Mutants Of Higher Plants Research Articles

A mutation in the promoter of the yellow stripe-like transporter gene in cucumber results in a yellow cotyledon phenotype

Leaf color mutants in higher plants are considered to be ideal materials for studying the chlorophyll biosynthesis, photosynthesis mechanism and chloroplast development. Herein, we identified a spontaneous mutant, yc412, in cultivated cucumber that exhibited yellow cotyledons. The yellow-lethal mutant was diagnosed with an abnormal chloroplast ultrastructure, and reduced photosynthetic capacity and pigment content. Through bulked segregant analysis-based whole-genome sequencing and fine genetic mapping, we narrowed the yellow cotyledons (yc) locus to a 96.8 kb interval on chromosome 3. By resequencing and molecular cloning, we showed that Csyc is a potential candidate gene, which encodes a yellow stripe-like (YSL) transporter. The T to C mutation in the promoter region of Csyc caused the yellow cotyledon phenotype in yc412. Compared to YZU027A (WT), the expression of Csyc was significantly downregulated in the cotyledons of yc412. Silencing of Csyc in cucumber via virus-induced gene silencing resulted in chlorotic leaves, mainly by suppressing the chlorophyll content. Furthermore, a comparative transcriptome analysis revealed that chloroplast-related genes and chlorophyll biosynthesis genes were significantly downregulated in yc412 cotyledons. Our results provide new insights into the molecular function of the YSL transporter in plant chloroplast development and chlorophyll synthesis.

Fine Mapping of CsVYL, Conferring Virescent Leaf Through the Regulation of Chloroplast Development in Cucumber.

Leaf color mutants in higher plants are ideal materials for investigating the structure and function of photosynthetic system. In this study, we identified a cucumber vyl (virescent-yellow leaf) mutant in the mutant library, which exhibited reduced pigment contents and delayed chloroplast development process. F2 and BC1 populations were constructed from the cross between vyl mutant and cucumber inbred line ‘Hazerd’ to identify that the vyl trait is controlled by a simply recessive gene designated as CsVYL. The CsVYL gene was mapped to a 3.8 cM interval on chromosome 4 using these 80 F2 individuals and BSA (bulked segregation analysis) approach. Fine genetic map was conducted with 1542 F2 plants and narrowed down the vyl locus to an 86.3 kb genomic region, which contains a total of 11 genes. Sequence alignment between the wild type (WT) and vyl only identified one single nucleotide mutation (C→T) in the first exon of gene Csa4G637110, which encodes a DnaJ-like zinc finger protein. Gene Expression analysis confirmed the differences in transcription level of Csa4G637110 between wild type and mutant plants. Map-based cloning of the CsVYL gene could accelerate the study of chloroplast development and chlorophyll synthesis of cucumber.

Chloroplast transformation reveals that tobacco ycf5 is involved in photosynthesis

Hypothetical chloroplast reading frame 5 (ycf5) is encoded by the chloroplast genome and potentially specifies a protein of 313 amino acids in tobacco. A northern blot analysis showed that ycf5 transcripts accumulated at substantial levels in the chloroplasts, but not in the proplastids of nonphotosynthetic cultured cells. In an attempt to determine the function of ycf5, we constructed mutant alleles for the targeted deletion of ycf5. The mutant allele was introduced into the tobacco chloroplast genome by biolistic chloroplast transformation to replace the corresponding wild-type ycf5 alleles by homologous recombination. Homoplasmic ycf5-knockout tobacco plants displayed apale-green phenotype. An analysis of the transient increase in chlorophyll fluorescence suggested that the electron flow around photosystem II (PSII) was completely impaired in the ycf5-deficient transformants. These findings indicate that ycf5 is indeed a functional gene, and that its gene product is involved in the generation of functional PSII units. This is the first report of the isolation and characterization of ycf5-knockout mutants in higher plants.

