Embryo-lethal mutants of Arabidopsis thaliana: analysis of mutants with a wide range of lethal phases

Summary Embryo-lethal mutants of Arabidopsis thaliana were isolated by treating mature seeds with an aqueous solution of ethyl methanesulfonate (EMS), screening the resulting M-1 plants for siliques containing 25% aborted seeds following self-pollination, and verifying the presence of induced mutations in subsequent generations. Thirty-two recessive lethals with a Mendelian pattern of inheritance were examined in detail. Developmental arrest of mutant embryos ranged from the zygotic stage of embryogenesis in mutant 53D-4A to the linear and curled cotyledon stages of development in mutants 112A-2A and 130B-A-2. These lethal phases did not change significantly when plants were grown at 18 °C rather than at 24 °C. Differences between mutant lines were found in the color of arrested embryos and aborted seeds, the percentage and distribution of aborted seeds in heterozygous siliques, the size of arrested embryos, and the extent of abnormal development. Unusual mutant phenotypes included the presence of unusually large suspensors, distorted and fused cotyledons, reduced hypocotyls, and arrested embryos without distinct cotyledons or hypocotyl tissue. The isolation of eight new mutants with a non-random distribution of aborted seeds in heterozygous siliques provides further evidence that many of the genes that control early stages of embryogenesis in plants are also expressed prior to fertilization.     

Analysis of storage proteins in normal and aborted seeds from embryo-lethal mutants of Arabidopsis thaliana

The major storage proteins isolated from wild-type seeds of Arabidopsis thaliana (L.) Heynh., strain Columbia, were studied by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both the hypocotyl and cotyledons of mature embryos contained abundant 12 S (cruciferin) and 2 S (arabin) proteins that appeared similar in size and subunit composition to the cruciferin (12 S) and napin (1.7 S) seed-storage proteins of Brassica napus. The 12 S protein from Arabidopsis was resolved by SDS-PAGE into two groups of subunits with approximate relative molecular weights of 22–23 kDa (kilodalton) and 30–34 kDa. These polypeptides accumulated late in embryo development, disappeared early in germination, and were not detected in other vegetative or reproductive tissues. Accumulation of the 12 S proteins in aborted seeds from nine embryo-lethal mutants with different patterns of abnormal development was studied to determine the extent of cellular differentiation in arrested embryos from each mutant line. Abundant 12 S proteins were found in arrested embryos from two mutants with late lethal phases, but not in seven other mutants with lethal phases ranging from the globular to the cotyledon stages of embryo development. These results indicate that the accumulation of seed-storage proteins in wild-type embryos of Arabidopsis is closely tied to morphogenetic changes that occur during embryo development. Embryo-lethal mutants may therefore be useful in future studies on the developmental regulation of storage-protein synthesis.Abbreviations kDa kilodalton - M_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Isolation and characterization of six embryo-lethal mutants of Arabidopsis thaliana

Mature seeds of Arabidopsis thaliana strain “Columbia” were soaked for 7.5 hr in an aqueous solution of the chemical mutagen ethyl methanesulfonate (0.05, 0.10, or 0.50%, v/v). Embryo-lethal mutants were identified in the resulting M-1 chimeral plants by screening the first five siliques of each plant and noting the frequency of aborted seeds. Three hundred sixty seeds were treated at each mutagen dose; the frequency of embryo-lethal mutants ranged from 1–3% of the M-1 plants grown from seeds exposed to 0.05% EMS, to 20–30% of the M-1 plants at the highest mutagen dose. Six embryo-lethal mutants identified through screening of M-1 plants were chosen for detailed studies in subsequent generations. All six mutants segregate as nonallelic, Mendelian recessive lethals, and are maintained as heterozygotes since homozygotes die as embryos. Fruits of heterozygous plants contain 25% aborted seeds and 75% phenotypically normal seeds ($\text{2}\text{3}$ heterozygotes and $\text{1}\text{3}$ wild type). Segregation ratios are not temperature sensitive; the same frequency of aborted seeds is found in plants grown at 18, 25, and 32°C. Embryo arrest and eventual lethality in each mutant occur at a characteristic stage of early embryo development: globular-heart, globular, early globular, or preglobular. Arrested embryos from five of the six mutants resemble normal embryos at early stages of development. Developmental arrest of the embryo proper in the remaining mutant is followed by abnormal growth of the suspensor, an embryonic structure that attaches the embryo proper to the maternal tissue.     

