Double Mutant Line Research Articles

Mono- and double-mutant mouse models of Parkinson's disease display severe mitochondrial damage

Mutations in the gene encoding alpha-synuclein (asyn) causes autosomal-dominant, in the parkin gene autosomal-recessive forms of Parkinson's disease (PD). The pathophysiology of PD is poorly understood, even though published evidence suggests a role for mitochondria in the pathogenesis. To gain insight into the influence of asyn and parkin on mitochondrial integrity and function, we have generated several mono-mutant mouse lines expressing doubly mutated human asyn (hm(2)asyn) under the control of two different promoters, or a targeted deletion of Parkin (Parkin-Exon3-knockout). Both mouse lines were crossed to generate the double-mutant. Here we compare the ultrastructure and functional properties of mitochondria in the substantia nigra (SN), the striatum, the cerebral cortex (Cx) and skeletal muscle of young (2-3 months) and aged (12-14 months) mono- and double-mutants mice. We observed severe genotype-, age- and region-dependent morphological alterations of mitochondria in neuronal somata. The number of structurally altered mitochondria was significantly increased in the SN of both double-mutants and in the Cx of one mono- and one double-mutant line. These alterations coincided with a reduced complex I capacity in the SN, but were neither accompanied by alterations in the number or the size of the mitochondria nor by leakage of cytochrome c, Smac/DIABLO or Omi/HtrA2. None of the transgenic animals developed any gross histopathological abnormalities or overt motor disabilities. Together our results provide compelling evidence that (i) both, asyn and parkin are relevant for mitochondrial integrity, (ii) the influence of these proteins on mitochondria are age- and tissue-specific and (iii) changes of mitochondrial morphology do not inevitably cause functional impairments.

Genetic Suppressors of the Lotus japonicus har1-1 Hypernodulation Phenotype

Lotus japonicus har1 mutants respond to inoculation with Mesorhizobium loti by forming an excessive number of nodules due to genetic lesions in the HAR1 autoregulatory receptor kinase gene. In order to expand the repertoire of mutants available for the genetic dissection of the root nodule symbiosis (RNS), a screen for suppressors of the L. japonicus har1-1 hypernodulation phenotype was performed. Of 150,000 M2 plants analyzed, 61 stable L. japonicus double-mutant lines were isolated. In the context of the har1-1 mutation, 26 mutant lines were unable to form RNS, whereas the remaining 35 mutant lines carried more subtle symbiotic phenotypes, either forming white ineffective nodules or showing reduced nodulation capacity. When challenged with Glomus intraradices, 18 of the 61 suppressor lines were unable to establish a symbiosis with this arbuscular mycorrhiza fungus. Using a combined approach of genetic mapping, targeting induced local lesions in genomics, and sequencing, all non-nodulating mutant lines were characterized and shown to represent new alleles of at least nine independent symbiotic loci. The class of mutants with reduced nodulation capacity was of particular interest because some of them may specify novel plant functions that regulate nodule development in L. japonicus. To facilitate mapping of the latter class of mutants, an introgression line, in which the har1-1 allele was introduced into a polymorphic background of L. japonicus ecotype MG20, was constructed.

Involvement of THH1, an Arabidopsis thaliana homologue of the TOM1 gene, in tobamovirus multiplication

The TOM1 and TOM3 genes of Arabidopsis thaliana encode homologous proteins that are required for tobamovirus multiplication. Although the A. thaliana genome encodes another TOM1-like gene, THH1, the tobamovirus coat protein (CP) does not accumulate to a detectable level in the tom1 tom3 double mutant. Here, double and triple mutants of tom1, tom3 and thh1 were generated to investigate whether THH1 functions to support tobamovirus multiplication. In the tom1 thh1 double mutant, the tobamovirus CP accumulated to a level that was detectable, but lower than that in the tom1 single mutant. In tom1 tom3 double-mutant lines overexpressing THH1, the tobamovirus CP accumulated to a level similar to that observed in wild-type plants. These results suggest that THH1 supports tobamovirus multiplication, but to a lesser extent than TOM1 and TOM3. The expression level of THH1 is lower than that of TOM1 and TOM3, which might explain the smaller contribution of THH1 to tobamovirus multiplication.

