Interactions between jointless and wild-type tomato tissues during development of the pedicel abscission zone and the inflorescence meristem.

The jointless mutation of tomato results in the formation of flower pedicels that lack an abscission zone and inflorescence meristems that revert to vegetative growth. We have analyzed periclinal chimeras and mericlinal sectors of jointless and wild-type tissue to determine how cells in different meristem layers (L1, L2, and L3) and their derivatives interact during these two developmental processes. Cells in the inner meristem layer, L3, alone determined whether the meristem maintained the inflorescence state or reverted to vegetative growth. Moreover, L3 derivatives determined whether a functional pedicel abscission zone formed. Limited and disorganized autonomous development of wild-type L2-derived cells occurred when they overlay mutant tissue. Adjacent mutant and wild-type L3-derived tissues in pedicels developed autonomously, indicating little or no lateral communication. Only the outermost L3-derived cells within the pedicel were capable of orchestrating normal pedicel development in overlying tissues, revealing the special status of those cells as coordinators of development for L1- and L2-derived cells, whereas the innermost L3-derived cells developed autonomously but did not influence the development of other cells.     

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.Abbreviation MF mutation frequency     

NARROW SHEATH1 functions from two meristematic foci during founder-cell recruitment in maize leaf development

The narrow sheath duplicate genes (ns1 and ns2) perform redundant functions during maize leaf development. Plants homozygous for mutations in both ns genes fail to develop wild-type leaf tissue in a lateral domain that includes the leaf margin. Previous studies indicated that the NS gene product(s) functions during recruitment of leaf founder-cells in a lateral, meristematic domain that contributes to leaf margin development. A mosaic analysis was performed in which the ns1-O mutation was exposed in hemizygous, clonal sectors in a genetic background already homozygous for ns2-O. Analyses of mutant, sectored plants demonstrate that NS1 function is required in L2-derived tissue layers for development of the narrow sheath leaf domain. NS1 function is not required for development of the central region of maize leaves. Furthermore, the presence of the non-mutant ns1 gene outside the narrow sheath domain cannot compensate for the absence of the non-mutant gene within the narrow sheath domain. NS1 acts non-cell autonomously within the narrow sheath-margin domain and directs recruitment of marginal, leaf founder cells from two discrete foci in the maize meristem. Loss of NS1 function during later stages of leaf development results in no phenotypic consequences. These data support our model for NS function during founder-cell recruitment in the maize meristem.     

COMPETITION AND ACCOMMODATION BETWEEN APICAL LAYERS AND THEIR DERIVATIVES IN THE ONTOGENY OF CHIMERAL SHOOTS OF PELARGONIUM X HORTORUM

Two mutant plastogenes in all possible chimeral combinations were followed in Pelargonium X hortorum Bailey (geranium) shoots. The part of stem, leaf, or other structure derived from each apical layer was clearly apparent on a cell to cell basis. Shoots typically were composed of derivatives of three apical layers but we found derivatives of as many as four apical layers in some leaves and of five layers in some stems. In chimeras with one of the mutants, Dpl W₁, the amount of tissue derived from the various apical layers was the same, whether the layer was mutant or wild type. We suggest that there are independent apical layers and cell lineages derived from them in nonchimeral shoots, and that their contribution in normal ontogeny is like that of the layers in Dpl W₁ chimeras. In chimeras carrying the second mutant, Dpl W₂, there was much less tissue derived from mutant than from wild-type apical layers. The phenotypic expression of the plastogenes was unchanged by their transmission through male or female gametes. Comparisons of the ontogeny of geranium plants carrying the W₁ or W₂ mutant suggested that, while there was competition between the apical layers and between their derivatives, the genome imposed a definite harmonious interaction or accommodation which led to a final normal morphology of all plant parts and organs through quite different ontogenetic pathways.     

