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A morphological and genetic analysis of conidiophore development in Neurospora crassa.

The filamentous fungus Neurospora crassa responds to nutrient deprivation and dessication by producing asexual spores, or conidia. These conidia are derived from differentiated aerial structures called conidiophores. The process of conidiation was analyzed in wild-type and morphological mutants using scanning electron microscopy (SEM) and specific fluorescent probes. The first discernible morphological step of conidiation is the transition from growth by hyphal tip elongation to growth by repeated apical budding, resulting in the formation of chains of proconidia that resemble beads on a string. The initial proconidial chains are morphologically distinct from those that form later and are capable of reverting to hyphal growth, whereas the later chains are committed to conidiation. As the proconidial chains are formed, nuclei migrate into the conidiophore, and cross-walls arise between adjoining proconidia in a series of steps that have been defined by staining with Calcofluor, a fluorescent chitin-binding probe. The chains ultimately disarticulate in several discrete stages into free, morphologically mature conidia. Different conidiation-defective mutants were shown to be blocked at distinct stages in conidiation. Our observations permit us to derive a developmental timeline of conidiation relating the occurrence of morphological changes and the stage blocked in specific mutants.

Gene Expression Regulation↗

Pattern-specific expression of the Drosophila decapentaplegic gene in imaginal disks is regulated by 3' cis-regulatory elements.

The pattern of structures on most of the adult cuticle of Drosophila is determined in the larval imaginal disks. The Drosophila growth factor homolog decapentaplegic (dpp) is believed to participate in pattern formation in imaginal disks, primarily along what will become the proximal-to-distal axis of adult appendages. We report that dpp expression in wing, leg, and eye-antennal imaginal disks is localized to a band of cells along the presumptive proximal-to-distal axis. The pattern and level of dpp expression in imaginal disks is affected by mutant lesions that remove 3' cis-regulatory sequences. We demonstrate that one portion of the 3' cis-regulatory region contains regulatory elements sufficient to activate gene expression in a subset of the cells that normally express dpp in the imaginal disks, allowing rescue of dpp mutant phenotypes. We propose that the complete dpp expression pattern is generated by an array of 3' regulatory elements that differ in their potency in specific disks and in certain positions within a disk. The identification of the factors that activate these elements should provide clues as to how positional information is encoded in imaginal disks.

Alcohol Dehydrogenase↗

Expression of con genes along the three sporulation pathways of Neurospora crassa.

The filamentous fungus Neurospora crassa produces three types of spores by using different developmental pathways: macroconidiation, microconidiation, and sexual spore (ascospore) formation. Several genes of unknown function have been cloned by virtue of their expression during macroconidiation but not during mycelial growth (con genes). It had been postulated that expression of the con genes was specific to macroconidiation. To test this assumption, protein extracts from macroconidia, microconidia, ascospores, and protoperithecia (sexual structures) were analyzed for the product of one of the con genes, con-10, by immunoblotting using a CON10-specific antiserum. CON10 was detected in all of these extracts. An immunologically related protein was detected in an extract from ascospores of a nonconidiating Neurospora species, N. africana. Total RNA isolated from the three types of N. crassa spores was analyzed for con gene mRNA by Northern blotting using five different con genes as probes. Transcripts for four of the genes were detected in all three spore types; mRNA for the fifth gene was detected in macroconidia and microconidia but not in ascospores. Analysis of aconidial and female sterile mutants showed that expression of the con genes along any one developmental pathway occurs when expression along another pathway is genetically blocked.

Blotting, Northern↗

Ethylene-dependent and -independent processes associated with floral organ abscission in Arabidopsis.

Abscission is an important developmental process in the life cycle of the plant, regulating the detachment of organs from the main body of the plant. This mechanism can be initiated in response to environmental cues such as disease or pathogen, or it can be a programmed shedding of organs that no longer provide essential functions to the plant. We have identified five novel dab (delayed floral organ abscission) mutants (dab1-1, dab2-1, dab3-1, dab3-2, and dab3-3) in Arabidopsis. These mutants each display unique anatomical and physiological characteristics and are governed by three independent loci. Scanning electron microscopy shows delayed development of the flattened fracture plane in some mutants and irregular elongation in the cells of the fracture plane in other mutants. The anatomical observations are also supported by breakstrength measurements that show high breakstrength associated with broken cells, moderate levels for the flattened fracture plane, and low levels associated with the initial rounding of cells. In addition, observations on the expression patterns in the abscission zone of cell wall hydrolytic enzymes, chitinase and cellulose, show altered patterns in the mutants. Last, we have compared these mutants with the ethylene-insensitive mutants etr1-1 and ein2-1 to determine if ethylene is an essential component of the abscission process and find that although ethylene can accelerate abscission under many conditions, the perception of ethylene is not essential. The role of the dab genes and the ethylene response genes during the abscission process is discussed.