The effects of genetic variation at r, rb and Tri loci in Pisum sativum L. on apparent ileal digestibility of amino acids in young broilers

AbstractThe nutritional effect of genetic variation at r and rb loci was investigated by determining apparent and true amino acid digestibility of four near‐isogenic lines of pea (RRRbRb, RRrbrb, rrRbRb, rrrbrb) fed to young broilers in a metabolism study. The results demonstrated that the round‐seeded line (RRRbRb) generally had the lowest amino acid content with, for example, 2.5 g cystine kg−1 dry matter (DM) and 1.6 g methionine kg−1 DM, compared with contents in the three wrinkled‐seeded lines (rrRbRb, RRrbrb and rrrbrb). The double mutant line (rrrbrb) generally had the highest amino acid content, with 3.2 g cystine kg−1 DM and 2.3 g methionine kg−1 DM, and an elevated amino acid content compared with the single mutant lines (RRrbrb, rrRbRb). However, the round‐seeded line (RRRbRb) consistently had the highest coefficient of true (CTID), and apparent (CAID), ileal digestibility for all amino acids (e.g. 0.959 and 0.928 respectively for CTID and CAID for methionine) when compared with the three wrinkled lines RRrbrb, rrRbRb and rrrbrb (0.853, 0.877 and 0.866, respectively, for CTID, and 0.862, 0.882 and 0.874 for CAID). A second experiment examined the digestibility of amino acids in a pair of lines, HC8 and LC8, near‐isogenic except for the locus controlling the concentration of seed trypsin inhibitor (H, high; L, low). The results showed that the CAID and CTID obtained for all amino acids were consistently higher for LC8 than for HC8 (for methionine: 0.905 and 0.709 for CTID, respectively, for LC8 and HC8; 0.887 and 0.729, respectively, for CAID), indicating the very specific effects of quantitative variation in these proteins on nutritional value of peas for poultry. Copyright © 2005 Society of Chemical Industry

Mutants of Arabidopsis Lacking a Chloroplastic Isoamylase Accumulate Phytoglycogen and an Abnormal Form of Amylopectin

Mutant lines defective for each of the four starch debranching enzyme (DBE) genes (AtISA1, AtISA2, AtISA3, and AtPU1) detected in the nuclear genome of Arabidopsis (Arabidopsis thaliana) were produced and analyzed. Our results indicate that both AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of DBE named Iso1, the major isoamylase activity found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content in both lines and to the accumulation of water-soluble polysaccharides whose structure is similar to glycogen. In addition, the residual amylopectin structure in the corresponding mutant lines displays a strong modification when compared to the wild type, suggesting a direct, rather than an indirect, function of Iso1 during the synthesis of amylopectin. Mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure. This result suggests that this isoamylase-type of DBE plays a major role during starch mobilization. The analysis of the Atpu1 single-mutant lines did not lead to a distinctive phenotype. However, Atisa2/Atpu1 double-mutant lines display a 92% decrease in starch content. This suggests that the function of pullulanase partly overlaps that of Iso1, although its implication remains negligible when Iso1 is present within the cell.

The Arabidopsis Pseudo-response Regulators, PRR5 and PRR7, Coordinately Play Essential Roles for Circadian Clock Function

In Arabidopsis thaliana, a number of clock-associated protein factors have been identified. Among them, TOC1 (TIMING OF CAB EXPRESSION 1) is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATOR, including PRR1/TOC1, PRR3, PRR5, PRR7 and PRR9. It has not been certain whether or not other PRR family members are also implicated in clock function per se. To clarify this problem, here we constructed a double mutant line, which is assumed to have severe lesions in both the PRR5 and PRR7 genes. Resulting homozygous prr5-11 prr7-11 young seedlings showed a marked phenotype of hyposensitivity to red light during de-etiolation. In addition, they displayed a phenotype of extremely late flowering under long-day photoperiod conditions, but not short-day conditions. The rhythms at the level of transcription of certain clock-controlled genes were severely perturbed in the double mutant plants when they were released into continuous light (LL) and darkness (DD). The observed phenotype was best interpreted as 'arrhythmic in both LL and DD' and/or 'very short period with markedly reduced amplitude'. Even under the light entrainment (LD) conditions, the mutant plants showed anomalous diurnal oscillation profiles with altered amplitude and/or phase with regard to certain clock-controlled genes, including the clock component CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) gene. Such events were observed even under temperature entrainment conditions, suggesting that the prr5-11 prr7-11 lesions cannot simply be attributed to a defect in the light signal input pathway. These pleiotropic circadian-associated phenotypes of the double mutant were very remarkable, as compared with those observed previously for each single mutant. Taking these results together, we propose for the first time that PRR5 and PRR7 coordinately (or synergistically) play essential clock-associated roles close to the central oscillator.

