LEAFY Interacts with Floral Homeotic Genes to Regulate Arabidopsis Floral Development.

In the leafy mutant of Arabidopsis, most of the lateral meristems that are fated to develop as flowers in a wild-type plant develop as inflorescence branches, whereas a few develop as abnormal flowers consisting of whorls of sepals and carpels. We have isolated several new alleles of leafy and constructed a series of double mutants with leafy and other homeotic mutants affecting floral development to determine how these genes interact to specify the developmental fate of lateral meristems. We found that leafy is completely epistatic to pistillata and interacts additively with agamous in early floral whorls, whereas in later whorls leafy is epistatic to agamous. Double mutants with leafy and either apetala1 or apetala2 showed a complete loss of the whorled phyllotaxy, shortened internodes, and suppression of axillary buds typical of flowers. Our results suggest that the products of LEAFY, APETALA1, and APETALA2 together control the differentiation of lateral meristems as flowers rather than as inflorescence branches.

The evolution of plant architecture.

The vascular plants have evolved from a simple body plan that has diversified into the vast array of architectures seen in plants today. Much architectural diversity results from the varied growth patterns of apical and axillary meristems. Current research is showing that meristem growth patterns are regulated genetically and hormonally, and the genes that control these processes are being identified and characterized.

1 L-myo-Inositol 1-Phosphate Synthase from Arabidopsis thaliana.

A recombinant phage containing an Arabidopsis thaliana cDNA sequence encoding a protein with 1L-myo-inositol 1-phosphate synthase (EC 5.5.1.4) activity has been isolated and used for transcriptional and translational studies. The identification of the recombinant phage relied on the observations that (a) the clone complements a mutation in the structural gene for 1L-myo-inositol 1-phosphate synthase in the yeast Saccharomyces cerevisiae, (b) the in vitro synthesized polypeptide enzymatically converts glucose 6-phosphate into inositol 1-phosphate, (c) in vitro transcription and translation of this cDNA sequence produces a polypeptide that is recognized by anti-yeast myo-inositol 1-phosphate synthase antiserum, and (d) inositol regulates the expression of the corresponding gene in Arabidopsis.

Cloning and characterization of an esophageal-gland-specific chorismate mutase from the phytoparasitic nematode Meloidogyne javanica.

Root-knot nematodes are obligate plant parasites that alter plant cell growth and development by inducing the formation of giant feeder cells. It is thought that nematodes inject secretions from their esophageal glands into plant cells while feeding, and that these secretions cause giant cell formation. To elucidate the mechanisms underlying the formation of giant cells, a strategy was developed to clone esophageal gland genes from the root-knot nematode Meloidogyne javanica. One clone, shown to be expressed in the nematode's esophageal gland, codes for a potentially secreted chorismate mutase (CM). CM is a key branch-point regulatory enzyme in the shikimate pathway and converts chorismate to prephenate, a precursor of phenylalanine and tyrosine. The shikimate pathway is not found in animals, but in plants, where it produces aromatic amino acids and derivative compounds that play critical roles in growth and defense. Therefore, we hypothesize that this CM is involved in allowing nematodes to parasitize plants.

Effect of lateral suppressor on petal initiation in tomato.

Flowers developing on tomato (Lycopersicon esculentum) plants homozygous for the lateral suppressor (ls) mutation lack petals. Scanning electron micrographs revealed that in ls plants no second whorl organs were initiated. The initiation of first, third, and fourth whorl organs were unaffected by this mutation. To investigate interactions between the cells in different layers of the floral meristem during organ initiation, a periclinal chimera between wild-type and ls tomato was generated. Flowers of the chimera having ls cells in the outer meristem layer (L1) and wild-type cells in internal layers (L2 and L3) developed normally, including the initiation of organ primordia that differentiated as petals in normal positions within the second whorl. L1 of the chimera developed in a non-autonomous manner during petal development. Thus, wild-type cells occupying the internal meristem layers provided developmental cues necessary for initiation of petal primordia at appropriate positions on the floral meristem. L1 cells carrying the lateral suppressor mutation were fully capable of responding to this information and differentiated appropriately.

Purification of an endopeptidase involved with storage-protein degradation in Phaseolus vulgaris L. cotyledons.

