Immunoelectrophoretic studies of heterosis effect in Zea mays

Summary The present report is based on tests using antigenic analysis of inbred lines and their hybrids for the prognosis of heterosis (hybrid vigour) in Zea mays.Inbred lines, whose hybrid vigour has been proved under field conditions, and their hybrids were characterized by the method of Grabar and Williams.The results of the electrophoretic and immunoelectrophoretic investigations show that the extracts from maize seeds of the inbred lines C-103, N-6 and WF-9 contain four protein fractions, while those of the hybrids N-6 x C-103 and WF-9 x N-6 also contain a fifth fraction.The immunoelectrophoretic pattern of the extracts from the hybrid seeds N-6 x C-103 shows that, with their homologous serum, they give a precipitation line (arc X) which is not obtained by the interaction of the parent line extracts with homologous and heterologous serums.In this case we probably have a protein synthesis in the hybrid which is caused by the deblocking of some links in the biosynthetic apparatus of the inbred lines.It was established that the extracts from the hybrid N-6 x C-103 gave a larger number of precipitation arcs with the heterologous serum anti C-103.The characterization of the antigenic structure of the inbred lines enables one to determine not only the fractions common to them but also the number of those fractions by which they differ from one another. Proceeding in this manner, one could accomplish an immunoelectrophoretic prognosis of heterosis and penetrate deeper into its essence.

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Zusammenfassung Die vorliegende Mitteilung basiert auf Versuchen, die Antigenanalyse von Inzuchtlinien und ihren Hybriden für eine Vorhersage des Auftretens von Heterosis bei Zea mays zu verwenden. Inzuchtlinien und ihre Hybriden, deren Heterosis unter Feldbedingungen erwiesen ist, wurden nach der Methode von Grabar und Williams geprüft.Die elektrophoretischen und immunelektrophoretischen Untersuchungen ergaben, daß die Extrakte von Maiskörnern der Inzuchtlinien C-103, N-6 und WF-9 vier Proteinfraktionen enthalten, während die Extrakte der Hybriden N-6 x C-103 und WF-9 x x N-6 noch eine fünfte Fraktion aufweisen.Das immunelektrophoretische Muster der Extrakte aus den Hybridkörnern von N-6 x C-103 zeigt, daß mit dem homologen Serum eine Präzipitationslinie, arc X, gebildet wird, die bei Reaktion der Extrakte aus den Elternlinien mit homologen und heterologen Seren nicht auftritt. Wahrscheinlich handelt es sich um eine zusätzliche Synthese von Protein, die auftritt, nachdem die Blockierung einiger Glieder im biosynthetischen Apparat der Inzuchtlinien aufgehoben worden ist.Es wurde festgestellt, daß die Extrakte der Hybride N-6 x C-103 mit heterologem Serum anti C-103 eine größere Anzahl Präzipitationslinien ergeben.Die Charakterisierung der Antigenstruktur der Inzuchtlinien ermöglicht nicht nur die Ermittlung von gemeinsamen, sondern auch von unterschiedlichen Fraktionen. Auf diese Weise könnten eine immunelektrophoretische Diagnose hinsichtlich Heterosis erreicht und Möglichkeiten zur eingehenderen Erforschung des Heterosisphänomens erschlossen werden.

     

Manifestation of heterosis during early maize (Zea mays L.) root development

Heterosis is typically detected in adult hybrid plants as increased yield or vigor compared to their parental inbred lines. Only little is known about the manifestation of heterosis during early postembryonic development. Objective of this study was to identify heterotic traits during early maize root development. Four German inbred lines of the flint (UH002 and UH005) and dent (UH250 and UH301) pool and the 12 reciprocal hybrids generated from these inbred lines were subjected to a morphological and histological analysis during early root development. Primary root length and width were measured daily in a time course between 3 and 7 days after germination (DAG) and displayed average midparent heterosis (MPH) of 17–25% and 1–7%, respectively. Longitudinal size of cortical cells in primary roots was determined 5 DAG and displayed on average 24% MPH thus demonstrating that enlarged primary roots of hybrids can mainly be attributed to elongated cortical cells. The number of seminal roots determined 14 DAG showed on average 18% MPH. Lateral root density of all tested hybrids was determined 5 DAG. This root trait showed the highest degree of heterosis with an average MPH value of 51%. This study demonstrated that heterosis is already manifesting during the very early stages of root development a few days after germination. The young root system is therefore a suitable model for subsequent molecular studies of the early stages of heterosis manifestation during seedling development.     

Somatic embryogenesis in Zea mays L.

