Genetic modification of the waxy character in barley after an injection of wild-type exogenous DNA. Analysis of the second seed generation

Preparations of the DNA extracted from the endospermal milk and leaves of normal distichous barley plants were injected into the grains of hexastichous barley at a milk-ripeness stage and carrying waxy mutation (i.e. having defective synthesis of amylose). Restoration of normal starch synthesis in plant cells was considered as an indication of the changes produced by the exogenous DNA. In normal barley, starch contains amylose and amylopectin and is stained blue-to-black in an iodine solution, whereas in the waxy mutant, starch has no amylose and is stained red-to-brown. To exclude any relationship of dominance the analysis was carried out at a haploid level, that is on the pollen harvested from the injected plants. This approach had the additional advantage of allowing us to work with individual cells instead of whole plants or their organs. Upon injection of the preparations of wild-type DNA a certain fraction of plants showed changes toward wild type. The seeds obtained from the altered plants were sown and the progeny examined. It was established that in the second generation most plants returned to the recepient type. A detailed analysis of the progeny of plant No. 506/19, in the first generation of which almost all pollen (over 99.5%) was of wild type, showed that in these plants the alterations were considerably more stable, only one fifth or one sixth of plants returning to the original recipient type. In half of the progeny of plant No. 506/19 the starch was of the donor type, and in 30% of the plants, as in the first generations, the bulk of the polen (99.98%) was stained black by iodine. In the second generation, segregation of plants for starch staining differed from the Mendelian one, which is probably indicative of non-chromosomal localization of the genetic determinants brought along with the injected DNA. Concurrent with the alterations of the starch structure, alterations of the type of spike (hexastichous barley plants modified distichous ones) were observed in those plants whose pollen became predominantly or wholly normal. Also, delayed manifestation of the synthesis of normal starch was noted. Certain plants which did not show any changes in the first generation yielded wild-type pollen grains in the second generation.     

Genetic change in the waxy trait of barley under the influence of wild type DNA. Analysis of the composition of starch and the electrophoretic spectrum of caryopsis hordein from altered plants and the retention of these changes to the fourth generation

Injection of DNA isolated from the wild type of barley into grains of recipient mutant plants (waxy mutants) at the milk stage of maturity leads to a change in starch synthesis; type of spikes and hordein composition. In the first generation of injected plants the wild type starch synthesis was observed in some separate plants (these observations were made at a haploid level in pollen cells). In the second generation of transformed plants along with the change in starch and hordein synthesis a modification of the type of spikes was also revealed. Recipient plants had six-rowded (hexastichous) spikes, and donor plants--two rowded (distichous) spikes. Disc-electrophoresis of hordeins of the wild type barley (Yuzhny var.), hordeins of the waxy mutant (defected in synthesis of normal starch) and barley plants transformed under the action of wild type exogenous DNA reveals differences in the protein spectrum between donor, recipient and transformants. In the second generation in many of the transformed plants starch synthesis reverted to the recipient mutant type. Simultaneously a reversion of hordein composition to the initial mutant type was observed, and the distichous pikes became hexastichous. Analysis of the components of starch revealed that donor plant that have amilose and amilopectin in starch, and the recipient plants that lack amilose, can be distinguished by the spectra of light absorption of starch. For characterizing these differences the plot of absoprtions at 490 versus that at 590 nm was used. The tangens of angles of these curves for the waxy mutant were equal to 1.05 +/- 0.07 and 1.81 +/- 0.04 for the wild type barley. All transformants have a 1.78 ratio and for revertants this value was 1.02.     

Hereditary variability of plants under the action of exogenous DNA

Summary Injection of exogenous barley donor DNA into grains of barley recipient plants at the milk maturity stage, with a specially designed syringe, led to the appearance of transformed plants. The transformation (in rare cases) was caused by the unsheared DNA since the DNA passing through the syringe needle remained relatively stable (10⁶ to 10⁷ daltons) as was confirmed by DNA sedimentation analysis.14 plants grown from seeds injected with highly polymeric DNA containing close to 30 per cent protein had transformed pollen grains. In the 2nd generation only 2 plants from the 8 studied preserved these changes. In the progeny of these two plants, i.e., in the 3rd seed generation after injection, 82.1 per cent of plants preserved the transformed characters. The next, 4th generation, preserved a transformed phenotype in 89.6 per cent of plants.It was also shown that reversion to a recipient-like state was not always constant. We found the reversion of transformed properties (i.e., normal starch and two-rowed spikes) in 40 per cent of the 4th generation descendants of one of the plants which had lost the phenotypical expression of these properties in the 3rd generation but had them in the 2nd generation.The study of the morphological properties of transformed plants showed that with respect to phenotypic expression some characters were changed towards the donor type, some remained as in the recipients and some were of the intermediate type.     

