Mutant characters of knotted maize leaves are determined in the innermost tissue layers

Knotted (Kn1), a dominant mutation in maize, perturbs normal leaf development. Mutant leaves have localized regions of extra growth called knots and, in addition to the normal ligule, ectopic fringes of ligule are found on the leaf blade. Previous clonal analysis showed that the epidermal genotype was immaterial in knot formation. To establish which inner leaf layer was required for formation of knots and ectopic ligule we used a closely linked albino mutation to mark X-ray-induced clonal sectors of wild type (kn) tissue in Kn1 plants. The sectors examined frequently changed in composition of layers in the leaf both transversely and longitudinally. We present results that show that both mutant characters are determined in the middle mesophyll-bundle sheath (MMBS) layer. We show that a lateral vein can produce a knot when only half the MMBS layer around the lateral vein contains the mutant gene. We also show that the ectopic ligule in Kn1 has contributions from both the adaxial epidermal and adaxial mesophyll layer.     

The Knotted leaf blade is a mosaic of blade,sheath, and auricle identities

The dominant Knotted-1 mutations in maize alter development of the leaf blade. Sporadic patches of localized growth, or knots, and fringes of ectopic ligule occur along lateral veins of mutant leaf blades. In addition, bundle sheaths do not completely encircle lateral veins on mutant leaf blades. We have compared mutant leaf blades with wild-type leaves to determine the precise nature of the perturbed regions. Our analysis includes characterization of epidermal cell shapes, localization of photosynthetic proteins and histology of the leaf. We show that mutant leaf blades are a mosaic of leaf organ components. Affected regions of mutant leaf blades resemble either sheath or auricle tissue in both external and internal features. This conversion of blade cells represents an acropetal shift of more basal parts of the leaf blade region and correlates with previously identified ectopic expression of the Knotted-1 protein in the leaf blade. We propose that inappropriate expression of Kn1 interferes with the process of establishment of cell identities, resulting in early termination of the normal blade development program or precocious expression of the sheath and auricle development programs. © 1994 Wiley-Liss, Inc.     

CHARACTERIZATION OF DEVELOPMENT IN MAIZE THROUGH THE USE OF MUTANTS. II. THE ABNORMAL GROWTH CONDITIONED BY THE KNOTTED MUTANT

The maize mutant Knotted (Kn) is characterized by hollow, finger-like outgrowths (knots) occurring mainly in the leaf blade. Portions of the ligule are displaced from the normal position to more distal locations within the blade. Knots apparently result from continued meristematic activity of isolated patches of cells surrounded by maturing tissue. Small knots appear to be centers of cell division. Epidermal cells overlying a small knot have been observed to undergo periclinal divisions. In addition to cell division, a reorientation of the axis of cell elongation is associated with knot formation. The pattern of knot distribution varies at different levels on the plant axis and within a leaf blade. From leaf 4 to leaf 10 or 11 the number of knots per leaf increases progressively, then declines in leaves initiated later. Knots always occur in association with lateral veins. The greatest number per vein occurs on the 3rd or 4th vein from the midrib. One plant developing from an X-rayed heterozygous seed possessed a sector of normal tissue bisecting the plant in a vertical plane passing through the midrib of each leaf except the top two. The normal sector was knot-free and had the ligule restored to the normal position. These observations suggest that cells with the characteristics of those from intercalary meristems occur throughout the blade in Knotted plants.     

A conceptual framework for maize leaf development.