Kanamycin-resistant alfalfa has a point mutation in the 16S plastid rRNA.

Genes conferring resistance to kanamycin are frequently used to obtain transgenic plants as spontaneous resistance to kanamycin is not known to exist in higher plants. Nevertheless, mutations conferring kanamycin resistance have been identified in Chlamydomonas reinhardtii, raising the question as to why kanamycin-resistant mutants have not been found in higher plants. While attempting plastid transformation of alfalfa, we obtained non-transgenic but kanamycin-resistant somatic embryos following 2 months of culture in the presence of 50 mg l(-1) kanamycin. Sequencing of the plastid DNA region corresponding to the decoding site of the 16S rRNA in ten independent resistant events revealed an A to C transversion at position 1357 of the 16S plastid rDNA, the same site at which an A to G conversion confers kanamycin resistance to C. reinhardtii by reducing the ability of the antibiotic to bind to its target site. All plants derived from the resistant embryos through additional cycles of somatic embryogenesis in the absence of kanamycin retained the mutant phenotype, suggesting that the mutation was homoplastomic. Resistant plants produced 85% less biomass than controls; their leaves were chlorotic during early development and over time slowly turned green. The absence of kanamycin- resistant mutants in higher plants might be explained by the requirement for a regeneration system capable of resulting in homoplastomic individuals, or it may be the result of the detrimental effect of the mutation on the phenotype.

Leaf-variegated mutants in higher plants and their responsible genes

Leaf-variegated mutants in higher plants and their responsible genes

An Arabidopsis T-DNA mutant affected in Nrt2 genes is impaired in nitrate uptake

Expression analyses of Nrt2 plant genes have shown a strict correlation with root nitrate influx mediated by the high-affinity transport system (HATS). The precise assignment of NRT2 protein function has not yet been possible due to the absence of heterologous expression studies as well as loss of function mutants in higher plants. Using a reverse genetic approach, we isolated an Arabidopsis thaliana knock-out mutant where the T-DNA insertion led to the complete deletion of the AtNrt2.1 gene together with the deletion of the 3′ region of the AtNrt2.2 gene. This mutant is impaired in the HATS, without being modified in the low-affinity system. Moreover, the de-regulated expression of a Nicotiana plumbaginifolia Nrt2 gene restored the mutant nitrate influx to that of the wild-type. These results demonstrate that plant NRT2 proteins do have a role in HATS.

Response in the inhibitor efficiency of substituted phenols on ps II activity in six mutants of the D1 protein subunit in Chlamydomonas reinhardtii

The D1 protein subunit of the reaction centre of photosystern II is also a herbicide-binding protein. Blockage of the Q B binding site in the D1 protein is the mode of action of many herbicides grouped as classical diuron atrazine type or as phenol group. Tolerance of photosynthetic electron flow in PS II to classical herbicides like diuron or atrazine is observed in several mutants of higher plants and algae with amino acid substitutions in the D1 protein. But in most of these mutants, the few phenol-typeinhibitors tested were not tolerated, some of them even displaying supersensitivity. This seemed to indicate a substantial change of orientation of the phenolic inhibitors in the Q B site when compared to the classical herbicides. However, we report here on the appreciable tolerance of mutants to phenol-type inhibitors of the D1 protein. We investigated the effect of 47 derivatives of 6-alkyl-substituted 2-halogeno-4-nitrophenols and of 2,4-dinitrophenols on photosynthetic electron flow in six mutants of Chlamydomonas reinhardtii. The mutants were obtained either by site-selected screening towards metribuzin or diuron or by site-directed mutagenesis. With the exception of an asn266thr mutant, all the mutants responded toward the phenol inhibitors either by decreased or enhanced sensitivity compared with the wild type. Greatest effects were observed with a val219ile mutant. Tolerance ranged from zero to 1.7 pI 50 units depending on the substituent at position 6 of the phenol ring. Similarly, but somewhat lower, is the tolerance in a phe255tyr mutant. A leu275phe and an ala251val mutant show a mixed behaviour of tolerance to some, and supersensitivity to other derivatives. Many phenols display supersensitivity in the ser264ala mutant, up to 100-fold, and there is never tolerance. It is possible to rationalize the cross resistance of phenol-type inhibitors of PS II with their substitution pattern, particularly in the val2l9ile mutant. The extent of tolerance depends entirely on the size and shape of the substituent in position 6. The maximum is found with 2-bromo-4-nitro-6 benzylphenol with a 50-fold decrease in inhibitor potency in this mutant. The data allow us to orient now also the phenol-type inhibitors in the herbicide binding niche in the D1 protein in a way not too dissimilar from that of the classical inhibitors. The concept that not only the classical herbicides but also phenol-type inhibitors act by binding to the D1 protein is now greatly supported by the observation of tolerance if the sequence of the D1 protein is changed. The principle difference of the phenols to the classical herbicides can now be specified clearly: it is the response to the ser264ala mutant where there is no tolerance of phenol-type inhibitors in contrast to the classical herbicides. This is not invalidated by observations that some phenols may bind to other subunits of PS II as well, nor that many of them are uncouplers. These are to be considered as secondary effects not contributing to the high affinity binding to the D1 protein.

