Leaf Mutant Research Articles

Biochemical and molecular characterization of photosystem I deficiency in the NCS6 mitochondrial mutant of maize

Interorganellar signaling interactions are poorly understood. The maize non-chromosomal stripe (NCS) mutants provide models to study the requirement of mitochondrial function for chloroplast biogenesis and photosynthesis. Previous work suggested that the NCS6 mitochondrial mutation, a cytochrome oxidase subunit 2 (cox2) deletion, is associated with a malfunction of Photosystem I (PSI) in defective chloroplasts of mutant leaf sectors (Gu et al., 1993). We have now quantified the reductions of photosynthetic rates and PSI activity in the NCS6 defective stripes. Major reductions of the plastid-coded PsaC and nucleus-coded PsaD and PsaE PSI subunits and of their corresponding mRNAs are seen in mutant sectors; however, although the psaA/B mRNA is greatly reduced, levels of PsaA and PsaB (the core proteins of PSI) are only slightly decreased. Levels of the PsaL subunit and its mRNA appear to be unchanged. Tested subunits of other thylakoid membrane complexes--PSII, Cyt b6/f, and ATP synthase, have minor (or no) reductions within mutant sectors. The results suggest that specific signaling pathways sense the dysfunction of the mitochondrial electron transport chain and respond to down-regulate particular PSI mRNAs, leading to decreased PSI accumulation in the chloroplast. The reductions of both nucleus and plastid encoded components indicate that complex interorganellar signaling pathways may be involved.

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.

Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA synthase.

Cuticular waxes play a pivotal role in limiting transpirational water loss across the plant surface. The correlation between the chemical composition of the cuticular waxes and their function as a transpiration barrier is still unclear. In the present study, intact tomato fruits (Lycopersicon esculentum) are used, due to their astomatous surface, as a novel integrative approach to investigate this composition- function relationship: wax amounts and compositions of tomato were manipulated before measuring unbiased cuticular transpiration. First, successive mechanical and extractive wax-removal steps allowed the selective modification of epi- and intracuticular wax layers. The epicuticular film consisted exclusively of very-long-chain aliphatics, while the intracuticular compartment contained large quantities of pentacyclic triterpenoids as well. Second, applying reverse genetic techniques, a loss-of-function mutation with a transposon insertion in a very-long-chain fatty acid elongase beta-ketoacyl-CoA synthase was isolated and characterized. Mutant leaf and fruit waxes were deficient in n-alkanes and aldehydes with chain lengths beyond C30, while shorter chains and branched hydrocarbons were not affected. The mutant fruit wax also showed a significant increase in intracuticular triterpenoids. Removal of the epicuticular wax layer, accounting for one-third of the total wax coverage on wild-type fruits, had only moderate effects on transpiration. By contrast, reduction of the intracuticular aliphatics in the mutant to approximately 50% caused a 4-fold increase in permeability. Hence, the main portion of the transpiration barrier is located in the intracuticular wax layer, largely determined by the aliphatic constituents, but modified by the presence of triterpenoids, whereas epicuticular aliphatics play a minor role.

Regulation and a conserved intron sequence of liguleless3/4 knox class-I homeobox genes in grasses.

The nine class-I maize (Zea mays L.) knox genes are putative transcription factors normally expressed in shoot apices, but not in leaves. knotted1 (kn1) seems to function in shoot apical meristem maintenance, and rough sheath1 (rs1)-like genes may act in internode elongation. The function of liguleless3 (lg3)-type genes is still unknown. Here, we characterized lg3 as well as the two most closely related genes liguleless4a (lg4a, formerly knox11) and liguleless4b (lg4b, formerly knox5). We termed this subclass of knox genes lg3/4 genes. We studied the expression patterns of lg3/4 genes and compared their sequences. We obtained knockout mutants of lg3 by finding Mu transposon insertions into exons. Our results show that lg3 was not essential for plant development, and that lg4a and lg4b were likely to encode the redundant function. In addition, lg4a but not lg4b was ectopically expressed in the Lg4-O mutant, suggesting that this mutant was affected at the lg4a locus. We found that the lg3 gene was unique among knox genes as it was co-induced in the leaves of leaf mutants that ectopically expressed knox genes in the leaves. The leaf phenotype expressed in the dominant Rs1-O mutant was not altered when lg3 function was removed using the knockout. Genomic sequence comparisons of lg3, lg4a and lg4b from maize and the two homologous genes, osh6 and osh71, from rice revealed a 14-bp phylogenetic footprint in intron II. This sequence was conserved in nucleotide composition, position and polarity in the lg3/4 genes of divergent grasses representing six Gramineae subfamilies. In an independent experiment, this same conserved sequence was found in a yeast reverse one-hybrid screen for putative binding sites of the LG3 homeodomain protein. Distribution of this 14-bp sequence was examined within the public rice database. The possible function of this sequence in regulation of lg3/4 genes is discussed.