What is and is not known about the maize leaf is reviewed. Analysis of genetic mosaics and direct observation with the SEM have broken leaf development into three distinct phases: recruitment of cells within the meristem, cell division into the 0.6-mm tall primordium, and postprimordial division and differentiation into the mature leaf. New data are presented that imply that cell division rates in the leaf are coordinated by inductive signals from the internal cells. Leaf cells that tend to divide more are held in check by slower growing neighbors; this complicates the search for developmental compartments. Experiments with recessive mutants that remove the ligule and auricle have been important in identifying an inducer signal with the specific meaning "make ligule-auricle." We have studied many dominant mutant alleles at seven different genes. Each mutant alters the position of the ligule boundary. We conclude the following. First, the mutants act in particular domains of the primordium. Second, the dominant mutants all move the ligule boundary in the same direction. Third, the mutants all retard developmental stage transitions. Fourth, three and probably four of the seven genes for which dominant mutants have been studied specify homeodomain proteins in the wrong place. The concept of "maturation schedule" is used to explain these data. All of the dominant mutant phenotypes are seen as consequences of immature cells being in the wrong place when inductive signals pass through the leaf. Several specific questions of leaf development and especially questions as to source of inductive signals or homologies among juvenile and adult organ parts are recast in light of this "maturation schedule" hypothesis.     

Cloning Knotted, the dominant morphological mutant in maize using Ds2 as a transposon tag

The Kn1-2F11 mutation causes protrusions or knots along the lateral veins of the first few leaves of the maize plant. The phenotype is visible when an unlinked gene, presumably Ac, is present in the genome. The mutation is closely linked to a genetically unstable Adh1 mutation that resulted from the insertion of a Ds2 element (Döring et al., 1984; Chen et al., 1986). Using a unique sequence from the Ds2 element as a hybridization probe, a genomic restriction fragment that cosegregated with the knotted phenotype was cloned. It carries the Kn1-2F11 locus by the following criteria. (i) Cosegregation of the fragment is tightly linked to the phenotype. (ii) Somatic and germinal excision produce a fragment which is the expected size of a revertant fragment; progeny containing the revertant size fragment are normal. (iii) The sequences that hybridize to this fragment are significantly altered in the chromosome containing the original knotted mutation, Kn1-O, (iv) The cloned fragment does not hybridize to a chromosome that contains a deletion of Kn1-O.     

Genetic analysis of mutations that alter cell fates in maize leaves: Dominant Liguleless mutations

The three major components of the maize leaf are the blade, the sheath, and at their junction, the ligular region. Each exhibits specific cell types and organization. Four dominant Liguleless (Lg) mutations (Lg3-O, Lg4-O, Lg*347, and Lg*9167) in at least three different genes cause a similar morphological phenotype in leaves, although each mutation affects a distinct domain of the blade. Mutant leaves display regions of altered cell fate in the blade, occompanied by elimination of ligule and auricle at their wild-type positions and development of ligule and auricle in the blade at the borders of the altered regions. The affected blade cells are transformed into sheath-like cells, as determined by morphological and genetic tests. Lg4-O expressivity is highly dependent on genetic background. For example, two different backgrounds may specify converse patterns of phenotypic expression. Lg4-O expressivity is also affected by the heterochronic mutation Teopod2 (Tp2). Gene dosage experiments indicate that Lg4-O is a neomorph. Interactions between recessive lg mutations (which eliminate ligular structures) and the dominant Lg mutations suggest that the lg⁺ genes act after the Lg mutations. Lg3-O and Lg4-O act semidominantly, and interact with each other and with other mutations in the Knotted1 (Kn1)-like family (a family in which dominant mutant alleles cause blade to sheath transformation phenotypes). These interactions suggest that the above Kn1-like mutations may function similarly in the leaf. We discuss the similarities between the Lg mutations and the other mutations of the Kn1-like family, which led us to postulate that lg3 and lg4 are members of a growing family of kn1-like (knox) homeobox genes that are identified by dominant mutant alleles causing leaf transformation phenotypes. We also propose that certain key characteristics of this family of dominant neomorphic mutations are important for generating meaningful morphological changes during evolution. © 1996 Wiley-Liss, Inc.     