C3–C4 intermediate photosynthesis: linking physiology to gene expression

C₃–C₄ intermediate photosynthesis: linking physiology to gene expression

Acetolactate Synthase and Ketol-Acid Reductoisomerase: Targets for Herbicides Obtained by Screening and de novo Design

Several major classes of herbicides, discovered by conventional screening techniques, have been found to inhibit the first common enzyme of branched-chain amino acid biosynthesis, acetolactate synthase, as their mode of action. These herbicides seem to bind to an evolutionary vestige of a quinone-binding site, extraneous to the active site, that is present due to the evolutionary history of this enzyme. Besides their herbicidal effect on sensitive plants, these compounds can effect stasis in the growth of bacteria and yeast. Recently is has been reported that an experimental herbicide from Hoechst. Hoe 704. that was discovered by conventional screening techniques, inhibits the second common enzyme of branched-chain amino acid bio- synthesis [Schultz etal., FEBS Lett. 238, 375-378 (1988)]. We have also recently designed novel reaction-intermediate analogs (e.g. N-isopropyl oxalylhydroxamate) that arc exceptionally potent (Ki = 22 pM: half-time for release approximately six days) and selective inhibitors of the second common enzyme, ketol-acid reductoisomerase. Both of these selective inhibitors of the second common enzyme will kill sensitive plants, but will only inhibit the growth (without killing) of bacteria. The effects in bacteria parallel those obtained by mutations in the relevant genes, where loss of either the first or second common enzyme in the pathway gives an organ- ism that is auxotrophic for branched-chain amino acids, but does not result in a conditionally lethal phenotype. Higher plant mutants have only been obtained to date that arc deficient in functional leucine-specific gene products (as yet uncharacterized), threonine deaminase (isoleucine specific), and dihydroxyacid dehydratase (common). The phenotypes of these mutants. at least at the level of cell culture, are similar to those of their bacterial counterparts, in that auxotrophy, but not conditional lethality, is obtained. These results highlight the potential non-equality of the enzymes of branched-chain amino acid biosynthesis as targets in herbicide design.

Phenolic Herbicides: Correlation between Lipophilicity and Increased Inhibitory Sensitivity of Thylakoids from Higher Plant Mutants

Abstract Pheonlic herbicides, being quite hydrophilic as compared to classical photosystem-II inhibitors do not exhibit a correlation between inhibitory activity and partition coefficient. Sensitivity against phenolic herbicides, however, as observed with atrazine-resistant thylakoids from higher plants mutants (Brassica napus) is increased by higher partition coefficients.

