Molecular characterization of rDt, a maize transposon of the “Dotted” controlling element system

Summary We have molecularly cloned the rDt transposon, one component of the classic Dotted two-element system of controlling elements. The rDt transposon was identified as a DNA insertion in each of two independent mutation events of the maize A1 gene, a gene necessary for the biosynthesis of anthocyanin pigment. Both mutant alleles result in a stable, anthocyaninless phenotype in all plant tissues. When the transposon Dotted, (Dt), is present in the genome each allele exhibits a characteristic mutable phenotype (spots of anthocyanin pigmentation). The DNA insertion has been designated rDt, for it responds to or is regulated by the Dt element to allow expression of the otherwise mutated gene, and it had not been named in earlier genetic studies. Sequence analysis revealed the rDt element to be an identical 704 bp insertion within the two mutable alleles, but in opposite orientation and in different exons of the gene. rDt contains an imperfect terminal inverted repeat with similarity to transposable elements of various species. A duplication of 8 bp of the target host site is formed upon integration of the element, and the element is excised from the locus in a germinal revertant. The difference in phenotype of the two unstable alleles, a1 and am-1:Cache, is discussed.     

Evaluation of three test statistics used to identify maize inbred lines with new favorable alleles not present in elite single cross

Summary The identification of inbred lines useful for improvement of an elite single cross hybrid is an essential part of a pedigree maize (Zea mays L.) breeding program. The objectives of this study were to identify lines that could be useful for improvement of hybrid B73 × Mo17 and to relate the values of estimators of new favorable alleles with test cross yields. Crosses of parents of hybrid B73 × Mo17 with 10 public lines from the United States (US), and 14 Maize Research Institute Zemun Polje proprietary lines (lines per se, and test crosses from 3 F₂ populations) were evaluated at 4 locations in Yugoslavia in 1986. Significant differences in grain yield were found among lines in minimally biased estimates of favorable alleles (G) present in a donor inbred but not present in a B73 × Mo17, in minimum upper bound (UBND) estimates and in predicted three-way performance (PTC). Twenty-one lines had a significant number of dominant favorable alleles for grain yield not present in B73 × Mo17. The highest values for all estimators of new favorable alleles were found for donor lines which belonged to different heterotic groups than the B73 and Mo17. For most of the inbreds, the (C + F) – (D + E) statistics agreed with predigree information. Simultaneous increases in grain yield and decreases in grain moisture content for B73 × Mo17 are possible with several donor inbred lines. All of the lines with a high number of new favorable alleles for grain yield not present in B73 × Mo17 had negative D (F)-G values for low plant height. Line N152 had the most new favorable alleles for grain yield not present in single cross B73 × Mo17. Population (N152 × Mo17) F₂ had the highest difference of observed test cross means from check mean, the most test crosses with significantly higher yields than the check, and the largest estimate of number of segregating loci.This project was partly supported by the United States Department of Agriculture and Republic Funds for Scientific Work of Serbia through funds available to the United States-Yugoslav Joint Board on Scientific and Technological Cooperation. Project No. JFP 662     

Ability of symbiotic and non-symbiotic rhizospheric microflora of maize (Zea mays) to weather micas and to promote plant growth and plant nutrition

Summary Maize was grown under axenic conditions in laboratory devices, in a K⁺-deficient medium, where biotite was the K⁺ source. In different treatments plants were inoculated by symbiotic (Glomus mosseae) and/or non symbiotic microflora. In those treatments inoculated byGlomus mosseae, the percentage of roots infection after 7 weeks plant growth was 65%. Rhizospheric bacterial population was approximately 10⁸/g (dry weight). Endomycorrhizae stimulated growth and K uptake. Non-symbiotic microflora increased also plant growth but promoted much more biotite weathering and K uptake. Endomycorrhizae and more particularly non-symbiotic microflora increased also Ca and Mg absorption by plants. Possible mechanisms involved and implications in plant growth and pedogenesis are discussed.     

Effect of inorganic pyrophosphate on respiration and oxidative phosphorylation in higher plants

In coupled mitochondria of maize, inorganic pyrophosphate has no effect on electron transport whereas it competitively inhibits state 3 (with addition of ADP) respiration. The degree of inhibition depends on the ADP concentration in the reaction medium. At 150 and 30O μM ADP, the inhibition constant (K_i) has a value of 1.1 × 10⁻⁴ M. Pyrophosphate either does not penetrate throucvh the membranes or penetrates through them in only very small amounts. It does not inhibit the exchange ³²PiPi; however, it undergoes an exchange with ADP (2 nmol PPi/mg protein for 10 min at 30°).     

