Dwarf Mutant Research Articles

Mutagenesis and digital image analysis of mutants for quality attributes of native Cynodon dactylon

The ability to capture information of turf grass in situ makes digital camera based image analysis, a viable tool to quantify turf grass (Cynodon dactylon Pers.) in field experiments. In addition to colour quantification, digital image analysis has been used successfully to quantify percentage turf grass cover and has also been proved to be useful in quantifying turf parameters such as weed infestation, disease incidence, herbicide toxicity, leaf area and recovery from injury. Colour is one of the major criteria used to evaluate the quality of turf and lawn. To generate variability in Bermuda grass to select genotypes responsive to low management, gamma-ray irradiation was used for induction of dwarfness and other quality attributes. Five dwarf mutant lines (DFR 440, DFR-C-444, DFR-C-445, DFR-C 446 and DFR-C-448) were isolated. In the present study, camera and image analysis technique is applied to measure turf colour by its reflectance in the HSB colour scale. The data depicts that the dwarf mutant lines had better quality of lower canopy height, shorter internodes and shorter leaves than the parent. It is demonstrated that image analysis is a suitable non-destructive tool to assess turf grass colour in a reproducible and calibrated manner, over a wide span of structural and colour attributes of turf grass.

Strigolactone signaling regulates rice leaf senescence in response to a phosphate deficiency.

Strigolactones (SLs) act as plant hormones that inhibit shoot branching and stimulate secondary growth of the stem, primary root growth, and root hair elongation. In the moss Physcomitrella patens, SLs regulate branching of chloronemata and colony extension. In addition, SL-deficient and SL-insensitive mutants show delayed leaf senescence. To explore the effects of SLs on leaf senescence in rice (Oryza sativa L.), we treated leaf segments of rice dwarf mutants with a synthetic SL analogue, GR24, and evaluated their chlorophyll contents, ion leakage, and expression levels of senescence-associated genes. Exogenously applied GR24 restored normal leaf senescence in SL-deficient mutants, but not in SL-insensitive mutants. Most plants highly produce endogenous SLs in response to phosphate deficiency. Thus, we evaluated effects of GR24 under phosphate deficiency. Chlorophyll levels did not differ of in the wild-type between the sufficient and deficient phosphate conditions, but increased in the SL-deficient mutants under phosphate deficiency, leading in the strong promotion of leaf senescence by GR24 treatment. These results indicate that the mutants exhibited increased responsiveness to GR24 under phosphate deficiency. In addition, GR24 accelerated leaf senescence in the intact SL-deficient mutants under phosphate deficiency as well as dark-induced leaf senescence. The effects of GR24 were stronger in d10 compared to d17. Based on these results, we suggest that SLs regulate leaf senescence in response to phosphate deficiency.

Selection of mutants obtained by gamma ray irradiation and analysis of genetic variation using RAPD markers in Acorus calamus L.

Acorus calamus is a perennial and medicinal hydrophyte belonging to the family Acoraceae. Irradiation of gamma ray was carried out to obtain mutants of A. calamus due to their limited genetic variation. In vitro explants of A. calamus were irradiated with various doses (20–500 Gy) of gamma rays and the irradiated explants were subcultured for three cycles (vM3). All these explants were cultured on MS medium (salts and vitamins) with 3% (w/v) sucrose, 0.8% (w/v) agar and 4 mg∙L−1 BA (6-benzyl amino purine). This system was performed to select various mutants such as dwarf and salt toleranct mutants. Four solid dwarf mutants were obtained from 3,844 explants and three salt tolerant mutants were selected among 2,800 explants. In selected mutants, polymorphic bands were obtained by using RAPD analysis with selected 9 random primers. The dwarf and salt tolerant mutants were differentiated as a result of cluster analysis. We obtained four dwarf and three salt tolerant mutants from gamma ray irradiation. We conclude that gamma ray irradiation applied to in vitro culture system is an effective way for inducing exclusive mutantationsof A. calamus and the mutants were cleanly differentiated from the wild-type control through RAPD analysis.