The chloroplast ribosomal protein L21 gene is essential for plastid development and embryogenesis in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Embryogenesis in higher plants is controlled by a complex gene network. Identification and characterization of genes essential for embryogenesis will provide insights into the early events in embryo development. In this study, a novel mutant with aborted seed development (asd) was identified in Arabidopsis. The asd mutant produced about 25% of albino seeds at the early stage of silique development. The segregation of normal and albino seeds was inherited as a single recessive embryo-lethal trait. The gene disrupted in the asd mutant was isolated through map-based cloning. The mutated gene contains a single base change (A to C) in the coding region of RPL21C (At1g35680) that is predicted to encode the chloroplast 50S ribosomal protein L21. Allele test with other two T-DNA insertion lines in RPL21C and a complementation test demonstrated that the mutation in RPL21C was responsible for the asd phenotype. RPL21C exhibits higher expression in leaves and flowers compared with expression levels in roots and developing seeds. The RPL21C–GFP fusion protein was localized in chloroplasts. Cytological observations showed that the asd embryo development was arrested at the globular stage. There were no plastids with normal thylakoids and as a result no normal chloroplasts formed in mutant cells, indicating an indispensable role of the ASD gene in chloroplasts biogenesis. Our studies suggest that the chloroplast ribosomal protein L21 gene is required for chloroplast development and embryogenesis in Arabidopsis.     

Induction and characterization of pigmentation mutants in Porphyra yezoensis (Bangiales, Rhodophyta)

Porphyra yezoensis Ueda conchospore germlings (1–4-cell stages) were treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) for inducing mutations. Three kinds of color-mutated gametophytic blades, which were composed of the mutated cells wholly, sectorially or spottedly, were obtained; and most of them were sectorially variegated blades. The highest frequency of these mutated blades was 1.3%. Four different pigmentation mutant strains were obtained by regenerating single cells and protoplasts that were enzymatically isolated from the mutated sectors of the sectorially variegated blades. The mutants were relatively stable in color in both gametophytic blade and conchocelis phases. In the two phases, each mutant strain showed characteristic differences in the in vivo absorption spectra, and had different pigment contents of major photosynthetic pigments (chlorophyll a, phycoerythrin and phycocyanin) as compared with the wild-type and with each other. The gametophytic blades from the four mutant lines showed significant differences in growth and photosynthetic rates, when they were cultured in the same conditions. By crossing the mutant with the wild-type, it was found that the color phenotypes of two mutants reported above, were resulted from two mutations in different genes, respectively. This revised version was published online in August 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">halfman</Emphasis>, an<Emphasis Type="Italic"> Arabidopsis</Emphasis> male gametophytic mutant associated with a 150 kb chromosomal deletion adjacent to an introduced<Emphasis Type="Italic"> Ds</Emphasis> transposable element

To identify genes that play an important gametophytic role during pollen development, an Arabidopsis transposon (DsE) mutagenised population was screened for marker segregation ratio distortion. We report the characterisation of a male gametophytic mutant termed halfman (ham) that results in an aborted pollen phenotype in mature anthers. The genetic transmission efficiency of DsE was 6.6% through the male and 98.4% through the female, which suggested that HAM may encode essential male-specific component(s) required for pollen development. Molecular analysis of the insertion site revealed a single copy of the DsE element inserted into the second exon of the RLK5 gene that was adjacent to a large (~150 kb) genomic deletion. The deleted region is predicted to encode 38 genes and to include one or more genes with important function(s) during pollen maturation and seed development.     

Functional disruption of the pentatricopeptide protein SLG1 affects mitochondrial RNA editing, plant development, and responses to abiotic stresses in Arabidopsis

Seed oil content is an important agronomic trait in rapeseed. However, our understanding of the regulatory processes controlling oil accumulation is still limited. Using two rapeseed lines (zy036 and 51070) with contrasting oil content, we found that maternal genotype greatly affects seed oil content. Genetic and physiological evidence indicated that difference in the local and tissue-specific photosynthetic activity in the silique wall (a maternal tissue) was responsible for the different seed oil contents. This effect was mimicked by in planta manipulation of silique wall photosynthesis. Furthermore, the starch content and expression of the important lipid synthesis regulatory gene WRINKLED1 in developing seeds were linked with silique wall photosynthetic activity. 454 pyrosequencing was performed to explore the possible molecular mechanism for the difference in silique wall photosynthesis between zy036 and 51070. Interestingly, the results suggested that photosynthesis-related genes were over-represented in both total silique wall expressed genes and genes that were differentially expressed between genotypes. A potential regulatory mechanism for elevated photosynthesis in the zy036 silique wall is proposed on the basis of knowledge from Arabidopsis. Differentially expressed ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-related genes were used for further investigations. Oil content correlated closely with BnRBCS1A expression levels and Rubisco activities in the silique wall, but not in the leaf. Taken together, our results highlight an important role of silique wall photosynthesis in the regulation of seed oil content in terms of maternal effects.     