Effect of salicylic acid on nitrite reductase and glutamate dehydrogenase activities in maize roots

The effects of 0.01 to 5 m M salicyclic acid on the increase in nitrite reductase or glutamate dehydrogenase activities in maize roots by nitrate or ammonium respectively, were examined. Nitrite reductase activity was inhibited by the highest concentration of the acid. The activity of NADH-glutamate dehydrogenase was stimulated slightly (but consistently) by the lowest concentration and was inhibited by higher concentrations. Total protein content was also inhibited at high concentrations. When the crude enzyme extract was stored at 25°C in light, the glutamate dehydrogenase activity in the control decreased after 4 h of incubation. Low concentrations of the acid had no effect on this decrease but higher concentration accelerated the process. The divalent cations Caz²⁺, Mn²⁺, Mg²⁺ and Zn²⁺ protected against loss of enzyme activity during storage, both in the absence and presence of the acid. The inhibitory effect of 5 m M salicylic acid on glutamate dehydrogenase activity is apparent due to interference with the activity of the enzyme rather than with its synthesis.     

Improved tissue culture response of an elite maize inbred through backcross breeding,and identification of chromosomal regions important for regeneration by RFLP analysis

Summary The frequency of initiation of friable, embryogenic callus from immature embryos of the elite maize inbred line B73 was increased dramatically by introgression of chromosomal segments from the inbred line A188 through classical backcross breeding. Less than 0.2% of the immature B73 embryos tested (5 of 3,710) formed embryogenic callus. The breeding scheme consisted of six generations of backcrossing to B73 with selection at each generation for high frequency initiation of embryogenic cultures. BC₆ individuals were selfed for four generations to select homozygous lines. The average embryogenic culture initiation frequency increased to 46% (256/561). Nearly all (91%) of the embryos from one BC₆ S₄ plant formed embryogenic cultures. RFLP analysis was used to determine the locations and effects of the introgressed A188 chromosomal segments. Five segments were retained through at least the fifth backcross generation. The hypothesis that one or more of these five regions contains genes controlling somatic embryogenesis in maize was tested using an F₂ population of the cross A188 X Mo17. A set of five DNA markers (three of them linked) explained 82% of the observed phenotypic variance for percentage of immature embryos forming embryognic callus. Four of the five markers were located in or near introgressed A188 chromosome segments.The region marked by probe c595 on the long arm of chromosome 9 was highly associated with several measures of in vitro culture response (percent embryogenic embryos, plants per embryo, and plants per embryogenic embryo). We propose that there is a major gene (or genes) in this region in A188 that promotes embryogenic callus initiation and plant regeneration in B73, Mo17, and probably many other recalcitrant inbred lines of maize.     

Dissection of a pollen-specific promoter from maize by transient transformation assays

We have previously reported the isolation and characterization of a gene (Zm 13) from Zea mays which shows a pollen-specific pattern of expression. Stably transformed tobacco plants containing a reporter gene linked to portions of the Zm 13 5 flanking region show correct temporal and spatial expression of the gene. Here we present a more detailed analysis of the 5 regions responsible for expression in pollen by utilizing a transient expression system. Constructs containing the -glucuronidase (GUS) gene under the control of various sized fragments of the Zm13 5 flanking region were introduced into Tradescantia and Zea mays pollen via high-velocity microprojectile bombardment, and monitored both visually and with a fluorescence assay. The results suggest that sequences necessary for expression in pollen are present in a region from –100 to –54, while other sequences which amplify that expression reside between –260 and –100. The replacement of the normal terminator with a portion of the Zm13 3 region containing the putative polyadenylation signal and site also increased GUS expression. While the –260 to –100 region contains sequences similar to other protein-binding domains reported for plants, the –100 to –54 region appears to contain no significant homology to other known promoter fragments which direct pollen-specific expression. The microprojectile bombardment of Tradescantia pollen appears to be a good test system for assaying maize and possibly other monocot promoter constructs for pollen expression.     

Characterization of protochlorophyllide and protochlorophyllide esters in roots of dark-grown plants

The protochlorophyll pools of roots of dark-grown wheat ( Triticum aestivum L. cv. Walde), maize ( Zea mays L. cv. Goldcrest) and wrinkledseeded pea ( Pisum sativum L. ssp. sativurh cv. Kelvedon Wonder) were investigated by high performance liquid chromatography (HPLC) and low temperature fluorescence spectroscopy. All roots contained protochlorophyllide and esterified protochlorophyllides (protochlorophylls) but with considerably larger relative amounts of the latter compared with etiolated leaves. The alcohol moieties of the 4 detected protochlorophylls were geranylgeraniol (GG), dihydrogeranylgeraniol (DHGG), tetrahydrogeranylgeraniol (THGG) and phytol. The relative amounts of the different protochlorophylls varied between the species. Protochlorophyllide and the 4 protochlorophylls all contained monovinyl forms. The divinyl forms could not be detected by our instruments. Wrinkledseeded pea contained in addition chlorophyll a , some unidentified chlorophylls and negligible amounts of chlorophyllide. Small amounts of carotenoids were found in roots of all investigated species. The carotenoids were the same as those found in green or etiolated leaves, but present in different relative amounts.