細胞分裂の縦横：シロイヌナズナ細葉変異体 angustifolia3 の解析

細胞分裂の縦横：シロイヌナズナ細葉変異体 angustifolia3 の解析

FIE and CURLY LEAF polycomb proteins interact in the regulation of homeobox gene expression during sporophyte development.

The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 homologue of Drosophila extra sex comb (ESC), regulates endosperm and embryo development and represses flowering during embryo and seedling development. As fie alleles are not transmitted maternally, homozygous mutant plants cannot be obtained. To study FIE function during the entire plant life cycle, we used Arabidopsis FIE co-suppressed plants. Low FIE level in these plants produced dramatic morphological aberrations, including loss of apical dominance, curled leaves, early flowering and homeotic conversion of leaves, flower organs and ovules into carpel-like structures. These morphological aberrations are similar to those exhibited by plants overexpressing AGAMOUS (AG) or CURLY LEAF (clf) mutants. Furthermore, the aberrant leaf morphology of FIE-silenced and clf plants correlates with de-repression of the class I KNOTTED-like homeobox (KNOX) genes including KNOTTED-like from Arabidopsis thaliana 2 (KNAT2) and SHOOTMERISTEMLESS (STM), whereas BREVIPEDICELLUS (BP) was upregulated in FIE-silenced plants, but not in the clf mutant. Thus, FIE is essential for the control of shoot and leaf development. Yeast two-hybrid and pull-down assays demonstrate that FIE interacts with CLF. Collectively, the morphological characteristics, together with the molecular and biochemical data presented in this work, strongly suggest that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Our findings demonstrate that the versatility of the plant FIE function, which is derived from association with different SET (SU (VAR)3-9, E (Z), Trithorax) domain PcG proteins, results in differential regulation of gene expression throughout the plant life cycle.

DCL is a plant-specific protein required for plastid ribosomal RNA processing and embryo development.

The defective chloroplast and leaf-mutable (dcl-m) mutation of tomato blocks chloroplast differentiation in leaf mesophyll cells and a signaling system that appears to be required for morphogenesis of palisade cells during leaf growth. To dissect the function of DCL, mutants with stable dcl alleles (dcl-s) were generated and examined for their phenotype. DCL/dcl-s plant produce dcl-s/dcl-s seeds with embryos arrested at the globular stage of development. The levels of several chloroplast- and nuclear-encoded proteins are strongly reduced in dcl-m mutant leaf sectors without significant changes in their corresponding mRNAs. The 4.5S rRNA fails to be processed efficiently, however, suggesting that DCL has a direct or indirect function in rRNA processing or correct ribosome assembly. Accordingly, chloroplasts in dcl-m sectors are impaired in polysome assembly, which can explain the reduced accumulation of chloroplast-encoded proteins. These results suggest that DCL is required for chloroplast rRNA processing, and emphasize the importance of plastid function during embryogenesis.

Roles for auxin during morphogenesis of the compound leaves of pea ( Pisum sativum).

The goal of this study was to explore the impact of the plant growth regulator auxin on the development of compound leaves in pea. Wildtype ( WT) plantlets, as well as those of two leaf mutants, acacia ( tl) and tendrilled acacia ( uni-tac) of pea ( Pisum sativum L.), were grown on media containing the auxin-transport inhibitors 2,3,5-triiodobenzoic acid (TIBA), N-(1-naphthyl)phthalamic acid (NPA), or the auxin antagonist, p-chlorophenoxyisobutyric acid (PCIB). The resulting plantlets were carefully analyzed morphologically, by scanning electron microscopy and for Uni gene expression using quantitative reverse transcription-polymerase chain reaction. Auxin transport was measured in WT leaf parts using [(14)C]indole-3-acetic acid. Relative Uni gene expression was determined in shoot tips of a range of leaf-form mutants. Morphological abnormalities were observed for all genotypes examined. The terminal tendrils on WT plants were converted to leaflets, stubs or were aborted. The number of pinna pairs produced on leaves was reduced, with the distal forms being eliminated before the proximal ones. Some leaves were converted to simple, including tri-and bilobed, forms. These treatments phenocopy the uni-tac and unifoliata ( uni) mutants of pea. In the most extreme situations, leaf blades were completely lost leaving only a pair of stipules or scale leaves. Polar auxin transport was basipetal for all leaf parts. Uni gene expression in shoot tips was significantly reduced in 60 microM NPA and TIBA. Uni mRNA was more abundant in tl, af and af tl and reduced in the uni mutants compared to WT. These results indicate that an auxin gradient plays fundamental roles in controlling morphogenesis in the compound leaves of pea and specifically it: (i). is the driving force for leaf growth and pinna determination; (ii). is necessary for pinna initiation; and (iii). controls subsequent pinna development.