Arabidopsis↗

petit1, a conditional growth mutant of Arabidopsis defective in sucrose-dependent elongation growth.

The hypocotyl of Arabidopsis is well suited for the analysis of cell elongation because it elongates without cell division. We have isolated a new class of recessive mutants, petit1 (pet1), which are defective in aspects of hypocotyl elongation. The short-hypocotyl phenotype of pet1 is caused by shortened cells. The cells of the elongation zone of the hypocotyl are often deformed. pet1 also shows defects in elongation of the roots, flower stalk, leaves, petals, pedicels, and siliques, and these defects cannot be repaired by the application of auxin, gibberellin, brassinolide, or an inhibitor of ethylene biosynthesis. The short-hypocotyl phenotype of pet1 is pronounced only in growth medium supplemented with sucrose, which has promotive effects on hypocotyl elongation. In pet1 this effect is much reduced, causing the sucrose-dependent short-hypocotyl phenotype of pet1. pet1 accumulates more soluble sugars than the wild type and also shows more intensive iodo-starch staining in the cotyledon and hypocotyl. These results indicate that PETIT1 is involved in a sugar-dependent elongation process that may include correct assembly of expanding cell wall architecture.

Arabidopsis↗

Clausa, a tomato mutant with a wide range of phenotypic perturbations, displays a cell type-dependent expression of the homeobox gene LeT6/TKn2.

Class I knox genes play an important role in shoot meristem function and are thus involved in the ordered development of stems, leaves, and reproductive organs. To elucidate the mechanism underlying the expression pattern of these homeobox genes, we studied a spontaneous tomato (Lycopersicon esculentum) mutant that phenotypically resembles, though is more extreme than, transgenic plants misexpressing class I knox genes. This mutant was found to carry a recessive allele, denoted clausa:shootyleaf (clau:shl)-a newly identified allele of clausa. Mutant plants exhibited abnormal leaf and flower morphology, epiphyllus inflorescences, fusion of organs, calyx asymmetry, and navel-like fruits. Analysis by scanning electron microscopy revealed that such fruits carried ectopic ovules, various vegetative primordia, as well as "forests" of stalked glandular trichomes. In situ RNA hybridization showed a peculiar expression pattern of the class I knox gene LeT6/TKn2; expression was restricted to the vascular system and palisade layer of mature leaves and to the inner part of ovules integuments. We conclude that CLAUSA regulates various aspects of tomato plant development, at least partly, by rendering the LeT6/TKn2 gene silent in specific tissues during development. Considering the expression pattern of LeT6/TKn2 in the clausa mutant, we suggest that the control over a given homeobox gene is maintained by several different regulatory mechanisms, in a cell type-dependent manner.

Base Sequence↗

Regulation of axis determinacy by the Arabidopsis PINHEAD gene.

Plants produce proximal-distal growth axes with two types of growth potential: they can be indeterminate, in which case growth continues indefinitely, or they can be determinate, in which case growth is limited to the production of a single organ or a discrete set of organs. The indeterminate shoot axes of Arabidopsis pinhead/zwille mutants frequently are transformed to a determinate state. PINHEAD (PNH) is expressed in the central domain of the developing plant: the provascular tissue, the shoot apical meristem, and the adaxial (upper) sides of lateral organ primordia. Here, we show that ectopic expression of PNH on the abaxial (lower) sides of lateral organs results in upward curling of leaf blades. This phenotype correlates with a loss of cell number coordination between the two surfaces of the blade, indicating that ectopic PNH can cause changes in cell division rates. More strikingly, moving PNH expression from the central to the peripheral domain of the embryo causes transformation of the determinate cotyledon axis to an indeterminate state. We propose that growth axes are specified as determinate versus indeterminate in a PNH-mediated step. Our results add to a growing body of evidence that radial positional information is important in meristem formation. These results also indicate that genes regulating cell division and axis determinacy are likely to be among PNH targets.

Arabidopsis↗

MADS-box protein complexes control carpel and ovule development in Arabidopsis.

The AGAMOUS (AG) gene is necessary for stamen and carpel development and is part of a monophyletic clade of MADS-box genes that also includes SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK). Here, we show that ectopic expression of either the STK or SHP gene is sufficient to induce the transformation of sepals into carpeloid organs bearing ovules. Moreover, the fact that these organ transformations occur when the STK gene is expressed ectopically in ag mutants shows that STK can promote carpel development in the absence of AG activity. We also show that STK, AG, SHP1, and SHP2 can form multimeric complexes and that these interactions require the SEPALLATA (SEP) MADS-box proteins. We provide genetic evidence for this role of the SEP proteins by showing that a reduction in SEP activity leads to the loss of normal ovule development, similar to what occurs in stk shp1 shp2 triple mutants. Together, these results indicate that the SEP proteins, which are known to form multimeric complexes in the control of flower organ identity, also form complexes to control normal ovule development.