Disruption of the two digalactosyldiacylglycerol synthase genes DGD1 and DGD2 in Arabidopsis reveals the existence of an additional enzyme of galactolipid synthesis.

Two genes (DGD1 and DGD2) are involved in the synthesis of the chloroplast lipid digalactosyldiacylglycerol (DGDG). The role of DGD2 for galactolipid synthesis was studied by isolating Arabidopsis T-DNA insertional mutant alleles (dgd2-1 and dgd2-2) and generating the double mutant line dgd1 dgd2. Whereas the growth and lipid composition of dgd2 were not affected, only trace amounts of DGDG were found in dgd1 dgd2. The growth and photosynthesis of dgd1 dgd2 were affected more severely compared with those of dgd1, indicating that the residual amount of DGDG in dgd1 is crucial for normal plant development. DGDG synthesis was increased after phosphate deprivation in the wild type, dgd1, and dgd2 but not in dgd1 dgd2. Therefore, DGD1 and DGD2 are involved in DGDG synthesis during phosphate deprivation. DGD2 was localized to the outer side of chloroplast envelope membranes. Like DGD2, heterologously expressed DGD1 uses UDP-galactose for galactosylation. Galactolipid synthesis activity for monogalactosyldiacylglycerol (MGDG), DGDG, and the unusual oligogalactolipids tri- and tetragalactosyldiacylglycerol was detected in isolated chloroplasts of all mutant lines, including dgd1 dgd2. Because dgd1 and dgd2 carry null mutations, an additional, processive galactolipid synthesis activity independent from DGD1 and DGD2 exists in Arabidopsis. This third activity, which is related to the Arabidopsis galactolipid:galactolipid galactosyltransferase, is localized to chloroplast envelope membranes and is capable of synthesizing DGDG from MGDG in the absence of UDP-galactose in vitro, but it does not contribute to net galactolipid synthesis in planta.

Analysis of Combinatorial Loss-of-Function Mutants in the Arabidopsis Ethylene Receptors Reveals That the ers1 etr1 Double Mutant Has Severe Developmental Defects That Are EIN2 Dependent

Ethylene responses in Arabidopsis are controlled by the ETR receptor family. The receptors function as negative regulators of downstream signal transduction components and fall into two distinct subfamilies based on sequence similarity. To clarify the levels of functional redundancy between receptor isoforms, combinatorial mutant lines were generated that included the newly isolated ers1-2 allele. Based on the etiolated seedling growth response, all mutant combinations tested exhibited some constitutive ethylene responsiveness but also remained responsive to exogenous ethylene, indicating that all five receptor isoforms can contribute to signaling and no one receptor subtype is essential. On the other hand, light-grown seedlings and adult ers1 etr1 double mutants exhibited severe phenotypes such as miniature rosette size, delayed flowering, and sterility, revealing a distinct role for subfamily I receptors in light-grown plants. Introduction of an ein2 loss-of-function mutation into the ers1 etr1 double mutant line resulted in plants that phenocopy ein2 single mutants, indicating that all phenotypes observed in the ers1 etr1 double mutant are EIN2 dependent.