Global gene expression analysis of a rice high-tillering dwarf mutant

Plant height and tiller number are indispensible for the establishment of grain production in rice (Oryza sativa L.). A new rice mutant high-tillering dwarf 3 (htd3) exhibiting more tiller number and shorter culm length than the wild-type Guichao 2 (GC2, an indica cultivar) was used to investigate the global gene expression patterns at days after germination 25 (DAG25) and DAG60. In this study, we identified 305 and 987 genes with at least twofold change in gene expression level at DAG25 and DAG60 respectively using the rice microarray chip. Gene ontology enrichment analysis of these twofold change regulated genes revealed that large numbers of genes were involved in binding activity, catalytic activity and metabolic process. The chip results also showed that some of the regulated genes involved in diverse molecular pathways, including gibberellin pathway, brassinosteroid pathway and auxin signal, had significant differences in gene expression abundance at DAG60. This genome-wide gene expression analysis could provide a new opportunity to uncover the regulation mechanisms of the development of culm and tiller, two important components of yields in rice.

Isolation and Characterization of the Brassinosteroid Receptor Gene (GmBRI1) from Glycine max

Brassinosteroids (BRs) constitute a group of steroidal phytohormones that contribute to a wide range of plant growth and development functions. The genetic modulation of BR receptor genes, which play major roles in the BR signaling pathway, can create semi-dwarf plants that have great advantages in crop production. In this study, a brassinosteroid insensitive gene homologous with AtBRI1 and other BRIs was isolated from Glycine max and designated as GmBRI1. A bioinformatic analysis revealed that GmBRI1 shares a conserved kinase domain and 25 tandem leucine-rich repeats (LRRs) that are characteristic of a BR receptor for BR reception and reaction and bear a striking similarity in protein tertiary structure to AtBRI1. GmBRI1 transcripts were more abundant in soybean hypocotyls and could be upregulated in response to exogenous BR treatment. The transformation of GmBRI1 into the Arabidopsis dwarf mutant bri1-5 restored the phenotype, especially regarding pod size and plant height. Additionally, this complementation is a consequence of a restored BR signaling pathway demonstrated in the light/dark analysis, root inhibition assay and BR-response gene expression. Therefore, GmBRI1 functions as a BR receptor to alter BR-mediated signaling and is valuable for improving plant architecture and enhancing the yield of soybean.

Genetic and gene expression analysis of dm1, a dwarf mutant from Cucurbita maxima Duch. ex Lam, based on the AFLP method

Wang, R., Huang, H., Lin, Y., Chen, Q., Liang, Z. and Wu, T. 2014. Genetic and gene expression analysis of dm1, a dwarf mutant from Cucurbita maxima Duch. ex Lam, based on the AFLP method. Can. J. Plant Sci. 94: 293–302. dm1, a dwarf mutant from Cucurbita maxima (Duch. ex Lam) by natural mutation, showed distinct dwarf phenotypes such as shorter vines and fewer and shorter internodes. Genetic analysis indicated that the dm1 mutation was recessive, and the dwarfing character was controlled by a single locus. DNA-AFLP analysis showed that a fragment (MCAG/ETT) was linked with the dwarfing character of dm1 and that the fragment contained 152 base pairs (bp). It was investigated in F2 populations of dm1 and vine plants, and the genetic distance between the MCAG/ETT fragment and dwarf gene in dm1 was 11.2 cM, calculated by JoinMap 3.0 software. In addition, the result of cDNA-AFLP analysis showed that there were 52 differential transcript derived fragments (TDFs) found between dm1 and vine plants. Only four TDFs, A16T12, A16T9, A6T14 and A6T16, were expressed stably and specifically in dm1 plants in subsequent investigation. The four fragments share 71, 79, 87 and 79% nucleic acid sequence similarity with the complete coding sequence of Arabidopsis thaliana histidine kinase 3 (AHK3) mRNA, nucleic acid sequence of Vitis vinifera dihydroflavonol-4-reductase-like (DFRL), nucleic acid sequence of Glycine max histone-lysine N-methyltransferase ATX4-like and nucleic acid sequence of Arabidopsis thaliana histidinol dehydrogenase (HDH), respectively. Bioinformatics analysis indicated that AHK3, DFRL and HDH were respectively related to Cytokinin signaling, indole acetic acid signaling and Ni accumulation, which played important roles in plant growth, so the expression of the four TDFs may contribute to form dwarfism in dm1.

