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.     

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.     

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 a dominant mutation for the liguleless trait: Aegilops tauschii liguleless (Lgt)

Background

Leaves of Poaceae have a unique morphological feature: they consist of a proximal sheath and a distal blade separated by a ligular region. The sheath provides structural support and protects young developing leaves, whereas the main function of the blade is photosynthesis. The auricles allow the blade to tilt back for optimal photosynthesis and determine the angle of a leaf, whereas the ligule protects the stem from the entry of water, microorganisms, and pests. Liguleless variants have an upright leaf blade that wraps around the culm. Research on liguleless mutants of maize and other cereals has led to identification of genes that are involved in leaf patterning and differentiation.

Results

We characterized an induced liguleless mutant (LM) of Aegilops tauschii Coss., a donor of genome D of bread wheat Triticum aestivum L.. The liguleless phenotype of LM is under dominant monogenic control (Lg^t). To determine precise position of Lg^t on the Ae. tauschii genetic map, highly saturated genetic maps were constructed containing 887 single-nucleotide polymorphism (SNP) markers derived via diversity arrays technology (DArT)seq. The Lg^t gene was mapped to chromosome 5DS. Taking into account coordinates of the SNP markers, flanking Lg^t, on the pseudomolecule 5D, a chromosomal region that contains this gene was determined, and a list of candidate genes was identified. Morphological features of the LM phenotype suggest that Lg^t participates in the control of leaf development, mainly, in leaf proximal–distal patterning, and its dominant mutation causes abnormal ligular region but does not affect reproductive development.

Conclusions

Here we report characterization of a liguleless Ae. tauschii mutant, whose phenotype is under control of a dominant mutation of Lg^t. The dominant mode of inheritance of the liguleless trait in a Triticeae species is reported for the first time. The position of the Lg^t locus on chromosome 5DS allowed us to identify a list of candidate genes. This list does not contain Ae. tauschii orthologs of any well-characterized cereal genes whose mutations cause liguleless phenotypes. Thus, the characterized Lg^t mutant represents a new model for further investigation of plant leaf patterning and differentiation.

     

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.     

Genetic Analysis of Rough Sheath1 Developmental Mutants of Maize

Maize Rough sheath1 (Rs1) mutants are dominant and cause a proliferation of sheath-like tissue at the base of the blade and throughout the ligular region. They also cause ligule displacement, a chaotic pattern of vasculature and abnormal cellular structure of vascular bundles. The affected region of Rs1-O leaves displays genetic and morphological attributes of both sheath and auricle, suggesting an overlap of these genetic programs. The rs1 locus maps approximately 26 map units distal to opaque2 (o2) on chromosome 7S, defining a new distal-most locus on the genetic map. Three mutant alleles, Rs1-O, Rs1-1025 and Rs1-Z, all display similar phenotypes. The mutations are completely dominant and the Rs1-O phenotype is not affected by dosage of the chromosome arm carrying the rs1(+) allele, indicating that these alleles are neomorphic. Analysis of genetic mosaics showed that the Rs1-O phenotype is non-cell-autonomous, suggesting that intercellular signals convey the phenotype. Rs1 mutant phenotypes are affected by modifiers present in particular genetic backgrounds. An enhancer of Rs1-O was identified; segregation data imply a single recessive gene, ers1. Rs1 mutants were also found to enhance the expression of unlinked rs2 and Rs4 mutants, suggesting that these mutations affect similar developmental processes. We discuss the phenotypic and genetic similarities between Rs1 and Knotted1 (Kn1) mutants that led to the identification of rs1 as a kn1-like homeobox gene (unpublished data).     

Interactions of Liguleless1 and Liguleless2 Function during Ligule Induction in Maize

The maize ligule is an adaxial membranous structure on the leaf that develops at the boundary of the sheath and blade. The ligule and the associated auricle are dispensable structures, amenable to genetic manipulation. We present here a genetic analysis of liguleless1 (lg1) and liguleless2 (lg2), the two genes known to be uniquely necessary for ligule and auricle development. We show that both reference mutant alleles, lg1-R and lg2-R, are null alleles. The double mutant phenotype suggests that lg1 and lg2 act in the same pathway. Indeed, the dosage of a functional allele at either gene affects the null phenotype of the other. While lg1 function has previously been shown to be cell-autonomous, here we show that the lg2-R phenotype is cell-nonautonomous, suggesting lg1 and lg2 play different roles in the ligule-auricle induction mechanism. We present a model in which early lg2 function specifies the precise position where ligule and auricle will develop. Later lg2 function interacts with lg1 function (either directly or indirectly) to transmit and receive a make-ligule-make-auricle inductive signal.     

