Inheritance of two independent isozyme variants in soybean plants derived from tissue culture

Summary Soybean [Glycine max (L.) Merr.] plants were regenerated via somatic embryogenesis from nine soybean cultivars. Our objective was to identify and characterize genetically novel mutations that would further our understanding of the soybean genome. Variant isozyme patterns were observed in two independent tissue culturederived lines. Genetic analyses were conducted on these two isozyme variants, and they were heritable. No variant isozyme patterns were evident in control (parental) soybean lines. In the cultivar BSR 101, a mutation of Aco2-b (aconitase) to a null allele was detected. The Aco2-bn mutant, Genetic Type T318, had not been previously observed in soybean. In the Chinese cultivar Jilin 3 (PI 427.099), a chlorophyll-deficient plant was identified that also lacked two mitochondrial malate-dehydrogenase (Mdh null) isozyme bands. These two mutant phenotypes, chlorophyll-deficient and Mdh null, were found to cosegregate. The Jilin 3 mutant, Mdh1-n (Ames 1) y20 (Ames 1) Genetic Type T317, was allelic to three chlorophyll-deficient, Mdh1 null mutants [Mdh1-n (Ames 2) y20 (Ames 2) (T323), Mdh1-n (Ames 3) y20 (Ames 3) (T324), and Mdh1-n (Ames 4) y20 (Ames 4) (T325)] previously identified from a transposon-containing soybean population, and to a chlorophyll-deficient, Mdh1 null mutant [Mdh1-n (Urbana) y20 (Urbana) k2, Genetic Type T253] which occurred spontaneously in soybean. The recovery of two isozyme variants from progeny of 185 soybean plants regenerated from somatic embryogenesis indicates the feasibility of selection for molecular variants.     

Conditional lethality involving a cytoplasmic mutant and chlorophyll-deficient malate dehydrogenase mutants in soybean

Summary Conditional lethality in soybean, Glycine max (L.) Merr., occurred in F₂ plants when cytoplasmicchlorophyll mutant Genetic Type T275 was the female parent and when either nuclear mutants T253 or T323 plants were the male parents. Mutant T253 [Mdh1-n (Urbana) y20 (Urbana) k2] is missing two of three mitochondrial malate dehydrogenase isozymes [Mdh1-n (Urbana)] and has yellowish-green leaves [y20 (Urbana)] and a tan-saddle pattern seed coat (k2). Mutant T323 [Mdh1-n (Ames 2) y20 (Ames 2)] also is missing two of three mitochondrial malate dehydrogenase isozymes [Mdh1-n (Ames 2)] and has yellowishgreen leaves [y20 (Ames 2)], but has yellow seed coat (K2). Mutants T275, T253, and T323 are viable both in the field and glasshouse. The genotypes cyt-Y2 Mdh1-n (Urbana) y20 (Urbana) k2/Mdh1-n (Urbana) y20 (Urbana) k2 and cyt-Y2 Mdh1-n (Ames 2) y20 (Ames 2)/Mdh1-n (Ames 2) y20 (Ames 2) are conditional lethals. These genotypes are lethal under field conditions, but plants survive in reduced light under shadecloth in the glasshouse. We do not know if their interaction with cyt-Y2 is due to Mdh1-n, y20, or Mdh1-n y20. The reciprocal cross (cyt-Y2 as male parent) gives viable genotypes. These conditional lethal genotypes should be useful for studies on the interaction between organelle and nuclear genomes.This is journal paper no. J-14777 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011-1010. Project 2985     

Genetic analysis of 4 new mutants at the unstable k2 Mdh1-n y20 chromosomal region in soybean

In soybean (Glycine max (L.) Merr.), a chromosomal region defined by 3 closely linked loci, k2 (tan-saddle seed coat), Mdh1-n (malate dehydrogenase 1 null), and y20 (yellow foliage), is highly mutable. A total of 31 mutants have been reported from this region. In this study, a mutation with tan-saddle seed coat was found from bulk-harvested seed of cultivar Kenwood. Genetic analysis established that this tan-saddle seed coat mutation is allelic to the k2 locus and inherited as a recessive gene. Simple sequence repeat analysis showed that this mutant is not a contaminant from other existing k2 mutants. The mutant was named Kenwood-k2. To test for genetic instability at the k2 Mdh1-n y20 chromosomal region, Kenwood-k2 was crossed reciprocally with cultivars Harosoy and Williams. No new mutants were found in F2 families. In the genetic instability tests of T239 (k2) with cultivar Williams, 3 new mutants with yellow foliage (y20) and malate dehydrogenase 1 null (Mdh1-n) were identified. In the genetic instability tests of T261 (k2 Mdh1-n) with cultivar Williams, no new mutants were found. The Kenwood-k2 and the 3 yellow-foliage, malate dehydrogenase 1-null mutants provide additional genetic materials to study chromosomal aberrations in this mutable/unstable chromosomal region.     