Brassinosteroids control the proliferation of leaf cells of Arabidopsis thaliana

The growth of leaves in the model plant, Arabidopsis thaliana (L.) Heynh., is determined by the extent of expansion of individual cells and by cell proliferation. Mutants of A. thaliana with known defects in the biosynthesis or perception of brassinosteroids develop small leaves. When the leaves of brassinosteroid-related mutants, det2 (de-etiolated2 = cro1) and dwf1 (dwarf1 = cro2) were compared to wild-type plants, an earlier cessation of leaf expansion was observed; a detailed anatomical analysis further revealed that the mutants had fewer cells per leaf blade. Treatment of the det2 mutants with the brassinosteroid, brassinolide, reversed the mutation and restored the potential for growth to that of the wild type. Restoration of leaf size could not be explained solely on the basis of an increase in individual cell volume, thus suggesting that brassinosteroids play a dual role in regulating cell expansion and proliferation.     

Investigation of the role of cell-cell interactions in division plane determination during maize leaf development through mosaic analysis of the tangled mutation

Most plant cells divide in planes that can be predicted from their shapes according to simple geometrical rules, but the division planes of some cells appear to be influenced by extracellular cues. In the maize leaf, some cells divide in orientations not predicted by their shapes, raising the possibility that cell-cell communication plays a role in division plane determination in this tissue. We investigated this possibility through mosaic analysis of the tangled (tan) mutation, which causes a high frequency of cells in all tissue layers to divide in abnormal orientations. Clonal sectors of tan mutant tissue marked by a closely linked albino mutation were examined to determine the phenotypes of cells near sector boundaries. We found that tan mutant cells always showed the mutant phenotype regardless of their proximity to wild-type cells, demonstrating that the wild-type Tan gene acts cell-autonomously in both lateral and transverse leaf dimensions to promote normally oriented divisions. However, if the normal division planes of wild-type cells depend on cell-cell communication involving the products of genes other than Tan, then aberrantly dividing tan mutant cells might send abnormal signals that alter the division planes of neighboring cells. The cell-autonomy of the tan mutation allowed us to investigate this possibility by examining wild-type cells near the boundaries of tan mutant sectors for evidence of aberrantly oriented divisions. We found that wild-type cells near tan mutant cells did not divide differently from other wild-type cells. These observations argue against the idea that the division planes of proliferatively dividing maize leaf epidermal cells are governed by short-range communication with their nearest neighbors.     

The dominant Basilicum Leaf mutation of sunflower controls leaf development multifariously and modifies the photosynthetic traits

Despite the remarkable morphological diversity in the leaf shape, usually higher plants maintain a flat lamina to ensure the desirable surface-to-volume ratio. The control of expansion of a flattened lateral organ is only partially known and the characterization of leaf mutants can be a useful strategy to add new information, as demonstrated in dicots with recessive mutants of model species. In sunflower, the dominant mutant Basilicum Leaf (BL) is characterized by prominent downward leaf curling. The central end of the present work is the detailed study of the BL phenotype to characterize the effects of this mutation, at organ and cellular levels, on both morphological and physiological traits. The geometry of mature leaves is obviously altered in BL plants because the lamina surfaces are downward curled and display a different length/width ratio with respect to wild type. The area of BL leaves is higher than that of wild type while the BL leaf perimeter is reduced. In BL leaves, the perimeter to the square root of area ratio is also abnormally low compared to the value of a flat sunflower lamina. These results suggest that the dominant mutation induces a deep change of the ratio between lamina and marginal growth. In addition, the epidermis and mesophyll of BL leaves comprised higher size pavement and palisade cells compared to those of wild type, indicating that the BL gene also control cell shape changes in the formation of the leaf lamina. Other leaf traits such as photosynthetic pigment content, pattern of minor vein architecture, margin type and stomatal density are affected by the mutation. In BL plants, the longitudinal axis of both cotyledons and ray flower corollas shows an unusual curvature while the diameter of shoot and inflorescence meristems is reduced. In mutant leaves, the gas exchange performance is influenced by low stomata conductance while the CO₂ assimilation rate is also depressed by a depleted Rubisco activity. Together, these data demonstrate the pleiotropic nature of the BL mutation, which affects a number of morphological aspects of flattened lateral organs as well as physiological parameters in sunflower.     