Summary Combining ability studies with respect to such green fodder quality characteristics as oxalic acid, calcium, sodium, potassium and green fodder yield were carried out in a 12 × 12 diallel cross set in pearl millet (Pennisetum typhoides (Burm) S. & H.). With regard to differential expression of gene effects, studies for quality traits were carried out in different seasons and on different plant parts. The relative proportions of general and specific combining variances indicated the preponderance of non-additive genetic variance. Parents possessing desirable fodder quality characteristics were identified on the basis of combining ability and per se performance, and selection criterion for crosses was discussed. It was recommended that leaf portion should be biochemically analysed and manipulated in an environment when the genes are expressed.Part of the Ph. D. dissertation submitted to the Punjab Agricultural University by the senior author in partial fulfilment of the requirements for the degree     

Leaf vasculature in Zea mays L.

The vascular system of the Zea mays L. leaf consists of longitudinal strands interconnected by transverse bundles. In any given transverse section the longitudinal strands may be divided into three types of bundle according to size and structure: small, intermediate, large. Virtually all of the longitudinal strands intergrade structurally however, from one bundle type to another as they descend the leaf. For example, all of the strands having large-bundle anatomy appear distally as small bundles, which intergrade into intermediates and then large bundles as they descend the leaf. Only the large bundles and the intermediates that arise midway between them extend basipetally into the sheath and stem. Most of the remaining longitudinal strands of the blade do not enter the sheath but fuse with other strands above and in the region of the blade joint. Despite the marked decrease in number of longitudinal bundles at the base of the blade, both the total and mean cross-sectional areas of sieve tubes and tracheary elements increase as the bundles continuing into the sheath increase in size. Linear relationships exist between leaf width and total bundle number, and between cross-sectional area of vascular bundles and both total and mean cross-sectional areas of sieve tubes and tracheary elements.     

Internode length in Zea mays L.

[¹³C, ³H]Gibberellin A₂₀ (GA₂₀) has been fed to seedlings of normal (tall) and dwarf-5 and dwarf-1 mutants of maize (Zea mays L.). The metabolites from these feeds were identified by combined gas chromatography-mass spectrometry. [¹³C, ³H]Gibberellin A₂₀ was metabolized to [¹³C, ³H]GA₂₉-catabolite and [¹³C, ³H]GA₁ by the normal, and to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₁ by the dwarf-5 mutant. In the dwarf-1 mutant, [¹³C, ³H]GA₂₀ was metabolized to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₂₉-catabolite; no evidence was found for the metabolism of [¹³C, ³H]GA₂₀ to [¹³C, ³H]GA₁. [¹³C, ³H]Gibberellin A₈ was not found in any of the feeds. In all feeds no dilution of ¹³C in recovered [¹³C, ³H]GA₂₀ was observed. Also in the dwarf-5 mutant, the [¹³C]label in the metabolites was apparently undiluted by endogenous [¹³C]GAs. However, dilution of the [¹³C]label in metabolites from [¹³C, ³H]GA₂₀ was observed in normal and dwarf-1 seedlings. The results from the feeding studies provide evidence that the dwarf-1 mutation of maize blocks the conversion of GA₂₀ to GA₁.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - RP reverse phase     

A Correlation between Structure and Function in the Root of Zea mays

The exudation rates of fluid and potassium ions from isolatedmaize roots were determined before and after excision of certainlengths of root tip. The results of this study suggest thatexcised maize roots possess the ability to absorb potassium(and presumably chloride) ions and concomitant amounts of waterover a considerable distance (10 cm) from the tip. Moreover,the observed power of absorption of ions and water into thetranslocatory pathway decreases in passing from the tip towardsthe base of the root. Both light and electron microscope techniques were used to examinethe anatomy of primary roots similar to those used in the physiologicalexperiments. The principal observation was that the xylem vesselsnear the root tip contain membrane-bounded cytoplasm with organelles.The number of mature xylem vessels, i.e. without cytoplasm,progressively increased in transverse sections cut from 1 to10 cm from the root tip; above 10 cm from the root tip all ofthe xylem vessels were found to be completely mature. It isevident that prima facie a connexion exists between this singleaspect of root anatomy and fluid exudation from excised roots. The uptake of tritiated water by roots and its transport intoexudates was examined. These data were analysed on the assumptionthat the exchange of external labelled water with the exudatewas achieved by the fluid exudation itself; this analysis indicatedthat an operational volume, similar to that of the total xylemvolume within the root, must become labelled during the formationof the exudate.     