Semidwarf gene sdk has pleiotropic effects on rice (Oryza sativa L.) plant architecture

AbstractIdeal plant architecture has become a significant objective in high‐yield rice breeding. In this study, a new semidwarf gene in sdk, a spontaneous dwarf mutant of ‘Kasalath’, was characterized and genetically mapped. The mutant has smaller panicles and seeds, reduced awns, and is 6–8 days earlier in heading than ‘Kasalath’. There was no significant difference in number (No.) of seeds and fertile seeds per panicle. Genetic analysis indicated that the dwarf character of sdk is controlled by a recessive gene, semidwarf k (sdk). From a segregating population of about 65 000 individuals, 4987 sdk‐type individuals were sampled. Gene sdk was located in a genomic region of 2622 kb flanked by markers R‐12 and R‐43 in the centromeric region of chromosome 2. The results provide an opportunity for gaining an understanding of the molecular mechanisms underlying sdk and opening possibilities for its use in rice breeding programmes.

Isolation and Characterization of a Dominant Dwarf Gene, D-h, in Rice

Plant height is an important agronomic trait that affects grain yield. Previously, we reported a novel semi-dominant dwarfmutant, HD1, derived from chemical mutagenesis using N-methyl-N-nitrosourea (MNU) on a japonica rice cultivar, Hwacheong. In this study, we cloned the gene responsible for the dwarf mutant using a map-based approach. Fine mapping revealed that the mutant gene was located on the short arm of chromosome 1 in a 48 kb region. Sequencing of the candidate genes and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) analysis identified the gene, d-h, which encodes a protein of unknown function but whose sequence is conserved in other cereal crops. Real-time (RT)-PCR analysis and promoter activity assays showed that the d-h gene was primarily expressed in the nodes and the panicle. In the HD1 plant, the d-h gene was found to carry a 63-bp deletion in the ORF region that was subsequently confirmed by transgenic experiments to be directly responsible for the gain-of-function phenotype observed in the mutant. Since the mutant plants exhibit a defect in GA response, but not in the GA synthetic pathway, it appears that the d-h gene may be involved in a GA signaling pathway.

A dwarfing mutant caused by deactivation function of alpha subunit of the heterotrimeric G-protein in rice

Dwarf mutants in plants are crucial for elucidating regulatory mechanisms for plant growth and development. Previous studies suggested that the heterotrimeric G-protein alpha subunit known as D1/RGA1 in rice was involved in deactivation function of the G protein. However, so far no partner has been analyzed the spatial structure change acting with D1. In this study, a dwarf mutant designated Mu101 was obtained in M2 population of rice indica cultivar M804 treated with 60Co γ-ray. Genetic analysis of Mu101 indicated that the dwarf phenotype was controlled by a single dwarf gene, and the dwarf mutant was insensitive to gibberellin (GA), which was named dwarf 89 (d89). Using a large F2 population derived from a cross between the d89 and a japonica rice variety, Taigeng16, the D89 gene was fine mapped into a 62.13 kb physical distance on chromosome 5, where eight open reading frames were predicted. Sequence analysis indicated that only one bp substitution (A-G) was found in LOC_Os05g26890 between M804 and the d89 mutant. The rice GA insensitive dwarf mutant DWARF1 gene was in this locus. The modeling analysis showed amino acid threonine to alanine mutation was likely to make the alpha helix short, and led to the G protein deactivation.

Mutation of a U-box E3 ubiquitin ligase results in brassinosteroid insensitivity in rice

Brassinosteroid (BR) is a natural product that promotes plant growth and development. In this paper, we report that the mutation of a U-box E3 ubiquitin ligase reduces BR sensitivity in rice (Oryza sativa L.). A spontaneous dwarf mutant, designated d1352, was identified from the maintainer line 1352. The plant height and grain length of d1352 decreased by 66 and 36 %, respectively, compared with those of wild-type (WT) rice. Anatomical investigation revealed that all mutant internodes were sharply shortened when compared with their WT counterparts, and the relative ratio of each internode suggested a d6-type mutation of d1352. Analysis using exogenous gibberellin (GA) and BR treatment showed that d1352 had normal sensitivity to GA but had reduced sensitivity to BR treatment compared with the WT. The mutant gene d1352 was located in a 98-kb region on rice chromosome 3 by employing a map-based cloning strategy. Sequencing comparison revealed a 24-bp insertion in the coding region of a putative U-box E3 ubiquitin ligase (LOC_Os03g13010), which led to the insertion of eight amino acids in the conserved domain of U-box. Linkage analysis in all populations showed that the insertion was co-segregated with the dwarf phenotype, indicating that the insertion of LOC_Os03g13010 was responsible for the mutant phenotype. The transcription levels of several BR-related genes were also significantly altered in d1352. In conclusion, D1352 has an important function in the BR signaling pathway.