Functional assessment of the pathogenesis-related protein PR-1b in barley

The pathogenesis-related protein 1 (PR-1b) of barley ( Hordeum vulgare L.) is a marker for the attack by the powdery mildew fungus ( Blumeria graminis f.sp. hordei, Bgh) and other pathogens. PR-1b consists of 164 amino acids and has a potential signal peptide for export into the cell wall. Here, we show that PR-1b is differentially expressed in near-isogenic barley lines exhibiting various forms of defence phenotypes including papilla formation and the hypersensitive cell death. To elucidate PR-1b function, we transiently silenced PR-1 expression by double stranded RNA (dsRNA) interference in the moderately susceptible barley double mutant line A89 (genotype: mlo5–ror1), which shows a papillae-based defence phenotype. Upon bombardment of leaf segments with PR-1b dsRNA and a GFP marker gene construct, Bgh slightly more frequently penetrated the plant cell wall of transformed epidermal cells relative to cells bombarded with human control dsRNA. We conclude that PR-1b contributes to penetration resistance to the powdery mildew fungus in barley. We also observed that PR-1b expression correlates with the production of H 2O 2 in responses to Bgh and Bipolaris sorokiniana and was induced upon infiltration of the H 2O 2 producing mixture of glucose and glucose oxidase.

Knock-down of the small G-protein RACB enhances penetration-resistance of barley against the powdery mildew fungus

Small G-proteins (RAC and RHO) are known to be involved in regulation of superoxide (O<sub>2</sub><sup>•–</sup>) production and the assembly of actin fibres. These processes are known to be crucial for accessibility and inaccessibility of barley cells to the biotrophic fungus Blumeria graminis f.sp. hordei (Bgh). Using a candidate RT-PCR approach six Rac-related cDNA-clones were isolated from barley. The transient knock-down of RacB led to a remarkably lower penetration efficiency of Bgh into susceptible barley lines (Mlo/Ror1). Surprisingly the inhibition of RacB expression had no effect in the double mutant line A89 (mlo5/ror1). This led us to the assumption that the RacB-dsRNA effect is dependent on functional ROR1 (such as mlo5-mediated resistance). Vice versa, overexpression of constitutive active RACB-V15 in the susceptible line Pallas resulted in hypersusceptibility to Bgh. Thus, we conclude that RACB is a signal transduction protein functional in the accessibility of epidermal barley cells to Bgh.

Cell death and salicylate- and jasmonate-dependent stress responses in Arabidopsis are controlled by single cet genes

The jasmonic acid (JA)-dependent regulation of the Thi2.1 gene had previously been exploited for setting up a genetic screen for the isolation of signal transduction mutants of Arabidopsis thaliana (L.) Heynh. that constitutively express the thionin gene. Several cet mutants had been isolated which showed a Constitutive Expression of the Thionin gene. These cet mutants, except for one, also showed spontaneous leaf cell necrosis and were up-regulated in the expression of the PR1 gene, reactions often associated with the systemic acquired resistance (SAR) pathway. Four of these cet mutants, cet1, cet2, cet3 and cet4.1 were crossed with the fad triple and coi1 mutants that are blocked at two steps within the JA-dependent signaling pathway, and with transgenic NahG plants that are deficient in salicylic acid (SA) and are unable to activate SAR. Analysis of the various double-mutant lines revealed that the four cet genes act within a signaling cascade at or prior to branch points from which not only JA-dependent signals but also SA-dependent signaling and cell death pathways diverge.

VGF is Required for Obesity Induced by Diet, Gold Thioglucose Treatment, and Agouti and is Differentially Regulated in Pro-Opiomelanocortin- and Neuropeptide Y-Containing Arcuate Neurons in Response to Fasting

Targeted deletion of the gene encoding the neuronal and neuroendocrine secreted polypeptide VGF (nonacronymic) produces a lean, hypermetabolic mouse. Consistent with this phenotype, VGF mRNA levels are regulated in the hypothalamic arcuate nucleus in response to fasting. To gain insight into the site(s) and mechanism(s) of action of VGF, we further characterized VGF expression in the hypothalamus. Double-label studies indicated that VGF and pro-opiomelanocortin were coexpressed in lateral arcuate neurons in the fed state, and that VGF expression was induced after fasting in medial arcuate neurons that synthesize neuropeptide Y (NPY). Like NPY, VGF mRNA induction in this region of the hypothalamus in fasted mice was inhibited by exogenous leptin. In leptin-deficient ob/ob and receptor-mutant db/db mice, VGF mRNA levels in the medial arcuate were elevated. To identify neural pathways that are functionally compromised by Vgf ablation, VGF mutant mice were crossed with obese A(y)/a (agouti) and ob/ob mice. VGF deficiency completely blocked the development of obesity in A(y)/a mice, whereas deletion of Vgf in ob/ob mice attenuated weight gain but had no impact on adiposity. Hypothalamic levels of NPY and agouti-related polypeptide mRNAs in both double-mutant lines were dramatically elevated 10- to 15-fold above those of wild-type mice. VGF-deficient mice were also found to resist diet- and gold thioglucose-induced obesity. These data and the susceptibility of VGF mutant mice to monosodium glutamate-induced obesity are consistent with a role for VGF in outflow pathways, downstream of hypothalamic and/or brainstem melanocortin 4 receptors, that project via the autonomic nervous system to peripheral metabolic tissues and regulate energy homeostasis.

