Morphogenetic effects of the interaction of floral mutations petal-sepal and anther in petal in Papaver somniferum l

The morphogenetic effects of the interaction of the floral mutations petal-sepal and anther in petal in Papaver somniferum L. with a monocarpic shoot were studied. During analysis of the mutations controlled by the genes ptsp and Ant, no plants of the double-mutant class were found in the second generation, in which microsporangia form on the corolla sepal structures. The ratio of phenotypic classes obtained in the experiment corresponds to that inheritance, when the genetic control of mutant characters is realized by nonallele nonlinked genes Ant and ptsp upon epistatic interaction of these genes. These data were confirmed by analysis of the genotypes of F2 plants from the phenotypic class petal-sepal, which include plants that carry both mutant genes Ant and ptsp. The results obtained suggest that the gene Ant, which controls the formation of microsporangia in the corolla metameres, is not expressed in the presence of a mutation of the gene ptsp; i.e., microsporangia are not formed in tissues with photosynthesizing cells. It is evident that the development of microsporangia is determined by the level of a product of the gene Ptsp. The role of flavonols (quercetin), inhibitors of photosynthesis, as a mechanism of regulation of activity of the genes controlling morphogenesis of the corolla elements and differentiation of microsporangia, is discussed.     

Involvement of the <Emphasis Type="Italic">Arabidopsis thaliana AtPMS1</Emphasis> gene in somatic repeat instability

Mismatch repair (MMR) genes participate in the maintenance of genome stability in all organisms. Based on its high degree of sequence conservation, it seems likely that the AtPMS1 gene of Arabidopsis thaliana is part of the MMR system in this model plant. To test this hypothesis, we aimed to disrupt AtPMS1 function by over-expressing mutated alleles expected to result in a dominant negative effect. To create one mutant allele we substituted two amino acids in the MutL-box, and for the other mutant allele we deleted 87 amino acids comprising the whole MutL-box. Contrary to published reports in some eukaryotes, transgenic plants expressing these alleles did not exhibit a decrease in fertility nor any other visible phenotype. To examine the impact of these mutations on microsatellite instability, the phenotype most often observed in organisms defective in MMR, reporter lines containing a uidA (GUS) gene inactivated by the insertion of a synthetic microsatellite (G₇ or G₁₆) were used. GUS gene function in these lines can be restored following the loss of one base or the gain of two bases in the repetitive tract. This results in the observation of blue sectors on a white background following histochemical staining. In a subset of the transformants, a significant increase (2- to 28-fold) in microsatellite instability was observed relative to wild-type. This report shows that MMR function can be disrupted via a dominant negative approach, and it is the first report to describe the phenotypic consequence of disrupting the function of a MutL homolog in plants.     

Identification of novel alleles induced by EMS-mutagenesis in key genes of kernel hardness and starch biosynthesis in wheat by TILLING

To identify novel allelic variations in key genes of wheat quality, the present study used the targeting induced local lesions in genomes platform to detect point mutations in target genes. The wheat variety Longfumai 17 was treated by the mutagen ethyl methanesulfonate to produce a bulk M₂ generation, and the population included 1122 plants. A total length of 3906.80 kb nucleotides was analyzed, and the average mutation density was 1/244.17 kb. The identified mutations included G>A substitutions (43.75%), C>T substitutions (31.25%), A insertions (12.50%), T insertions (6.25%), and deletions (6.25%). These point mutations led to changes in amino acids and thus the encoded protein sequences, ultimately producing 18.75% of missense mutations, 12.50% of frame shift mutations, 6.25% of nonsense mutations, 25.00% of silent mutations and 37.50% of non-coding region mutations. In the kernel hardness gene Pinb and 3 starch synthesis genes waxy, Agp2 and SSIIa-A, we detected 16 different point mutations in 25 mutant lines. The Pinb gene harbored two missense mutations and a nonsense mutation; the C>T missense mutation resulted in a novel allele, this novel allele and the nonsense mutation alerted protein 3D structure; the waxy gene presented missense and frame shift mutations; the Agp2 gene carried a missense mutation; the SSIIa-A incurred a missense mutation and a frame shift mutation that resulted in premature protein termination. All the frame shift mutations, nonsense mutations and the Pinb novel allele resulted in allelic variation of their corresponding genes, which in turn affected their gene functions. The identified mutant lines can be used as intermediate materials in wheat quality improvement schemes.     

Combining next‐generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M₂) family derived from a heterozygous (M₁) parent were identified using whole‐genome shotgun (WGS) sequencing of a small number of (M₂) individuals with mutant and wild‐type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self‐pollinated (M₃) offspring. Finally, we filtered for segregating EMS‐induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M₂) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non‐synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.     