Correlation of Blue Light-Induced Phosphorylation to Phototropism in Zea mays L

The physiology of light-induced phototropic curvature has been studied extensively in coleoptiles of grasses, particularly in Avena and Zea mays L. In Z. mays L., we have found that, in addition to curvature, blue light also induces rapid phosphorylation of a 114-kD protein in the tips of coleoptiles, and, in a previous report, we reported several characteristics of the phosphorylated substrate protein and kinase (J.M. Palmer, T.W. Short, S. Gallagher, W.R. Briggs [1993] Plant Physiol 102: 1211-1218). Here, we compare the phosphorylation response to several known aspects of phototropism physiology. Blue light-induced phosphorylation occurs only in the upper portion of the coleoptile and is absent from the node and mesocotyl. The specific activity of phosphorylation is highest in the extreme apical portion of the tip, which is also the site of maximal sensitivity to phototropic stimuli (A. W. Galston [1959] In Physiology of Movements, Encyclopedia of Plant Physiology, Springer, Berlin). Fluence-response determinations indicate that light dosage levels that stimulate curvature also stimulate phosphorylation. However, the threshold for inducing detectable phosphorylation in maize cannot be matched to the threshold for curvature induction. The recovery of sensitivity to phototropic stimuli after exposure to high fluences of light occurs with kinetics that are very similar to those for recovery of the phosphorylation response after a previous high-fluence light exposure. In addition, wavelengths of light in the blue and near-ultraviolet regions of the spectrum that maximally stimulate phototropic curvature also maximally stimulate in vitro phosphorylation in maize. The pattern of stimulation matches the absorption spectra of flavoproteins, which have been proposed as candidates for blue light photoreceptors.     

Biochemical characterization of maize (Zea mays L.) for salt tolerance

The inherent differences for salt tolerance in two maize cultivars (Agatti-2002 and Sahiwal-2002) were evaluated in pot experiments. Plants were grown in half-strength of Hoagland nutrient solution added with 0, 80, 100, 120, 140 and 160 mM of NaCl. Salt stress markedly reduced the shoot and root lengths and fresh and dry masses. Reduction in growth attributes was more pronounced in cv. Agatti-2002 than cv. Sahiwal-2002. Both maize cultivars exhibited significant perturbations in important biochemical attributes being employed for screening the crops for salt tolerance. Cultivar Sahiwal-2002 was found salt tolerant as compared to cv. Agatti-2002 because it exhibited lower levels of H₂O₂, malondialdehyde (MDA) and higher activities of antioxidant enzymes. In addition, cultivar Sahiwal-2002 exhibited less salt-induced degradation of photosynthetic pigments, lower levels of toxic Na⁺ and Cl^?  and higher endogenous levels of K⁺ and K⁺/Na⁺ ratio. The results indicate that salt stress induced a marked increase in MDA, H₂O₂, relative membrane permeability, total soluble proteins and activities of antioxidant enzymes (superoxide dismutase, peroxidase, catalase andascorbate peroxidase). Moreover, increase in endogenous levels of Na⁺ and Cl^?  and decrease in K⁺ and K⁺/Na⁺ ratio and photosynthetic pigments were recorded in plants grown under salinity regimes.     

Identification and characterization of a repertoire of genes differentially expressed in developing top ear shoots between a superior hybrid and its parental inbreds in Zea mays L.

Heterosis has been widely used in crop breeding and production; however, little is known about the genes controlling trait heterosis. The shortage of genes known to function in heterosis significantly limits our understanding of the molecular basis underlying heterosis. Here, we report 748 genes differentially expressed (DG) in the developing top ear shoots between a maize heterotic F₁ hybrid (Mo17 × B73) and its parental inbreds identified using maize microarrays containing 28,608 unigene features. Of the 748 DG, over 600 were new for the inbred and hybrid combination. The DG were enriched for 35 of the total 213 maize gene ontology (GO) terms, including those describing photosynthesis, respiration, DNA replication, metabolism, and hormone biosynthesis. From the DG, we identified six genes involved in glycolysis, three genes in the citrate cycle, and four genes in the C4-dicarboxylic acid cycle. We mapped 533 of the 748 DG to the maize B73 genome, 298 (55.9 %) of which mapped to the QTL intervals of 11 maize ear traits. Moreover, we compared the repertoire of the DG with that of 14-day seedlings of the same inbred and hybrid combination. Only approximately 5 % of the DG was shared between the two organs and developmental stages. Furthermore, we mapped 417 (55.7 %) of the 748 maize DG to the QTL intervals of 26 rice yield-related traits. Therefore, this study provides a repertoire of genes useful for identification of genes involved in maize ear trait heterosis and information for a better understanding of the molecular basis underlying heterosis in maize.     

Calcium-induced sperm fusion in Zea mays L.

