LEAFY COTYLEDON1 Is an Essential Regulator of Late Embryogenesis and Cotyledon Identity in Arabidopsis

LEAFY COTYLEDON1 (LEC1) is an embryo defective mutation that affects cotyledon identity in Arabidopsis. Mutant cotyledons possess trichomes that are normally a leaf trait in Arabidopsis, and the cellular organization of these organs is intermediate between that of cotyledons and leaves from wild-type plants. We present several lines of evidence that indicate that the control of late embryogenesis is compromised by the mutation. First, mutant embryos are desiccation intolerant, yet embryos can be rescued before they dry to yield homozygous recessive plants that produce defective embryos exclusively. Second, although many genes normally expressed during embryonic development are active in the mutant, at least one maturation phase-specific gene is not activated. Third, the shoot apical meristem is activated precociously in mutant embryos. Fourth, in mutant embryos, several genes characteristic of postgerminative development are expressed at levels typical of wild-type seedlings rather than embryos. We conclude that postgerminative development is initiated prematurely and that embryonic and postgerminative programs operate simultaneously in mutant embryos. The pleiotropic effects of the mutation indicate that the LEC1 gene plays a fundamental role in regulating late embryogenesis. The role of LEC1 and its relationship to other genes involved in controlling late embryonic development are discussed.     

Cell wall glucomannan in Arabidopsis is synthesised by CSLA glycosyltransferases, and influences the progression of embryogenesis

Mannans are hemicellulosic polysaccharides that have previously been implicated as structural constituents of cell walls and as storage reserves but which may serve other functions during plant growth and development. Several members of the Arabidopsis cellulose synthase-like A (CSLA) family have previously been shown to synthesise mannan polysaccharides in vitro when heterologously expressed. It has also been found that CSLA7 is essential for embryogenesis, suggesting a role for the CSLA7 product in development. To determine whether the CSLA proteins are responsible for glucomannan synthesis in vivo , we characterised insertion mutants in each of the nine Arabidopsis CSLA genes and several double and triple mutant combinations. csla9 mutants showed substantially reduced glucomannan, and triple csla2csla3csla9 mutants lacked detectable glucomannan in stems. Nevertheless, these mutants showed no alteration in stem development or strength. Overexpression of CSLA2, CSLA7 and CSLA9 increased the glucomannan content in stems. Increased glucomannan synthesis also caused defective embryogenesis, leading to delayed development and occasional embryo death. The embryo lethality of csla7 was complemented by overexpression of CSLA9 , suggesting that the glucomannan products are similar. We conclude that CSLA2, CSLA3 and CSLA9 are responsible for the synthesis of all detectable glucomannan in Arabidopsis stems, and that CSLA7 synthesises glucomannan in embryos. These results are inconsistent with a substantial role for glucomannan in wall strength in Arabidopsis stems, but indicate that glucomannan levels affect embryogenesis. Together with earlier heterologous expression studies, the glucomannan deficiency observed in csla mutant plants demonstrates that the CSLA family encodes glucomannan synthases.     

The auxin-insensitive bodenlos mutation affects primary root formation and apical-basal patterning in the Arabidopsis embryo

In Arabidopsis embryogenesis, the primary root meristem originates from descendants of both the apical and the basal daughter cell of the zygote. We have isolated a mutant of a new gene named BODENLOS (BDL) in which the primary root meristem is not formed whereas post-embryonic roots develop and bdl seedlings give rise to fertile adult plants. Some bdl seedlings lacked not only the root but also the hypocotyl, thus resembling monopteros (mp) seedlings. In addition, bdl seedlings were insensitive to the auxin analogue 2,4-D, as determined by comparison with auxin resistant1 (axr1) seedlings. bdl embryos deviated from normal development as early as the two-cell stage at which the apical daughter cell of the zygote had divided horizontally instead of vertically. Subsequently, the uppermost derivative of the basal daughter cell, which is normally destined to become the hypophysis, divided abnormally and failed to generate the quiescent centre of the root meristem and the central root cap. We also analysed double mutants. bdl mp embryos closely resembled the two single mutants, bdl and mp, at early stages, while bdl mp seedlings essentially consisted of hypocotyl but did form primary leaves. bdl axr1 embryos approached the mp phenotype at later stages, and bdl axr1 seedlings resembled mp seedlings. Our results suggest that BDL is involved in auxin-mediated processes of apical-basal patterning in the Arabidopsis embryo.     

