Genetic Variability of Natural Populations of Cotton Leaf Curl Geminivirus, a Single-Stranded DNA Virus

Reports on the genetic variability and evolution of natural populations of DNA viruses are scarce in comparison with the abundant information on the variability of RNA viruses. Geminiviruses are plant viruses with circular ssDNA genomes that are replicated by the host plant DNA polymerases. Whitefly-transmitted geminiviruses (WTG) are the agents of important diseases of crop plants and best exemplify emerging plant viruses. In this report we have analyzed the genetic diversity of cotton leaf curl geminivirus (CLCuV), a typical emerging WTG. No genetic differentiation was observed between isolates from different host plant species or geographic regions. Thus, the analyzed isolates represented a unique, undifferentiated population. Genetic variability, estimated as nucleotide diversities at synonymous positions in open reading frames (ORFs) for the AC1 (=replication) protein and coat protein (CP = AV1), was very high, exceeding the values reported for different genes in several plant and animal RNA viruses. This was unexpected in a virus that uses the DNA replication machinery of its eukaryotic host. Diversities at nonsynonymous positions, on the other hand, indicated that variability may be constrained in the genome of CLCuV. The ratio of nonsynonymous-to-synonymous substitutions varied for the different ORFs: they were higher for CP than for AC1 and lower still for the AC4 and AV2 ORFs, which overlap AC1 and CP ORFs, respectively. Analysis of nucleotide diversities at synonymous and nonsynonymous positions of the AC4 and AV2 ORFs suggest that their evolution is constrained by AC1 and CP, respectively. Data suggest that AC4 and AV2 are new genes that may have originated by overprinting on the preexistent AC1 and CP genes. Evidence for recombination was found for the AC1 and CP ORFs and for the noncoding intergenic region (IR). Data indicate that the origin of replication is a major recombination point in the IR, but not the only one. Analyses of the IR also suggest that recombinants may be frequent in the population and that recombination may have an important role in the generation of CLCuV variability. Received: 26 February 1999 / Accepted: 31 May 1999     

The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): Genetics and evolution of plasmid-associated anthranilate synthase (trpEG) within the aphididae

The bacterial endosymbionts (Buchnera) from the aphids Rhopalosiphum padi, R. maidis, Schizaphis graminum, and Acyrthosiphon pisum contain the genes for anthranilate synthase (trpEG) on plasmids made up of one or more 3.6-kb units. Anthranilate synthase is the first as well as the rate-limiting enzyme in the tryptophan biosynthetic pathway. The amplification of trpEG on plasmids may result in an increase of enzyme protein and overproduction of this essential amino acid, which is required by the aphid host. The nucleotide sequence of trpEG from endosymbionts of different species of aphids is highly conserved, as is an approximately 500-bp upstream DNA segment which has the characteristics of an origin of replication. Phylogenetic analyses were performed using trpE and trpG from the endosymbionts of these four aphids as well as from the endosymbiont of Schlechtendalia chinensis, in which trpEG occurs on the chromosome. The resulting phylogeny was congruent with trees derived from sequences of two chromosome-located bacterial genes (part of trpB and 16S ribosomal DNA). In turn, trees obtained from plasmid-borne and bacterial chromosome-borne sequences were congruent with the tree resulting from phylogenetic analysis of three aphid mitochondrial regions (portions of the small and large ribosomal DNA subunits, as well as cytochrome oxidase II). Congruence of trees based on genes from host mitochondria and from bacteria adds to previous support for exclusively vertical transmission of the endosymbionts within aphid lineages. Congruence with trees based on plasmid-borne genes supports the origin of the plasmid-borne trpEG from the chromosomal genes of the same lineage and the absence of subsequent plasmid exchange among endosymbionts of different species of aphids. Received: 22 August 1995 / Accepted: 6 September 1995     

Defense-related signaling by interaction of arabinogalactan proteins and beta-glucosyl Yariv reagent inhibits gibberellin signaling in barley aleurone cells