Nitrate reductase is not accumulated in chloroplast-ribosome-deficient mutants of higher plants

The activities of nitrite reductase (EC 1.7.7.1) are 60-70% of wild-type activity in pigment-deficient leaves of the chloroplast-ribosomedeficient mutants 'albostrians' (Hordeum vulgare) and 'iojap' (Zea mays). The activity and apoprotein of nitrate reductase (EC 1.6.6.1.) are lacking in the barley mutant. Only very low activities of nitrate reductase can be extracted from leaves of the maize mutant. The molybdenum cofactor of nitrate reductase and xanthine dehydrogenase (EC 1.2.3.2) is present in maize and barley mutant plants. However, it is not inducible by nitrate in pigment-deficient leaves of 'albostrians'. From these results we conclude: (i) Nitrite reductase (a chloroplast enzyme) is synthesized in the cytoplasm and does not need the presence of nitrate reductase for the induction and maintenance if its activity. (ii) The loss or low activity of nitrate reductase is a consequence of the inability of the mutants to accumulate the apoprotein of this enzyme. (iii) The chloroplasts influence the accumulation (i.e. most probably the synthesis) of the nonchloroplast enzyme, nitrate reductase. The accumulation of nitrate reductase needs a chloroplast factor which is not provided by mutant plastids blocked at an early stage of their development.

Postmeiotic mitoses without chromosome replication in a mutagen-sensitive Neurospora mutant

In the recessive mutagen-sensitive mutant mus-8, no ascospores are formed. Ascospore delimiting membranes may be defective. Multiple postmeiotic mitoses occur, apparently without chromosome replication (at least at the first two mitoses). This results in many small nuclei, most of which contain only a few chromosomes or a single chromosome. These events resemble those in certain higher-plant mutants such as polymitotic in Zea mays.

Analysis of Photosynthesis with Mutants of Higher Plants and Algae

Analysis of Photosynthesis with Mutants of Higher Plants and Algae

Analysis of Photosynthesis with Mutants of Higher Plants and Algae

Analysis of Photosynthesis with Mutants of Higher Plants and Algae

Comparative herbicide binding by photosynthetic membranes from resistant mutants

Resistance patterns against herbicides inhibiting photosynthetic electron transport have been obtained from several mutants of higher plants and algae. While most of the herbicides assayed show varying resistance patterns with different mutants, resistance against metribuzin is generally very high and always present. On the other hand, no resistance is measured using the phenol-type herbicide ioxynil. With both herbicides replacement experiments vs 14C-labeled atrazine yield a mixed-type competition, i.e., loss of binding sites (receptors) and increasing binding constant. Contrary to ioxynil, the phenol-type herbicide bromonitrothymol gives normal binding and (competitive) replacement data. A mutant of Bumilleriopsis filiformis was isolated which is resistant against bromonitrothymol. Resistance generally does not lead to total loss of binding of a certain inhibitor. A model with two independent receptors at a binding protein is presented, which may explain the nonspecific binding at high herbicide concentrations.

Altered Glycine Decarboxylation Inhibition in Isonicotinic Acid Hydrazide-Resistant Mutant Callus Lines and in Regenerated Plants and Seed Progeny

Isonicotinic acid hydrazide (INH), an inhibitor of the photorespiratory pathway blocking the conversion of glycine to serine and CO(2), has been used as a selective agent to obtain INH-resistant tobacco (Nicotiana tabacum) callus cells. Of 22 cell lines that were INH-resistant, none were different from wild-type cells in their ability to take up [(3)H]INH or to oxidize INH to isonicotinic acid. In 7 of the 22 cell lines, INH resistance was associated with decreased inhibition of NAD-dependent glycine decarboxylation activity in isolated mitochondrial preparations. In the cell line that was most extensively investigated (I 24), this biochemical phenotype (exhibiting a 3-fold higher K(i) with INH) was observed in leaf mitochondria of regenerated plants and of plants produced from them by self-fertilization. After crosses between resistant and sensitive plants, the decreased inhibition of glycine decarboxylation was observed among F(2) and backcross progeny only in those plants previously identified as INH-resistant by callus growth tests. In contrast, in siblings identified as INH-sensitive, glycine decarboxylation was inhibited by INH at the wild-type level. This demonstration of the transfer of an altered enzyme property from callus to regenerated plants and through seed progeny fulfills an important requirement for the use of somatic cell genetics to produce biochemical mutants of higher plants.