AGAMOUS Protein, Arabidopsis↗

FILAMENTOUS FLOWER controls the formation and development of arabidopsis inflorescences and floral meristems.

Phenotypic analysis of single and multiple mutants as well as in situ localization analysis of the expression patterns of floral genes have revealed that the FIL AMENTOUS FLOWER (FIL) gene plays important roles in establishing the inflorescence in Arabidopsis. As previously reported, the fil mutant generates clusters of both filamentous structures and flowers with floral organs of altered number and shape. The structural resemblance of the filamentous structures to peduncles and the expression pattern of the APETALA1 (AP1) gene have shown that these filamentous structures are underdeveloped flowers that fail to form receptacles and floral organs, indicating that one of the roles of the FIL gene is to support the development of the floral meristem. That FIL also is involved in fate determination in the floral meristem is demonstrated by the homeotic conversion of flowers to inflorescences in fil ap1 double mutants and in fil ap1 cauliflower triple mutants. In double mutants with flowering-time loci (i.e., f t or f wa), leafy (lf y), and unusual floral organs (ufo), filamentous structures are formed, but very few or no flowers with floral organs develop. The enhanced phenotype in the fil ap1 and the fil lfy double mutants suggests that the FIL protein may work together with AP1 and LFY proteins. The FIL gene also may be involved in the cell fate determination of floral organ primordia, possibly by controlling the spatial expression patterns of the class A and C floral organ identity genes.

Arabidopsis↗

Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, cochleata, afila, and tendril-lessn.

The compound leaf primordium of pea represents a marginal blastozone that initiates organ primordia, in an acropetal manner, from its growing distal region. The UNIFOLIATA (UNI) gene is important in marginal blastozone maintenance because loss or reduction of its function results in uni mutant leaves of reduced complexity. In this study, we show that UNI is expressed in the leaf blastozone over the period in which organ primordia are initiated and is downregulated at the time of leaf primordium determination. Prolonged UNI expression was associated with increased blastozone activity in the complex leaves of afila (af), cochleata (coch), and afila tendril-less (af tl) mutant plants. Our analysis suggests that UNI expression is negatively regulated by COCH in stipule primordia, by AF in proximal leaflet primordia, and by AF and TL in distal and terminal tendril primordia. We propose that the control of UNI expression by AF, TL, and COCH is important in the regulation of blastozone activity and pattern formation in the compound leaf primordium of the pea.

Epistasis, Genetic↗

AINTEGUMENTA promotes petal identity and acts as a negative regulator of AGAMOUS.

The Arabidopsis AINTEGUMENTA (ANT) gene has been shown previously to be involved in ovule development and in the initiation and growth of floral organs. Here, we show that ANT acts in additional processes during flower development, including repression of AGAMOUS (AG) in second whorl cells, promotion of petal epidermal cell identity, and gynoecium development. Analyses of ap2-1 ant-6 double mutants reveal that ANT acts redundantly with AP2 to repress AG in second whorl cells. The abaxial surface of ant petals contains features such as stomata and elongated, interdigitated cells that are not present on wild-type petals. The loss of petal identity in these second whorl cells does not result from ectopic AG expression, suggesting that ANT acts in a pathway promoting petal cell identity that is independent of its role in repression of AG. These data suggest that ANT may function as a class A gene.

AGAMOUS Protein, Arabidopsis↗

Regulation of SUP expression identifies multiple regulators involved in arabidopsis floral meristem development.

During the course of flower development, floral homeotic genes are expressed in defined concentric regions of floral meristems called whorls. The SUPERMAN (SUP, also called FLO10) gene, which encodes a C2H2-type zinc finger protein, is involved in maintenance of the stamen/carpel whorl boundary (the boundary between whorl 3 and whorl 4) in Arabidopsis. Here, we show that the regulation of SUP expression in floral meristems is complex, consisting of two distinct phases, initiation and maintenance. The floral meristem identity gene LEAFY (LFY) plays a role in the initiation phase through at least two pathways, which differ from each other in the involvement of two homeotic genes, APETALA3 (AP3) and PISTILLATA (PI). AP3, PI, and another homeotic gene, AGAMOUS (AG), are further required for SUP expression in the later maintenance phase. Aside from these genes, there are other as yet unidentified genes that control both the temporal and spatial patterns of SUP expression in whorl 3 floral meristems. SUP appears to act transiently, probably functioning to trigger a genetic circuit that creates the correct position of the whorl 3/whorl 4 boundary.