Division and differentiation during normal and liguleless-1 maize leaf development

The maize leaf is composed of a blade and a sheath, which are separated at the ligular region by a ligule and an auricle. Mutants homozygous for the recessive liguleless-1 (lg1) allele exhibit loss of normal ligule and auricle. The cellular events associated with development of these structures in both normal and liguleless plants are investigated with respect to the timing of cell division and differentiation. A new method is used to assess orientation of anticlinal division planes during development and to determine a division index based on recent epidermal cross-wall deposition. A normal leaf follows three stages of development: first is a preligule stage, in which the primordium is undifferentiated and dividing throughout its length. This stage ends when a row of cells in the preligule region divides more rapidly in both transverse and longitudinal anticlinal planes. During the second stage, ligule and auricle form, blade grows more rapidly than sheath, divisions in the blade become exclusively transverse in orientation, and differentiation begins. The third stage is marked by rapid increase in sheath length. The leaf does not have a distinct basal meristem. Instead, cell divisions are gradually restricted to the base of the leaf with localized sites of increased division at the preligule region. Divisions are not localized to the base of the sheath until near the end of development. The liguleless-1 homozygote shows no alteration in this overall pattern of growth, but does show distinct alteration in the anticlinal division pattern in the preligule region. Two abnormal patterns are observed: either the increase in division rate at the preligule site is absent or it exhibits loss of all longitudinal divisions so that only transverse (or cell-file producing) divisions are present. This pattern is particularly apparent in developing adult leaves on older lg1 plants, in which sporadic ligule vestiges form. From these and results previously published (Becraft et al. (1990) Devl Biol. 14), we conclude that the information carried by the Lg1+ gene product acts earlier in development than formation of the ligule proper. We hypothesize that Lg1+ may be effective at the stage when the blade-sheath boundary is first determined.     

Leaf permease1 gene of maize is required for chloroplast development.

Adjacent bundle sheath and mesophyll cells cooperate for carbon fixation in the leaves of C4 plants. Mutants with compromised plastid development should reveal the degree to which this cooperation is obligatory, because one can assay whether mesophyll cells with defective bundle sheath neighbors retain C4 characteristics or revert to C3 photosynthesis. The leaf permease1-mutable1 (lpe1-m1) mutant of maize exhibits disrupted chloroplast ultrastructure, preferentially affecting bundle sheath choroplasts under lower light. Despite the disrupted ultrastructure, the metabolic cooperation of bundle sheath and mesophyll cells for C4 photosynthesis remains intact. To investigate this novel mutation, the Activator transposon-tagged allele and cDNAs corresponding to the Lpe1 mRNA from wild-type plants were cloned. The Lpe1 gene encodes a polypeptide with significant similarity to microbial pyrimidine and purine transport proteins. An analysis of revertant sectors generated by Activator excision suggests that the Lpe1 gene product is cell autonomous and can be absent up to the last cell divisions in the leaf primordium without blocking bundle sheath chloroplast development.     

Sectors of liguleless-1 tissue interrupt an inductive signal during maize leaf development.

The ligule and auricles separate the blade and sheath of normal maize leaves and are absent in liguleless-1 (lg1) mutant leaves. We induced chromosome breakage using X-rays to create plants genetically mosaic for lg1. In genetically mosaic leaves, when an lg1 mutant sector interrupts the normal ligule, the ligule is often displaced basipetally on the marginal side of the sector. Therefore, lg1 mutant sectors not only fail to induce ligule and auricle, but are also disrupting some form of intercellular communication that is necessary for the normally coordinated development of the ligular region. Our data are consistent with a model in which an inductive signal originates near the midvein, cannot traverse the lg1 mutant sector, and reinitiates in the wild-type tissue across the sector toward the leaf margin. The lg1 gene product, therefore, appears to be required for the transmission of this signal and could be involved with reception.     