Starch-synthesizing Enzymes in the Endosperm and Pollen of Maize

Two mutations, amylose-extender and waxy, which affect the proportion of amylose and amylopectin of starch synthesized in the endosperm of maize (Zea mays L.) seeds, are also expressed in the pollen. However, most mutations that affect starch synthesis in the maize endosperm are not expressed in the pollen. In an attempt to understand the nonconcordance between the endosperm and pollen, extracts of mature pollen grains were assayed for a number of the enzymes possibly implicated in starch synthesis in the endosperm. Sucrose synthetase (sucrose-UDP glucosyl transferase, EC 2.4.1.13) activity was not detectable in either mature or immature pollen grains of nonmutant maize, but both bound and soluble invertase (EC 3.2.1.26) exhibited much greater specific activity (per milligram protein) in pollen extracts than in 22-day-old endosperm extracts. Phosphorylase (EC 2.4.1.1) activity was also higher in pollen than in endosperm extracts. ADP-Glucose pyrophosphorylase (EC 2.7.7.27) activity was much lower in pollen than endosperm extracts, but mutations that drastically reduced ADP-glucose pyrophosphorylase activity in the endosperm (brittle-2 and shrunken-2) did not markedly affect enzymic activity in the pollen. Specific activities of other enzymes implicated in starch synthesis were similar in endosperm and pollen extracts.     

phlF− mutant of Pseudomonas fluorescens J2 improved 2,4-DAPG biosynthesis and biocontrol efficacy against tomato bacterial wilt

Pseudomonas fluorescens J2 can produce 2,4-diacetylphloroglucinol (2,4-DAPG) as the main antibiotic compound and effectively inhibits the wilt pathogens Ralstonia solanacearum and Fusarium oxysporum. The phlF which negatively regulates the 2,4-DAPG synthesis in strain J2 was disrupted by homologous recombination to construct a mutant strain J2-phlF⁻. The mutant J2-phlF⁻ produced much more 2,4-DAPG and showed higher inhibitory effect on R. solanacearum than the wild type strain J2 in vitro. The mutant J2-phlF⁻ also showed more colonization of tomato roots and higher inhibition to R. solanacearum in soil than wild type strain J2. The biocontrol efficiency of mutant J2-phlF⁻ was higher against tomato bacterial wilt than wild type strain J2, but the differences were not significant. However, the application of both strains with organic fertilizer improved the colonization and biocontrol efficiency against tomato bacterial wilt and mutant strain J2-phlF⁻ showed higher biocontrol efficiency against tomato bacterial wilt than wild type strain J2. Both strains, J2 and J2-phlF⁻, could also promote the growth of tomato plants.     

Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana

Arabidopsis has three cytokinin receptors genes: CRE1, AHK2 and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma and female gametophyte formation and maturation.Key words: cytokinin, cytokinin receptor, female gametophyte, male gametophyte, stigma     

Boron deficiency in maize

Boron (B) deficiency depresses wheat, barley and triticale yield through male sterility. On the basis of field responses to B fertilization, maize (Zea mays L.) is affected by B deficiency in five continents. In a series of sand culture trials with maize subject to B0 (nil added B) and B20 (20???M added B) treatments, we described how B deficiency depressed maize grain yield while showing an imperceptible effect on vegetative dry weight. With manual application of pollen to the silk of each plant, B0 plants produced 0.4 grain ear^?1 compared with 410 grains ear^?1 in B20 plants. Symptoms of B deficiency was observed only in B0 plants, which exhibited symptoms of narrow white to transparent lengthwise streaks on leaves, multiple but small and abnormal ears with very short silk, small tassels with some branches emerging dead, and small, shrivelled anthers devoid of pollen. Tassels, silk and pollen of B0 plants contained only 3?C4?mg B kg^?1 DW compared with twice or more B in these reproductive tissues in B20 plants. A cross-fertilization experiment showed that, although the tassels and pollen were more affected, the silk was more sensitive to B deficiency. Pollen from B20 plants applied to B0 silk produced almost no grains, while pollen from B0 on B20 silk increased the number of grains to 37% of the 452 grains plant^?1 produced from B20 pollen on B20 silk. Therefore, the silk of the first ear may be targeted for precise diagnosis of B status at maize reproduction, for timely correction by foliar B application, and even for B-efficient genotype selection.     

OsMLO12, encoding seven transmembrane proteins,is involved with pollen hydration in rice

Key message

MLO mediates pollen hydration.