Phaseolin, the major seed storage protein of Phaseolus vulgaris L., is degraded in the cotyledons in the first 7-10 d following seed germination. We assayed cotyledon extracts for protease activity by using [(3)H]phaseolin as a substrate and then fractionated the digestion mixtures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in order to identify the cleavage products. The cotyledons of 4-d-old seedlings contain an endopeptidase which cleaves the polypeptides of [(3)H]phaseolin (apparent molecular weights=51 000, 48 000, 46 000 and 43 000) into three discrete clusters of proteolytic fragments (M rs=27 000, 25 000 and 23 000). Endopeptidase activity is not detected in the cotyledons until the protein content of these organs starts to decline, shortly after the first day of seedling growth. Endopeptidase activity increases to a maximum level in the cotyledons of 5-d-old seedlings and then declines to a minimum value by day 10. The enzyme was purified 335-fold by ammonium-sulfate precipitation, organomercurial-agarose chromatography, gel filtration and ion-exchange chromatography. The endopeptidase constitutes 0.3% of the protein content in the cotyledons of 4-d-old seedlings. It is a cysteine protease with a single polypeptide chain (M r=30 000). Optimum hydrolysis of [(3)H]phaseolin occurs at pH 5. The enzyme is irreversibly inactivated at pH values above 7 and at temperatures above 45° C. The endopeptidase attacks only a limited number of peptide bonds in [(3)H]phaseolin, without causing any appreciable change in the native molecular weight of the storage protein. The endopeptidase is also able to hydrolyze the bean-seed lectin, phytohemagglutinin. Thus, this enzyme may play a general role in degrading cotyledon proteins of P. vulgaris following seed germination.

Patterns of protein synthesis in dormant and growing vegetative buds of pea.

Lateral buds on intact pea plants (Pisum sativum L. cv. Alaska) remain dormant until they are stimulated to develop by decapitating the terminal bud. Using two-dimensional gel electrophoresis, we have examined the protein content of terminal and lateral buds from intact plants and from plants at various times after decapitation. Silver-staining and in-vivo-labeling demonstrated very different sets of proteins. The level of expression of 18 stained and 25 labeled proteins was altered when growth was stimulated; this represents 3.4% and 9.1% of the total proteins detected by each method, respectively. Within 24 h of being stimulated, lateral buds doubled in length and their protein content was qualitatively nearly the same as that of terminal buds. Six hours after decapitation, before the onset of detectable growth, the overall pattern of protein synthesis in lateral buds was more like that of growing lateral buds or of terminal buds than that of dormant lateral buds. Direct application of N(6)-furfurylaminopurine (kinetin) to buds on intact plants stimulated their growth and resulted in the same pattern of protein synthesis as did decapitation. Inhibition of bud growth by addition of indole-3-acetic acid to the stumps of decapitated plants resulted in the synthesis of dormancy-related proteins. Lateral buds at all stages of development incorporated labeled amino acids at similar rates, indicating that metabolic activity is not a component of dormancy in these buds.

Regulation of chlorophyll and Rubisco levels in embryonic cotyledons of Phaseolus vulgaris.

While deep within the maternal tissues (pods and testa), cotyledons of the bean (Phaseolus vulgaris L.) green and the plastids differentiate as chloroplasts. At the time of seed maturation the chloroplasts dedifferentiate and the green color is lost. We have used Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and chlorophyll to study chloroembryo development. Chlorophyll levels and Rubisco activity increase early in embryonic development then decline as the cotyledons enter the maturation phase. Rubisco accumulation follows a strong temporal pattern over the course of embryo development, and furthermore, occurs in total darkness. Therefore, accumulation of Rubisco during embryogenesis may occur in response to developmental signals. In embryos developed in total darkness, Rubisco accumulation was uncoupled from chlorophyll accumulation. Exposure of isolated cotyledons to abscisic acid (ABA) resulted in loss of chlorophyll and decline in Rubisco levels comparable to those seen in normal embryogenesis. This indicates that the decline in Rubisco in chloroembryos in vivo results from factors such as ABA that signal the onset of maturation. The results show that ABA not only enhances the accumulation of some proteins (e.g. storage proteins), but also depresses the accumulation of others during embryogeny.