The high mutagenic effectiveness of MNUA in inducing a diversity of dwarf and semidwarf forms of spring barley

By the modified method of mutagenic MNUA treatment consisting in a short interincubation germination, a very high frequency of point mutations was obtained in many varieties and stocks of spring barley. In the obtained collection of dwarf and semidwarf mutants a rich variability of many traits not connected with the plant height was noted. Mutations concerned the productivity of the plants, their tillering, the length and width of leaves, the habitus, distribution of leaves on the stalk and the morphology of the ear. The diversity of forms described in the collection of dwarf and semidwarf mutants characterises MNUA as a mutagen inducing a very high frequency of mutations in initial cells.

100 Gy 60 Co γ-ray induced novel mutations in tetraploid wheat.

10 accessions of tetraploid wheat were radiated with 100 Gy 60Co γ-ray. The germination energy, germination rate, special characters (secondary tillering, stalk with wax powder, and dwarf), meiotic process, and high-molecular-weight glutenin subunits (HMW-GSs) were observed. Different species has different radiation sensibility. With 1 seed germinated (5%), T. dicoccum (PI434999) is the most sensitive to this dose of radiation. With a seed germination rate of 35% and 40%, this dose also affected T. polonicum (As304) and T. carthlicum (As293). Two mutant dwarf plants, T. turgidum (As2255) 253-10 and T. polonicum (As302) 224-14, were detected. Abnormal chromosome pairings were observed in pollen mother cells of both T. dicoccoides (As835) 237-9 and T. dicoccoides (As838) 239-8 with HMW-GS 1Ax silent in seeds from them. Compared with the unirradiated seed of T. polonicum (As304) CK, a novel HMW-GS was detected in seed of T. polonicum (As304) 230-7 and its electrophoretic mobility was between 1By8 and 1Dy12 which were the HMW-GSs of Chinese Spring. These mutant materials would be resources for wheat breeding.

Strigolactone and Cytokinin Act Antagonistically in Regulating Rice Mesocotyl Elongation in Darkness

Strigolactones (SLs) are a group of phytohormones that control plant growth and development including shoot branching. Previous studies of the phenotypes of SL-related rice (Oryza sativa) dwarf (d) mutants demonstrated that SLs inhibit mesocotyl elongation by controlling cell division. Here, we found that the expression of cytokinin (CK)-responsive type-A RESPONSE REGULATOR (RR) genes was higher in d10-1 and d14-1 mutants than in the wild type. However, CK levels in mesocotyls of the d mutants were not very different from those in the wild type. On the other hand, application of a synthetic CK (kinetin) enhanced mesocotyl elongation in the d mutants and the wild type. d10-1 and d14-1 mesocotyls were more sensitive to CK than wild-type mesocotyls, suggesting that the up-regulation of the CK-responsive type-A RR genes and the higher elongation of mesocotyls in the d mutants are mainly due to the increased sensitivity of the d mutants to CK. Co-treatment with kinetin and a synthetic SL (GR24) confirmed the antagonistic functions of SL and CK on mesocotyl elongation. OsTCP5, which encodes a transcription factor belonging to the cell division-regulating TCP family, was also regulated by SL and CK and its expression was negatively correlated with mesocotyl length. These findings suggest that OsTCP5 contributes to the SL- and CK-controlled mesocotyl elongation in darkness.