An expanded role for the TWN1 gene in embryogenesis: defects in cotyledon pattern and morphology in the twn1 mutant of Arabidopsis (Brassicaceae)

The suspensor is a specialized basal structure that differentiates early in plant embryogenesis to support development of the embryo proper. Suspensor differentiation in Arabidopsis is maintained in part by the TWIN1 (TWN1) gene, which suppresses embryogenic development in suspensor cells: twn1 mutants produce supernumerary embryos via suspensor transformation. To better understand mechanisms of suspensor development and further investigate the function of TWN1, we have characterized late-embryo and post-embryonic development in the twn1 mutant, using seedling culture, microscopy, and genetics. We report here that the twn1 mutation disrupts cotyledon number, arrangement, and morphology and occasionally causes partial conversion of cotyledons into leaves. These defects are not a consequence of suspensor transformation. Thus, in addition to its basal role in suspensor differentiation, TWN1 influences apical pattern and morphology in the embryo proper. To determine whether other genes can similarly affect both suspensor and cotyledon development, we looked for twinning in Arabidopsis mutants previously identified by their abnormal cotyledon phenotypes. One such mutant, amp1, produced a low frequency of twin embryos by suspensor transformation. Our results suggest that mechanisms that maintain suspensor identity also function later in development to influence organ formation at the embryonic shoot apex. We propose that TWN1 functions in cell communication pathways that convey local positional information in both the apical and basal regions of the Arabidopsis embryo.     

Condensin and cohesin knockouts in Arabidopsis exhibit a titan seed phenotype

The titan (ttn) mutants of Arabidopsis exhibit striking alterations in chromosome dynamics and cell division during seed development. Endosperm defects include aberrant mitoses and giant polyploid nuclei. Mutant embryos differ in cell size, morphology and viability, depending on the locus involved. Here we demonstrate that three TTN genes encode chromosome scaffold proteins of the condensin (SMC2) and cohesin (SMC1 and SMC3) classes. These proteins have been studied extensively in yeast and animal systems, where they modulate chromosome condensation, chromatid separation, and dosage compensation. Arabidopsis contains single copies of SMC1 and SMC3 cohesins. We used forward genetics to identify duplicate T-DNA insertions in each gene. These mutants (ttn7 and ttn8) have similar titan phenotypes: giant endosperm nuclei and arrested embryos with a few small cells. A single SMC2 knockout (ttn3) was identified and confirmed by molecular complementation. The weak embryo phenotype observed in this mutant may result from expression of a related gene (AtSMC2) with overlapping functions. Further analysis of titan mutants and the SMC gene family in Arabidopsis should provide clues to chromosome mechanics in plants and insights into the regulation of nuclear activity during endosperm development.     

Synergistic repression of the embryonic programme by SET DOMAIN GROUP 8 and EMBRYONIC FLOWER 2 in Arabidopsis seedlings

