A Single Nucleotide Deletion in Gibberellin20-oxidase1 Causes Alpine Dwarfism in Arabidopsis

Alpine dwarfism is widely observed in alpine plant populations and often considered a high-altitude adaptation, yet its molecular basis and ecological relevance remain unclear. In this study, we used map-based cloning and field transplant experiments to investigate dwarfism in natural Arabidopsis (Arabidopsis thaliana) accessions collected from the Swiss Alps. A loss-of-function mutation due to a single nucleotide deletion in gibberellin20-oxidase1 (GA5) was identified as the cause of dwarfism in an alpine accession. The mutated allele, ga5-184, was found in two natural Arabidopsis populations collected from one geographic region at high altitude, but was different from all other reported ga5 null alleles, suggesting that this allele has evolved locally. In field transplant experiments, the dwarf accession with ga5-184 exhibited a fitness pattern consistent with adaptation to high altitude. Across a wider array of accessions from the Swiss Alps, plant height decreased with altitude of origin, but fitness patterns in the transplant experiments were variable and general altitudinal adaptation was not evident. In general, our study provides new insights into molecular basis and possible ecological roles of alpine dwarfism, and demonstrates the importance of the GA-signaling pathway for the generation of ecologically relevant variation in higher plants.Adaptation to environmental conditions is one of the evolutionary processes that can lead to lineage divergence and the generation of biodiversity (Savolainen et al., 2013). Local adaptation within species may evolve in spatially heterogeneous environments if the strength of divergent selection among populations overrides other evolutionary forces (e.g. genetic drift, gene flow), and is manifested in an increased fitness of local genotypes in the local habitat when compared with foreign genotypes in the local habitat and when compared with fitness of the local genotypes in foreign habitats (Kawecki and Ebert, 2004; Savolainen et al., 2013). It is often a challenge for classic studies of local adaptation to identify traits mediating local adaptation and the underlying genetic architectures. To date, our knowledge on the molecular basis of local adaptation remains remarkably limited, even though this topic has been extensively studied in recent decades (Bergelson and Roux, 2010; Barrett and Hoekstra, 2011). A particular difficulty in studying the molecular basis of local adaptation comes from the need to combine meaningful field experiments together with relevant molecular studies to provide solid evidence on the adaptive value of phenotypes and associated genotypes. Therefore, a complete study of molecular mechanisms of adaptation ideally requires (1) identifying candidate traits under natural selection or relevant for adaptation, (2) isolating genes/loci that influence candidate traits, and (3) quantifying the fitness (adaptive value) conferred by different alleles under natural conditions (Bergelson and Roux, 2010). In the model plant Arabidopsis (Arabidopsis thaliana) and its relatives, as well as in several animal systems, great advances in detecting the molecular basis of adaptation have been made (e.g. Linnen et al., 2009; Fournier-Level et al., 2011; Hancock et al., 2011). Nevertheless, discussions on adaptionist storytelling or of producing molecular spandrels have been recurrent (for review, see Barrett and Hoekstra, 2011).Alpine habitats are characterized by a fine mosaic of heterogeneous and often extreme environmental conditions (Körner, 2003; Byars et al., 2007; Pico, 2012). Along altitudinal gradients, changes in environmental conditions are often steep, and plants growing in alpine areas have developed a variety of morphological and physiological adaptations that allow them to cope with extreme conditions. Alpine plant dwarfism, that is, reduced plant stature with increasing altitude, is one of the most common characteristics observed in plant populations originating from high altitudes (Clausen et al., 1948; Körner, 2003). Alpine dwarfism is thought to help alpine plants take advantage of the higher ambient temperature close to the soil surface, allocate more resources to reproduction, decrease damage from strong wind, and reduce evaporation (Turesson, 1922; Körner, 2003). Therefore, alpine dwarfism is widely considered as adaptive in plants (Turesson, 1922; Clausen et al., 1948; Körner, 2003; Byars et al., 2007; Gonzalo-Turpin and Hazard, 2009). However, whether alpine dwarfism is indeed adaptive and how it is genetically controlled remain unknown in many species. This may be partially attributable to the limited genomic resources that are available for alpine plant species, which are usually nonmodel organisms. Hence, studying the molecular basis of this ecologically important trait in model organisms such as Arabidopsis can give much-needed insights (Bergelson and Roux, 2010).Dwarfism and semidwarfism are important and well-studied traits in agriculture because they help overcome lodging and thus substantially contributed to the green revolution in the last century, the unprecedented increase in crop yields due to the adoption of genetically improved crop varieties (Peng et al., 1999; Khush, 2001). It has repeatedly been found in a diversity of plants that dwarfism and semidwarfism were due to deficiencies in either signaling or biosynthesis of GA (e.g. Spray et al., 1996; Peng et al., 1999; Monna et al., 2002). Dwarfism and dwarfing alleles have also been reported in natural Arabidopsis accessions (Barboza et al., 2013), and other recent studies have shown evidence of population differentiation and climatic adaptation along altitudinal gradients in this model plant (Montesinos et al., 2009; Méndez-Vigo et al., 2011, 2013; Montesinos-Navarro et al., 2011; Pico, 2012; Suter et al., 2014; Luo et al., 2015). In this study, we have investigated plant dwarfism in natural Arabidopsis accessions collected in the Swiss Alps (Supplemental Table S1) and identified a loss-of-function mutation in gibberellin20-oxidase1 (GA5, also called GA20ox1) as the cause of dwarfism in an Arabidopsis accession collected from high altitude (2,012 m above sea level [a.s.l.]). In field transplant experiments, this accession displayed fitness patterns consistent with altitudinal adaptation. Across a larger set of regional accessions, including 10 further accessions without the dwarfing mutation, plant height decreased with altitude of origin; however, this pattern could not be tied to adaptive differentiation along altitude.     