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.     

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.     

中华水韭叶舌和缘膜的发生及其发育进程研究

采用石蜡切片技术,以人工培养的中华水韭幼苗的最初几枚叶至成熟植株的叶为实验材料,连续解剖观察其叶舌和缘膜的发生、发育进程,并分析其发育进程与孢子囊和叶片的关系.结果显示:(1)中华水韭叶舌与叶片在其个体发育早期来自于同一原基,但叶舌最初的发育速度快于叶片.(2)中华水韭的苗龄达到15枚叶时开始有孢子囊发生,此时的叶舌下方有明显的缘膜结构.(3)当中华水韭的孢子体达到30枚叶片以上时,早期产生于植株外围的孢子囊已经发育成熟,可以清楚地区分出大、小孢子囊,其中在已经成熟的大孢子叶上,叶舌相对于孢子囊的长度变短,下唇萎缩,缘膜消失;成熟小孢子叶的叶舌比大孢子叶的叶舌长,上翻程度大,下唇萎缩程度不如大孢子叶明显,缘膜也退化消失.研究认为,缘膜是水韭系统发育早期的普遍结构,而演化后期一些地区的缘膜则显著退化甚至消失;对于系统发育初期的中华水韭,其叶舌与叶片的差异并不像现代水韭那么明显.     

CRISPR/Cas9 mediated targeted mutagenesis of LIGULELESS-1 in sorghum provides a rapidly scorable phenotype by altering leaf inclination angle

Sorghum (Sorghum bicolor L. Moench) is one of the world's most cultivated cereal crops. Biotechnology approaches have great potential to complement traditional crop improvement. Earlier studies in rice and maize revealed that LIGULELESS-1 (LG1) is responsible for formation of the ligule and auricle, which determine the leaf inclination angle. However, generation and analysis of lg1 mutants in sorghum has so far not been described. Here, we describe CRISPR/Cas9 mediated targeted mutagenesis of LG1 in sorghum and phenotypic changes in mono- and bi-allelic lg1 mutants. Genome editing reagents were co-delivered to sorghum (var. Tx430) with the nptII selectable marker via particle bombardment of immature embryos followed by regeneration of transgenic plants. Sanger sequencing confirmed a single nucleotide insertion in the sgRNA LG1 target site. Monoallelic edited plantlets displayed more upright leaves in tissue culture and after transfer to soil when compared to wild type. T1 progeny plants with biallelic lg1 mutation lacked ligules entirely and displayed a more severe reduction in leaf inclination angle than monoallelic mutants. Transgene-free lg1 mutants devoid of the genome editing vector were also recovered in the segregating T1 generation. Targeted mutagenesis of LG1 provides a rapidly scorable phenotype in tissue culture and will facilitate optimization of genome editing protocols. Altering leaf inclination angle also has the potential to elevate yield in high-density plantings.     

Developmental genetics of mutants that specify knotted leaves in maize

Of seven dominant knotted-leaf mutants tested, six mapped at or near Kn1 on the long arm of chromosome 1, and one was not linked to Kn1. Comparisons of phenotypes among these mutants allowed us to focus on a systematic abnormality: the parenchyma cells associated with lateral veins do not fully differentiate into bundle sheath, mesophyll or upper sclerenchyma. The more dramatic expression of Kn1 mutants—knots, ligule alterations and twisting—is sporadic and dependent on the time when the mutant acts in leaf primordium development. Using lw to mark leaf sectors that lose Kn1 following X-irradiation, we show that the knotted-leaf phenotype encoded by chromosome 1L is autonomous. Analysis of sectors lacking a particular Kn1 gene ( Kn1-N2) suggests that Kn1 itself, rather than a linked modifier gene, is autonomous in the leaf primordium. Aneuploid studies using various translocations involving 1L and marked by Adh1 allozymes are compared. The Kn1 mutant appears to encode a "new" function or a considerable overproduction of an extant product in the leaf. Kn1/- 1L hypoploids either express knotted poorly or not at all; transvection is ruled out, but the cause for this modification of Kn1 expression is not yet known.—Our working hypothesis is that Kn1 mutants permit the expression of a product that is usually not produced in leaf primordial cells. We suggest that this product interferes with the early cell-type commitments of cells near lateral veins. Thus, relatively uncommitted cells are present in more mature blades, where they may divide unexpectedly into knots or may induce bits of ligule.