A mutation in the nuclear-encoded plastid ribosomal protein S9 leads to early embryo lethality in maize

Seeds of the lethal embryo 1 (lem1) mutant in maize (Zea mays) display a non-concordant lethal phenotype: whereas the embryo aborts very early, before the transition stage, the endosperm develops almost normally. The mutant was identified in a collection of maize lines that carried the transposon Activation (Ac) at different locations in the genome. Co-segregation and reversion analysis showed that lem1 was tagged by Ac. The lem1 gene encodes a protein that is highly similar to the rice plastid 30S ribosomal protein S9 (PRPS9). lem1 maps to chromosome 1L and appears to be the only copy of prps9 in the maize genome. Green fluorescent protein (GFP) fusion constructs containing only the putative transit peptide (TP) of LEM1 localize exclusively to the plastids, confirming that the LEM1 protein is a PRP. In contrast, GFP fusion constructs containing the entire LEM1 protein co-localize to the plastids and to the nucleus, suggesting a possible dual function for this protein. Two alternative, although not mutually exclusive, explanations are considered for the lem phenotype of the lem1 mutant: (i) functional plastids are required for normal embryo development; and (ii) the PRPS9 has an extra-ribosomal function required for embryogenesis.     

Dual targeting of organellar seryl-tRNA synthetase to maize mitochondria and chloroplasts

Aminoacyl-tRNA synthetases (AARSs) play a critical role in translation and are thus required in three plant protein-synthesizing compartments: cytosol, mitochondria and plastids. A systematic study had previously shown extensive sharing of organellar AARSs from Arabidopsis thaliana, mostly between mitochondria and chloroplasts. However, distribution of AARSs from monocot species, such as maize, has never been experimentally investigated. Here we demonstrate dual targeting of maize seryl-tRNA synthetase, SerZMo, into both mitochondria and chloroplasts using combination of complementary methods, including in vitro import assay, transient expression analysis of green fluorescent protein (GFP) fusions and immunodetection. We also show that SerZMo dual localization is established by the virtue of an ambiguous targeting peptide. Full-length SerZMo protein fused to GFP is targeted to chloroplast stromules, indicating that SerZMo protein performs its function in plastid stroma. The deletion mutant lacking N-terminal region of the ambiguous SerZMo targeting peptide was neither targeted into mitochondria nor chloroplasts, indicating the importance of this region in both mitochondrial and chloroplastic import.     

The Arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome chromophore biosynthesis as a result of a mutation in a plastid heme oxygenase

The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme oxygenases and contains a possible transit peptide for transport to plastids. Heme oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme oxygenase necessary for phytochrome chromophore biosynthesis.     

Dual targeting to mitochondria and plastids of AtBT1 and ZmBT1, two members of the mitochondrial carrier family

Zea mays and Arabidopsis thaliana Brittle 1 (ZmBT1 and AtBT1, respectively) are members of the mitochondrial carrier family. Although they are presumed to be exclusively localized in the envelope membranes of plastids, confocal fluorescence microscopy analyses of potato, Arabidopsis and maize plants stably expressing green fluorescent protein (GFP) fusions of ZmBT1 and AtBT1 revealed that the two proteins have dual localization to plastids and mitochondria. The patterns of GFP fluorescence distribution observed in plants stably expressing GFP fusions of ZmBT1 and AtBT1 N-terminal extensions were fully congruent with that of plants expressing a plastidial marker fused to GFP. Furthermore, the patterns of GFP fluorescence distribution and motility observed in plants expressing the mature proteins fused to GFP were identical to those observed in plants expressing a mitochondrial marker fused to GFP. Electron microscopic immunocytochemical analyses of maize endosperms using anti-ZmBT1 antibodies further confirmed that ZmBT1 occurs in both plastids and mitochondria. The overall data showed that (i) ZmBT1 and AtBT1 are dually targeted to mitochondria and plastids; (ii) AtBT1 and ZmBT1 N-terminal extensions comprise targeting sequences exclusively recognized by the plastidial compartment; and (iii) targeting sequences to mitochondria are localized within the mature part of the BT1 proteins.     

Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient mutants of the moss Ceratodon purpureus

 In protonemal tip cells of the moss Ceratodon purpureus (Hedw.) Brid., phototropism and chlorophyll accumulation are regulated by the photoreceptor phytochrome. The mutant ptr116 lacks both responses as a result of a defect in the biosynthesis of phytochromobilin, the chromophore of phytochrome, at the point of biliverdin formation. The rescue of the phototropic response and of chlorophyll synthesis were tested by injecting different substances into tip cells of ptr116. Microinjection was first optimised with the use of fluorescent dyes and an expression plasmid containing a green fluorescent protein (GFP) gene. Injected phycocyanobilin, which substitutes for phytochromobilin, rescued both the phototropic response and light-induced chlorophyll accumulation in ptr116. The same results were obtained when expression plasmids with heme oxygenase genes of rat (HO-1) and Arabidopsis thaliana (L.) Heynh. (HY1) were injected. Heme oxygenase catalyses the conversion of heme into biliverdin. Whereas HY1 has a plastid target sequence and is presumably transferred to plastids, HO-1 is proposed to be cytosolic. The data show that ptr116 lacks heme oxygenase enzyme activity and indicate that heme oxygenases of various origin are active in Ceratodon bilin synthesis. In addition, it can be inferred from the data that the intracellular localisation of the expressed heme oxygenase is not important since the plastid enzyme can be replaced by a cytosolic one. Received: 8 March 1999 / Accepted: 30 July 1999     