Gibberellin biosynthesis and its regulation

The GAs (gibberellins) comprise a large group of diterpenoid carboxylic acids that are ubiquitous in higher plants, in which certain members function as endogenous growth regulators, promoting organ expansion and developmental changes. These compounds are also produced by some species of lower plants, fungi and bacteria, although, in contrast to higher plants, the function of GAs in these organisms has only recently been investigated and is still unclear. In higher plants, GAs are synthesized by the action of terpene cyclases, cytochrome P450 mono-oxygenases and 2-oxoglutarate-dependent dioxygenases localized, respectively, in plastids, the endomembrane system and the cytosol. The concentration of biologically active GAs at their sites of action is tightly regulated and is moderated by numerous developmental and environmental cues. Recent research has focused on regulatory mechanisms, acting primarily on expression of the genes that encode the dioxygenases involved in biosynthesis and deactivation. The present review discusses the current state of knowledge on GA metabolism with particular emphasis on regulation, including the complex mechanisms for the maintenance of GA homoeostasis.     

Gibberellin biosynthesis: its regulation by endogenous and environmental signals

The hormone gibberellin (GA) plays an essential role in many aspects of plant growth and development, such as seed germination, stem elongation and flower development. In recent years, exciting progress has been made in understanding how the biosynthesis of this hormone is regulated by endogenous and environmental factors. This has resulted from isolation of genes encoding enzymes involved in GA biosynthesis and metabolism, which also enabled us to manipulate the pathway by modifying the expression of these genes in transgenic plants. In addition, new GA response mutants provided information about how signaling components are involved in feedback regulation of the GA biosynthetic pathway.     

Gibberellin production by bacteria and its involvement in plant growth promotion and yield increase

This review focuses on studies with bacteria for which biosynthesis/production of the plant hormones gibberellins have been demonstrated. Actual data on gibberellin metabolism by bacteria are analyzed in comparison with the biosynthetic pathways known for vascular plants and fungi. The potential involvement of gibberellins produced by symbiotic and soil-endophytic microorganisms in plant growth promotion and yield increase is also discussed.     

Maternal Obesity Caused by Overnutrition Exposure Leads to Reversal Learning Deficits and Striatal Disturbance in Rats

Maternal obesity caused by overnutrition during pregnancy increases susceptibility to metabolic risks in adulthood, such as obesity, insulin resistance, and type 2 diabetes; however, whether and how it affects the cognitive system associated with the brain remains elusive. Here, we report that pregnant obesity induced by exposure to excessive high fatty or highly palatable food specifically impaired reversal learning, a kind of adaptive behavior, while leaving serum metabolic metrics intact in the offspring of rats, suggesting a much earlier functional and structural defects possibly occurred in the central nervous system than in the metabolic system in the offspring born in unfavorable intrauterine nutritional environment. Mechanically, we found that above mentioned cognitive inflexibility might be associated with significant striatal disturbance including impaired dopamine homeostasis and disrupted leptin signaling in the adult offspring. These collective data add a novel perspective of understanding the adverse postnatal sequelae in central nervous system induced by developmental programming and the related molecular mechanism through which priming of risk for developmental disorders may occur during early life.     