DWARF 53 acts as a repressor of strigolactone signalling in rice

Strigolactones (SLs) are a group of newly identified plant hormones that control plant shoot branching. SL signalling requires the hormone-dependent interaction of DWARF 14 (D14), a probable candidate SL receptor, with DWARF 3 (D3), an F-box component of the Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. Here we report the characterization of a dominant SL-insensitive rice (Oryza sativa) mutant dwarf 53 (d53) and the cloning of D53, which encodes a substrate of the SCF(D3) ubiquitination complex and functions as a repressor of SL signalling. Treatments with GR24, a synthetic SL analogue, cause D53 degradation via the proteasome in a manner that requires D14 and the SCF(D3) ubiquitin ligase, whereas the dominant form of D53 is resistant to SL-mediated degradation. Moreover, D53 can interact with transcriptional co-repressors known as TOPLESS-RELATED PROTEINS. Our results suggest a model of SL signalling that involves SL-dependent degradation of the D53 repressor mediated by the D14-D3 complex.

The maize d2003, a novel allele of VP8, is required for maize internode elongation

The d2003 is a natural dwarf mutant from maize inbred line K36 and has less than one-third of K36 plant height with severely shortened internodes. In this study, we reported the cloning of d2003 gene using positional cloning. The results showed that there was a single-base insertion in the coding region of Viviparous8 (VP8) in d2003 mutant, which resulted in a premature stop codon. Further genetic allelism tests confirmed that d2003 mutation is a novel allele of VP8. VP8 is mainly expressed in the stem apex, young leaves, and developing vascular tissues, and its expression levels in nodes are significantly higher than that in internodes at 12-leaf stage. Subcellular localization demonstrated that the VP8 protein is localized to the endoplasmic reticulum and the N-terminal 26 amino acids (aa) of VP8 protein are essential to its localization in ER. Further transgenic experiments showed that lack of the 26 aa leads to loss of VP8 function in Arabidopsis amp1 phenotype rescue. These results strongly suggested that the N-terminal 26 aa is critical for VP8 protein localization, and the correct protein localization of VP8 in ER is necessary for its function.

Characterization of a dwarf mutant allele of Arabidopsis MDR-like ABC transporter AtPGP1 gene

Asymmetric auxin distribution caused by polar auxin transport (PAT) regulates many plant developmental and physiological processes. Plant two closely ABC (ATP-binding cassette) transporter, AtPGP1 and AtPGP19 (AtMDR1), have been implicated in auxin transport. However, unlike atpgp19 mutant and atpgp1 atmdr1 double mutant show decreased apical dominance, reduced growth, and impaired basipetal auxin transport, atpgp1 mutant exhibit no significant difference from wild type. We report a new allele of atpgp1 mutants, designated as atpgp1-2, which showed shorter hypocotyl and dwarf phenotype under long-day condition. Auxin transport activity was greatly impaired and NPA-sensitivity was decreased in the mutant. Moreover, we detected transcript in the atpgp1 mutants reported previously, but not in atpgp1-2. These results suggest a direct involvement of AtPGP1 in auxin transport processes controlling plant growth.

Characterization and genetic mapping of a Photoperiod-sensitive dwarf 1 locus in rice (Oryza sativa L.)

Plant height is an important agronomic trait for crop architecture and yield. Most known factors determining plant height function in gibberellin or brassinosteroid biosynthesis or signal transduction. Here, we report a japonica rice (Oryza sativa ssp. japonica) dominant dwarf mutant, Photoperiod-sensitive dwarf 1 (Psd1). The Psd1 mutant showed impaired cell division and elongation, and a severe dwarf phenotype under long-day conditions, but nearly normal growth in short-day. The plant height of Psd1 mutant could not be rescued by gibberellin or brassinosteroid treatment. Genetic analysis with R1 and F2 populations determined that Psd1 phenotype was controlled by a single dominant locus. Linkage analysis with 101 tall F2 plants grown in a long-day season, which were derived from a cross between Psd1 and an indica cultivar, located Psd1 locus on chromosome 1. Further fine-mapping with 1017 tall F2 plants determined this locus on an 11.5-kb region. Sequencing analysis of this region detected a mutation site in a gene encoding a putative lipid transfer protein; the mutation produces a truncated C-terminus of the protein. This study establishes the genetic foundation for understanding the molecular mechanisms regulating plant cell division and elongation mediated by interaction between genetic and environmental factors.