Abstract

Hydration is the first step in pollen germination. However, the process is not well understood. OsMLO12 is highly expressed in mature pollen grains; plants containing alleles caused by transfer DNA insertions do not produce homozygous progeny. Reciprocal crosses between wild-type and OsMLO12/osmlo12 plants showed that the mutant alleles were not transmitted through the male gametophyte. Microscopic observations revealed that, although mutant grains became mature pollen with three nuclei, they did not germinate in vitro or in vivo due to a failure in hydration. The OsMLO12 protein has seven transmembrane motifs, with an N-terminal extracellular region and a C-terminal cytosolic region. We demonstrated that the C-terminal region mediates a calcium-dependent interaction with calmodulin. Our findings suggest that pollen hydration is regulated by MLO12, possibly through an interaction with calmodulin in the cytosol.     

Arabidopsis mutant of AtABCG26, an ABC transporter gene, is defective in pollen maturation

In plants, pollen is the male gametophyte that is generated from microspores, which are haploid cells produced after meiosis of diploid pollen mother cells in floral anthers. In normal maturation, microspores interact with the tapetum, which consists of one layer of metabolically active cells enclosing the locule in anthers. The tapetum plays several important roles in the maturation of microspores. ATP-binding cassette (ABC) transporters are a highly conserved protein super-family that uses the energy released in ATP hydrolysis to transport substrates. The ABC transporter gene family is more diverse in plants than in animals. Previously, we reported that an Arabidopsis half-size type ABC transporter gene, COF1/AtWBC11/AtABCG11, is involved in lipid transport for the construction of cuticle layers and pollen coats in normal organ formation, as compared to CER5/AtWBC12/AtABCG12. However, physiological functions of most other ABCG members are unknown. Here, we identified another family gene, AtABCG26, which is required for pollen development in Arabidopsis. An AtABCG26 mutant developed very few pollen grains, resulting in a male-sterile phenotype. By investigating microspore and pollen development in this mutant, we observed that there was a slight abnormality in tetrad morphology prior to the formation of haploid microspores. At a later stage, we could not detect exine deposition on the microspore surface. During pollen maturation, many grains in the mutant anthers got aborted, and surviving grains were found to be defective in mitosis. Transmission of the mutant allele through male gametophytes appeared to be normal in genetic transmission analysis, supporting the view that the pollen function was disturbed by sporophytic defects in the AtABCG26 mutant. AtABCG26 can be expected to be involved in the transport of substrates such as sporopollenin monomers from tapetum to microspores, which both are plant-specific structures critical to pollen development.     

Plant food subsistence in the human diet of the Bronze Age Caspian and Low Don steppe pastoralists: archaeobotanical,isotope and <Superscript>14</Superscript>C data

The paper presents the result of analysis of charred food on the interior part of the vessels from the graves of the East Manych and West Manych Catacomb archaeological cultures (2500–2350 cal bc). The phytolith and pollen analyses identified pollen of wild steppe plants and phytoliths of domesticated gramineous plants determined as barley phytoliths. Direct ¹⁴С dating of one of the samples demonstrates that barley spikelets and stems were used in funeral rites by local steppe communities. However, there are no data suggesting that steppe inhabitants of the Lower Don Region were engaged in agriculture in the mid-3000 bc. Supposedly, barley could have reached the steppes through seasonal migrations of mobile pastoralists to the south, use of North Caucasus grasslands in the economic system of seasonal moves and exchange with local people. Nevertheless, presence of carbonized barley seeds in the occupation layers at North Caucasus settlements of 4000–3000 bc requires confirmation by direct ¹⁴С dating of such samples.     

A Ds-mutation of the Adh1 gene in Zea mays L.

Summary An unstable spontaneous mutation in the maize Adh1 gene, coding for alcohol dehydrogenase, was selected by allyl alcohol poisoning of wild type Adh1 pollen from a maize line carrying Ds at the Bz2 locus and one copy of Ac in an unknown position. The mutant has a null phenotype. No wild type pollen grains were detected in strains devoid of Ac, but in the presence of Ac, wild type pollen grains were detected with a frequency of between 10^-4 and 10^-3. In addition, events have been identified in the aleurone in which reversions of both bz2-m and the unstable adh1 mutation occurred in the same patch of tissue, presumably in response to an alteration of Ac. By these criteria, the Adh1 mutant is caused by Ds. DNA blotting experiments have shown the presence of a 1.3 kb insertion in the Adh1 gene. All or part of this Ds insertion is transcribed, and is detected as an insertion within the ADH1-mRNA. The longer mRNA hybridizes to an authentic Ds probe.This Ds element differs in size from other known Ds insertions.     