Selection of biochemical mutants in plant cell cultures: Some considerations

The availability of diverse biochemical mutants for use as experimental probes in higher plants has been limited. Nevertheless, the importance of mutants in furthering the understanding of plant processes and in crop improvement has encouraged the development of numerous mutation selection methodologies that exploit the advantages of the plant cell culture technologies; examples of successful efforts to select mutants in plant cell culture systems are accumulating. We have also recognized that the mutation selection methodologies that have proven useful with microbes may not be as successful when applied to plant cell culture systems. It is necessary to establish the basis (whether genetic, epigenetic, or a sorting out of a specific cell type) of the variant phenotype. Thorough characterization of the frequency (with and without mutagenic treatment), stability and biochemical properties of a variant cell line will aid in establishing the basis of the altered phenotype, even in the absence of a direct genetic analysis through meiosis. Further research on the selection of specific types of biochemical mutants using various plant cell, tissue and organ will likely produce additional useful higher plant mutants.

Cytogenetics of synaptic mutants in higher plants

Cytogenetics of synaptic mutants in higher plants

Herbicide resistance in a mutant of the microalga Bumilleriopsis filiformis

A DCMU* (diuron)-resistant algal mutant was selected and characterized. Chlorophyll content, growth, and photosystem-I activity are as in the wild-type. Growth in liquid medium with 3 μM DCMU present is half of the control. Apparently only the herbicide-binding site is affected within the redox chain. In contrast to the wild-type, trypsin treatment of isolated chloroplast material completely abolishes photosynthetic electron transport inhibition by DCMU or atrazine.DCMU resistance of chloroplasts is accompanies by tolerance to triazinones and phenylpyridazinones, but not to symmetric triazines. Sensitivity to diphenylethers, DBMIB or o-phenanthroline is not altered.Data on this algal mutant combined with those from triazine-resistant mutants of higher plants give direct evidence of overlapping binding sites at a (hypothetical) binding protein located at the reducing side of photosytem II.

Биохимические мутан ты высших растений ка к объекты для химических и физиологических исс ледований. — Обзор

A report is given on biochemical mutants in higher plants with emphasis on metabolic mutants. Since the discovery of thiamine-defective mutants inArabidopsis thaliana andLycopersicon esculentum some other mutants in different species became available. The first higher plant with an absolute requirement for an amino acid is a proline-deficient mutant inZea mays. Inchloronerva ofLycopersicon esculentum synthesis of nicotianamine is blocked. This is the first instance where a hitherto unknown function is attributed to an already known compound by work with a mutant of higher plants. Nicotianamine is distributed throughout all vascular plants and appears to be linked with short-distance iron transport. The wilty mutantflacca inL. esculentum lacks abscisic acid with some consequences to different parts of metabolism. In the same species the mutant T 3238btl/btl, fer/fer carries two separate, independent mutations which affect the transport of boron and iron, respectively. Selection for chlorate resistance led to the discovery of mutants with impaired nitrate reductase activity and nitrate uptake inArabidopsis thaliana and in consequence added new information to our knowledge of the regulation of nitrate assimilation. Other mutants of similar kind were detected inNicotiana tabacum andHordeum vulgare. Mutants with defects in the photosynthetic system were discovered inZea mays andHordeum vulgare. Plastome mutants inOenothera revealed a lack of ribulose-1,5-biphosphate carboxylase activity and showed interference between the genetic system of the plastids and that of the nucleus.