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination.     

Phenotypic Characterization of the Komeda Miniature Rat Ishikawa, an Animal Model of Dwarfism Caused by a Mutation in Prkg2

The Komeda miniature rat Ishikawa (KMI) is a spontaneous animal model of dwarfism caused by a mutation in Prkg2, which encodes cGMP-dependent protein kinase type II (cGKII). This strain has been maintained as a segregating inbred strain for the mutated allele mri. In this study, we characterized the phenotype of the KMI strain, particularly growth traits, craniofacial measurements, and organ weights. The homozygous mutant (mri/mri) animals were approximately 70% to 80% of the size of normal, heterozygous (mri/+) animals in regard to body length, weight, and naso-occipital length of the calvarium, and the retroperitoneal fat of mri/mri rats was reduced greatly. In addition, among progeny of the (BN×KMI-mri/mri)F1×KMI-mri/mri backcross, animals with the KMI phenotype (mri/mri) were easily distinguished from those showing the wild-type phenotype (mri/+) by using growth traits such as body length and weight. Genetic analysis revealed that all of the backcrossed progeny exhibiting the KMI phenotype were homozygous for the KMI allele in the 1.2-cM region between D14Rat5 and D14Rat80 on chromosome 14, suggesting strongly that mri acts in a completely recessive manner. The KMI strain is the first and only rat model with a confirmed mutation in Prkg2 and is a valuable model for studying dwarfism and longitudinal growth traits in humans and for functional studies of cGKII.Abbreviations: cGKII, cGMP-dependent protein kinase type II; CNP, C-type natriuretic peptide; KMI, Komeda miniature rat IshikawaDwarfism is caused by both endocrinologic and nonendocrinologic defects. Most instances of dwarfism, including normal variants, are nonendocrinologic, and subjects retain growth hormone secretion. Although spontaneous rodent models of dwarfism with confirmed mutations have been reported—Snell dwarf mice with Pou1f1 (Pit1) mutation,¹⁴ Ames dwarf mice with Prop1 mutation,²² little mice with Ghrhr mutation,¹⁵ pygmy mice (also known as mini-mice) with Hmga2 (HMGI-C) mutation,²⁶ spontaneous dwarf rats with Gh mutation,²³ and rdw rats with Tg mutation^9,11—most of these are models of endocrinologic dwarfism. A few models of nonendocrinologic dwarfism have been produced by gene manipulation techniques, such as transgenic and knockout strategies, and include Col2a1-transgenic mice,^7,24 Col10a1-transgenic mice,¹⁰ and Fgfr3-knock-in mice.¹³A novel spontaneous dwarf mutation, miniature rat Ishikawa (mri), was discovered in a closed colony of Wistar rats at Ishikawa Animal Laboratory (Saitama, Japan) and has been maintained on the genetic background of Wistar rats. This mutant strain, previously termed Miniature Rat Ishikawa (MRI), has recently been established as a segregating inbred strain on the Wistar genetic background, designated Komeda Miniature rat Ishikawa (KMI). The breeding record suggested that the mutation was inherited in an autosomal recessive mode. KMI rats show no abnormality in the basal amounts or distribution of several hormones, including growth hormone, luteinizing hormone, follicle-stimulating hormone, prolactin, thyroid-stimulating hormone, and adrenocorticotropic hormone, but growth hormone response to growth hormone releasing hormone is decreased.²¹Using positional candidate cloning of mri, we recently identified a deletion mutation in Prkg2, which encodes cGMP-dependent protein kinase type II (cGKII), and clarified a role of cGKII as a molecular switch that couples cessation of proliferation and the start of hypertrophic differentiation of chondrocytes.² Longitudinal skeletal growth is achieved by endochondral ossification in the growth plate, in which chondrocyte hypertrophic differentiation is an important step. Due to the impaired coupling of proliferation and hypertrophic differentiation in the growth plate chondrocytes, homozygous mutant (mri/mri) animals show longitudinal growth retardation.In this study, we further characterize the phenotype of the KMI strain, including body length, body weight, organ weight, and craniofacial measurements. Furthermore, we describe phenotypic characteristics of the progeny produced from the (BN×KMI-mri/mri)F1×KMI-mri/mri backcross and provide updated genetic, physical, and comparative maps of the mri region.     