The ANGULATA7 gene encodes a DnaJ‐like zinc finger‐domain protein involved in chloroplast function and leaf development in Arabidopsis

The characterization of mutants with altered leaf shape and pigmentation has previously allowed the identification of nuclear genes that encode plastid‐localized proteins that perform essential functions in leaf growth and development. A large‐scale screen previously allowed us to isolate ethyl methanesulfonate‐induced mutants with small rosettes and pale green leaves with prominent marginal teeth, which were assigned to a phenotypic class that we dubbed Angulata. The molecular characterization of the 12 genes assigned to this phenotypic class should help us to advance our understanding of the still poorly understood relationship between chloroplast biogenesis and leaf morphogenesis. In this article, we report the phenotypic and molecular characterization of the angulata7‐1 (anu7‐1) mutant of Arabidopsis thaliana, which we found to be a hypomorphic allele of the EMB2737 gene, which was previously known only for its embryonic‐lethal mutations. ANU7 encodes a plant‐specific protein that contains a domain similar to the central cysteine‐rich domain of DnaJ proteins. The observed genetic interaction of anu7‐1 with a loss‐of‐function allele of GENOMES UNCOUPLED1 suggests that the anu7‐1 mutation triggers a retrograde signal that leads to changes in the expression of many genes that normally function in the chloroplasts. Many such genes are expressed at higher levels in anu7‐1 rosettes, with a significant overrepresentation of those required for the expression of plastid genome genes. Like in other mutants with altered expression of plastid‐encoded genes, we found that anu7‐1 exhibits defects in the arrangement of thylakoidal membranes, which appear locally unappressed.     

Co-suppression of the petunia homeotic gene fbp2 affects the identity of the generative meristem

The function of the petunia MADS box gene fbp2 in the control of floral development has been investigated. Inhibition of fbp2 expression in transgenic plants by a co-suppression approach resulted in the development of highly aberrant flowers with modified whorl two, three and four organs. This mutant flower phenotype inherited as a single Mendelian trait. The flowers possess a green corolla which is reduced in size. Furthermore, the stamens are replaced by green petaloid structures and the inner gynoecial whorl is dramatically reduced. No ovules or placenta are formed and instead two new inflorescences developed in the axils of the carpels. These homeotic transformations are accompanied by a complete down-regulation of the petunia MADS box gene fbp6 which is highly homologous to the Arabidopsis and Antirrhinum genes agamous (ag) and plena (ple). In contrast to this, two other petunia MADS box genes, exclusively expressed in whorls two and three, are still transcribed. Our results indicate that the fbp2 gene belongs to a new class of morphogenesis genes involved in the determination of the central part of the generative meristem.     

A corolla-and carpel-abundant,non-specific lipid transfer protein gene is expressed in the epidermis and parenchyma of Gerbera hybrida var. Regina (Compositae)

We are examining the floral organ differentiation in Compositae by isolating and characterizing corolla abundant genes. Differential screening of a cDNa library made from the ray floret corolla of Gerbera hybrida var. Regina revealed an abundant cDNA clone which is expressed in the corolla but not in leaves. This cDNA (gltp1) codes for a polypeptide similar to non-specific lipid transfer proteins of the plants. The gltp1 gene is expressed only in the corolla and carpels and is developmentally regulated during corolla development. The gltp1 mRNA accumulates both in epidermal cell layers and in the mesophyll of the corolla. In the stylar part of the carpel, the gltp1 mRNA can be detected in the epidermal and in parenchymal cells but not in the transmitting tissue. Analogous patterns of gltp1 expression in the corolla and carpel may indicate that similar genetic programmes operate during the development of these two tissues.     

Evolution of microsporangium numbers in Microseris (Asteraceae: Lactuceae)

The genus Microseris contains species with disporangiate stamens and species with tetrasporangiate stamens. We determined the number of microsporangia per stamen in serial sections of heads from all 13 species of Microseris, its close relative Uropappus lindleyi, and the two allopolyploid species of Slebbinsoseris. Four Microseris species, three diploid and one tetraploid, have two microsporangia per stamen; all other species investigated have four. The most parsimonious assumption is that the disporangiate condition is derived and arose once in the evolution of Microseris. The inheritance of the number of microsporangia per stamen in crosses between M. bigelovii (disporangiate) and M. douglasii (tetrasporangiate) was determined. Segregation of microsporangium number per stamen in F2s derived from these crosses is quantitative rather than Mendelian. The average number of microsporangia per stamen in the F2 plants ranges from 2.0 to 4.0. There is a predominance of tetrasporangiate stamens in the F1 and in most F2 plants. The observed pattern of inheritance suggests a major gene with dominance and quantitative modifiers.     

Highly efficient multiplex editing: one-shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene-free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T₁ and T₂, respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on the production of toxic 5-fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T₁, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher-order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.     