Regeneration of chlorophyll chimeras from leaf explants ofNicotiana tabacum L.

Chimeral plants with variegated leaf blades were obtained by induction of organogenesis in primocultures of leaf explants ofNicotiana tabacum L. cv. Burley 49, chlorophyll mutation White Seedling. Only green plants regenerated from primocultures of explants taken from dark green leaf areas of the chimeras. The possibility of a multicellular initiation of chimera regeneration from tissue cultures is discussed.     

Clonal analysis of leaf development in cotton

Clonal analysis has been used to describe the cellular parameters of leaf development in American Pima cotton (Gossypium barbadense). Sectors (clones) induced before leaf initiation indicate that the leaf primordium arises from ~100 cells on the flank of the shoot meristem. An analysis of sector frequency during the period of leaf expansion suggests that the rate of cell division is fairly uniform throughout the length of the leaf, but is lower at the margin of the lamina than in intercalary regions. The shapes of marginal sectors indicate that the orientation of cell division (as defined by the orientation of the new cell wall) in this region is more often parallel to the margin than perpendicular to it, although the degree of polarization varies along the length of the margin. There is a slight gradient in the duration of cell division along the length of of the lamina late in development, with cell division ceasing progressively from the lamina tip to the base over two cell cycles. The parameters of cell division in cotton are therefore similar to those described for tobacco with the notable exception of the behavior of cells at the leaf margin.     

FLEXIBILITY IN ONTOGENY AS SHOWN BY THE CONTRIBUTION OF THE SHOOT APICAL LAYERS TO LEAVES OF PERICLINAL CHIMERAS

The development of leaves on apically stable, periclinal chimeras was studied in a number of dicot genera. The mutant cell layers of the shoot apex and the tissues derived from them were as active developmentally as the normal layers. Ontogeny was the same in these chimeras as in nonchimeras, and growth of their leaves can be outlined as follows. Formation of the buttress, the axis, and the lamina of simple dicot leaves were independent events. In each the first growth included derivatives of the apical layers, usually three in number, found in the apex of the shoot and the lateral buds. Most cell divisions in the outer layers (L-I and L-II) were anticlinal relative to the new structures. Therefore, in the proximal regions of the buttress, axis (petiole and midrib), and lamina, the derivative cells of L-I and L-II were usually present in single layers. The rest of the internal tissue was from L-III. As formation of the axis and the lamina proceeded, derivatives of L-II replaced L-III internally in the distal and marginal regions leaving cells of L-III behind. Both the determinate growth of leaves and the pattern of cell divisions at and near the leading edges of growth meant that no cells in the leaf were comparable to the initial cells of the shoot apex. As the lamina extended, there were extensive intercalary cell divisions, both anticlinal and periclinal, so that in any given region of a leaf the layers of internal cells were from either L-II or L-III. At any point along the axis, L-III participated or did not participate in laminar extension. At any given stage in laminar growth either of two sister cells in any internal layer divided either a few times or extensively. The extreme variability in direction and frequency of cell division during leaf development was under an overriding genetic control, which resulted in the normal or typical size, shape and thickness of leaves.     