THIS1 is a putative lipase that regulates tillering, plant height, and spikelet fertility in rice

Proper branching and successful reproductive growth is of great importance for rice productivity. Substantial progress has been made in uncovering the molecular mechanisms underlying tillering control and spikelet sterility. However, rice tillering is developmentally controlled, and how it is regulated coordinately with reproductive growth remains unclear. This study characterized a rice mutant, the most obvious phenotypes of which are high tillering, reduced height, and infertile spikelets (named this1). Similarly to the high tiller number and dwarf mutants in rice, the increased tiller number of this1 plants is ascribed to the release of tiller bud outgrowth rather than to increased tiller bud formation. In the this1 mutant, however, the accelerated rate of branching was delayed until the stem elongation stage, while other mutants lost the ability to control branching at all developmental stages. The seed-setting rate of this1 was less than half that of the wild type, owing to defects in pollen maturation, anther dehiscence, and flower opening. Histological analyses showed that the mutation in this1 resulted in anisotropic cell expansion and cell division. Using a map-based cloning approach, This1 was found to encode a class III lipase. Homology searches revealed that THIS1 is conserved in both monocots and eudicots, suggesting that it plays fundamental role in regulating branch and spikelet fertility, as well as other aspects of developmental control. The relative change in expression of marker genes highlighted the possibility that This1 is involved in phytohormone signalling pathways, such as those for strigolactone and auxin. Thus, This1 provides joint control between shoot branching and reproductive development.

Cloning and Expression of Gene Responsible for High-Tillering Dwarf Phenotype in Indica Rice Mutant gsor23

High-tillering dwarf mutant gsor23 was generated from an indica rice variety Indica9 radiatied by γ-ray. Genetic analysis showed that this phenotype was controlled by one single recessive gene, which was mapped within a physical distance of 386 kb between two insertion-deletion (InDel) markers C1-WT2 and C1-WT4 on the long arm of chromosome 1. There is a known gene D10 within this region, the mutation of which causes high-tillering in rice. Sequence analysis of the D10 allele in gsor23 revealed that the base cytosine (C) at the 404th position in the coding region was deleted, which would cause frameshift mutation after the 134th amino acids. The mutation site and indica background of gsor23 were different from the previously reported japonica mutants d10-1 and d10-2. Therefore, gsor23 is a novel allelic mutant of D10 which encodes the carotenoid-cleaving dioxygenase 8 (CCD8), a key enzyme involved in the biosynthesis of the new plant hormone strigolactones (SLs). After treatment with GR24, a synthetic analogue of SLs, the high-tillering phenotype of gsor23 was restored to normal. Real-time RT-PCR analysis showed that D10 expression was high in roots, but low in leaves. Compared with the wild type Indica9, the expression of the SL biosynthesis gene D10 was upregulated, while genes likely involved in the SL signal transduction pathway such as D3 and D14 were down-regulated in the gsor23 mutant.

Isolation and characterisation of a dwarf rice mutant exhibiting defective gibberellins biosynthesis

We have isolated a severe dwarf mutant derived from a Ds (Dissociation) insertion mutant rice (Oryza sativa var. japonica c.v. Dongjin). This severe dwarf phenotype, has short and dark green leaves, reduced shoot growth early in the seedling stage, and later severe dwarfism with failure to initiate flowering. When treated with bioactive GA3 , mutants are restored to the normal wild-type phenotype. Reverse transcription PCR analyses of 22 candidate genes related to the gibberellin (GA) biosynthesis pathway revealed that among 22 candidate genes tested, a dwarf mutant transcript was not expressed only in one OsKS2 gene. Genetic analysis revealed that the severe dwarf phenotype was controlled by recessive mutation of a single nuclear gene. The putative OsKS2 gene was a chromosome 4-located ent-kaurene synthase (KS), encoding the enzyme that catalyses an early step of the GA biosynthesis pathway. Sequence analysis revealed that osks2 carried a 1-bp deletion in the ORF region of OsKS2, which led to a loss-of-function mutation. The expression pattern of OsKS2 in wild-type cv Dongjin, showed that it is expressed in all organs, most prominently in the stem and floral organs. Morphological characteristics of the dwarf mutant showed dramatic modifications in internal structure and external morphology. We propose that dwarfism in this mutant is caused by a point mutation in OsKS2, which plays a significant role in growth and development of higher plants. Further investigation on OsKS2 and other OsKS-like proteins is underway and may yield better understanding of the putative role of OsKS in severe dwarf mutants.