Inheritance and mapping of a yellow leaf mutant of carrot (Daucus carota)

AbstractA yellow leaf mutant at a locus named YEL was selected in a population of the cultivated carrot. Genetic analysis of segregating F2 progenies and corresponding F3 families, indicates that the phenotype expressed is controlled by a single recessive nuclear gene. The mutant is stably inherited and is associated with a reduced leaf‐biomass of approximately 30% compared with the wild‐type. Amplified fragment length polymorphism markers were developed and used in bulked segregant analysis. Seventeen marker candidates were detected by using 45 primer pairs. Ten of these could be linked with the YEL locus and mapped in a linkage group with a total length of 33.2 cM. Application of the yellow leaf mutant in carrot research is discussed.

PIE1, an ISWI family gene, is required for FLC activation and floral repression in Arabidopsis.

Proper control of the floral transition is critical for reproductive success in flowering plants. In Arabidopsis, FLOWERING LOCUS C (FLC) is a floral repressor upon which multiple floral regulatory pathways converge. Mutations in PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1) suppress the FLC-mediated delay of flowering as a result of the presence of FRIGIDA or of mutations in autonomous pathway genes. PIE1 is required for high levels of FLC expression in the shoot apex, but it is not required for FLC expression in roots. PIE1 is similar to ATP-dependent, chromatin-remodeling proteins of the ISWI and SWI2/SNF2 family. The role of PIE1 as an activator of FLC is consistent with the general role of ISWI and SWI2/SNF2 family genes as activators of gene expression. The pie1 mutation also causes early flowering in noninductive photoperiods independently of FLC; thus, PIE1 appears to be involved in multiple flowering pathways. PIE1 also plays a role in petal development, as revealed by the suppression of petal defects of the curly leaf mutant by the pie1 mutation.

Induction of somatic instability in stable yellow leaf mutant of rice by ion beam irradiation

Any class II type active transposons have not been discovered in rice though transposon (mobile element) is very useful for gene isolation in several plant species. In order to capture somatic instability induced by an endogenous active transposon in rice, stable yellow leaf plants derived from a variegated yellow leaf ( yl-v) mutant found in F2 of a cross between distantly related rice varieties were irradiated with carbon and helium ion beams. In M1 plants derived from the seeds irradiated with 50 Gy of 220 MeV carbon ions, a variegated yl plant was generated and this plant showed small or large sectors in leaves expanded later. Most of panicle-row M2 lines segregated into variegated and stable yl plants. In total, the ratio of variegated to stable yl plants was 3:1, suggesting that clear variegation observed on M1 plants might be caused by activation of a cryptic inactive autonomous element by carbon ion beam irradiation.

Identification of Mutator insertional mutants of starch-branching enzyme 1 (sbe1) in Zea mays L.

Starch-branching enzymes (SBE) alter starch structure by breaking an alpha-1,4 linkage and attaching the reducing end of the new chain to a glucan chain by an alpha 1,6 bond. In maize, three isoforms of SBE have been identified. In order to examine the function of the SBEI isoform, a reverse-genetics PCR-based screen was used to identify a mutant line segregating for a Mutator transposon within Sbe1. Compared to wild-type controls, Sbe1 transcripts accumulate at extremely low levels in leaves of the homozygous mutant. Antibodies failed to detect SBEI in leaf tissue of mutants or wild-type controls. In contrast, the level of SBEI in endosperm is undetectable in homozygous mutants while easily detected in wild-type controls. Starches extracted from mutant leaves and endosperm have structures indistinguishable from starches of wild-type controls as determined by size-exclusion chromatography (SEC) of intact starch and high-performance SEC of debranched starch. To investigate the possibility of compensation for the lack of SBEI by expression of the homologous sequence reported by Kim etal. (1998), agenomic fragment (Sbe1b) of this sequence was cloned. Northern hybridizations of mutant leaf, root, tassel, endosperm and embryo tissues with non-specific Sbelb probes failed to reveal expression of the homologous sequence. These results suggest that the homologous sequence is not compensating for a lack of SBEI in sbe1::Mu mutants. Further study of this sbel mutation in the presence of other genetic mutations may help to understand the role of SBEI in determining starch structure in leaves and endosperm.