Cell lineage patterns in the shoot meristem of the sunflower embryo in the dry seed.

We mapped the fate of cells in the shoot meristem of the dry-seed embryo of sunflower, Helianthus annuus L. cv. Peredovic, using irradiation-induced somatic sectors. We analyzed 249 chlorophyll-deficient or glabrous (hairless) sectors generated in 236 plants. Most sectors observed in the inflorescence extended into vegetative nodes. Thus cell lineages that ultimately gave rise to reproductive structures also contributed to vegetative structures. No single sector extended the entire length of the shoot. Thus the shoot is not derived from one or a few apical initials. Rather, the position, vertical extent, and width of the sectors at different levels of the shoot suggest that the shoot is derived from three to four circumferential populations of cells in each of three cell layers of the embryo meristem. Sectors had no common boundaries even in plants with two or three independent sectors, but varied in extent and overlapped along the length of the shoot. Thus individual cells in a single circumferential population behaved independently to contribute lineages of different vertical extents to the growing shoot. The predicted number of circumferential populations of cells as well as the apparent cell number in each population was consistent with the actual number of cells in the embryo meristem observed in histological sections.

Morphogenesis in selaginella: auxin transport in the stem.

Selaginella willdenovii Baker is a prostrate vascular cryptogam with a dorsiventral stem. At each major branching of the stem apex a dorsal and a ventral angle meristem is formed. The ventral meristem becomes determined as a root, and the dorsal meristem as a shoot. The present investigation examined the distribution and transport of (14)C-indoleacetic acid through stem tissues as a basis for the pattern of meristem determination. Externally applied indoleacetic acid is transported into receiver blocks with a velocity of 12 millimeters per hour. Much of the auxin becomes immobilized in the tissue and is not transported. The polar ratio of auxin transport is approximately 2. Auxin is transported equally on the dorsal and the ventral sides of the stem axis, and the auxin flux in vascular tissue is twice that in the cortex. In the branch junctions twice as much auxin is transported on the dorsal side as on the ventral side, and this is held to be the consequence of the lateral branch vascular tissue connecting with the dorsal and median, but not with the ventral vascular strand of the stem axis.

Expression of a ribosomal protein gene in axillary buds of pea seedlings.

Axillary buds of intact pea seedlings (Pisum sativum L. cv Alaska) do not grow and are said to be dormant. Decapitation of the terminal bud promotes the growth of these axillary buds, which then develop in the same manner as terminal buds. We previously showed that unique sets of proteins are expressed in dormant and growing buds. Here we describe the cloning, sequencing, and expression of a cDNA clone (pGB8) that is homologous to ribosomal protein L27 from rat. RNA corresponding to this clone increases 13-fold 3 h after decapitation, reaches a maximum enhancement of about 35-fold after 12 h, and persists at slightly reduced levels at later times. Terminal buds, root apices, and elongating internodes also contain pGB8 mRNA but fully expanded leaflets and fully elongated internodes do not. In situ hybridization analysis demonstrates that pGB8 mRNA increases in all parts of the bud within 1 h of decapitation. Under appropriate conditions, growing buds can be made to stop growing and become dormant; these buds subsequently can grow again. Therefore, buds have the capacity to undergo multiple cycles of growth and dormancy. RNA gel blots show that pGB8 expression is reduced to dormancy levels as soon as buds stop growing. However, in situ hybridization experiments show that pGB8 expression continues at growing-bud levels in the apical meristem for 2 d after it is reduced in the rest of the bud. When cultured stems containing buds are treated with indoleacetic acid at concentrations >/=10 mum, bud growth and expression of pGB8 in the buds are inhibited.

Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro.