Plantlet formation via somatic embryogenesis and LC ESI Q-TOF MS determination of secondary metabolites in <Emphasis Type="Italic">Butea monosperma</Emphasis> (Lam.) Kuntze

Medicinal properties of Butea monosperma (BM) and overexploitation of bark as a rich source of flavonoids for different biological activities, development of efficient method for high frequency somatic embryos and in vitro synthesis of bioactive secondary metabolites using plant tissue culture technology is important. Initially, callus was induced from leaf explants of BM on Murashige and Skoog (MS) medium containing 0.25 mg L^?1 2,4-d-dichlorophenoxyacetic acid (2,4-d) with 0.1 mg L^?1 kinetin (Kn) and ascorbic acid (AA). MS half strength macronutrients and full strength micronutrients containing 0.25 mg L^?1 2,4-d with 0.1 mg L^?1 Kn, and 0.5 mg L^?1 AA provided fragile callus with 84.0 ± 1.00 % optimal growth response. Shoot formation occurred via somatic embryogenesis through an intermediary callus phase. However, 2.1 mg L^?1 thidiazuron with 0.5 mg L^?1 AA provides high frequency (79.6 ± 2.02 %) of somatic embryogenesis within 5 weeks. Developed embryos when transferred to woody plant medium containing 0.5 mg L^?1 AA with 3.0 mg L^?1 Kn and 0.5 mg L^?1 α naphthalene acetic acid responded 44.0 ± 0.00 % embryo maturation, whereas 0.5 mg L^?1 Kn, 0.3 mg L^?1 indole-3-butyric acid, and 0.25 mg L^?1 AA induced rooting within 6 and 8 weeks, respectively. Liquid chromatography electro spray ionization quadrupole time of flight mass spectrometry (LC ESI Q-TOF MS) analysis of in vitro cultures showed similarity to those compounds identified in wild grown leaf samples known for osteogenic activity. Histological investigation through scanning electron microscopy demonstrates the developmental stages of somatic embryos, shoot bud formation, and induction of root primordial.     

黎平玉山竹，贵州竹亚科一新种

描述了产自贵州的竹亚科一新种:黎平玉山竹(Yushania lipingensis Z. X. Zhang, Y. H. TongZ. Yang)。本种形态上与显耳玉山竹(Y. auctiaurita T. P. Yi)接近,但区别在于该种箨鞘背面密被向上的黄褐色或紫色疣基刺毛,箨耳及叶耳明显弯曲呈镰刀形,鞘口繸毛发达,通常呈放射状,箨舌先端截平,不圆拱,边缘密生短纤毛,箨片腹面被微柔毛,叶片次脉通常5～6对。     

The establishment of axial patterning in the maize leaf

The maize leaf consists of four distinct tissues along its proximodistal axis: sheath, ligule, auricle and blade. liguleless1 (lg1) functions cell autonomously to specify ligule and auricle, and may propagate a signal that correctly positions the blade-sheath boundary. The dominant Wavy auricle in blade (Wab1) mutation disrupts both the mediolateral and proximodistal axes of the maize leaf. Wab1 leaf blades are narrow and ectopic auricle and sheath extend into the blade. The recessive lg1-R mutation exacerbates the Wab1 phenotype; in the double mutants, most of the proximal blade is deleted and sheath tissue extends along the residual blade. We show that lg1 is misexpressed in Wab1 leaves. Our results suggest that the Wab1 defect is partially compensated for by lg1 expression. A mosaic analysis of Wab1 was conducted in Lg1+ and lg1-R backgrounds to determine if Wab1 affects leaf development in a cell-autonomous manner. Normal tissue identity was restored in all wab1+/- sectors in a lg1-R mutant background, and in three quarters of sectors in a Lg1+ background. These results suggest that lg1 can influence the autonomy of Wab1. In both genotypes, leaf-halves with wab1+/- sectors were significantly wider than non-sectored leaf-halves, suggesting that Wab1 acts cell-autonomously to affect lateral growth. The mosaic analysis, lg1 expression data and comparison of mutant leaf shapes reveal previously unreported functions of lg1 in both normal leaf development and in the dominant Wab1 mutant.     