Enzymes of the heme biosynthetic pathway in the nonphotosynthetic alga Polytomella sp

Heme biosynthesis involves a number of enzymatic steps which in eukaryotes take place in different cell compartments. Enzyme compartmentalization differs between photosynthetic and nonphotosynthetic eukaryotes. Here we investigated the structures and subcellular localizations of three enzymes involved in the heme pathway in Polytomella sp., a colorless alga evolutionarily related to the green alga Chlamydomonas reinhardtii. Functional complementation of Escherichia coli mutant strains was used to isolate cDNAs encoding three heme biosynthetic enzymes, glutamate-1-semialdehyde aminotransferase, protoporphyrinogen IX oxidase, and ferrochelatase. All three proteins show highest similarity to their counterparts in photosynthetic organisms, including C. reinhardtii. All three proteins have N-terminal extensions suggestive of intracellular targeting, and immunoblot studies indicate their enrichment in a dense cell fraction that is enriched in amyloplasts. These results suggest that even though the plastids of Polytomella sp. are not photosynthetically active, they are the major site of heme biosynthesis. The presence of a gene for glutamate-1-semialdehyde aminotransferase suggests that Polytomella sp. uses the five-carbon pathway for synthesis of the heme precursor 5-aminolevulinic acid.     

A soybean coproporphyrinogen oxidase gene is highly expressed in root nodules

In plants the enzyme coproporphyrinogen oxidase catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX in the heme and chlorophyll biosynthesis pathway(s).We have isolated a soybean coproporphyrinogen oxidase cDNA from a cDNA library and determined the primary structure of the corresponding gene. The coproporphyrinogen oxidase gene encodes a polypeptide with a predicted molecular mass of 43 kDa. The derived amino acid sequence shows 50% similarity to the corresponding yeast amino acid sequence. The main difference is an extension of 67 amino acids at the N-terminus of the soybean polypeptide which may function as a transit peptide.A full-length coproporphyrinogen oxidase cDNA clone complements a yeast mutant deleted of the coproporphyrinogen oxidase gene, thus demonstrating the function of the soybean protein.The soybean coproporphyrinogen oxidase gene is highly expressed in nodules at the stage where several late nodulins including leghemoglobin appear. The coproporphyrinogen oxidase mRNA is also detectable in leaves but at a lower level than in nodules while no mRNA is detectable in roots.The high level of coproporphyrinogen oxidase mRNA in soybean nodules implies that the plant increases heme production in the nodules to meet the demand for additional heme required for hemoprotein formation.     

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots ( Arabidopsis , broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.     

Transcription of yeast COX6, the gene for cytochrome c oxidase subunit VI, is dependent on heme and on the HAP2 gene

The COX6 gene encodes subunit VI of cytochrome c oxidase. Previously, this gene and its mRNAs were characterized, and its expression has been shown to be subject to glucose repression/derepression. In this study we have examined the effects of heme and the HAP1 (CYP1) and HAP2 genes on the expression of COX6. By quantitating COX6 RNA levels and assaying beta-galactosidase activity in yeast cells carrying COX6-lacZ fusion genes, we have found that COX6 is regulated positively by heme and HAP2, but is unaffected by HAP1. Through 5' deletion analysis we have also found that the effects of heme and HAP2 on COX6 are mediated by sequences between 135 and 590 base pairs upstream of its initiation codon. These findings identify COX6 as the fourth respiratory protein gene that is known to be regulated positively by heme and HAP2. The other three, CYC1, COX4, and COX5a, encode iso-1-cytochrome c, cytochrome c oxidase subunit IV, and an isolog, Va, of cytochrome c oxidase subunit V, respectively. Thus, it appears that the biogenesis of two interacting proteins, cytochrome c and cytochrome c oxidase, in the mitochondrial respiratory chain, are under the control of common factors.     

A cryptic algal group unveiled: a plastid biosynthesis pathway in the oyster parasite Perkinsus marinus

