Characterization of the first dominant dwarf maize mutant carrying a single amino acid insertion in the VHYNP domain of the <Emphasis Type="Italic">dwarf8</Emphasis> gene

The “green revolution” involving mainly wheat and rice was based on the use by breeders of semidominant mutations involved in the signal transduction pathway of Gibberellin (GA). In particular, mutations in the Reduced height (Rht) gene of wheat have been used to reduce plant height and consequently to avoid storm damage and lodging. These genes have been cloned and they encode for DELLA proteins which contain an N-terminal DELLA and a VHYNP domain essential for GA-dependent degradation of these proteins. In maize several mutations have been isolated which affect gibberellin biosynthesis and perception and in particular, mutations in Dwarf8 (D8) gene cause a severe dwarfing phenotype. D8 gene has been identified as an orthologue of Rht (Reduced height), Slr1(Slender rice 1) and Gibberellic Acid Insensitive (GAI) genes, this latter is a negative regulator of GA response in Arabidopsis. In this work, for the first time, we isolated and characterized a single amino acid insertion in the VHYNP domain of D8 maize gene causing the appearance of a dominant dwarf mutation. This spontaneous mutation, named D8-1023, showed a phenotype which is less severe in comparison with the other D8 mutants previously isolated which have modifications in the DELLA domain. This mutant appears to be an useful tool either to study the mechanism of GA-modulated growth in plants or to lower the height of maize tropical germplasm for breeding purposes.     

A novel dwarfing mutation in a green revolution gene from Brassica rapa

Mutations in the biosynthesis or signaling pathways of gibberellin (GA) can cause dwarfing phenotypes in plants, and the use of such mutations in plant breeding was a major factor in the success of the Green Revolution. DELLA proteins are GA signaling repressors whose functions are conserved in different plant species. Recent studies show that GA promotes stem growth by causing degradation of DELLA proteins via the ubiquitin-proteasome pathway. The most widely utilized dwarfing alleles in wheat (Triticum aestivum; e.g. Rht-B1b and Rht-D1b) encode GA-resistant forms of a DELLA protein that function as dominant and constitutively active repressors of stem growth. All of the previously identified dominant DELLA repressors from several plant species contain N-terminal mutations. Here we report on a novel dwarf mutant from Brassica rapa (Brrga1-d) that is caused by substitution of a conserved amino acid in the C-terminal domain of a DELLA protein. Brrga1-d, like N-terminal DELLA mutants, retains its repressor function and accumulates to high levels, even in the presence of GA. However, unlike wild-type and N-terminal DELLA mutants, Brrga1-d does not interact with a protein component required for degradation, suggesting that the mutated amino acid causes dwarfism by preventing an interaction needed for its degradation. This novel mutation confers nondeleterious dwarf phenotypes when transferred to Arabidopsis (Arabidopsis thaliana) and oilseed rape (Brassica napus), indicating its potential usefulness in other crop species.     

A single nucleotide mutation in GID1c disrupts its interaction with DELLA1 and causes a GA‐insensitive dwarf phenotype in peach

Plant stature is one important factor that affects the productivity of peach orchards. However, little is known about the molecular mechanism(s) underlying the dwarf phenotype of peach tree. Here, we report a dwarfing mechanism in the peach cv. FenHuaShouXingTao (FHSXT). The dwarf phenotype of ‘FHSXT’ was caused by shorter cell length compared to the standard cv. QiuMiHong (QMH). ‘FHSXT’ contained higher endogenous GA levels than did ‘QMH’ and did not response to exogenous GA treatment (internode elongation). These results indicated that ‘FHSXT’ is a GA‐insensitive dwarf mutant. A dwarf phenotype‐related single nucleotide mutation in the gibberellic acid receptor GID1 was identified in ‘FHSXT’ (GID1c^S191F), which was also cosegregated with dwarf phenotype in 30 tested cultivars. GID1c^S191F was unable to interact with the growth‐repressor DELLA1 even in the presence of GA. ‘FHSXT’ accumulated a higher level of DELLA1, the degradation of which is normally induced by its interaction with GID1. The DELLA1 protein level was almost undetectable in ‘QMH’, but not reduced in ‘FHSXT’ after GA₃ treatment. Our results suggested that a nonsynonymous single nucleotide mutation in GID1c disrupts its interaction with DELLA1 resulting in a GA‐insensitive dwarf phenotype in peach.     