Duplicate chlorophyll-deficient loci in soybean.

Three lethal-yellow mutants have been identified in soybean (Glycine max (L.) Merr.), and assigned genetic type collection numbers T218H, T225H, and T362H. Previous genetic evaluation of T362H indicated allelism with T218H and T225H and duplicate-factor inheritance. Our objectives were to confirm the inheritance and allelism of T218H and T225H and to molecularly map the locus and (or) loci conditioning the lethal-yellow phenotype. The inheritance of T218H and T225H was 3 green : 1 lethal yellow in their original parental source germplasm of Glycine max 'Illini' and Glycine max 'Lincoln', respectively. In crosses to unrelated germplasm, a 15 green : 1 lethal yellow was observed. Allelism tests indicated that T218H and T225H were allelic. The molecular mapping population was Glycine max 'Minsoy' x T225H and simple sequence repeat (SSR) markers were used. The first locus, designated y18-1, was located on soybean molecular linkage group B2, between SSR markers Satt474 and Satt534, and linked to each by 4.4 and 13.4 cM, respectively. The second locus, designated y18-2, was located on soybean molecular linkage group D2, between SSR markers Satt543 and Sat-001, and linked to each by 2.2 and 4.4 cM, respectively.     

Instability at the k2 Mdh1-n y20 chromosomal region in soybean

Ten mutants have been reported at the k2 (tan saddle seed coat) Mdh1-n (mitochondrial malate dehydrogenase 1 null) y20 (yellow foliage) chromosomal region in soybean [Glycine max (L.) Merr.]. The precise genetic mechanism(s) responsible for generating these mutants is (are) not known. The objective of this study was to determine whether chromosomal instability exists at this region. We introduced the w4-m and Y18-m mutable systems into the three independent sources of tan saddle seed coat mutants, T239 (k2), T261 (k2 Mdh1-n), and L67-3483 (k2). A total of 12 bright yellow mutants were isolated with tan saddle seed coat, malate dehydrogenase 1 null phenotypes. Of these, 11 were found in 11 F2 mutant families out of a total of 977 derived by crossing T239 (k2), T261 (k2 Mdh1-n), and L67-3483 (k2) with six lines suspected to contain active transposable elements. One was found in the F3 generation derived from the cross A1937?×?T239 (k2). Of the 11 F2 mutant families, 10 (out of a total of 381 F2 families) were associated with the T239 (k2) genetic background, and one out of 323 was associated with the T261 (k2 Mdh1-n) genetic background. But no mutation events were found among the 273 families with the L67-3483 (k2) genetic background. Allelism and inheritance studies indicated that these 12 bright yellow mutants were new mutants in the k2 Mdh1-n y20 chromosomal region. Thus, on introducing the w4-m and Y18-m mutable systems into T239 (k2) and T261 (k2 Mdh1-n) genetic backgrounds, chromosomal instability was induced in this region. In addition, 21 greenish yellow mutants were identified in the total of 977 F2 families. All 21 greenish yellow mutants were associated with the T239 (k2) genetic background. The mutations for greenish yellow foliage affected foliage color only at the seedling stage. Cosegregation of the tan saddle seed coat character with greenish yellow foliage were observed for these 21 greenish yellow mutants, suggesting that the greenish yellow phenotype may be due to a pleiotropic effect of the k2 allele in T239 or to chromosomal rearrangements at or near the k2 allele in T239. Finally, we believe that the genetic mechanism responsible for this high frequency of instability at the k2 Mdh1-n y20 chromosomal region involves receptor element activities present at this chromosomal region, which may contain complex chromosomal rearrangements in T239 and T261.     