The late pollen actins are essential for normal male and female development in Arabidopsis

In angiosperms the late pollen actins (LPAs) are strongly expressed in mature pollen and pollen tubes and at much lower levels in ovules. Four Arabidopsis lines with homozygous knockout mutations in the four individual LPA genes displayed normal flowers, pollen, and seed set. However, when all four LPAs were silenced simultaneously with a single RNA interference (RNAi) construct targeting the 3′UTR of each mRNA, obvious reproductive defects were observed. Western analysis of various Late Pollen actin RNA interference (LPRi) epialleles showed total LPA protein and RNA expression levels were knocked down from 0% to 95% compared to wild-type levels. Reciprocal crosses with the RNAi lines demonstrated that lowered LPA expression was associated with defects in both male and female fertility. Strong epialleles showed significant reductions in normal silique and seed production and were nearly sterile. Dissection of the siliques from moderate LPRi epialleles revealed many unfertilized ovules, increased numbers of aborted seeds, and decreased numbers of healthy seeds. Microscopic analysis of LPRi pollen indicated that the pollen shape and size were normal, but pollen germinated poorly. While multiple LPA genes may have some functional redundancy, the combined expression of multiple LPA genes appears essential to normal male and female reproductive development.     

Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis

Long distance transport of amino acids is mediated by several families of differentially expressed amino acid transporters. The two genes AAP1 and AAP2 encode broad specificity H⁺-amino acid co-transporters and are expressed to high levels in siliques of Arabidopsis, indicating a potential role in supplying the seeds with organic nitrogen. The expression of both genes is developmentally controlled and is strongly induced in siliques at heart stage of embryogenesis, shortly before induction of storage protein genes. Histochemical analysis of transgenic plants expressing promoter-GUS fusions shows that the genes have non-overlapping expression patterns in siliques. AAP1 is expressed in the endosperm and the cotyledons whereas AAP2 is expressed in the vascular strands of siliques and in funiculi. The endosperm expression of AAP1 during early stages of seed development indicates that the endosperm serves as a transient storage tissue for organic nitrogen. Amino acids are transported in both xylem and phloem but during seed filling are imported only via the phloem. AAP2, which is expressed in the phloem of stems and in the veins supplying seeds, may function in uptake of amino acids assimilated in the green silique tissue, in the retrieval of amino acids leaking passively out of the phloem and in xylem-to-phloem transfer along the path. The promoters provide excellent tools to study developmental, hormonal and metabolic control of nitrogen nutrition during development and may help to manipulate the timing and composition of amino acid import into seeds.     

ABNORMAL DEVELOPMENT OF THE SUSPENSOR IN AN EMBRYO-LETHAL MUTANT OF ARABIDOPSIS THALIANA

Developmental arrest of the embryo proper in aborted seeds from mutant 50B, a recessive embryo-lethal mutant of Arabidopsis thaliana, was shown to be followed by abnormal growth of the suspensor. Each of the 12 aborted seeds examined in sectioned material contained an abnormally large suspensor and an embryo proper arrested at a preglobular stage of development. Analysis of serial sections revealed that mutant suspensors contained 15–150 cells whereas wild-type suspensors were composed of only six to eight cells. Development of the mutant endosperm continued to a late nuclear or early cellular stage even in the absence of further development of the embryo proper. These results suggest that the missing gene product in mutant 50B is required for development of the embryo proper but not for continued growth of the suspensor or endosperm tissue. The pattern of abnormal development observed in this mutant provides further evidence that continued growth of the suspensor during normal development is inhibited by the developing embryo proper and that the full developmental potential of cells in the suspensor is expressed only when this inhibitory effect is removed through a mutation or experimental treatment that is lethal only to cells of the embryo proper.     

Distorted segregation and linkage of alcohol dehydrogenase genes in Camellia japonica L. (Theacease)

Alcohol dehydrogenase isozymes in Camellia japonica are encoded by two genes, Adh-1 and Adh-2. Both loci are expressed in seeds, and their products randomly associate into intragenic and intergenic dimers. Electrophoresis of leaf extracts reveals only the products of Adh-2. Formal genetic analysis indicated that the two Adh loci are tightly linked (combined estimate of r=0.004). Most segregations fit expected Mendelian ratios, but in some families distorted segregation was observed at Adh-1, Adh-2, or both loci. The deficient progeny class varied across families, and in two apparent backcrosses three rather than two phenotypic classes were recovered. The mechanism underlying these distortions is not known, but evidence is presented that suggests that the phenomenon is genic or segmental in nature. Plausible hypotheses include linkage of the Adh structural genes with a gametophytic self-incompatibility locus, translocation heterozygosity involving the segment bearing Adh-1 and Adh-2, or a combination of these two mechanisms.     