Isolation and characterization of a <Emphasis Type="Italic">GAI/RGA</Emphasis>-like gene from <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

The aim of the investigation reported here was to assess the role of gibberellin in cotton fiber development. The results of experiments in which the gibberellin (GA) biosynthesis inhibitor paclobutrazol (PAC) was tested on in vitro cultured cotton ovules revealed that GA is critical in promoting cotton fiber development. Plant responses to GA are mediated by DELLA proteins. A cotton nucleotide with high sequence homology to Arabidopsis thaliana GAI (AtGAI) was identified from the GenBank database and analyzed with the BLAST program. The full-length cDNA was cloned from upland cotton (Gossypium hirsutum, Gh) and sequenced. A comparison of the putative protein sequence of this cDNA with all Arabidopsis DELLA proteins indicated that GhRGL is a putative ortholog of AtRGL. Over-expression of this cDNA in Arabidopsis plants resulted in the dwarfed phenotype, and the degrees of dwarfism were related to the expression levels of GhRGL. The deletion of 17 amino acids, including the DELLA domain, resulted in the dominant dwarf phenotype, demonstrating that GhRGL is a functional protein that affects plant growth. Real-time quantitative PCR results showed that GhRGL mRNA is highly expressed in the cotton ovule at the elongation stage, suggesting that GhRGL may play a regulatory role in cotton fiber elongation.     

Dbest1, a drosophila homolog of human bestrophin,is not required for viability or photoreceptor integrity

Summary: Best macular dystrophy (BMD) is an autosomal dominant human disease characterized by macular degeneration with juvenile onset (OMIM 153700). The disease is most often associated with mutations in Bestrophin, which encodes a novel protein with four putative transmembrane domains. However, complete loss‐of‐function mutations in Bestrophin have not been reported in humans or mice. We have identified three homologs of human Bestrophin in the Drosophila genome (dbest1‐3). The protein products of these three genes share significant homology to a 364 amino acid N‐terminal domain of human Bestrophin. We used P‐element mutagenesis to delete dbest1, which encodes a protein with the highest amino acid similarity to Bestrophin. Three independent dbest1 mutants were recovered from the mutagenesis screen. Homozygous null mutations in dbest1 do not significantly alter the viability or fertility of mutant flies. Moreover, dbest1 mutants have normal photoreceptor morphology and function. genesis 31:130–136, 2001. © 2001 Wiley‐Liss, Inc.     

Conditional and domain‐specific inactivation of the Tsc2 gene in neural progenitor cells

Tuberous sclerosis complex (TSC) is a genetic disease characterized by multiorgan benign tumors as well as neurological manifestations. Epilepsy and autism are two of the more prevalent neurological complications and are usually severe. TSC is caused by mutations in either the TSC1 (encodes hamartin) or the TSC2 (encodes tuberin) genes with TSC2 mutations being associated with worse outcomes. Tuberin contains a highly conserved GTPase‐activating protein (GAP) domain that indirectly inhibits mammalian target of rapamycin complex 1 (mTORC1). mTORC1 dysregulation is currently thought to cause much of the pathogenesis in TSC but mTORC1‐independent mechanisms may also contribute. We generated a novel conditional allele of Tsc2 by flanking exons 36 and 37 with loxP sites. Mice homozygous for this knock‐in Tsc2 allele are viable and fertile with normal appearing growth and development. Exposure to Cre recombinase then creates an in‐frame deletion involving critical residues of the GAP domain. Homozygous conditional mutant mice generated using Emx1^Cre have increased cortical mTORC1 signaling, severe developmental brain anomalies, seizures, and die within 3 weeks. We found that the normal levels of the mutant Tsc2 mRNA, though GAP‐deficient tuberin protein, appear unstable and rapidly degraded. This novel animal model will allow further study of tuberin function including the requirement of the GAP domain for protein stability. genesis 51:284–292. © 2013 Wiley Periodicals, Inc.     