The seed maturation programme occurs only during the late phase of embryo development, and repression of the maturation genes is pivotal for seedling development. However, mechanisms that repress the expression of this programme in vegetative tissues are not well understood. A genetic screen was performed for mutants that express maturation genes in leaves. Here, it is shown that mutations affecting SDG8 (SET DOMAIN GROUP 8), a putative histone methyltransferase, cause ectopic expression of a subset of maturation genes in leaves. Further, to investigate the relationship between SDG8 and the Polycomb Group (PcG) proteins, which are known to repress many developmentally important genes including seed maturation genes, double mutants were made and formation of somatic embryos was observed on mutant seedlings with mutations in both SDG8 and EMF2 (EMBRYONIC FLOWER 2). Analysis of histone methylation status at the chromatin sites of a number of maturation loci revealed a synergistic effect of emf2 and sdg8 on the deposition of the active histone mark which is the trimethylation of Lys4 on histone 3 (H3K4me3). This is consistent with high expression of these genes and formation of somatic embryos in the emf2 sdg8 double mutants. Interestingly, a double mutant of sdg8 and vrn2 (vernalization2), a paralogue of EMF2, grew and developed normally to maturity. These observations demonstrate a functional cooperative interplay between SDG8 and an EMF2-containing PcG complex in maintaining vegetative cell identity by repressing seed genes to promote seedling development. The work also indicates the functional specificities of PcG complexes in Arabidopsis.     

Sulfurtransferases 1 and 2 play essential roles in embryo and seed development in Arabidopsis thaliana

Sulfurtransferases (STRs) catalyze the transfer of a sulfur atom from a donor to a suitable acceptor molecule. The Arabidopsis thaliana genome encodes 20 putative STR proteins. The biological functions of most are unclear. We found that STR1 and STR2 play important roles in embryo/seed development. Mutation of STR1 alone resulted in a shrunken seed phenotype, although growth and development of vegetative and reproductive organs were not affected. The shrunken seed phenotype was associated with the delayed/arrested embryo development, in most cases, at the heart stage. The embryo defect of str1 mutant is not fully penetrant. Approximately 12.5% of embryos developed further and formed normal looking seeds. In severely shrunken seeds, no embryo could be identified after seed collection. Partially shrunken seeds that contained viable embryos could still germinate. However, cotyledons of the seedlings from such seeds were abnormal. An STR1-GUS fusion reporter revealed that the STR1 gene was universally expressed, with high levels of expression in specific tissues/organs including embryos. The incomplete penetrance of str1 embryo/seed phenotype is a result of functional STR2. Single str2 mutant had no phenotype. However, no str1(-/-)/str2(-/-) double mutant embryos were able to develop past the heart stage. Furthermore, STR2 is haplo-insufficient in str1 mutant background, and str1(-/-)/str2(+/-) embryos were 100% lethal. These data provide new insights into the biological functions of the ubiquitous sulfurtransferase in Arabidopsis embryogenesis and seed development.     

Genetic Characterization of Tube and Pelle, Genes Required for Signaling between Toll and Dorsal in the Specification of the Dorsal-Ventral Pattern of the Drosophila Embryo

tube and pelle are two of the maternally transcribed genes required for dorsal-ventral patterning of the Drosophila embryo. Females homozygous for strong alleles of tube or pelle produce embryos that lack all ventral and lateral embryonic pattern elements. By analyzing the phenotypes caused by 24 pelle and 9 tube alleles, we have defined characteristic features of the two genes, including the extremely variable phenotypes of a number of tube alleles and the antimorphic character of a number of pelle alleles. Double mutant females carrying dominant ventralizing alleles of Toll and dorsalizing alleles of tube or pelle produce dorsalized embryos, suggesting that tube and pelle act downstream of the membrane protein Toll in the signaling pathway that defines the embryonic dorsal-ventral pattern. Both tube and pelle are also important zygotically for survival: at least 30% of the zygotes lacking either tube or pelle die before adult stages, while 90-95% of tube(-) pelle(-) double mutant zygotes die. We discuss the phenotypes of tube-pelle double mutants in the context of whether the two proteins interact directly.     

Maturation of arabidopsis seeds is dependent on glutathione biosynthesis within the embryo

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, gamma-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoterbeta-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.     