VGF is required for obesity induced by diet, gold thioglucose treatment, and agouti and is differentially regulated in pro-opiomelanocortin- and neuropeptide Y-containing arcuate neurons in response to fasting.

Targeted deletion of the gene encoding the neuronal and neuroendocrine secreted polypeptide VGF (nonacronymic) produces a lean, hypermetabolic mouse. Consistent with this phenotype, VGF mRNA levels are regulated in the hypothalamic arcuate nucleus in response to fasting. To gain insight into the site(s) and mechanism(s) of action of VGF, we further characterized VGF expression in the hypothalamus. Double-label studies indicated that VGF and pro-opiomelanocortin were coexpressed in lateral arcuate neurons in the fed state, and that VGF expression was induced after fasting in medial arcuate neurons that synthesize neuropeptide Y (NPY). Like NPY, VGF mRNA induction in this region of the hypothalamus in fasted mice was inhibited by exogenous leptin. In leptin-deficient ob/ob and receptor-mutant db/db mice, VGF mRNA levels in the medial arcuate were elevated. To identify neural pathways that are functionally compromised by Vgf ablation, VGF mutant mice were crossed with obese A(y)/a (agouti) and ob/ob mice. VGF deficiency completely blocked the development of obesity in A(y)/a mice, whereas deletion of Vgf in ob/ob mice attenuated weight gain but had no impact on adiposity. Hypothalamic levels of NPY and agouti-related polypeptide mRNAs in both double-mutant lines were dramatically elevated 10- to 15-fold above those of wild-type mice. VGF-deficient mice were also found to resist diet- and gold thioglucose-induced obesity. These data and the susceptibility of VGF mutant mice to monosodium glutamate-induced obesity are consistent with a role for VGF in outflow pathways, downstream of hypothalamic and/or brainstem melanocortin 4 receptors, that project via the autonomic nervous system to peripheral metabolic tissues and regulate energy homeostasis.

Characterization of Retinoic Acid Receptor-deficient Keratinocytes

Retinoids are essential for normal epidermal growth and differentiation and show potential for the prevention or treatment of various epithelial neoplasms. The retinoic acid receptors (RARalpha, -beta, and -gamma) are transducers of the retinoid signal. The epidermis expresses RARgamma and RARalpha, both of which are potential mediators of the effects of retinoids in the epidermis. To further investigate the role(s) of these receptors, we derived transformed keratinocyte lines from wild-type, RARalpha, RARgamma, and RARalphagamma null mice and investigated their response to retinoids, including growth inhibition, markers of growth and differentiation, and AP-1 activity. Our results indicate that RARgamma is the principle receptor contributing to all-trans-retinoic acid (RA)-mediated growth arrest in this system. This effect partially correlated with inhibition of AP-1 activity. In the absence of RARs, the synthetic retinoid N-(4-hydroxyphenyl)-retinamide inhibited growth; this was not observed with RA, 9-cis RA, or the synthetic retinoid (E)-4-[2-(5, 5, 8, 8 tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl] benzoic acid. Finally, both RARalpha and RARgamma differently affected the expression of some genes, suggesting both specific and overlapping roles for the RARs in keratinocytes.

The whorl-specific action of a petunia class B floral homeotic gene.