GmPIN-dependent polar auxin transport is involved in soybean nodule development

To overcome nitrogen deficiency, legume roots establish symbiotic interactions with nitrogen-fixing rhizobia that are fostered in specialized organs (nodules). Similar to other organs, nodule formation is determined by a local maximum of the phytohormone auxin at the primordium site. However, how auxin regulates nodule development remains poorly understood. Here, we found that in soybean, (Glycine max), dynamic auxin transport driven by PIN-FORMED (PIN) transporter GmPIN1 is involved in nodule primordium formation. GmPIN1 was specifically expressed in nodule primordium cells and GmPIN1 was polarly localized in these cells. Two nodulation regulators, (iso)flavonoids trigger expanded distribution of GmPIN1b to root cortical cells, and cytokinin rearranges GmPIN1b polarity. Gmpin1abc triple mutants generated with CRISPR-Cas9 showed the impaired establishment of auxin maxima in nodule meristems and aberrant divisions in the nodule primordium cells. Moreover, overexpression of GmPIN1 suppressed nodule primordium initiation. GmPIN9d, an ortholog of Arabidopsis thaliana PIN2, acts together with GmPIN1 later in nodule development to acropetally transport auxin in vascular bundles, fine-tuning the auxin supply for nodule enlargement. Our findings reveal how PIN-dependent auxin transport modulates different aspects of soybean nodule development and suggest that the establishment of auxin gradient is a prerequisite for the proper interaction between legumes and rhizobia.

In soybean, nodule primordium formation involves GmPIN1-mediated polar auxin transport within primordium cells, and nodule enlargement involves the collaboration of GmPIN9d and GmPIN1-dependent auxin transport within nodule vasculature.     

Characterization of T4 Mutants That Partially Suppress the Inability of T4rII to Grow in Lambda Lysogens

In the course of isolating viable T4 deletions that affect plaque morphology (Homyk and Weil 1974), two closely linked point mutants, sip1 and sip2, were obtained. They map between genes t and 52, cause a reduction in plaque size and burst size, and partially suppress the lethality of rII mutants for growth in lambda lysogens. These characteristics demonstrate that sip1 and sip2 are similar to mutants previously reported by Freedman and Brenner (1972). In addition, D. Hall (personal communication) has shown that sip1 and sip2 are similar to the mutant farP85, which affects the regulation of a number of early genes ( Chace and Hall 1975).——Sip suppression of rII mutants can be demonstrated in one-step growth experiments, even when both rII genes are completely deleted. This indicates that sip mutants do not simply reduce the level of rII gene products required for growth in a lambda lysogen. Instead, they alter the growth cycle so as to partially circumvent the need for any rII products.——Mutations at two other sites, designated L₁ and L₂, reverse the poor phage growth caused by sip and, in the one case tested, reverse the rII-suppressing ability of sip.     

Structure and Function in the Grass Ligule: Optical and Electron Microscopy of the Membranous Ligule of Lolium temulentum L.

Aspects of the structure and ultrastructure of the membranousligule of mature leaves of Lolium temulentum L. are described.In transverse section the ligule was lens-shaped and wedge-shapedin longitudinal section, 6 or 7 cells wide near the base and1 or 2 cells wide at the edges. Two uniseriate epidermes encloseda chlorenchymatous mesophyll tissue of varying thicknesses;both epidermes were continuous with the leaf adaxial epidermis.The cells comprising these three issues all appeared like typicalgrass epidermal long cells; elongate papillate cells were presentat the edges. No stomata, trichomes, intercellular spaces orvascular tissue were found in the ligule. A marked polarizationof ultrastructural complexity existed from the large-vacuolateabaxial epidermis to the ‘densely cytoplasmic’ small-vacuolateadaxial epidermis. Cells of the latter tissue contained numerousmitochondria, hypersecretory dictyosomes and abundant strandsof rough endoplasmic reticulum. Fluorescence microscopy providedevidence for the accumulation of a polysaccharide-containingmaterial within the periplasmic space next to the outer tangentialwall of adaxial epidermal cells. The ligule is considered tobe a highly organized and differentiated leaf organ with a pholosyntheticmesophyll and an adaxial epidermis active in the synthesis ofprotein and polysaccharide. Darnel, fluorescence microscopy, ligule, Lolium temulentum L., Poaceae, ultrastructure     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.