Immature embryos of Brassica napus were cultured in vitro with and without various concentrations of germination inhibitors, and the progress of embryogeny was monitored by comparing accumulation of storage proteins in culture with the normal accumulation in seeds. The two major B. napus storage proteins (12S and 1.7S) were purified from seed extracts and analyzed by rocket immunoelectrophoresis (12S protein) or by sodium lauryl sulfate polyacrylamide gel electrophoresis (1.7S protein). During embryo development within seeds both the 12S and 1.7S proteins were first detected when the cotyledons were well developed (embryo dry weight, 0.4 mg), and each storage protein accumulated at an average rate of 26 µg d(-1) during maximum deposition. Accumulation of the 1.7S protein stopped when the water content of the embryo began to decline (embryo DW, 2.7 mg), but accumulation of the 12S protein continued until seed maturity (embryo DW, 3.6 mg). At the end of embryo development the 12S and the 1.7S proteins comprised approx. 60 and 20% of the total salt-soluble protein, respectively. When embryos were removed from seeds at day 27, just as storage protein was starting to accumulate, and placed in culture on a basal medium, they precociously germinated within 3d, and incorporation of amino acids into the 12S storage protein dropped from 3% of total incorporation to less than 1%. If 10(-6) M abscisic acid (ABA) was included in the medium, amino-acid incorporation into the 12S protein increased from 3% of total incorporation when embryos were placed into culture to 18%, 5d later, and the accumulation rate (27.1±2.6 µg embryo(-1) d(-1)) matched the maximum rate observed in the seed. High osmotica, such as 0.29 M sucrose or mannitol, added to the basal medium, also inhibited precocious germination, but there was a lag period before 12S-protein synthesis rates equaled the rates on ABA media. These results indicate that some factor in the seed environment is necessary for storage-protein synthesis to proceed, and that ABA is a possible candidate.

Isolation of abscisic acid-resistant variants from tobacco cell cultures : I. Physiological bases for selection.

The goal of this work was to begin a genetic study of the molecular mode of action of abscisic acid (ABA), by isolating variant cultured cells resistant to the hormone, or to a factor which induces ABA synthesis, namely water stress. Cell cultures of Nicotiana tabacum L. cv. Wisconsin 38 and N. silvestris Speg. and Comes were chosen as the experimental materials. Studies of the effects of the two stresses on the growth of the cultures demonstrated that ABA or water stress imposed by mannitol could completely inhibit growth. These effects arose in both cases from a constant reduction of the growth rate of the cells throughout the culture period. Mannitol also induced an increase in ABA content of the cells and media of suspension cultures, although not to the concentrations required to achieve the same degree of growth inhibition when the hormone was applied exogenously.

Isolation of abscisic acid-resistant variants from tobacco cell cultures : II. Selection and characterization of variants.

Variant clones were isolated from Nicotiana silvestris Speg. et Comes cell cultures at low frequencies following severe abscisic-acid (ABA) or mannitol-induced water-stress treatments of plated cells. N. tabacum L. variants were not recovered. Variants from the ABA selection experiments exhibited a 10-fold increase in resistance to the hormone. This trait was stable in non-selective conditions for as long as was tested (200 days), but did not alter the response of the cells to water stress. Cell lines from the waterstress selection were not more resistant to mannitol than the parent line, and had a wide range of response to ABA.

The developmental morphology and growth dynamics of the tobacco leaf.

The developmental morphology and growth dynamics of the leaf of Nicotiana tabacum L. cv. Xanthi Nc. are described. Epidermal and internal cell patterns indicate that the leaf axis arises from approx. 100 cells in four layers of the shoot apex, while the lamina arises from several rows of cells in each of three layers of the leaf axis. Cell patterns at the apex and margin of the leaf do not support the classical view that these regions have a specialized meristematic function. Instead the development of the leaf appears to be largely dependent on intercalary growth. The pattern of growth within the lamina is surprisingly complex. In addition to a proximal-distal gradient in the duration of growth and cell division during development, localized transitory changes in the rate of these processes also occur. These observations are discussed in reference to previous discriptions of leaf development in tobacco.

The cellular parameters of leaf development in tobacco: a clonal analysis.