Leaf and Flower Development in Pea (Pisum sativum L.): Mutants cochleata andunifoliata

The stipule mutant cochleata(coch) and the simple-leaf mutantunifoliata (uni) are utilized to increase understanding of the control of compound leaf and flower development in pea. The phenotype of the coch mutant, which affects the basal stipules of the pea leaf, is described in detail. Mutant coch flowers have supernumerary organs, abnormal fusing of flower parts, mosaic organs and partial male and female sterility. The wild-type Coch gene is shown to have a role in inflorescence development, floral organ identity and in the positioning of leaf parts. Changes in meristem size may be related to changes in leaf morphology. In the coch mutant, stipule primordia are small and their development is retarded in comparison with that of the first leaflet primordia. The diameter of the shoot apical meristem of the uni mutant is approx. 25% less than that of its wild-type siblings. This is the first time that a significant difference in apical meristem size has been observed in a pea leaf mutant. Genetic controls in the basal part of the leaf are illustrated by interactions between coch and other mutants. The mutantcoch gene is shown to change stipules into a more ‘compound leaf-like’ identity which is not affected by thestipules reduced mutation. The interaction of coch and tendril-less(tl) genes reveals that the expression of the wild-type Tl gene is reduced at the base of the leaf, supporting the theories of gradients of gene action.

Interactions between venation pattern formation genes in Arabidopsis thaliana

Aiming to contribute to the understanding of the genetic mechanisms underlying venation pattern formation in the vegetative leaves of Arabidopsis thaliana, we have previously analyzed the natural variability of this trait and searched for venation pattern mutants with normally shaped leaves after chemical and insertional mutagenesis. The rarity of such mutants suggested that vein patterning and leaf morphogenesis might not be independent processes. To test this hypothesis, we have examined 97 abnormally shaped leaf mutants, each one belonging to a different complementation group, and found 2 whose venation patterns differed from that of the wild type. We have focused our attention on one of these mutants, rotunda1 (ron1), and in a recessive monogenic trait, hemivenata (hve), which was identified in an ecotype. Our study reveals that the Hve phenotype is pleiotropic, including an extremely simple venation pattern, increased stem branching, altered root waving, and low fertility. We have obtained double mutants involving mutations at several genes known to participate in vascular development, and/or auxin transport and perception. Linkage analysis has allowed us to assign the HVE gene to a 200 kb interval in the short arm of chromosome 2. Further genetic and molecular analyses are in progress to identify HVE among the available candidate genes.

Genetic analysis of mutations at loci controlling leaf form in cowpea (Vigna unguiculata.

Mutations affecting leaflet number and shape occurred at high frequencies in some cowpea crosses. Mutant plants were nonpetiolate and unifoliolate as opposed to normal plants which were petiolate and trifoliolate. Two types of unifoliolate mutants were distinguishable on the basis of leaf shape which was ovate in one mutant and orbicular in the other. The nonpetiolate and the unifoliolate traits in the two mutants are each controlled by single recessive genes, but the genes controlling the traits in the different mutants were nonallelic. The orbicular leaf shape was also under the control of a single recessive gene. In the F2 and subsequent generations of the cross IBS 2497 x IBS 2625, orbicular-shaped unifoliolate leaf mutants were regularly produced, although the two parents involved in the cross were both trifoliolate. Linkage tests showed that the genes pt-1 for nonpetiolate trait, un-2 for unifoliolate leaf, and orb for orbicular leaf shape in one of the mutants were located on the same chromosome, while the genes pt-3 and un-3 in the other were also linked on a different chromosome. The results of this study provide further evidence indicating the involvement of transposable elements in the mutations observed in the cowpea lines used in this study.

Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis.

Molecular and genetic characterizations of mutants have led to a better understanding of many developmental processes in the model system Arabidopsis thaliana. However, the leaf development that is specific to plants has been little studied. With the aim of contributing to the genetic dissection of leaf development, we have performed a large-scare screening for mutants with abnormal leaves. Among a great number of leaf mutants we have generated by T-DNA and transposon tagging and ethyl-methae sulfonate (EMS) mutagenesis, four independent mutant lines have been identified and studied genetically. Phenotypes of these mutant lines represent the defects of four novel nuclear genes designated LL1 (LOTUS LEAF 1), LL2 (LOTUS LEAF 2), URO (UPRIGHT ROSETTE). and EIL (ENVIRONMENT CONDITION INDUCED LESION). The phenotypic analysis indicates that these genes play important roles during leaf development. For the further genetic analysis of these genes and the map-based cloning of LL1 and LL2, we have mapped these genes to chromosome regions with an efficient and rapid mapping method.