Instability at the k2 Mdh1-n y20 chromosomal region in soybean

Ten mutants have been reported at the k2 (tan saddle seed coat) Mdh1-n (mitochondrial malate dehydrogenase 1 null) y20 (yellow foliage) chromosomal region in soybean [Glycine max (L.) Merr.]. The precise genetic mechanism(s) responsible for generating these mutants is (are) not known. The objective of this study was to determine whether chromosomal instability exists at this region. We introduced the w4-m and Y18-m mutable systems into the three independent sources of tan saddle seed coat mutants, T239 (k2), T261 (k2 Mdh1-n), and L67-3483 (k2). A total of 12 bright yellow mutants were isolated with tan saddle seed coat, malate dehydrogenase 1 null phenotypes. Of these, 11 were found in 11 F2 mutant families out of a total of 977 derived by crossing T239 (k2), T261 (k2 Mdh1-n), and L67-3483 (k2) with six lines suspected to contain active transposable elements. One was found in the F3 generation derived from the cross A1937 × T239 (k2). Of the 11 F2 mutant families, 10 (out of a total of 381 F2 families) were associated with the T239 (k2) genetic background, and one out of 323 was associated with the T261 (k2 Mdh1-n) genetic background. But no mutation events were found among the 273 families with the L67-3483 (k2) genetic background. Allelism and inheritance studies indicated that these 12 bright yellow mutants were new mutants in the k2 Mdh1-n y20 chromosomal region. Thus, on introducing the w4-m and Y18-m mutable systems into T239 (k2) and T261 (k2 Mdh1-n) genetic backgrounds, chromosomal instability was induced in this region. In addition, 21 greenish yellow mutants were identified in the total of 977 F2 families. All 21 greenish yellow mutants were associated with the T239 (k2) genetic background. The mutations for greenish yellow foliage affected foliage color only at the seedling stage. Cosegregation of the tan saddle seed coat character with greenish yellow foliage were observed for these 21 greenish yellow mutants, suggesting that the greenish yellow phenotype may be due to a pleiotropic effect of the k2 allele in T239 or to chromosomal rearrangements at or near the k2 allele in T239. Finally, we believe that the genetic mechanism responsible for this high frequency of instability at the k2 Mdh1-n y20 chromosomal region involves receptor element activities present at this chromosomal region, which may contain complex chromosomal rearrangements in T239 and T261. Received: 7 January 1998 / Accepted: 7 July 1998     

Inheritance of malate dehydrogenase nulls in soybean

Three chlorophyll-deficient mutants (CD-1, CD-2, and CD-3), derived from the progeny of independent germinal revertants from the w4-mutable soybean line [Glycine max (L.) Merrill], were characterized genetically. Electrophoretic analyses indicated that these lines lacked two of three mitochondrial malate dehydrogenase isozymes (MDH-). The absence of two MDH bands was conditioned by a recessive allele at a locus designated Mdh1. All three CDs were allelic to each other and to T253, a Harosoy isoline y20-k2 MDH- from the Genetic Type Collection. The MDH- phenotype and the yellow-green plant phenotype were each inherited as single recessive alleles. No recombination between the two traits was found in nine F2 populations from crosses of the CDs by wild-type soybean lines. Complete linkage of the Mdh1 and y20 loci suggested that the mutations in the chlorophyll-deficient lines were deletions. Phenotypic differences among the CDs suggested that the deletions may have different endpoints. The chromosomal aberrations were not large enough to affect transmission of y20 and Mdh1 mutant alleles through the pollen or ovule. CD-1, CD-2, and CD-3 were added to the Soybean Genetic Type Collection as T323, T324, and T325, respectively.     

Molecular mapping of k2 Mdh1-n y20, an unstable chromosomal region in soybean [Glycine max (L.) Merr.]