Developmental potential and survival of glycolysis-deficient cells in fetal mouse chimeras

Mouse embryos homozygous for a null allele of Gpi1 fail to complete gastrulation and die around E7.5. We produced E12.5 chimeric mouse conceptuses, composed of wild-type and homozygous Gpi1m/m null mutant cells to test whether the presence of wild-type cells allowed mutant cells to survive and, if so, whether they survived better in some tissue locations than others. Fourteen homozygous Gpi1m/m<-->Gpi1c/c chimeras were identified and these contained low levels of homozygous mutant cells in most tissues tested. Homozygous Gpi1m/m cells contributed better to the yolk sac endoderm and placenta than to the epiblast derivatives tested (retinal pigment epithelium, brain, tail, amnion, and yolk sac mesoderm). The depletion of mutant cells confirms that the gene acts cell autonomously, but the GPI deficiency is not always cell-lethal. When mixed with wild-type cells in chimeras, homozygous mutant cells can differentiate into many different cell types and survive until at least E12.5.     

Dissection of enhanced cell expansion processes in leaves triggered by a defect in cell proliferation, with reference to roles of endoreduplication

Leaf development relies on cell proliferation, post-mitotic cell expansion and the coordination of these processes. In several Arabidopsis thaliana mutants impaired in cell proliferation, such as angustifolia3 (an3), leaf cells are larger than normal at their maturity. This phenomenon, which we call compensated cell enlargement, suggests the presence of such coordination in leaf development. To dissect genetically the cell expansion system(s) underlying this compensation seen in the an3 mutant, we isolated and utilized 10 extra-small sisters (xs) mutant lines that show decreased cell size but normal cell numbers in leaves. In the xs single mutants, the palisade cell sizes in mature leaves are about 20-50% smaller than those of wild-type cells. Phenotypes of the palisade cell sizes in all combinations of xs an3 double mutants fall into three classes. In the first class, the compensated cell enlargement was significantly suppressed. Conversely, in the second class, the defective cell expansion conferred by the xs mutations was significantly suppressed by the an3 mutation. The residual xs mutations had effects additive to those of the an3 mutation on cell expansion. The endopolyploidy levels in the first class of mutants were decreased, unaffected or increased, as compared with those in wild-type, suggesting that the abnormally enhanced cell expansion observed in an3 could be mediated, at least in part, by ploidy-independent mechanisms. Altogether, these results clearly showed that a defect in cell proliferation in leaf primordia enhances a part of the network that regulates cell expansion, which is required for normal leaf expansion.     

YUCCA genes are expressed in response to leaf adaxial-abaxial juxtaposition and are required for leaf margin development

During leaf development, the formation of leaf adaxial-abaxial polarity at the primordium stage is crucial for subsequent leaf expansion. However, little is known about the genetic control from polarity establishment to blade outgrowth. The leaf margin, comprising elongated margin cells and hydathodes, is thought to affect leaf expansion. Here, we show that mutants with defective leaf polarity or with loss of function in the multiple auxin-biosynthetic YUCCA (YUC) genes exhibited a similar abnormal leaf margin and less-expanded leaves. Leaf margins of these mutants contained fewer hydathodes and an increased number of cell patches in which the patterns of epidermal cells resembled those of hydathodes. The previously characterized leaf-abaxialized asymmetric leaves2 (as2) revoluta (rev) and leaf-adaxialized kanadi1 (kan1) kan2 double mutants both produce finger-shaped, hydathode-like protrusions on adaxial and abaxial leaf surfaces, respectively. YUCs are required for formation of the protrusions, as those produced by as2 rev and kan1 kan2 were absent in the yuc1 yuc2 yuc4 triple mutant background. Expressions of YUC1, YUC2, and YUC4 were spatially regulated in the leaf, being associated with hydathodes in wild-type leaves and protrusions on as2 rev and kan1 kan2 leaves. In addition, inhibition of auxin transport by treatment of seedlings with N-(1-naphtyl) phtalamic acid or disruption of the auxin gradient by transforming plants with the 35S:YUC1 construct also blocked leaf margin development. Collectively, our data show that expressions of YUCs in the leaf respond to the adaxial-abaxial juxtaposition, and that the activities of auxin mediate leaf margin development, which subsequently promotes blade outgrowth.     