Mice with endogenous TDP‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

TDP‐43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP‐43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C‐terminal domain of TDP‐43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP‐43 loss‐ and gain‐of‐function effects. TDP‐43 gain‐of‐function effects in these mice reveal a novel category of splicing events controlled by TDP‐43, referred to as “skiptic” exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain‐of‐function mutation in endogenous Tardbp causes an adult‐onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain‐of‐function and skiptic exons in ALS patient‐derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP‐43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.     

Dwarfing genes in the genus Lens Mill.

 Dwarfing genes were detected following intra- and interspecific hybridization in Lens. Dwarf phenotypes are controlled by two complementary dominant genes, Df ₁ and Df ₂. These two genes are suppressed by the dominant allele of the dwarf inhibitor genes, Dfi. The dominant allele of the Df gene was detected in L. ervoides from Ethiopia and Uganda and in a cultivated line of L. culinaris from Ethiopia, that of the Df ₂ gene in a L. ervoides accession from Israel. The dominant allele of the Dfi gene was detected in segregating populations of hybrids between L. ervoides accessions from Israel and Uganda. Using the homozygous dwarf, dfidfi, Df ₁ Df ₁, Df ₂ Df ₂ as the parent in interspecific crosses, we detected the dominant allele of the Dfi gene in one accession of L. nigricans and another of L. lamottei. The appearance of dwarf plants in segregating populations of hybrids between the cultivated line from Ethiopia and L. ervoides from Israel indicate that the cultivated line possesses the dominant allele of the Dfi gene. Dwarf plants were characterized by short internodes, a short leaf axis and smaller, convex leaflets. Spraying the dwarf plants with gibberellic acid induced internode and lead-axis elongation but had no effect on leaflet shape and size. When the dwarfs and their parental lines were grown in the dark they had the same internode length. Received: 12 April 1997 / Accepted: 25 June 1997     

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.     

A Gly65Val substitution in an actin,GhACT_LI1, disrupts cell polarity and F‐actin organization resulting in dwarf,lintless cotton plants

Actin polymerizes to form part of the cytoskeleton and organize polar growth in all eukaryotic cells. Species with numerous actin genes are especially useful for the dissection of actin molecular function due to redundancy and neofunctionalization. Here, we investigated the role of a cotton (Gossypium hirsutum) actin gene in the organization of actin filaments in lobed cotyledon pavement cells and the highly elongated single‐celled trichomes that comprise cotton lint fibers. Using mapping‐by‐sequencing, virus‐induced gene silencing, and molecular modeling, we identified the causative mutation of the dominant dwarf Ligon lintless Li₁ short fiber mutant as a single Gly65Val amino acid substitution in a polymerization domain of an actin gene, GhACT_LI1 (Gh_D04G0865). We observed altered cell morphology and disrupted organization of F‐actin in Li₁ plant cells by confocal microscopy. Mutant leaf cells lacked interdigitation of lobes and F‐actin did not uniformly decorate the nuclear envelope. While wild‐type lint fiber trichome cells contained long longitudinal actin cables, the short Li₁ fiber cells accumulated disoriented transverse cables. The polymerization‐defective Gly65Val allele in Li₁ plants likely disrupts processive elongation of F‐actin, resulting in a disorganized cytoskeleton and reduced cell polarity, which likely accounts for the dominant gene action and diverse pleiotropic effects associated with the Li₁ mutation. Lastly, we propose a model to account for these effects, and underscore the roles of actin organization in determining plant cell polarity, shape and plant growth.     