In the soybean genome, a chromosomal region covering three tightly linked genes, k2, Mdh1-n, and y20, was found very unstable. It was suspected that the instability of the k2 Mdh1-n y20 chromosomal region was caused by a non-autonomous transposable element residing adjacent to or in this region. In this study, we located and mapped this region with simple sequence repeat (SSR) markers on the soybean integrated map using five mapping populations. The k2 Mdh1-n y20 chromosomal region was located on molecular linkage group H. The integrated map from five mapping populations consisted of 13 loci in the order Satt541, Satt469, Sat_122, Satt279, Satt253, Satt314, Mdh1-n,y20, k2, Satt302, Satt142, Satt181, and Satt434. The k2 Mdh1-n y20 chromosomal region was very close to Satt314, Satt253, and Satt279. The genetic distance between the Mdh1-n gene and Satt314 was less than 1 cM. The results of the mapping study were consistent with the results from previous studies that the Mdh1-n mutation in T261 (k2 Mdh1-n) and the Mdh1-n y20 mutation in T317 (Mdh1-n y20) were caused by deletions. In addition, another putative deletion was found in the genome of T261 which covered three SSR markers (Satt314, Satt253, and Satt279). This is a joint contribution of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 3769, and from the USDA, Agricultural Research Service, Corn Insects and Crop Genetics Research Unit, and supported by the Hatch Act and the State of Iowa. The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by Iowa State University or the USDA, and the use of the name by Iowa State University or the USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Nuclear-cytoplasmic interaction in chlorophyll-deficient soybean,Glycine max (Fabaceae)

In higher plants, plastids and mitochondria are the predominant carriers of extrachromosomal genetic information. There is interplay between the plastids, the mitochondria, and the nuclear genome. In soybean, Glycine max (L.) Merr., both nuclearly and maternally inherited chlorophyll-deficient mutants have been described. Conditional lethality previously was reported in soybean when maternally inherited chlorophyll-deficient mutant (Genetic Type T275) was crossed with nuclearly inherited yellow foliar malate dehydrogenase null mutants (Genetic Types T253 and T323). Our objective was to test for conditional lethality when maternally inherited yellow foliar mutants T278, T314, T315, T316, T319, and T320 were female parents and nuclearly inherited yellow foliar malate dehydrogenase null mutants T253 and T323 were male parents. Our results indicated conditional lethality in the F₂ generation when any of the six cytoplasmically inherited yellow foliar mutants were female parents and either T253 or T323 were male parents. The physiological nature of conditional lethality is not known. Data indicate a common basis in soybean for conditional lethality among the cytoplasmically inherited yellow foliar mutants when crossed with the nuclearly inherited yellow foliar malate dehydrogenase null mutants. No interactions were observed between cytoplasmically inherited or nuclearly inherited green seed embryo mutants as female parents and either T253 or T323 as male parents.     

The ms1 mutation in soybean: involvement of gametes in crosses with tetraploid soybean

Summary Previous studies indicated that ms1ms1 malesterile female-fertile soybean (Glycine max [L.] Merr.) plants can produce seeds with different ploidy levels. The codominant chlorophyll-deficient mutant y11 was used in attempts to understand the embryo-endosperm relationship in seed production in ms1ms1 plants and to determine the mechanism of gamete formation in the ms1 mutation. Crosses were conducted between yellow-green male-sterile plants (ms1ms1Y11y11) and green fertile tetraploid cultivars (Ms1Ms1Ms1Ms1Y11Y11Y11Y11) in the greenhouse in the summers of 1987 and 1988. A total of 2,007 cross-pollinations were made. Thirty hybrid seeds were obtained, and plants were analyzed for chromosome number, fertility, and color. All the hybrid seedlings were tetraploid and fertile. No triploids were found. Among the 30 F₁ plants, 7 were green (Y11Y11Y11Y11), 17 were green-yellow (Y11Y11Y11y11), and 6 were yellow-green (Y11Y11y11y11). The segregation ratio was close to the expected 1 green: 2 green-yellow: 1 yellow-green (X² = 0.38; 0.90>p>0.75). From the results of this experiment, we conclude that: (1) triploids were not produced by crossing diploid ms1ms1 soybean plants with tetraploid plants; (2) tetraploid progeny can be produced from these crosses by the fusion of 2n ms1 eggs, or fusion of other 2n gametophyte cells in the embryo sac with a 2x sperm from tetraploid plants; (3) the megaspore mother cell of male-sterile plants undergoes meiotic division without cytokinesis after telophase II and forms more than the normal number of gametes, which can fuse with each other to generate tetraploid gametophyte cells.Joint contribution: U.S. Department of Agriculture, Agricultural Research Service, Cereal and Soybean Research Improvement Unit, Midwest Area, and Journal Paper No.-13838 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa     

Genetic and cytological analyses of three lethal ovule mutants in soybean (Glycine max; Leguminosae).