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins present at the plasma membrane and in extracellular spaces. A synthetic chemical, beta-glucosyl Yariv reagent (beta-GlcY), binds specifically to AGPs. We previously reported that gibberellin signaling is specifically inhibited by beta-GlcY treatment in barley aleurone protoplasts. In the present study, we found that beta-GlcY also inhibited gibberellin-induced programmed cell death (PCD) in aleurone cells. We examined the universality and specificity of the inhibitory effect of beta-GlcY on gibberellin signaling using microarray analysis and found that beta-GlcY was largely effective in repressing gibberellin-induced gene expression. In addition, >100 genes were up-regulated by beta-GlcY in a gibberellin-independent manner, and many of these were categorized as defense-related genes. Defense signaling triggered by several defense system inducers such as jasmonic acid and a chitin elicitor could inhibit gibberellin-inducible events such as alpha-amylase secretion, PCD and expression of some gibberellin-inducible genes in aleurone cells. Furthermore, beta-GlcY repressed the gibberellin-inducible Ca2+-ATPase gene which is important for gibberellin-dependent gene expression, and induced known repressors of gibberellin signaling, two WRKY genes and a NAK kinase gene. These effects of beta-GlcY were also phenocopied by the chitin elicitor and/or jasmonic acid. These results indicate that gibberellin signaling is under the regulation of defense-related signaling in aleurone cells. It is also probable that AGPs are involved in the perception of stimuli causing defense responses.     

Positional Preferences of Polypurine/Polypyrimidine Tracts in Saccharomyces cerevisiae Genome: Implications for cis Regulation of Gene Expression

The complete genome of the baker's yeast S. cerevisiae was analyzed for the presence of polypurine/polypyrimidine (poly[pu/py]) repeats and their occurrences were classified on the basis of their location within and outside open reading frames (ORFs). The analysis reveals that such sequence motifs are present abundantly both in coding as well as noncoding regions. Clear positional preferences are seen when these tracts occur in noncoding regions. These motifs appear to occur predominantly at a unit nucleosomal length both upstream and downstream of ORFs. Moreover, there is a biased distribution of polypurines in the coding strands when these motifs occur within open reading frames. The significance of the biased distribution is discussed with reference to the occurrence of these motifs in other known mRNA sequences and expressed sequence tags. A model for cis regulation of gene expression is proposed based on the ability of these motifs to form an intermolecular triple helix structure when present within the coding region and/or to modulate nucleosome positioning via enhanced histone affinity when present outside coding regions. Received: 14 November 1996 / Accepted: 7 May 1997     

Evolution of MADS-box gene induction by FLO/LFY genes

Some MADS-box genes function as floral homeotic genes. The Arabidopsis LFY gene is a positive regulator of floral homeotic genes, and homologs of the FLO/LFY gene family in other angiosperms and gymnosperms are likely to have a similar function. To investigate the origin of the floral homeotic gene regulatory cascade involving the FLO/LFY gene, FLO/LFY homologs were cloned from a leptosporangiate fern (Ceratopteris richardii), two eusporangiate ferns (Angiopteris lygodiifolia and Botrychium multifidum var. robustum), three fern allies (Psilotum nudum, Equisetum arvense, and Isoetes asiatica), and a moss (Physcomitrella patens). The FLO/LFY gene phylogenetic tree indicates that both duplication and loss of FLO/LFY homologs occurred during the course of vascular plant evolution. The expression patterns of the Ceratopteris LFY genes (CrLFY1 and 2) were assessed. CrLFY1 expression was prominent in tissues including shoot tips and circinate reproductive leaves, but very weak in other tissues examined. Expression of CrLFY2 was also prominent in tissues, including shoot tips and circinate reproductive leaves. These patterns of expression are dissimilar to that of any Ceratopteris MADS-box gene previously reported, suggesting that the induction of MADS-box genes by FLO/LFY is not established at the stage of ferns. Received: 4 January 2001 / Accepted: 28 February 2001     

Algae or Protozoa: Phylogenetic Position of Euglenophytes and Dinoflagellates as Inferred from Mitochondrial Sequences