The cellular parameters of leaf development in tobacco (Nicotiana tabacum L.) have been characterized using clonal analysis, an approach that provides unequivocal evidence of cell lineage. Our results indicate that the tobacco leaf arises from a group of around 100 cells in the shoot apical meristem. Each of these cells contributes to a unique longitudinal section of the axis and transverse section of the lamina. This pattern of cell lincage indicates that primordial cells contribute more or less equally to the growth of the axis, in contrast to the more traditional view of leaf development in which the leaf is pictured as arising from a group of apical initials. Clones induced prior to the initiation of the lamina demonstrate that the subepidermal layer of the lamina arises from at least six files of cells. Submarginal cells usually divide with their spindles parallel to the margin, and therefore contribute relatively little to the transverse expansion of the lamina. During the expansion of the lamina the orientation and frequency of cell division are highly regulated, as is the duration of meristematic growth. Initially, cell division is polarized so as to produce lineages that are at an oblique angle to the midrib; later cell division is in alternating perpendicular planes. The distribution of clones generated by irradiation at various stages of development indicates that cell division ceases at the tip of the leaf when the leaf is about one tenth its final size, and then ceases in progressively more basal regions of the lamina. Variation in the mutation frequency within the lamina reflects variation in the frequency of mitosis. Prior to the mergence of the leaf the frequency of mutation is maximal near the tip of the leaf and extremely low at its base; after emergence, the frequency of mutation increases at the base of the leaf. In any given region of the lamina the frequency of mutation is highest in interveinal regions, and is relatively low near the margin. Thus, both the orientation and frequency of cell division at the leaf margin indicate that this region plays a minor role in the growth of the lamina.

Genomic amplification in the cotyledon parenchyma of common bean.

The cotyledon parenchyma cells of common bean (Phaseolus vulgaris L.) produce large quantities of storage proteins during embryo maturation; throughout this period, these cells also accumulate nuclear DNA (nDNA). To investigate the basis of this nDNA accumulation, we have measured storage protein mRNA pools, nDNA mass, and gene copy number at specific stages of cotyledon development. RNA blotting and hybridization show that transcripts encoding the major embryo-specific storage proteins are present very early in cotyledon development, accumulate in coordinate fashion to peak during mid-maturation, and fall in abundance prior to the onset of dormancy. During this same period, nDNA mass per parenchyma cell nucleus, as measured by Feulgen microspectrophotometry, increases from 2C-4C to about 64C (C being the haploid germ cell genomic complement). The nDNA values do not cluster around integral multiples of the diploid 2C amount. DNA blotting and hybridization are used to evaluate the relative representations of different classes of the bean genome in DNA samples isolated from vegetative tissues, from cotyledons beginning to accumulate storage proteins, and from cotyledons of late maturation embryos entering dormancy. The results demonstrate that the observed DNA accumulation in the cotyledon parenchyma is due to overlapping rounds of replication of the complete genome and not to disproportionate amplification of specific sequences nor to random DNA synthesis.

Function of the apetala-1 gene during Arabidopsis floral development.

We have characterized the floral phenotypes produced by the recessive homeotic apetala 1-1 (ap1-1) mutation in Arabidopsis. Plants homozygous for this mutation display a homeotic conversion of sepsis into brachts and the concomitant formation of floral buds in the axil of each transformed sepal. In addition, these flowers lack petals. We show that the loss of petal phenotype is due to the failure of petal primordia to be initiated. We have also constructed double mutant combinations with ap1 and other mutations affecting floral development. Based on these results, we suggest that the AP1 and the apetala 2 (AP2) genes may encode similar functions that are required to define the pattern of where floral organs arise, as well as for determinate development of the floral meristem. We propose that the AP1 and AP2 gene products act in concert with the product of the agamous (AG) locus to establish a determinate floral meristem, whereas other homeotic gene products are required for cells to differentiate correctly according to their position. These results extend the proposed role of the homeotic genes in floral development and suggest new models for the establishment of floral pattern.

The internal meristem layer (L3) determines floral meristem size and carpel number in tomato periclinal chimeras.

Cell-cell interactions are important during plant development. We have generated periclinal chimeras between plants that differ in the number of carpels per flower to determine the roles of cells occupying specific positions in the floral meristem in determining the number of carpels initiated. Intraspecific chimeras were generated between tomato (Lycopersicon esculentum) expressing the mutation fasciated, which causes an increased number of floral organs per whorl, and tomato wild type for fasciated. Interspecific chimeras were generated between tomato and L. peruvianum, which differ in number of carpels per flower. In both sets of chimeras, carpel number as well as the size of the floral meristem during carpel initiation were not determined by the genotype of cells in the outer two layers of the meristem (L1 and L2) but were determined by the genotype of cells occupying the inner layer (L3) of the meristem. We concluded from these experiments that during floral organ initiation, cells in certain layers of the meristem respond to information supplied to them from other cells in the meristem.