Soybean partially sterile mutants 2, 3, and 4 (PS-2, PS-3, and PS-4), recovered from a gene-tagging experiment, were studied to clarify their inheritance, linkage, allelism, and reproductive biology. The PS-2, PS-3, and PS-4 mutants were maintained as heterozygotes and upon self-pollination segregated l fertile: l partially-sterile. For inheritance and linkage tests, all three PS mutants were crossed to flower color mutant Harosoy-w4 and to chlorophyll-deficient (CD) mutants CD-1 and CD-5, also recovered from the tagging study. For allelism tests, reciprocal crosses were made among the three partially sterile mutants. Linkage results indicated that the gene for partial sterility in the PS-2, PS-3, and PS-4 mutants was not linked either to the w4 locus or to the genes for chlorophyll deficiency. Studies of pollen development, pollen viability, and pollen-tube germination indicated no difference between normal and partially sterile genotypes. Linkage and allelism tests indicated that the gene in the three partially sterile mutants was not transmitted through the female when they were used as a female parent. A study of megagametogenesis indicated that the ovules from partially sterile plants had normal embryo sac development. Ovule abortion was due to failure of fertilization.     

Positioning 3 qualitative trait loci on soybean molecular linkage group E

In soybean (Glycine max [L.] Merr.), 3 qualitative trait loci (Pb, Y9, and Y17) are located on classical linkage group 14, which corresponds to molecular linkage group (MLG) E. The Pb locus conditions sharp/blunt pubescence tip; the y9 and y17 loci condition green/chlorotic foliage. The gene order is not known. Our objective was to determine the gene order on soybean MLG E of the Pb, Y9, and Y17 loci using previously mapped simple sequence repeat (SSR) markers. Allelism tests between y9 and y17 gave normal green foliage F(1) plants, indicating nonallelism. Our F(2) data from the allelism test could not distinguish between a 1:1 or a 9:7 ratio. The F(2:3) family segregation indicated a very close genetic linkage between the y9 and the y17 loci. Two molecular mapping populations were developed. Population-1 segregated for Pb and y9, and population-2 segregated for Pb and y17. The gene order on soybean MLG E, using SSR markers, was Pb, Y9, and Y17.     

Genetics and cytology of a genic male-sterile, female-sterile mutant from a transposon-containing soybean population

A male-sterile, female-sterile soybean mutant (w4-m sterile) was identified among progeny of germinal revertants of a gene-tagging study. Our objectives were to determine the genetics (inheritance, allelism, and linkage) and the cytology (microsporogenesis and microgametogenesis) of the w4-m sterile. The mutant was inherited as a single recessive nuclear gene and was nonallelic to known male-sterile, female-sterile mutants st2 st2, st3 st3, st4 st4, st5 st5, and st6 st6 st7 st7. No linkage was detected between the w4-m sterile and the w4w4, y10 y10, y11 y11, y20 y20, fr1 fr1, and fr2 fr2 mutants. Homologous chromosome pairing was complete in fertile plants. Chromosome pairing, as observed in squash preparation, was almost completely absent in sterile plants. Developmentally microsporogenesis proceeded normally in both the fertile and the w4-m sterile through the early microspore stage. Then the tapetal cells of the w4-m sterile surrounding the young microspores developed different-size vacuoles. These tapetal cells became smaller in size and separated from each other. Some of the microspores of the w4-m sterile also became more vacuolate prematurely and sometimes they collapsed, usually by the late microspore stage. In the w4-m sterile the microspore walls remained thinner and structurally different from the microspore walls of fertile plants. No pollen was formed in the mutant plants, even though some of the male cells reached the pollen stage, although without normal filling. The w4-m sterile was designated st8st8 and assigned Soybean Genetic Type Collection number T352.     

浅绿叶水稻突变体的特性与遗传分析

在簇生稻与粳稻日本晴杂交后代F8世代中发现一个能稳定遗传的浅绿叶色突变体(pgl,pale green leaf).与野生型相比,突变体pgl株高、剑叶宽、主穗粒数和千粒重均显著下降.从幼苗开始,突变体pgl叶片都表现为浅绿色.在苗期和抽穗期突变体叶片的叶绿素含量都极显著低于野生型,其中叶绿素b的含量极低,仅为0.00...     