The Arabidopsis histidine phosphotransfer proteins are redundant positive regulators of cytokinin signaling

Arabidopsis thaliana histidine phosphotransfer proteins (AHPs) are similar to bacterial and yeast histidine phosphotransfer proteins (HPts), which act in multistep phosphorelay signaling pathways. A phosphorelay pathway is the current model for cytokinin signaling. To assess the role of AHPs in cytokinin signaling, we isolated T-DNA insertions in the five AHP genes that are predicted to encode functional HPts and constructed multiple insertion mutants, including an ahp1,2,3,4,5 quintuple mutant. Single ahp mutants were indistinguishable from wild-type seedlings in cytokinin response assays. However, various higher-order mutants displayed reduced sensitivity to cytokinin in diverse cytokinin assays, indicating both a positive role for AHPs in cytokinin signaling and functional overlap among the AHPs. In contrast with the other four AHPs, AHP4 may play a negative role in some cytokinin responses. The quintuple ahp mutant showed various abnormalities in growth and development, including reduced fertility, increased seed size, reduced vascular development, and a shortened primary root. These data indicate that most of the AHPs are redundant, positive regulators of cytokinin signaling and affect multiple aspects of plant development.     

Cell division events are essential for embryo patterning and morphogenesis: studies on dominant-negative cdc2aAt mutants of arabidopsis

During plant development, cell division events are coordinately regulated, leading to specific growth patterns. Experimental evidence indicates that the morphogenetic controls that act at the vegetative plant growth stage are flexible and tolerate distortions in patterns and frequencies of cell division. To address questions concerning the relationship between cell division and embryo formation, a novel experimental approach was used. The frequencies of cell division were reduced exclusively during embryo development of Arabidopsis by the expression of a dominant cdc2a mutant. The five independent transgenic lines with the highest levels of the mutant cdc2a affected embryo formation. In the C13 line, seeds failed to germinate. The C1, C5 and C12 lines displayed a range of distortions on the apical-basal embryo pattern. In the C3 line, the shoot apical meristem of the seedlings produced leaves defective in growth and with an incorrect phyllotactic pattern. The results demonstrate that rates of cell division do not dictate cellular differentiation of embryos. Nevertheless, whereas cell divisions are uncoupled from vegetative development, they are instrumental in elaborating embryo structures and modulating embryo and seedling morphogenesis.     

Mutation of E1-CONJUGATING ENZYME-RELATED1 decreases RELATED TO UBIQUITIN conjugation and alters auxin response and development

The ubiquitin-like protein RELATED TO UBIQUITIN (RUB) is conjugated to CULLIN (CUL) proteins to modulate the activity of Skp1-Cullin-F-box (SCF) ubiquitylation complexes. RUB conjugation to specific target proteins is necessary for the development of many organisms, including Arabidopsis (Arabidopsis thaliana). Here, we report the isolation and characterization of e1-conjugating enzyme-related1-1 (ecr1-1), an Arabidopsis mutant compromised in RUB conjugation. The ecr1-1 mutation causes a missense change located two amino acid residues from the catalytic site cysteine, which normally functions to form a thioester bond with activated RUB. A higher ratio of unmodified CUL1 relative to CUL1-RUB is present in ecr1-1 compared to wild type, suggesting that the mutation reduces ECR1 function. The ecr1-1 mutant is resistant to the auxin-like compound indole-3-propionic acid, produces fewer lateral roots than wild type, displays reduced adult height, and stabilizes a reporter fusion protein that is degraded in response to auxin, suggesting reduced auxin signaling in the mutant. In addition, ecr1-1 hypocotyls fail to elongate normally when seedlings are grown in darkness, a phenotype shared with certain other RUB conjugation mutants that is not general to auxin-response mutants. The suite of ecr1-1 molecular and morphological phenotypes reflects roles for RUB conjugation in many aspects of plant growth and development. Certain ecr1-1 elongation defects are restored by treatment with the ethylene-response inhibitor silver nitrate, suggesting that the short ecr1-1 root and hypocotyl result from aberrant ethylene accumulation. Further, silver nitrate supplementation in combination with various auxins and auxin-like compounds reveals that members of this growth regulator family may differentially rely on ethylene signaling to inhibit root growth.     