Thermodormancy in Celery Seeds and its Removal by Cytokinins and Gibberellins

Imbibition of celery (Apium graveolens L.) seeds at 32°C for up to 96 h lowered the upper temperature limit for germination. If this high temperature treatment was given in the light, these seeds germinated slightly earlier than those treated in the dark although the final percentage germination was similar for both treatments. The inhibitory effect of the high temperature treatment was completely removed by allowing the seeds to imbibe in a mixture of the gibberellins A₄ and A₇ (GA_4/7) and partially removed by the cytokinin N⁶-benzylaminopurine (BA). GA_4/7 was less effective when added before rather than after the high temperature treatment, whereas the opposite was true of BA. At constant temperatures more GA_4/7 was required to promote germination as the temperature was raised but addition of BA reduced the concentration of GA_4/7 required. A model is proposed for the control of celery seed germination by light and temperature through the action of endogenous cytokinins and gibberellins.     

A Cucumber Disease Caused by Alternaria alternata and its Control

A severe leaf spot disease of cucumber caused by a pathotype of Alternaria alternata (Fr.) Keissler was recorded recently in plastic houses in Crete. Lesions ranged in size of a pin point to over 5 cm in diameter, with necrotic tissue on most of their area and a surrounding yellow zone. The pathogen grew satisfactorily on PDA at temperatures between 5 °C–40 °C and spore germination occurred in the range less than 10 °C to over 37 °C. Optimum temperature in both cases was near 26 °C. Of,13 fungicides tested in vitro, sodium omadine, etem, dichlofluanid, captan and folpet were the most inhibitory on spore germination, and iprodione, sodium omadine and dichlofluanid on mycelial growth. Of 25 fungicides applied on two leaf cucumber plants 24 h before inoculation, maneb, etem, dichlofluanid and chlorothalonil were the most effective. When the last fungicides, plus mancozeb, were applied 24 h after inoculation only maneb was effective. In greenhouse experiments, iprodione, prochloraz-manganese-complex, chlorothalonil, dichlofluanid, guazatine, maneb and etem were the most effective for disease control, while mancozeb was less effective. The local cucumber cv. Knossos and the Dutch F₁ hybrids Evadan, Frella, Herta, Malfa, Mazourka, Pepinex 69 and Renova were all susceptible to infection.     

Rescue from Dwarfism by Thyroid Function Compensation in rdw Rats.

The rdw rat was initially reported as having hereditary dwarfism caused by pituitary dysfunction. Subsequent studies on the rdw rat, however, have demonstrated that the primary cause of rdw dwarfism is present in the thyroid gland but not in the pituitary gland. The primary cause of rdw rat disorders is a missense mutation of the thyroglobulin (Tg) gene by a one-point mutation. In the present study, we attempted to rescue the dwarfism of the rdw rats using a diet supplemented with thyroid powder (T-powder) and a thyroid graft (T-graft). The infants of the rdw rat were successfully raised to a mature stage body weight, accompanied by elevation of serum growth hormone (GH) and prolactin (PRL), by the T-powder. Furthermore, the T-graft successfully increased the body weight with fertility. The serum GH and PRL levels in the T-graft rdw rat significantly increased. The serum thyroid-stimulating hormone (TSH) levels in the T-graft rdw rat were significantly decreased but were significantly higher than those in the control rat. The GH and PRL mRNA expression in the rdw rat with the T-graft was virtually the same as that of the control, but the TSH beta mRNA differed from that of the control rats. Thus, the dwarfism in the rdw rat is rescued by thyroid function compensation, such as that afforded by T-powder and T-graft.     

Extent of Deterioration in Physical Condition during Postoperative Bed Rest and its Reversal by Rehabilitation

A study has been made of changes in physical condition associated with bed rest and rehabilitation in hospital patients. It was found that after two weeks recumbent bed rest there was a significant decline in physical condition. It was also found that the better the initial physical condition the greater the decline. Rehabilitation was associated with an improvement in physical condition, which seemed to be related to the increased level of daily activity rather than to the absolute levels of physical condition.     