Expression of Mdh1 locus alleles in apozygotic progenies of sugar beet (Beta vulgaris L.)

Expression of Mdh1 alleles has been studied in 60 apozygotic (agamospermic) sugar beet progenies. Seed progenies were obtained by uniparental (pollen less) mode of seed reproduction: selfing of pollen-sterile plants isolated with paper bags. The apozygotic seed progenies demonstrate a disomic gamete autosegregation, i.e., the ratio between genotypes in the progenies correspond to the gamete segregation in a duplex heterozygote of an autotetraploid. It was shown that the ratio between the Mdh1 phenotypes in apozygotic progenies is strongly affected by spontaneous inactivation of one of the alleles. In most progenies, the excess of FF phenotypes and the deficit of SS phenotypes were observed. In our opinion, such deviations in genotype and phenotype frequencies result from conversion of the active Mdh1-S into the inactive Mdh1-S0 allele (epigenetic gene inactivation). The spontaneous inactivation of one allele results in extremely variable frequencies of heterozygous Mdh1-F/Mdh1-S genotypes and phenotypes in the apozygotic seed progenies. The empirical distribution of the frequencies of heterozygous genotypes in the apozygotic seed progenies is given by a negative binomial distribution describing the expected time of occurrence of random events.     

Genetic and cytological analyses of a partial-female-sterile mutant (PS-1) in soybean (Glycine max; Leguminosae)

Soybean partial-female-sterile mutant 1 (PS-1) was recovered from a gene-tagging study. The objectives were to study the inheritance, linkage, allelism, and certain aspects of the reproductive biology of the PS-1 mutant. For inheritance and linkage tests, PS-1 was crossed to flower color mutant Harosoy-w4 and to chlorophyll-deficient mutant CD-1, also recovered from the gene-tagging study. For allelism tests, reciprocal crosses were made with PS-1 and three other partial-sterile mutants (PS-2, PS-3, and PS-4) recovered from the same gene-tagging study. The PS-1 mutant is inherited in a 3:1 ratio and is a single recessive gene. Linkage results indicated that the gene for partial sterility in PS-1 is not linked either to the w4 locus or to the CD-1 locus. Allelism tests showed that the gene in PS-1 is nonallelic to the gene in PS-2, PS-3, and PS-4. Investigations of developing and mature pollen indicated no differences in morphology, stainability, or fluorescence between normal and partial-sterile genotypes. The PS-1 mutant is completely male fertile. Confocal scanning laser microscopy was used to determine that early embryo abortion in PS-1 is due indirectly to abnormal migration of the fused polar nucleus, which prevented it from being fertilized. Subsequent absence of endosperm development leads directly to abortion of the proembryo.     

Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase

Gastric tissues from amphibian Rana perezi express the only vertebrate alcohol dehydrogenase (ADH8) that is specific for NADP(H) instead of NAD(H). In the crystallographic ADH8-NADP+ complex, a binding pocket for the extra phosphate group of coenzyme is formed by ADH8-specific residues Gly223-Thr224-His225, and the highly conserved Leu200 and Lys228. To investigate the minimal structural determinants for coenzyme specificity, several ADH8 mutants involving residues 223 to 225 were engineered and kinetically characterized. Computer-assisted modeling of the docked coenzymes was also performed with the mutant enzymes and compared with the wild-type crystallographic binary complex. The G223D mutant, having a negative charge in the phosphate-binding site, still preferred NADP(H) over NAD(H), as did the T224I and H225N mutants. Catalytic efficiency with NADP(H) dropped dramatically in the double mutants, G223D/T224I and T224I/H225N, and in the triple mutant, G223D/T224I/H225N (kcat/KmNADPH = 760 mm-1 min-1), as compared with the wild-type enzyme (kcat/KmNADPH = 133330 mm-1 min-1). This was associated with a lower binding affinity for NADP+ and a change in the rate-limiting step. Conversely, in the triple mutant, catalytic efficiency with NAD(H) increased, reaching values (kcat/KmNADH = 155000 mm-1 min-1) similar to those of the wild-type enzyme with NADP(H). The complete reversal of ADH8 coenzyme specificity was therefore attained by the substitution of only three consecutive residues in the phosphate-binding site, an unprecedented achievement within the ADH family.     