Targeted disruption of mouse Coch provides functional evidence that DFNA9 hearing loss is not a COCH haploinsufficiency disorder

Dominant progressive hearing loss and vestibular dysfunction DFNA9 is caused by mutations of the human COCH gene. COCH encodes cochlin, a highly abundant secreted protein of unknown function in the inner ear. Cochlin has an N-terminal LCCL domain followed by two vWA domains, and all known DFNA9 mutations are either missense substitutions or an amino acid deletion in the LCCL domain. Here, we have characterized the auditory phenotype associated with a genomic deletion of mouse Coch downstream of the LCCL domain. Homozygous Coch ^−/− mice express no detectable cochlin in the inner ear. Auditory brainstem responses to click and pure-tone stimuli (8, 16, 32 kHz) were indistinguishable among wild type and homozygous Coch ^−/− mice. A Coch-LacZΔneo reporter allele detected Coch mRNA expression in nonsensory epithelial and stromal regions of the cochlea and vestibular labyrinth. These data provide functional evidence that DFNA9 is probably not caused by COCH haploinsufficiency, but via a dominant negative or gain-of-function effect, in nonsensory regions of the inner ear.Tomoko Makishima, Clara I. Rodriguez contributed equally     

Molecular cloning and expression analysis of a RGA-like gene responsive to plant hormones in Brassica napus

DELLA proteins are negative regulators of GA-induced growth. DELLA protein family is characterized by a DELLA domain essential for GA-dependent proteasomal degradation of DELLA repressors. A full-length cDNA encoding a putative DELLA protein with high sequence homology to Arabidopsis thaliana RGA (AtRGA), designated as BnRGA, was isolated from Brassica napus. The full-length cDNA of BnRGA contained a 1,740 bp open reading frame (ORF) encoding a precursor protein of 579 amino acid residues. Comparative and bioinformatics analyses revealed that BnRGA showed a high degree of homology with DELLA proteins and contained the DELLA domain, TVHYNP domain, VHIID domain and RVER domain. Using real-time PCR, the expression patterns of BnRGA and two our previously isolated genes, BnGID1a and BnSLY1 in B. napus, were analyzed by adding exogenous gibberellins acid-3 (GA₃), GA biosynthetic inhibitor paclobutrazol (PAC) and abscisic acid (ABA). The results showed that the expression of BnGID1a and BnSLY1 was down-regulated after treated by GA₃ and induced by PAC and ABA. These results suggest that the expression of BnGID1a and BnSLY1 may be negatively regulated by the level of endogenous GA in B. napus. Moreover, BnRGA was not significantly regulated by GA₃, PAC and ABA in the low concentrations. These suggest that GA-GID1-SCF-DELLA complex may have a mechanism of self-regulation, thereby preserving the stability of the expression level of BnRGA in B. napus.     

Proteolysis-independent downregulation of DELLA repression in Arabidopsis by the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1

This article presents evidence that DELLA repression of gibberellin (GA) signaling is relieved both by proteolysis-dependent and -independent pathways in Arabidopsis thaliana. DELLA proteins are negative regulators of GA responses, including seed germination, stem elongation, and fertility. GA stimulates GA responses by causing DELLA repressor degradation via the ubiquitin-proteasome pathway. DELLA degradation requires GA biosynthesis, three functionally redundant GA receptors GIBBERELLIN INSENSITIVE DWARF1 (GID1a, b, and c), and the SLEEPY1 (SLY1) F-box subunit of an SCF E3 ubiquitin ligase. The sly1 mutants accumulate more DELLA proteins but display less severe dwarf and germination phenotypes than the GA biosynthesis mutant ga1-3 or the gid1abc triple mutant. Interestingly, GID1 overexpression rescued the sly1 dwarf and infertility phenotypes without decreasing the accumulation of the DELLA protein REPRESSOR OF ga1-3. GID1 rescue of sly1 mutants was dependent on the level of GID1 protein, GA, and the presence of a functional DELLA motif. Since DELLA shows increasing interaction with GID1 with increasing GA levels, it appears that GA-bound GID1 can block DELLA repressor activity by direct protein-protein interaction with the DELLA domain. Thus, a SLY1-independent mechanism for GA signaling may function without DELLA degradation.     