AGAMOUS Protein, Arabidopsis↗

Expression of an N-terminal fragment of COP1 confers a dominant-negative effect on light-regulated seedling development in Arabidopsis.

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is an essential regulatory gene that plays a role in light control of seedling development in Arabidopsis. The COP1 protein possesses three recognizable structural domains: a RING finger zinc binding domain near the N terminus, followed by a coiled-coll domain and a domain with WD-40 repeats in the C-terminal half. To determine whether COP1 acts specifically as a light-inactivable repressor of photomorphogenic development and to elucidate the functional roles of the specific structural domains, mutant cDNAs encoding the N-terminal 282 amino acids (N282) of COP1 were expressed and analyzed in transgenic plants. High-level expression of the N282 fragment caused a dominant-negative phenotype similar to that of the loss-of-function cop1 mutants. The phenotypic characteristics include hypersensitivity of hypocotyl elongation to inhibition by white, blue, red, and far-red light stimuli. In the dark, N282 expression led to pleiotropic photomorphogenic cotyledon development, including cellular differentiation, plastid development, and gene expression, although it has no significant effect on the hypocotyl elongation. However, N282 expression had a minimal effect on the expression of stress- and pathogen-inducible genes. These observations support the hypothesis that COP1 is directly involved in the light control of seedling development and that it acts as a repressor of photomorphogenesis. Further, the results imply that the N282 COP1 fragment, which contains the zinc binding and colled-coil domains, is capable of interacting with either downstream targets or with the endogenous wild-type COP1, thus interfering with normal regulatory processes. The fact the N282 is able to interact with N282 and full-length COP1 in yeast provided evidence for the latter possibility.

Arabidopsis↗

Determination of Arabidopsis floral meristem identity by AGAMOUS.

Determinate growth of floral meristems in Arabidopsis requires the function of the floral regulatory gene AGAMOUS (AG). Expression of AG mRNA in the central region of floral meristems relies on the partially overlapping functions of the LEAFY (LFY) and APETALA1 (AP1) genes, which promote initial floral meristem identity. Here, we provide evidence that AG function is required for the final definition of floral meristem identity and that constitutive AG function can promote, independent of LFY and AP1 functions, the determinate floral state in the center of reproductive meristems. Loss-of-function analysis showed that the indeterminate central region of the ag mutant floral meristem undergoes conversion to an inflorescence meristem when long-day-dependent flowering stimulus is removed. Furthermore, gain-of-function analysis demonstrated that ectopic AG function results in precocious flowering and the formation of terminal flowers at apices of both the primary inflorescence and axillary branches of transgenic Arabidopsis plants in which AG expression is under the control of the 35S promoter from cauliflower mosaic virus. Similar phenotypes were also observed in lfy ap1 double mutants carrying a 35S-AG transgene. Together, these results indicate that AG is a principal developmental switch that controls the transition of meristem activity from indeterminate to determinate.

AGAMOUS Protein, Arabidopsis↗

Alanine-scanning mutagenesis of the beta-sheet region of phage T4 lysozyme suggests that tertiary context has a dominant effect on beta-sheet formation.

In general, alpha-helical conformations in proteins depend in large part on the amino acid residues within the helix and their proximal interactions. For example, an alanine residue has a high propensity to adopt an alpha-helical conformation, whereas that of a glycine residue is low. The sequence preferences for beta-sheet formation are less obvious. To identify the factors that influence beta-sheet conformation, a series of scanning polyalanine mutations were made within the strands and associated turns of the beta-sheet region in T4 lysozyme. For each construct the stability of the folded protein was reduced substantially, consistent with removal of native packing interactions. However, the crystal structures showed that each of the mutants retained the beta-sheet conformation. These results suggest that the structure of the beta-sheet region of T4 lysozyme is maintained to a substantial extent by tertiary interactions with the surrounding parts of the protein. Such tertiary interactions may be important in determining the structures of beta-sheets in general.

Alanine↗

Cell surface of a tetrads-forming mutant of Micrococcus luteus: chemical treatment of the cells and teichuronic acids on the surface.

Tetrads-forming mutant MT cells of Micrococcus luteus, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of the cells with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the cells, mediated with the antiserum, was also observed with SEM. A tetrad surface, not surface of each of four "unit monococci" constituting a tetrad, consisted of two or three smooth areas with borders. The difference in the surface features between M. luteus wild-type IFO 3333 (Monodane et al, Microbiol. Immunol. 33: 165-174, 1989) and the mutant MT cells is discussed.

Cell Wall↗