GREEN PETAL (GP) is thought to be a petunia class B floral homeotic gene, because the gp mutant flower displays a severe homeotic conversion of petals into sepals in the second whorl. However, since the third whorl stamens remain unaffected in the gp null mutant, gp is different from class B mutants in Arabidopsis and Antirrhinum, which also show a conversion of the third whorl stamens into the carpelloid tissue. BLIND (BL) is thought to be a petunia class A floral homeotic gene, because the bl mutant flower displays homeotic conversions of sepals into the stigmatoid tissue in the first whorl and of the corolla limb into antheroid structures in the second whorl. A double mutant line homozygous for both bl and gp mutations was constructed. The bl gp double mutant flower displays homeotic conversions of sepals into the stigmatoid tissue in the first whorl and of the corolla limb into antheroid structures with stigmatoid tips in the second whorl. In the third and fourth whorls of the mutant flower, organs remained unchanged. In the gp flower, a petunia B-type gene FBP1 is expressed strongly in the third whorl organs, but much more weakly in the second whorl organs. In the bl gp flower, FBP1 was found to be expressed strongly in the second whorl organs as well as in the third whorl organs. Petunia has a class B gene other than GP that determines organ identities, both in the second and third whorls of the double mutant flower, and the action of the postulated class B gene (here called PhBX) is prevented by the BL gene in the second whorl of the gp flower. PhBX appears to be a gene that specifically interacts with the FBP1 gene, and is involved in the up-regulation of FBP1.

Toxicity of Bread Wheat Lines Lacking Prolamins Encoded by theGli-B1/Gli-B5/Glu-B3andGli-D1/Glu-D3Loci in Coeliac Disease as Determined by their Agglutinating Activity

Peptic-tryptic (PT) digests of alcohol-soluble proteins from the flour of three mutant lines of bread wheat, lacking γ-gliadins, γ-gliadins and low molecular-weight glutenin subunits encoded by the Gli-B1/Gli-B5/Glu-B3 loci (line S. Pastore 4A), the Gli-D1/Glu-D3 loci (line Alpe 1I-) or both groups of loci (line DM 22166), were compared with those of the normal cultivars S. Pastore and Alpe 1 I for their agglutinating activities on human myelogenous leukemia K562(S) cells, agglutination being strongly associated with toxicity for the coeliac intestine. All of the genotypes tested contained A-type α-gliadins, which constituted about 19% of the gliadins in the S. Pastore and Alpe 1I cultivars, 24.5% in the S. Pastore 4A and Alpe 1I null lines, and 34.8% in the double mutant line, DM 22166, as determined by densitometric scanning of their acid polyacrylamide gel electrophoresis patterns. The minimal concentrations of PT digest required to agglutinate 100% of K562(S) cells were 73mg/l and 96mg/l, in the S. Pastore and Alpe 1I cultivars, respectively, compared with 146mg/l, 138mg/l and 200mg/l in the “null” lines, S. Pastore 4A, Alpe 1 I-and DM 22166, respectively. The results indicated that proteins other than α-gliadins are involved in the gluten-sensitive enteropathy.

Characterization of immature thymocyte lines derived from T-cell receptor or recombination activating gene 1 and p53 double mutant mice.

The T-cell receptor (TCR) beta chain is instrumental in the progression of thymocyte differentiation from the CD4-CD8- to the CD4+CD8+ stage. This differentiation step may involve cell surface expression of novel CD3-TCR complexes. To facilitate biochemical characterization of these complexes, we established cell lines from thymic lymphomas originating from mice carrying a mutation in the p53 gene on the one hand and a mutation in TCR-alpha, TCR-beta, or the recombination activating gene 1 (RAG-1) on the other hand. The cell lines were CD4+CD8+ and appeared to be monoclonal. A cell line derived from a RAG-1 x p53 double mutant thymic lymphoma expressed low levels of CD3-epsilon, -gamma, and -delta on the surface. TCR-alpha x p53 double mutant cell lines were found to express complexes consisting of TCR-beta chains associated with CD3-epsilon, -gamma, and -delta chains and CD3-zeta zeta dimers. These lines will be useful tools to study the molecular structure and signal transducing properties of partial CD3-TCR complexes expressed on the surface of immature thymocytes.