Plastids are widespread in plant and algal lineages. They are also exploited by some nonphotosynthetic protists, including malarial parasites, to support their diverse modes of life. However, cryptic plastids may exist in other nonphotosynthetic protists, which could be important in studies on the diversity and evolution of plastids. The parasite Perkinsus marinus, which causes mass mortality in oyster farms, is a nonphotosynthetic protist that is phylogenetically related to plastid-bearing dinoflagellates and apicomplexans. In this study, we searched for P. marinus methylerythritol phosphate (MEP) pathway genes, responsible for de novo isoprenoid synthesis in plastids, and determined the full-length gene sequences for 6 of 7 of these genes. Phylogenetic analyses revealed that each P. marinus gene clusters with orthologs from plastid-bearing eukaryotes, which have MEP pathway genes with essentially the same mosaic pattern of evolutionary origin. A new analytical method called sliding-window iteration of TargetP was developed to examine the distribution of targeting preferences. This analysis revealed that the sequenced genes encode bipartite targeting peptides that are characteristic of proteins targeted to secondary plastids originating from endosymbiosis of eukaryotic algae. These results support our idea that Perkinsus is a cryptic algal group containing nonphotosynthetic secondary plastids. In fact, immunofluorescent microscopy indicated that 1 of the MEP pathway enzymes, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, was localized to small compartments near mitochondrion, which are possibly plastids. This tiny organelle seems to contain very low quantities of DNA or may even lack DNA entirely. The MEP pathway genes are a useful tool for investigating plastid evolution in both of the photosynthetic and nonphotosynthetic eukaryotes and led us to propose the hypothesis that ancestral "chromalveolates" harbored plastids before a secondary endosymbiotic event.     

Subcellular localization of two types of ferrochelatase in cucumber

It is widely believed that ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous ion into protoporphyrin IX to form protoheme, exists in both plastids and mitochondria of higher plants. By in vitro import assay with isolated pea (Pisum sativum L.) organelles, it has been proposed that one of two isoforms of ferrochelatase (type 1) is dual-targeted into both plastids and mitochondria, and functions for heme biosynthesis in the both organelles. Recently, however, mitochondrial targeting of ferrochelatase is being disputed since pea mitochondria appeared to accept a variety of chloroplast proteins including the type-1 ferrochelatase of Arabidopsis thaliana (L.) Heynh. To clarify the precise subcellular localization of ferrochelatase in higher plants, here we investigated the subcellular localization of two types of ferrochelatase (CsFeC1 and CsFeC2) in cucumber (Cucumis sativus L.). In cotyledons, a significant level of specific ferrochelatase activity was detected in thylakoid membranes, but only a trace level of activity was detectable in mitochondria. Western blot analysis with specific antibodies showed that anti-CsFeC2 antiserum cross-reacted with plastids in photosynthetic and non-photosynthetic tissues. Anti-CsFeC1 did not cross-react with mitochondria, but CsFeC1 was clearly detectable in plastids from non-photosynthetic tissues. In situ transient-expression assays using green fluorescent protein demonstrated that, as well as CsFeC2, the N-terminal transit peptide of CsFeC1 targeted the fusion protein solely into plastids, but not into mitochondria. These results demonstrated that in cucumber both CsFeC1 and CsFeC2 are solely targeted into plastids, but not into mitochondria. Screening of a cucumber genomic or cDNA library did not allow any other ferrochelatase homologous gene to be isolated. The data presented here imply the reconsideration of mitochondrial heme biosynthesis in higher plants.     

Visualisation of plastids in endosperm,pollen and roots of transgenic wheat expressing modified GFP fused to transit peptides from wheat SSU RubisCO,rice FtsZ and maize ferredoxin III proteins

The ability to target marker proteins to specific subcellular compartments is a powerful research tool to study the structure and development of organelles. Here transit sequences from nuclear-encoded, plastid proteins, namely rice FtsZ, maize non-photosynthetic ferredoxin III (FdIII) and the small subunit of RubisCO were used to target a modified synthetic GFP (S65G, S72A) to plastids. The localisations of the fusion proteins expressed in transgenic wheat plants and under the control of the rice actin promoter were compared to an untargeted GFP control. GFP fluorescence was localised to non-green plastids in pollen, roots and seed endosperm and detected in isolated leaf chloroplasts using a GFP-specific antibody. Transit peptides appeared to influence the relative fluorescence intensities of plastids in different tissues. This is consistent with differential targeting and/or turnover of GFP fusion proteins in different plastid types. Replacement of GFP sequences with alternative coding regions enables immediate applications of our vectors for academic research and commercial applications.     

SEMAPHORE1 functions during the regulation of ancestrally duplicated knox genes and polar auxin transport in maize

The expression of class 1 knotted1-like homeobox (knox) genes affects numerous plant developmental processes, including cell-fate acquisition, lateral organ initiation, and maintenance of shoot apical meristems. The SEMAPHORE1 gene product is required for the negative regulation of a subset of maize knox genes, the duplicated loci rough sheath 1 and gnarley1 (knox4). Recessive mutations in semaphore1 result in the ectopic expression of knox genes in leaf and endosperm tissue. Genetic analyses suggest that SEMAPHORE1 may regulate knox gene expression in a different developmental pathway than ROUGH SHEATH2, the first-identified regulator of knox gene expression in maize. Mutations at semaphore1 are pleiotropic, disrupting specific domains of the shoot. However, unlike previously described mutations that cause ectopic knox gene expression, semaphore1 mutations affect development of the embryo, endosperm, lateral roots, and pollen. Moreover, polar transport of the phytohormone auxin is significantly reduced in semaphore1 mutant shoots. The data suggest that many of the pleiotropic semaphore1 phenotypes result from defective polar auxin transport (PAT) in sem1 mutant shoots, and support models correlating down-regulated knox gene expression and PAT in maize shoots.