The Role of Arabidopsis ABCG9 and ABCG31 ATP Binding Cassette Transporters in Pollen Fitness and the Deposition of Steryl Glycosides on the Pollen Coat

The pollen coat protects pollen grains from harmful environmental stresses such as drought and cold. Many compounds in the pollen coat are synthesized in the tapetum. However, the pathway by which they are transferred to the pollen surface remains obscure. We found that two Arabidopsis thaliana ATP binding cassette transporters, ABCG9 and ABCG31, were highly expressed in the tapetum and are involved in pollen coat deposition. Upon exposure to dry air, many abcg9 abcg31 pollen grains shriveled up and collapsed, and this phenotype was restored by complementation with ABCG9_pro:GFP:ABCG9. GFP-tagged ABCG9 or ABCG31 localized to the plasma membrane. Electron microscopy revealed that the mutant pollen coat resembled the immature coat of the wild type, which contained many electron-lucent structures. Steryl glycosides were reduced to about half of wild-type levels in the abcg9 abcg31 pollen, but no differences in free sterols or steryl esters were observed. A mutant deficient in steryl glycoside biosynthesis, ugt80A2 ugt80B1, exhibited a similar phenotype. Together, these results indicate that steryl glycosides are critical for pollen fitness, by supporting pollen coat maturation, and that ABCG9 and ABCG31 contribute to the accumulation of this sterol on the surface of pollen.     

Ribosomal RNA cistron number in a polyploid series of plants

A comparison has been made of the amount of DNA coding for ribosomal RNA present in a polyploid series of plants of Datura innoxia. The plants which were produced by in vitro culture of pollen grains were haploid, diploid, triploid, tetraploid and hexaploid. In all instances there was a similar proportion of DNA coding for ribosomal RNA. The implications of these results are discussed, and the data compared with that available from other series of plants of different DNA content or ploidy.     

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H⁺ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 μM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H⁺ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H⁺ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen. Received: 20 June 2000 / Accepted: 2 October 2000     

Growth characteristics, grain filling, and assimilate transport in a shrunken endosperm mutant of barley

The reported inheritance pattern of the seg1 shrunken endosperm mutant of barley (Hordeum vulgare L. cv Betzes) suggests that some defective process in the maternal plant tissues, and not in the endosperm, prevents normal grain filling in the mutant. To identify the physiological mechanism of the mutation, we compared growth, carbon exchange, and assimilate transport of Betzes and seg1 plants. Betzes and seg1 plants did not differ in mean relative growth rate, mean net assimilation rate, or carbon exchange rate. The rate and duration of grain growth of seg1 was lower than Betzes on intact plants and on detached, cultured spikes. Increasing the supply of sucrose in culture media up to 300 mm sucrose did not eliminate differences between normal and mutant grain growth. Translocation of ¹⁴C-labeled assimilates into seg1 grains ceased by 21 days after anthesis, and assimilates were diverted to lower plant parts. In contrast, assimilates were still entering Betzes grains at 29 days after anthesis. Evidence suggests that some maternal spike or grain tissue is affected by the mutation after the onset of grain filling. Identification of the specific seg1 defect may provide information about the cessation of normal grain filling.     

C]Sucrose Uptake and Labeling of Starch in Developing Grains of Normal and segl Barley

Previous work showed that the segl mutant of barley (Hordeum vulgare cv Betzes) did not differ from normal Betzes in plant growth, photosynthesis, or fertility, but it produced only shrunken seeds regardless of pollen source. To determine whether defects in sucrose uptake or starch synthesis resulted in the shrunken condition, developing grains of Betzes and segl were cultured in [¹⁴C]sucrose solutions after slicing transversely to expose the endosperm cavity and free space. In both young grains (before genotypes differed in dry weight) and older grains (17 days after anthesis, when segl grains were smaller than Betzes), sucrose uptake and starch synthesis were similar in both genotypes on a dry weight basis. To determine if sucrose was hydrolyzed during uptake, spikes of Betzes and segl were allowed to take up [fructose-U-¹⁴C]sucrose 14 days after anthesis and the radioactivity of endosperm sugars was examined during 3 hours of incubation. Whereas less total radioactivity entered the endosperm and the endosperm cavity (free space) of segl, in both genotypes over 96% of the label of endosperm sugars was in sucrose, and there was no apparent initial or progressive randomization of label among hexose moieties of sucrose as compared to the free space sampled after 1 hour of incubation. We conclude that segl endosperms are capable of normal sucrose uptake and starch synthesis and that hydrolysis of sucrose is not required for uptake in either genotype. Evidence suggests abnormal development of grain tissue of maternal origin during growth of segl grains.