gurke and pasticcino3 mutants affected in embryo development are impaired in acetyl-CoA carboxylase

Normal embryo development is required for correct seedling formation. The Arabidopsis gurke and pasticcino3 mutants were isolated from different developmental screens and the corresponding embryos exhibit severe defects in their apical region, affecting bilateral symmetry. We have recently identified lethal acc1 mutants affected in acetyl-CoA carboxylase 1 (ACCase 1) that display a similar embryo phenotype. A series of crosses showed that gk and pas3 are allelic to acc1 mutants, and direct sequencing of the ACC1 gene revealed point mutations in these new alleles. The isolation of leaky acc1 alleles demonstrated that ACCase 1 is essential for correct plant development and that mutations in ACCase affect cellular division in plants, as is the case in yeast. Interestingly, significant metabolic complementation of the mutant phenotype was obtained by exogenous supply of malonate, suggesting that the lack of cytosolic malonyl-CoA is likely to be the initial factor leading to abnormal development in the acc1 mutants.     

The role of ABI3 and FUS3 loci in Arabidopsis thaliana on phase transition from late embryo development to germination

Arabidopsis abi3 and fus3 mutants are defective in late embryo development and their embryos show precocious growth. To understand the function and role of ABI3 and FUS3, we analyzed expression patterns of genes which were normally activated during late embryo development and germination in these mutants. Using the differential display method, both upregulated and downregulated genes were observed in immature siliques of the abi3 fus3 double mutant. Four clones having more abundant expression in the abi3 fus3 double mutant than in wild type were isolated. These genes were activated during wild-type germination, suggesting that some genes that are activated during wild-type germination are precociously activated in the abi3 fus3 mutant during late embryo development. Also, genes that were activated during wild-type germination were isolated and their expression patterns during late embryo development in the wild type and in abi3, fus3, and abi3 fus3 mutants were analyzed. Sixteen such clones were found, and 11 of these showed derepression or precocious activation of gene expression in the mutants. These results indicate that ABI3 and FUS3 negatively regulate a particular set of genes during late embryo development. We also showed that immature fus3 siliques accumulated one-third of the wild-type level of abscisic acid (ABA), but mature fus3 siliques accumulated ABA at a level comparable to that in the wild type. The possible mechanisms of controlling developmental timing in late embryo development as well as collaborative and distinct roles of ABI3 and FUS3 are discussed.     

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     

Late embryo-defective mutants of Arabidopsis

The embryo-defective (emb) mutants of Arabidopsis constitute a large and diverse group of mutants disrupted in a broad range of embryonic processes, including morphogonesis, cell differentiation, and maturation programs. This report describes a subset of these mutants, the late embryo defectives, which develop beyond the globular stage of embryogenesis but fail to complete normal morphogenesis. A representative sample of 12 late mutants was chosen for this study, patterns of morphogenesis were characterized, the germination potential of mutant seeds was investigated, and additional mutant alleles within the collection were identified. Morphological defects in mutant embryos became apparent during the heart stage of development, when embryos normally begin the rapid cell division and expansion required for the completion of morphogenesis. Despite their morphological abnormalities, mutant embryos often germinated from dry seed, demonstrating that genetic programs required for the establishment of desiccation tolerance remained intact. Mutant seedlings displayed a wide range of developmental abnormalities, including altered morphology, lack of pigmentation, dwarfism, and disorganized vegetative growth. One late mutant was found to be allelic to an early embryo defective that arrests at the globular stage. These results suggest that a number of late EMB genes encode basic cellular and metabolic functions needed for cell division, enlargement, and embryonic growth. The rapid growth and metabolic changes that occur at the heart stage may present a barrier to normal development in the late mutants, resulting in altered embryo morphology and other developmental defects. It is proposed that many Arabidopsis mutants with abnormal embryo and seedling morphology are not defective in the regulation of pattern formation or morphogenesis, but rather in fundamental physiological and cellular processes required for the completion of normal growth and development. © 1995 Wiley-Liss, Inc.