Mapping genes essential for embryo development in Arabidopsis thaliana.

Summary We have previously isolated and characterized over 90 recessive mutants of Arabidopsis thaliana defective in embryo development. These emb mutants have been shown to differ in lethal phase, extent of abnormal development, and response in culture. We demonstrate in this report the value and efficiency of mapping emb genes relative to visible and molecular markers. Sixteen genes essential for embryo development were mapped relative to visible markers by analyzing progeny of selfed F₁ plants. Embryonic lethals are now the most common type of visible marker included on the linkage map of Arabidopsis. Backcrosses were used in several cases to orient genes relative to adjacent markers. Three genes were located to chromosome arms with telotrisomics by screening for a reduction in the percentage of aborted seeds produced by F₁ plants. A restriction fragment length polymorphism (RFLP) mapping strategy that utilizes pooled EMB/EMB F₂ plants was devised to increase the efficiency of mapping embryonic lethals relative to molecular markers. This strategy was tested by demonstrating that the biol locus of Arabidopsis is within 0.5 cM of an existing RFLP marker. Mapping embryonic lethals with both visible and molecular markers may therefore help to identify large numbers of genes with essential functions in Arabidopsis.     

Nodulin gene expression during soybean (Glycine max) nodule development

In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be divided into two classes according to the time of expression during nodule development. Class A comprises at least 4 nodulin mRNAs which are found when a globular meristem is present in the root cortex. These class A nodulin genes have a transient expression. Class B nodulin genes are expressed when the formation of a nodule structure has been completed. Bradyrhizobium japonicum nod ⁺ fix^-mutants, with large deletions spanning the nif H,DK region, still induced nodules showing normal expression of all nodulin genes, indicating that the nif H,DK region is not involved in the induction of nodulin genes. In nodules induced by Bradyrhizobium japonicum nod ⁺ fix^-mutant HS124 the bacteria are rarely released from the infection thread and the few infected cells appear to be collapsed. All class A and class B nodulin genes are expressed in HS124 nodules with the exception of 5 class B genes.     

Gametophytic expression of genes controlling endosperm development in maize

Summary An indirect approach was adopted to select viable mutants affecting the male gametophytic generation in maize. This approach consists of a selection of endosperm defective mutants followed by a test for gametophytic gene expression, based on the distortion from mendelian segregation and on the measurement of pollen size and pollen sterility. The material used consisted of 34 endosperm defective viable mutants introgressed in B37 genetic background. Complementation tests indicated that the mutation in the collection of mutants affected different genes controlling endosperm development. The study of the segregation in F2 revealed four classes of de (defective endosperm) mutants: (1) mutants in which the mutation does not affect either gametophytic development or function; (2) mutants in which the effect on the gametophyte affects pollen development processes; (3) mutants showing effects on both pollen development and function, and (4) mutants where only pollen tube growth rate is affected. Positive and negative interactions between pollen and style were detected by means of mixed pollination (pollen produced by de/de plants and pollen from an inbred line used as a standard and carrying genes for colored aleurone), on de/de and de/ + plants. Positive interactions were interpreted as methabolic complementation between defective pollen and normal styles.     

A role for LORELEI,a putative glycosylphosphatidylinositol‐anchored protein,in Arabidopsis thaliana double fertilization and early seed development

In plants, double fertilization requires successful sperm cell delivery into the female gametophyte followed by migration, recognition and fusion of the two sperm cells with two female gametes. We isolated a null allele (lre‐5) of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)‐anchored protein implicated in reception of the pollen tube by the female gametophyte. Although most lre‐5 female gametophytes do not allow pollen tube reception, in those that do, early seed development is delayed. A fraction of lre‐5/lre‐5 seeds underwent abortion due to defect(s) in the female gametophyte. The aborted seeds contained endosperm but no zygote/embryo, reminiscent of autonomous endosperm development in the pollen tube reception mutants scylla and sirene. However, unpollinated lre‐5/lre‐5 ovules did not initiate autonomous endosperm development and endosperm development in aborted seeds began after central cell fertilization. Thus, the egg cell probably remained unfertilized in aborted lre‐5/lre‐5 seeds. The lre‐5/lre‐5 ovules that remain undeveloped due to defective pollen tube reception did not induce synergid degeneration and repulsion of supernumerary pollen tubes. In ovules, LORELEI is expressed during pollen tube reception, double fertilization and early seed development. Null mutants of LORELEI‐like‐GPI‐anchored protein 1 (LLG1), the closest relative of LORELEI among three Arabidopsis LLG genes, are fully fertile and did not enhance reproductive defects in lre‐5/lre‐5 pistils, suggesting that LLG1 function is not redundant with that of LORELEI in the female gametophyte. Our results show that, besides pollen tube reception, LORELEI also functions during double fertilization and early seed development.     