An Analysis of Seed Development in Pisum sativum XVII. The Effect of the rb Locus Alone and in Combination with r on the Growth and Development of the Seed

Near-isogenic lines have been produced in pea to examine the effects of genes at the r and rb loci on the growth and development of the seed. The rb alleles were found to have an effect on the growth of the testa as well as on that of the embryo in contrast to those at the r locus which only affect the latter. The relationship between the fresh and dry weights of the embryos of rb isoline and the double mutant differed from that of the round-seeded wild-type line. Furthermore, for this relationship, the double mutant line very closely resembled the rr isoline.

Combining mutations for the two homoeologous pairing suppressor genes Ph1 and Ph2 in common wheat and in hybrids with alien Triticeae

Two lines of common wheat cv. Chinese Spring, carrying simultaneous mutations for the two major homoeologous pairing wheat suppressor genes Ph1 and Ph2 have been developed and their pairing behaviour compared with that of the ph1b mutant of the same cultivar. Besides carrying the ph1b mutation, the first double mutant line lacked the chromosome arm pair 3DS, containing Ph2, whereas the second had a euploid constitution and carried the ph1b allele on 3DS. Hybrids of Aegilops variabilis and Secale cereale with mono-5B (ph1b) and 3D/3DL plants have also been obtained, where the 3D versus 3DL presence marked the two pairing alternatives (ph1b only and ph1b + Ph2−, respectively). In the wheat x Ae. variabilis hybrids, an 8% increase in total chromosome pairing, almost entirely ascribable to an increment of multivalent associations, was observed in the 2n = 34 + t plants with respect to their 2n = 35 sibs. The number of bivalents showed no significant difference, but a tendency towards a decrease, which was significant for the rod types, was exhibited by the Ph2− plants. A weaker but similar effect was observed in wheat itself. The different mutants, in fact, showed a similar percentage of paired chromosomes but varied in their pairing pattern. A significant reduction in the number of bivalents, owing to a decrease of the rings, only partly compensated for by an increase of the rods, was observed in the double mutants. They also exhibited an increase in the multivalent fraction, which was significant for the most complex associations. In both common wheat and its hybrids with Ae. variabilis the addition of a ph2 mutation thus seems to reinforce the ph1b effect in promoting homoeologous pairing. On the other hand, such an effect was not noticed in the wheat x S. cereale hybrids. However, possible quantitative differences could have been masked by the considerable plant-to-plant variation and potential differences in relative incidence of wheat-wheat versus wheat-rye associations were undetectable in the Feulgen-stained materials analyzed.

Expression of alcohol-soluble endosperm proteins in maize single and double mutants

Many maize (Zea mays L.) mutant genes exist. Some affect protein content or composition, while others modify carbohydrates or kernel phenotype. In doublemutant lines, two mutant genes are present. We know little about interactions of such genes, however. We therefore examined a normal maize inbred, B37, 10 near-isogenic single mutants and 46 double mutants to analyze quantitative effects on alcohol-soluble endosperm proteins. Proteins were extracted with 70% ethanol0.5% sodium acetate-5% mercaptoethanol, and fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). Early peaks were alcohol-soluble glutelin (ASG) subunits, while late peaks contained zein. Results were quantified and statistically analyzed. In many double mutants, protein compositions differed significantly from averages of compositions of corresponding single mutants. For example, a high-methionine, water-insoluble ASG is absent when the opaque-2 (o2) gene combines with shrunken-1 (sh1) or surgary-1 (su1). Another water-insoluble ASG nearly doubled when floury-2 (fl2) andsu1 combined. A high-proline, high-histidine, water-soluble ASG nearly doubled in combinations offl2 witho2,su1 and sugary-2 (su2). Zein was about half its expected value wheno2 combined with amylose-extender (ae), floury-1 (fl1), soft-starch (h),sh1 andsu1. Thus, rapid protein extraction and quantitative RP-HPLC showed major new epistatic and synergistic effects of several mutant genes on protein composition. Unexpectedly, these effects often involve genes that primarily affect starch composition or kernel phenotype. Alcohol-soluble proteins often vary in amount, as ino2 lines. They also differ in nutritional value. Thus, RP-HPLC analysis of these proteins can identify nutritionally superior genotypes, and may help explain the basis of such quality.