The chloroplasts of euglenophytes and dinoflagellates have been suggested to be the vestiges of endosymbiotic algae acquired during the process of evolution. However, the evolutionary positions of these organisms are still inconclusive, and they have been tentatively classified as both algae and protozoa. A representative gene of the mitochondrial genome, cytochrome oxidase subunit I (coxI), was chosen and sequenced to clarify the phylogenetic positions of four dinoflagellates, two euglenophytes and one apicomplexan protist. This is the first report of mitochondrial DNA sequences for dinoflagellates and euglenophytes. Our COXI tree shows clearly that dinoflagellates are closely linked to apicomplexan parasites but not with algae. Euglenophytes and algae appear to be only remotely related, with euglenophytes sharing a possible evolutionary link with kinetoplastids. The COXI tree is in general agreement with the tree based on the nuclear encoded small subunit of ribosomal RNA (SSU rRNA) genes, but conflicts with that based on plastid genes. These results support the interpretation that chloroplasts present in euglenophytes and dinoflagellates were captured from algae through endosymbioses, while their mitochondria were inherited from the host cell. We suggest that dinoflagellates and euglenophytes were originally heterotrophic protists and that their chloroplasts are remnants of endosymbiotic algae. Received: 24 March 1997 / Accepted: 21 April 1997     

Mitochondrial DNA Phylogeny and the Evolution of Host-Plant Use in Palearctic Chrysolina (Coleoptera, Chrysomelidae) Leaf Beetles

The genus Chrysolina consists of specialized phytophagous leaf-beetles (Coleoptera, Chrysomelidae) with feed on several plant families. There is no explicit phylogenetic hypothesis available for this genus, which includes 65 subgenera and more than 400 species with a wide distribution. We obtained 839-bp sequence data from the 16S rDNA and cytochrome oxidase subunit I (COI) mitochondrial genes. Thirty Chrysolina taxa representing eight host–plant affiliations, two species of the closely related genus Oreina, and two outgroups were sampled. These data sets were used separately and combined to obtain the mitochondrial cladogram of the group using maximum-parsimony and maximum-likelihood criteria. The results were compared to current proposals for Chrysolina systematics that are based on morphological, ecological, and karyological data. The trees obtained were in the most part congruent with the proposed ancestral association of Chrysolina to Lamiaceae based on chromosome number in several lineages. A minimum of five host-plant switches from the ancestral state inferred at the family level and two at the subclass level suggests the absence of parallel evolution of beetles and their host plants. Another switch leading to oligophagy at the family level was deduced to have occurred in the lineage of the subgenus Chrysolina s.str. Received: 22 May 1998 / Accepted: 16 September 1998     

Codon Usage in Plastid Genes Is Correlated with Context, Position Within the Gene, and Amino Acid Content

Highly expressed plastid genes display codon adaptation, which is defined as a bias toward a set of codons which are complementary to abundant tRNAs. This type of adaptation is similar to what is observed in highly expressed Escherichia coli genes and is probably the result of selection to increase translation efficiency. In the current work, the codon adaptation of plastid genes is studied with regard to three specific features that have been observed in E. coli and which may influence translation efficiency. These features are (1) a relatively low codon adaptation at the 5′ end of highly expressed genes, (2) an influence of neighboring codons on codon usage at a particular site (codon context), and (3) a correlation between the level of codon adaptation of a gene and its amino acid content. All three features are found in plastid genes. First, highly expressed plastid genes have a noticeable decrease in codon adaptation over the first 10–20 codons. Second, for the twofold degenerate NNY codon groups, highly expressed genes have an overall bias toward the NNC codon, but this is not observed when the 3′ neighboring base is a G. At these sites highly expressed genes are biased toward NNT instead of NNC. Third, plastid genes that have higher codon adaptations also tend to have an increased usage of amino acids with a high G + C content at the first two codon positions and GNN codons in particular. The correlation between codon adaptation and amino acid content exists separately for both cytosolic and membrane proteins and is not related to any obvious functional property. It is suggested that at certain sites selection discriminates between nonsynonymous codons based on translational, not functional, differences, with the result that the amino acid sequence of highly expressed proteins is partially influenced by selection for increased translation efficiency. Received: 21 July 1999 / Accepted: 5 November 1999