Extended leaf longevity in the ore4-1 mutant of Arabidopsis with a reduced expression of a plastid ribosomal protein gene

The longevity of plant leaf organs is genetically determined. However, the molecular mechanisms underlying the control of longevity are still largely unknown. Here, we describe a T-DNA-insertional mutation of Arabidopsis thaliana that confers extended leaf longevity. The mutation, termed ore4-1, delays a broad spectrum of age-dependent leaf senescence, but has little effect on leaf senescence artificially induced by darkness, abscisic acid (ABA), methyl jasmonate (MeJA), or ethylene. The T-DNA was inserted within the promoter region of the plastid ribosomal small subunit protein 17 (PRPS17) gene, and this insertion dramatically reduced PRPS17 mRNA expression. In the ore4-1 mutant, the leaf growth rate is decreased, while the maturation timing is similar to that of wild-type. In addition, the activity of the photosystem I (PSI) is significantly reduced in the ore4-1 mutant, as compared to wild-type. Thus, the ore4-1 mutation results in a deficiency in various chloroplast functions, including photosynthesis, which may decrease leaf growth. Our results suggest a possible link between reduced metabolism and extended longevity of the leaf organs in the ore4-1 mutation.     

The tobacco plastid accD gene is essential and is required for leaf development

Angiosperm plastid genomes typically encode approximately 80 polypeptides, mainly specifying plastid-localized functions such as photosynthesis and gene expression. Plastid protein synthesis and expression of the plastid clpP1 gene are essential for development in tobacco, indicating the presence of one or more plastid genes whose influence extends beyond the plastid compartment. The plastid accD gene encodes the beta-carboxyl transferase subunit of acetyl-CoA carboxylase and is present in the plastids of most flowering plants, including non-photosynthetic parasitic plants. We replaced the wild-type accD gene with an aadA-disrupted mutant allele using homologous recombination. Persistent heteroplasmy in the presence of antibiotics indicated that the wild-type accD allele was essential. The phenotype of the accD knockout was revealed in plastid transformants grown in the absence of antibiotics. Leaves contained pale green sectors and lacked part or all of the leaf lamina due to arrested division or loss of cells. Abnormal structures were present in plastids found in mutant plants, indicating that accD might be required to maintain the plastid compartment. Loss of the plastid compartment would be expected to be lethal. These results provide genetic evidence showing the essential role of plastid ACCase in the pathway leading to the synthesis of products required for the extra-plastidic processes needed for leaf development.     

Mutations in oxyR resulting in peroxide resistance in Xanthomonas campestris

A spontaneous Xanthomonas campestris pv. phaseoli H(2)O(2)-resistant mutant emerged upon selection with 1 mM H(2)O(2). In this report, we show that growth of this mutant under noninducing conditions gave high levels of catalase, alkyl hydroperoxide reductase (AhpC and AhpF), and OxyR. The H(2)O(2) resistance phenotype was abolished in oxyR-minus derivatives of the mutant, suggesting that elevated levels and mutations in oxyR were responsible for the phenotype. Nucleotide sequence analysis of the oxyR mutant showed three nucleotide changes. These changes resulted in one silent mutation and two amino acid changes, one at a highly conserved location (G197 to D197) and the other at a nonconserved location (L301 to R301) in OxyR. Furthermore, these mutations in oxyR affected expression of genes in the oxyR regulon. Expression of an oxyR-regulated gene, ahpC, was used to monitor the redox state of OxyR. In the parental strain, a high level of wild-type OxyR repressed ahpC expression. By contrast, expression of oxyR5 from the X. campestris pv. phaseoli H(2)O(2)-resistant mutant and its derivative oxyR5G197D with a single-amino-acid change on expression vectors activated ahpC expression in the absence of inducer. The other single-amino-acid mutant derivative of oxyR5L301R had effects on ahpC expression similar to those of the wild-type oxyR. However, when the two single mutations were combined, as in oxyR5, these mutations had an additive effect on activation of ahpC expression.