Phloem loading in the sucrose-export-defective (SXD-1) mutant maize is limited by callose deposition at plasmodesmata in bundle sheath—vascular parenchyma interface

Using Lucifer Yellow we have demonstrated that the phloem-loading pathway from the mesophyll to the bundle sheath—vascular parenchyma interface inZea mays source leaves follows a symplasmic route in small and intermediate vascular bundles in control as well as in the green sections of mutant sucrose-export-defective (SXD-1) plants. In the anthocyanin-rich mutant leaf sections, Lucifer Yellow transport was prohibited along the same path, at the bundle sheath—vascular parenchyma interface in particular. Plasmodesmata at the latter interface in SXD-1 anthocyanin-rich leaf sections appear to be structurally altered through callose deposition at the plasmodesmal orifices. We suggest that a transport bottleneck at the bundle sheath—vascular parenchyma interface is thus orchestrated and regulated through callose formation, preventing symplasmic transport across this important loading interface.

038 Characterization of an Architectural Mutant of Bean (Phaseolus vulgaris L.)

Genetic and morphological characteristics of an architectural mutant in common beans were studied. The mutant had shiny, dark green leaves, overlapping leaflets, short petioles and a terminal reproductive bud even though the line did not carry the fin gene. Branching was nearly absent, resulting in a single stem vine. This is a new form of determinancy in common bean. Inheritance studies demonstrated that the mutant trait was controlled by a single recessive gene. Allelism tests were performed between the mutant and a previously reported similar mutants, which were overlapping leaflets mutant (ol), and dark green savoy leaf mutant (dgs). Results showed that the mutant trait was not allelic to ol and dgs. As a temporary designation, the name “”opiary” describing its compact and neat appearance is being used. Linkage was tested for growth habit (fin), shiny leaf, cross-sectional shape of pods, striped pod (prpst) and pod suture strings (st) with the topiary mutant. No linkages were detected between either the mutant and marker genes or among the marker genes. The topiary mutant has potential for improving common beans. Its single stem growth habit may allow closer row spacing leading to higher planting populations and may enhance the efficiency of mechanical harvest. Pod formation at higher nodes may escape disease. Currently, the thin stems cause lodging. Development of thick and upright forms will be the subject of future studies.

A mutational analysis of leaf morphogenesis in Arabidopsis thaliana.

As a contribution to a better understanding of the developmental processes that are specific to plants, we have begun a genetic analysis of leaf ontogeny in the model system Arabidopsis thaliana by performing a large-scale screening for mutants with abnormal leaves. After screening 46,159 M2 individuals, arising from 5770 M1 parental seeds exposed to EMS, we isolated 1926 M2 putative leaf mutants, 853 of which yielded viable M3 inbred progeny. Mutant phenotypes were transmitted with complete penetrance and small variations in expressivity in 255 lines. Most of them were inherited as recessive monogenic traits, belonging to 94 complementation groups, which suggests that we did not reach saturation of the genome. We discuss the nature of the processes presumably perturbed in the phenotypic classes defined among our mutants.

A new CMS source in Nicotiana developed via somatic cybridization between N. tabacum and N. alata

Cytoplasmic somatic hybrids (cybrids) between the two sexually incompatible species Nicotiana tabacum and Nicotiana alata were constructed. A total of 33 green regenerants were obtained after fusion of protoplasts from a tobacco cytoplasmic chlorophyll-deficient mutant and gamma irradiation-inactivated leaf protoplasts of N. alata. Twenty nine of them were male sterile and displayed an altered stamen morphology (formation of petaloid and stigmoid structures instead of stamens). Southern-blot analyses of eight CMS plants using N. alata-specific nuclear repetitive DNA and cpDNA probes revealed that they contained nuclear genetic material of N. tabacum and chloroplasts from N. alata. Restriction-enzyme analysis of mitochondrial DNAs of the cybrids in question showed different patterns consisting of an incomplete mix of mtDNA fragments from both parents, as well as new fragments. Southern-blot analysis of mtDNAs with a sunflower atpA probe gave the same recombinant hybridization pattern for all analyzed cybrids, indicating that high-frequency specific recombination occurs in the atpA region. Analysis of the progeny from three successive backcrosses of the studied cybrids with N. tabacum demonstrated a strict cytoplasmic inheritance of the male-sterile phenotype.