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Processing of double‐stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL‐effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi‐allelic double mutant for the two soya bean paralogous Double‐stranded RNA‐binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9‐generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ‐line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer‐like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer‐like3 gene and the GmHen1a gene was observed in the T₀ generation, but these mutations failed to transmit to the T₁ generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole‐genome sequencing to reveal a spectrum of non‐germ‐line‐targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.     

Association between inbreeding depression and floral traits in a generalist‐pollinated plant

Individual variation in the magnitude of inbreeding depression (ID) in plants and its association with phenotypic traits may have important consequences for mating system evolution. This association has been investigated only scarcely, and always considering traits functionally related to autogamy. Here, we explore the association between individual variation in ID and plant traits associated with pollinator attractiveness (related to plant size, corolla size and corolla shape) in two populations of Erysimum mediohispanicum (Brassicaceae). ID was calculated along the entire life cycle of the plants. In addition, we also explored the relationship between phenotypic traits and the individual levels of heterozygosity. We found significant associations between ID and corolla diameter and stalk height, being taller plants with larger corollas those undergoing a lower intensity of ID. Furthermore, we found a negative relationship between corolla diameter and heterozygosity, suggesting that plants with large flowers have purged their genetic load. Finally, we found a significant effect of corolla diameter on the intrapopulation genetic structure. All these findings suggest that plants with large flowers have secularly suffered frequent inbreeding in the study populations. Because corolla diameter is a trait frequently selected by pollinators in E. mediohispanicum, we believe that the observed relationship between this trait and ID could be mediated by pollinators, probably throughout an increasing in biparental inbreeding, geitonogamy or autogamy.     

High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction

Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene‐edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high‐throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high‐throughput quantitative real‐time (qPCR)‐based method. The qPCR‐based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild‐type and a gene‐edited mutant. We showed that the qPCR‐based method can accurately distinguish CRISPR/Cas9‐induced mutants from the wild‐type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR‐based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T₀ transgenic plants. In a 384‐well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post‐polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T₀ transgenic plants, which will be widely used in the area of plant gene editing.     

Variegation mutants and mechanisms of chloroplast biogenesis

Variegated plants typically have green‐ and white‐sectored leaves. Cells in the green sectors contain normal‐appearing chloroplasts, whereas cells in the white sectors lack pigments and appear to be blocked at various stages of chloroplast biogenesis. Variegations can be caused by mutations in nuclear, chloroplast or mitochondrial genes. In some plants, the green and white sectors have different genotypes, but in others they have the same (mutant) genotype. One advantage of variegations is that they provide a means of studying genes for proteins that are important for chloroplast development, but for which mutant analysis is difficult, either because mutations in a gene of interest are lethal or because they do not show a readily distinguishable phenotype. This paper focuses on Arabidopsis variegations, for which the most information is available at the molecular level. Perhaps the most interesting of these are variegations caused by defective nuclear gene products in which the cells of the mutant have a uniform genotype. Two questions are of paramount interest: (1) What is the gene product and how does it function in chloroplast biogenesis? (2) What is the mechanism of variegation and why do green sectors arise in plants with a uniform (mutant) genotype? Two paradigms of variegation mechanism are described: immutans (im) and variegated2 (var2). Both mechanisms emphasize compensating activities and the notion of plastid autonomy, but redundant gene products are proposed to play a role in var2, but not in im. It is hypothesized that threshold levels of certain activities are necessary for normal chloroplast development.     

ECOLOGICAL CONSEQUENCES AND PHENOTYPIC CORRELATES OF PETAL SIZE VARIATION IN WILD RADISH,RAPHANUS SATIVUS (BRASSICACEAE)

In this paper we examine some ecological consequences and phenotypic correlates of flower size variation in wild radish, Raphanus sativus. Mean corolla diameter varied significantly among individuals within natural populations of R. sativus in California. On the average, almost 40% of flower biomass was allocated to corolla tissue. In field experiments, pollinator visitation increased significantly with corolla size. Large flowers also accumulated more nectar when pollinators were excluded from plants. In three populations, corolla size was positively correlated with allocation to pollen per flower (either anther weight or pollen grain number), but there was usually no phenotypic relationship between corolla size and several measures of female allocation (ovule number per flower, proportion fruit set, and total seed mass per fruit). Plants growing in the field produced fewer large flowers per unit of stem, and stem biomass was negatively related to corolla size for plants grown under controlled greenhouse conditions. Male and female fitness may covary differently with allocation to attractive floral features in species such as R. sativus, where seed production is often limited by resources rather than by pollen.     