Petunia actin-depolymerizing factor is mainly accumulated in vascular tissue and its gene expression is enhanced by the first intron

Actin-depolymerizing factor (ADF) is one of the actin cytoskeleton-modulating proteins. We have characterized the accumulation pattern of petunia ADF proteins. PhADF proteins are accumulated in every petunia organ and their accumulation is differentially regulated by developmental signals. Their cellular localization is vascular tissue-preferential in vegetative organs, whereas somewhat different in reproductive organs. In reproductive organs, PhADFs are present in outer integument, endocarp of ovary wall, transmitting tissue of style, and epidermis and endothecium of young anther. From a petunia genomic library, we have isolated a genomic clone encoding PhADF1. Comparison to complementary DNA sequence revealed that the coding region of PhADF1 gene consists of three exons and two introns. Analysis of chimeric gene expression using beta-glucuronidase as a reporter gene in transgenic Arabidopsis revealed that PhADF1 was strongly expressed in every vegetative tissue except petal. In addition, expression of the gene was highly enhanced by its first intron. These results suggest that PhADF1 gene of petunia is mainly expressed in vascular tissues and its expression is regulated by intron-mediated enhancement mechanism.     

The Inhibition of Growth in Higher Plants by a Homologous Series of Guanidines and its Reversal by Spermine

The effects of guazatine, synthalins A and B and a homologousseries of aliphatic monoguanidines on the growth of cress, barleyand oat seedlings, and apple cell suspension cultures have beenstudied. In the homologous series of aliphatic monoguanidines[NH₂C(=NH)NH(CH₂)_x–1CH₃] greatest inhibition was foundwith x = 8-10 for cress, barley and oats and x= 10–14for apple cells. Spermine partially reversed the inhibitionin the light for cress and barley but in the dark no reversalwas found. Technical guazatine inhibited growth to a greaterextent than pure guazatine, and was comparable in toxicity tosynthalin B in cress, barley and oats. Reversal by spermineof inhibition due to guazatine and synthalin B was greater inthe light than in the dark in these plants. Calcium ions didnot reverse the toxicity of guazatine, synthalin B or dodine.Reversal of the inhibition by guazatine, synthalin B and dodineof the growth of the apple cells was considerably greater withspermine than with spermidine. Lepidium sativumcress, Hordeum vulgarebarley, Avena sativaoat, Malus sylvestrisapple, guanidines, guazatine, synthalins, dodine, spermine, spermidine     

Reversal of ABCG2-mediated multidrug resistance by human cathelicidin and its analogs in cancer cells

Multidrug resistance (MDR) of cancer cells to a wide spectrum of anticancer drugs is a major obstacle to successful chemotherapy. It is usually mediated by the overexpression of one of the three major ABC transporters actively pumping cytotoxic drugs out of the cells. There has been great interest in the search for inhibitors toward these transporters with an aim to circumvent resistance. This is usually achieved by screening from natural product library and the subsequent structural modifications. This study reported the reversal of ABCG2-mediated MDR in drug-selected resistant cancer cell lines by a class of host defense antimicrobial peptides, the human cathelicidin LL37 and its fragments. The effective human cathelicidin peptides (LL17-32 and LL13-37) were found to increase the accumulation of mitoxantrone in cancer cell lines with ABCG2 overexpression, thereby circumventing resistance to mitoxantrone. At the effective concentrations of the cathelicidin peptides, cell proliferation of the parental cells without elevated ABCG2 expression was not affected. Result from drug efflux and ATPase assays suggested that both LL17-32 and LL13-37 interact with ABCG2 and inhibit its transport activity in an uncompetitive manner. The peptides were also found to downregulate ABCG2 protein expression in the resistant cells, probably through a lysosomal degradation pathway. Our data suggest that the human cathelicidin may be further developed for sensitizing resistant cancer cells to chemotherapy.     

Dwarfism and male sterility in interspecific hybrids of Epilobium

Summary Reciprocal differences for male sterility, dwarfism and morphological traits have been studied in intra- and interspecific crosses of five Epilobium species. Male sterility occurred in two interspecific hybrids with E. montanum as the male parent while dwarfism has been found to varying degrees in three interspecific crosses with E. watsonni. In contrast to transient differences in plant height and leaf morphology in reciprocal hybrids of the cross between E. hirsutum and E. parviflorum, male sterility and dwarfism persistently occur as reciprocally different traits which may be influenced by determinants of the cytoplasm. The molecular characterization of the plastid DNA of the parental lines and the F₁ hybrids indicate that the plastome of male sterile and dwarf plants is identical to that of the female parents. Furthermore, in spite of these developmental disturbances, the expression of plastid genes coding for polypeptides of thylakoid-membrane complexes is unchanged. Thus, it seems unlikely that the genetic compartement of the plastids is responsible for the expression of the male sterile or the dwarfed phenotype.