Alfalfa transposable elements and variegation

Alfalfa with unstable anthocyanin pigmentation has been independently discovered on six occasions since 1958. Genetic studies showed that each of the six unstable stocks was due to an allele mutable at the basic anthocyanin locus C2 in alfalfa. The alleles are designated c2-m1 through c2-m6. Variegated phenotypes of m1, m2, and m3 are similar and express reversion from the recessive to the dominant state. This reversion produces streaks and sectors of pigment in flower petals and seeds that are otherwise white. Reversion occurs at various times in development and may result in periclinal chimeras. The c2-m4 allele is unique in that it arose during tissue culture, whereas the other mutables were discovered in plant populations. Interestingly, m4 is very stable in planta and only rarely produces a sectored flower, but is very unstable in vitro as measured by about 23% revertant plants regenerated from tissue cultures. Most m4 reversion occurs relatively early in development and results in completely pigmented in vitro revertants, and in large sectors on in planta revertants. Alleles m5 and m6 are phenotypically and genetically similar. Their flowers are basic purple with white streaks thus representing mutation from dominant purple to recessive white. White progeny of m5 and m6 are very stable both in planta and in vitro; reversion of white to purple was never observed. Thus, the loss of function of the dominant allele results in a stable recessive or a deficiency. The absolute stability of m5 white derivatives favors the deficiency model, because transposable element mutations might show reversion. Finally, several mutations are described that reoccur in the mutable populations. It is speculated that they are recent mutations due to transposition of transposable elements.     

The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation

The nuclear DELLA proteins are highly conserved repressors of hormone gibberellin (GA) signaling in plants. In Arabidopsis thaliana, GA derepresses its signaling pathway by inducing proteolysis of the DELLA protein REPRESSOR OF ga1-3 (RGA). SLEEPY1 (SLY1) encodes an F-box-containing protein, and the loss-of-function sly1 mutant has a GA-insensitive dwarf phenotype and accumulates a high level of RGA. These findings suggested that SLY1 recruits RGA to the SCFSLY1 E3 ligase complex for ubiquitination and subsequent degradation by the 26S proteasome. In this report, we provide new insight into the molecular mechanism of how SLY1 interacts with the DELLA proteins for controlling GA response. By yeast two-hybrid and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with RGA and GA INSENSITIVE (GAI, a closely related DELLA protein) via their C-terminal GRAS domain. The rga and gai null mutations additively suppressed the recessive sly1 mutant phenotype, further supporting the model that SCFSLY1 targets both RGA and GAI for degradation. The N-terminal DELLA domain of RGA previously was shown to be essential for GA-induced degradation. However, we found that this DELLA domain is not required for protein-protein interaction with SLY1 in yeast (Saccharomyces cerevisiae), suggesting that its role is in a GA-triggered conformational change of the DELLA proteins. We also identified a novel gain-of-function sly1-d mutation that increased GA signaling by reducing the levels of the DELLA protein in plants. This effect of sly1-d appears to be caused by an enhanced interaction between sly1-d and the DELLA proteins.     

Mitofusin gain and loss of function drive pathogenesis in Drosophila models of CMT2A neuropathy

Charcot–Marie–Tooth disease type 2A (CMT2A) is caused by dominant alleles of the mitochondrial pro‐fusion factor Mitofusin 2 (MFN2). To address the consequences of these mutations on mitofusin activity and neuronal function, we generate Drosophila models expressing in neurons the two most frequent substitutions (R94Q and R364W, the latter never studied before) and two others localizing to similar domains (T105M and L76P). All alleles trigger locomotor deficits associated with mitochondrial depletion at neuromuscular junctions, decreased oxidative metabolism and increased mtDNA mutations, but they differently alter mitochondrial morphology and organization. Substitutions near or within the GTPase domain (R94Q, T105M) result in loss of function and provoke aggregation of unfused mitochondria. In contrast, mutations within helix bundle 1 (R364W, L76P) enhance mitochondrial fusion, as demonstrated by the rescue of mitochondrial alterations and locomotor deficits by over‐expression of the fission factor DRP1. In conclusion, we show that both dominant negative and dominant active forms of mitofusin can cause CMT2A‐associated defects and propose for the first time that excessive mitochondrial fusion drives CMT2A pathogenesis in a large number of patients.