In vitro morphogenesis of arrested embryos from lethal mutants of Arabidopsis thaliana

Summary Arrested embryos from lethal (emb) mutants of Arabidopsis thaliana were rescued on a nutrient medium designed to promote plant regeneration from immature wild-type cotyledons. The best response was observed with mutant embryos arrested at the heart to cotyledon stages of development. Embryos arrested at a globular stage produced callus but failed to turn green or form normal shoots in culture. Many of the mutant plants produced in culture were unusually pale with abnormal leaves, rosettes, and patterns of reproductive development. Other plants were phenotypically normal except for the presence of siliques containing 100% aborted seeds following self-pollination. These results demonstrate that genes with essential functions during plant embryo development differ in their pattern of expression at later stages of the life cycle. Most of the 15 genes examined in this study were essential for embryogenesis but were required again for subsequent stages of development. Only EMB24 appeared to be limited in function to embryo development. These differences in the response of mutant embryos in culture may facilitate the classification of embryonic lethals and the identification of genes with developmental rather than housekeeping functions.     

Induction and expression of seed-specific promoters in Arabidopsis embryo-defective mutants

In order to assess the importance of morphogenesis on the induction of promoter markers for storage and Lea programmes, advantage was taken of the emb mutations producing embryos arrested at a wide range of developmental stages in Arabidopsis. These embryos are viable during their stage of developmental arrest and continue to divide further, but apparently without further differentiation into the main organs and tissues of the normal embryos. Eight independent emb mutants arrested in their development prior to the cotyledon stage were selected. These emb embryos lack the normal morphology of the wild-type embryos when the synthesis of storage and Lea proteins are normally initiated. The 2S1-uidA chimeric gene, representative of the maturation programme and the Em 1-uidA chimeric gene, representative of the desiccation programme were introduced by crosses into the emb background. In the eight emb lines, the expression of the GUS reporter gene directed by the 2S1 and Em 1 promoters was observed in the aborted seeds irrespective of their stage of developmental arrest. The time of induction of the expression of both promoters was the same in the arrested embryos as compared with the normal embryos within the same silique. Thus, the activation of these two promoters is triggered by the same signal and can occur in the absence of morphogenesis. However, in the absence of normal organ formation, the expression of the reporter gene under the control of the 2S1 and Em 1 promoters was evident throughout the whole seed tissues. In normal seed development, the hormone abscisic acid (ABA) activates the promoters of the 2S1 and Em 1 genes. One of the important members of the signal transduction pathway of ABA is the ABI3 protein. It has been shown previously that this protein is a prerequisite for the induction of Em 1 by ABA in seeds. A good correlation with the expression of the ABI3 promoter and the 2S1 and Em 1 promoters was found in emb seeds tissues. This observation suggests that the promoters of the 2S1 and the Em 1 genes are expressed in the mutant seeds not at a basal level, but are probably induced by ABA, as in normal seed development.     

Potential sites of bioactive gibberellin production during reproductive growth in Arabidopsis

Gibberellin 3-oxidase (GA3ox) catalyzes the final step in the synthesis of bioactive gibberellins (GAs). We examined the expression patterns of all four GA3ox genes in Arabidopsis thaliana by promoter-beta-glucuronidase gene fusions and by quantitative RT-PCR and defined their physiological roles by characterizing single, double, and triple mutants. In developing flowers, GA3ox genes are only expressed in stamen filaments, anthers, and flower receptacles. Mutant plants that lack both GA3ox1 and GA3ox3 functions displayed stamen and petal defects, indicating that these two genes are important for GA production in the flower. Our data suggest that de novo synthesis of active GAs is necessary for stamen development in early flowers and that bioactive GAs made in the stamens and/or flower receptacles are transported to petals to promote their growth. In developing siliques, GA3ox1 is mainly expressed in the replums, funiculi, and the silique receptacles, whereas the other GA3ox genes are only expressed in developing seeds. Active GAs appear to be transported from the seed endosperm to the surrounding maternal tissues where they promote growth. The immediate upregulation of GA3ox1 and GA3ox4 after anthesis suggests that pollination and/or fertilization is a prerequisite for de novo GA biosynthesis in fruit, which in turn promotes initial elongation of the silique.