Expression of Alleles of the Mdh1Locus in Apozygotic Progenies of Sugar Beet Beta vulgarisL.

Expression of Mdh1alleles has been studied in 60 apozygotic (agamospermic) sugar beet progenies. Seed progenies were obtained by uniparental (pollenless) mode of seed reproduction: selfing of pollen-sterile plants isolated with paper bags. The apozygotic seed progenies demonstrate a disomic gamete autosegregation, i.e., the ratio between genotypes in the progenies correspond to the gamete segregation in a duplex heterozygote of an autotetraploid. It was shown that the ratio between theMdh1phenotypes in apozygotic progenies is strongly affected by spontaneous inactivation of one of the alleles. In most progenies, the excess of FF phenotypes and the deficit of SS phenotypes were observed. In our opinion, such deviations in genotype and phenotype frequencies result from conversion of the active Mdh1-Sinto the inactive Mdh1-S ⁰allele (epigenetic gene inactivation). The spontaneous inactivation of one allele results in extremely variable frequencies of heterozygous Mdh1-F/Mdh1-Sgenotypes and phenotypes in the apozygotic seed progenies. The empirical distribution of the frequencies of heterozygous genotypes in the apozygotic seed progenies is given by a negative binomial distribution describing the expected time of occurrence of random events.     

Regulation of photosynthesis in developing leaves of soybean chlorophyll-deficient mutants

In this report we examine the factors that regulate photosynthesis during leaf ontogeny in y3y3 and Y11y11, two chlorophyll-deficient mutants of soybean. Photosynthetic rates were similar during wild type and Y11y11 leaf development, but the senescence decline in photosynthesis was accelerated in y3y3. Photosynthetic rates fell more rapidly than chlorophyll concentrations during senescence in wild type leaves, indicating that light harvesting is not strongly limiting for photosynthesis during this phase of leaf development. Chlorophyll concentrations in Y11y11, though significantly lower than normal, were able to support normal photosynthetic rates throughout leaf ontogeny. Chlorophyll a/b ratios were constant during leaf development in the wild type, but in the mutants they progressively increased (y3y3) or decreased (Y11y11). In all three sets of plants, photosynthetic rates were directly proportional to Rubisco contents and activities, suggesting that Rubisco plays a dominant role in regulating photosynthesis throughout leaf ontogeny in these plants. The expression of some photosynthetic proteins, such as Rubisco activase, was coordinately regulated with that of Rubisco in all three genotypes, i.e. an early increase, coincident with leaf expansion, followed by a senescence decline in the fully-expanded leaf. On the other hand, the light harvesting chlorophyll a/b-binding proteins of PS II (the CAB proteins), while they showed a profile similar to that of Rubisco in the wild type and y3y3, progressively increased in amount during Y11y11 leaf development. We conclude that Y11y11 may be defective in the accumulation of a component required for LHC II assembly or function, while y3y3 has more global effects and may be a regulatory factor that controls the duration of senescence.     

Improved thermostability and the optimum temperature of Rhizopus arrhizus lipase by directed evolution

To expand the functionality of lipase from Rhizopus arrhizus (RAL) we have used error-prone PCR and DNA shuffling methods to create RAL mutants with improved thermostability and the optimum temperature. One desirable mutant with three amino acids substitution was obtained. The mutated lipase was purified and characterized. The optimum temperature of the mutant lipase was higher by 10 °C than that of the wild-type RAL (WT-RAL). In addition, the thermostability characteristic of the mutant was also improved as the result of directed evolution. The half-life (T_1/2) at 50 °C of the mutant exceeded those of WT-RAL by 12-fold. To confirm which substitution contributed to enhance thermostability and the optimum temperature for lipase activity, three chimeric lipases: chimeric lipase 1(CL-1; A9T), chimeric lipase 2 (CL-2; E190V) and chimeric lipase 3 (CL-3; M225I) from the WT-RAL gene were constructed. Each of the chimeric enzymes was purified and characterized. Amino acid substitution at position 190 was determined to be critical for lipase thermostability and the optimum temperature, while the residue at position 9 and 225 had only marginal effect. The mutational effect is interpreted according to a simulated three-dimensional structure for the mutant lipase.