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T₀ plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T₁ generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T₀ plants, we could remove foreign DNA easily by genetic segregation in the T₁ generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

     

Cloning of cDNA coding for dihydroflavonol-4-reductase (DFR) and characterization of dfr expression in the corollas of Gerbera hybrida var. Regina (Compositae)

We are approaching corolla differentiation in Compositae by studying the regulation of flavonoid pathway genes during inflorescence development in gerbera. We have cloned a dfr cDNA from a ray floret corolla cDNA library of Gerbera hybrida var. Regina by a PCR technique based on homologies found in genes isolated from other plant species. The functionality of the clone was tested in vivo by complementing the dihydrokaempferol accumulating petunia mutant line RL01. By Southern blot analysis, G. hybrida var. Regina was shown to harbour a small family of dfr genes, one member of which was deduced to be mainly responsible for the DFR activity in corolla. Dfr expression in corolla correlates with the anthocyanin accumulation pattern: it is basipetally induced, epidermally specific and restricted to the ligular part of corolla. By comparing the dfr expression in different floret types during inflorescence development, we could see that dfr expression reflects developmental schemes of the outermost ray and trans florets, contrasted with that of the disc florets.     

New vector system for induction of gene expression in dicotyledonous plants

A system of two vectors, pEnLox and pCre, was developed. The pEnLox vector is used to induce insertional mutations, while pCre is used to obtain transgenic plants expressing the Cre recombinase gene. The T-DNA enhancer is flanked by the loxP sites in the pEnLox vector. As an Arabidopsis thaliana insertional mutant obtained by transformation with pEnLox is crossed with another transgenic line carrying the cre gene, the enhancer sequence is eliminated. The vector system makes it possible to induce insertional mutations and to determine whether the mutant phenotype is caused by the loss-of-function insertional mutation or by overespression of nearby genes in response to the enhancer contained in the insert.     

Effect of gibberellic acid treatments, environmental conditions, and genetic background on the expression of theparthenocarpic fruit mutation in tomato

Summary Theparthenocarpic fruit (pat) allele causes a complex syndrome affecting different aspects of tomato reproductive development. This mutation affects stamen (reduced length and carpelloidy), ovule (arrested integument growth and unviability), and ovary (autonomous growth, i.e., parthenocarpy) development;pat mutant plants therefore have reduced male and female fertility. We studied the phenotypic expression patterns of thepat gene after treatments with gibberellic acid (GA₃) and under different growth seasons (late spring and autumn) and genetic backgrounds (backcross [BC] population after interspecific cross). GA₃ treatments were only effective in restoring carpelloid anthers to the wild-type phenotype. Compared to late spring, mutant plants grown in autumn had a lower frequency of carpelloid anthers and aberrant ovules and a higher seed set. Inflorescence position also affected thepat expression; upper inflorescences had low frequency of short anthers and aberrant ovules and an increased tendency to set seeds,pat expressivity was more variable in BC₁ plants segregating after interspecific cross withLycopersicon pennellii than in the originalL. esculentum line. Therefore, a role for minor genes that modify the quantitative expression of thepat mutation is postulated and discussed.Abbreviations ANOVA analysis of variance - BC backcross - GA gibberellic acid     

Altered expression of flowering class B and class C genes in the Appendix tobacco mutant

 In the tobacco (Nicotiana tabacum) Appendix mutant, anthers are tipped by a miniature style and stigma. The outgrowth appears on the anther when it is already differentiating and follows the developmental timing of the central carpel. The Appendix mutation thus represents a late homeotic transformation suggesting that the APPENDIX (APX) gene either could be a misregulated organ identity gene or could be involved in regulating the expression of such genes. RFLP analysis with two class B (TM6 and NTGLO) and a class C (NAG) probes revealed that the Appendix phenotype is not caused by a mutation in one of these genes. However, in situ hybridization showed important changes in the expression of NTGLO and NAG in the mutant when compared with wild-type tobacco. Surprisingly, although no phenotypic alteration other than the style and stigma outgrowth is observed in the Appendix mutant, changes in class B and class C gene expession were not restricted to the anther tip cells from which the outgrowth originates. As expected, NAG was expressed in the Appendix outgrowth but it was also overexpressed in the normal third and fourth whorl organs at the time the outgrowth, as well as the central styles and stigmas, differentiated. Overexpression of a class C gene is probably responsible for the Appendix phenotype. In normal and mutant flowers, NTGLO was expressed in the second, third and fourth whorls up to the time of carpel fusion. Expression of this class B gene then ceased in the fourth whorl organs but was reactivated at later stages only in the styles and stigmas as well as in the outgrowths of the mutant. It thus seems that the function of the APX gene is either to regulate the late expression of organ identity genes or to control cell proliferation in such a way that, in the mutant, some cells are in a state where they respond in an unusual way to developmental signals. Received: 17 October 1997 / Revision accepted: 24 March 1998