 In this study, we examined morphological changes of isolated maize (Zea mays L.) sperm cells in the presence of Brewbaker and Kwack salts (BKS) or the individual components of BKS using light, transmission electron and scanning electron microscopy. Freshly isolated sperms are 7.5 μm in diameter. Treatment with BKS for 5 h resulted in large cells with a diameter up to 41 μm. Staining of sperm nuclei with 4′, 6-diamidino-2-phenylindole (DAPI) revealed two or more nuclei in a single cell, suggesting that BKS induces cell fusion. Treatment with each BKS component showed that cell fusion occurs only in the presence of calcium nitrate. Use of several calcium salts showed the same results, suggesting that the calcium ion, alone, is responsible for the observed cell fusion. Further studies were conducted to examine the relationship between calcium distribution and sperm location in pollen tubes using chlorotetracycline and DAPI. Growing maize pollen tubes exhibited a high membrane calcium region within 20–50 μm from the tip. The Sperms are found no closer than 90 μm to the tip of the tube, suggesting that sperms are located in a low calcium region prior to being released to the degenerating synergid. Received: 12 August 1996 / Revision accepted: 6 December 1996     

Possible Linkage between NADH-Oxidation and Proton Secretion in Zea mays L. Roots

A possible involvement of two different systems in proton translocationand the correlation of this factor to growth rates were measuredsimultaneously by means of a pH stat and an optical system.Ferricyanide, which can accept electrons at the plasmalemma,led to an immediate increase of net H⁺ -efflux but also decreasedroot growth rate. The reduced form, ferrocyanide, inhibitednet H⁺ -effluxwithout changing the growth rate. Thus, corn rootgrowth was not determined by proton secretion exclusively. Vanadatestrongly inhibited net H⁺ -efflux by the roots but did not preventthe stimulating effect of fcrricyanide. Moreover, the extentof enhancement of net H⁺ -effluxby ferricyanide was exactlythe same in vanadate pretreated as in untreated roots. Alcoholswere used to try to increase the intracellular NADH level throughthe action of the cytoplasmic alcohol dehydrogenase presentin the roots and coleoptiles. Alcohols, known to be substratesfor alcohol dehydrogenase such as propan- 1-ol, ethanol andbutan-l -ol increased net H⁺ -effluximmediately but methanoland secondary alcohols which are not substrates had no effecton proton secretion. The K_m values of alcohol dehydrogenasefor the alcohols correspond only partly to their effects onproton secretion. However, the specificlty observed suggeststhat increased H⁺ -efflux arose from reduction of endogenousNAD by ADH and consequent increased membrane NADH-oxidasc activitytrans locating protons and electrons out of the cells. Decreased oxygen concentrations slowed proton secretion at valuesfar higher than are necessary to saturate cytochrome c oxidase.The results of these experiments suggest two distinct systemscontributing to proton efflux. Key words: ADH, proton transport, redox chain     

Seed development and vivipary in Zea mays L.

Seed development was investigated in kernels of developing wild-type and viviparous (vp-1) Zea mays L. Embryos and endosperm of wild-type kernels began to dehydrate at approx. 35 d after pollination (DAP); viviparous embryos did not desiccate but accumulated fresh weight via coleoptile growth in the caryopses. Concentrations of endogenous abscisic acid (ABA) in the embryo were relatively high early in development, being approx. 150 ng·g^-1 fresh weight at 20 DAP. The ABA content declined thereafter, falling to approx. 50 ng·g^-1 at 30 DAP. Endosperm ABA content was always low, being less than 20 ng·g^-1. There were no differences between wild-type and vp-1 tissues. Immature kernels did not germinate when removed from the ear until late in development. The ability to germinate was correlated with decreasing moisture content in the endosperm at the time of removal; premature drying of immature kernels resulted in greatly increased germination following imbibition. Excised embryos germinated precociously when removed from the endosperm as early as 25 DAP. Such germination could be prevented by treatment with 10^-5 M ABA or by lowering the solute potential (_s) of the medium with 0.3 M mannitol. Treatment of excised embryos with ABA led to internal ABA concentrations comparable to those in embryos in which germination was inhibited in situ. Mannitol treatment did not have this effect, although water-deficit stress of excised embryos resulted in substantial ABA production. Germinated vp-1 embryos were less sensitive to growth inhibition by ABA or mannitol than germinating wild-type embryos. The vp-1 seedlings were not wilty and their transpiration rates were reduced in response to ABA or water shortage.Abbreviations and symbols ABA abscisic acid - DAP days after pollination - FW fresh weight - vp-1 viviparous genotype - _s solute potential     

玉米(Zea mays L.)叶脉发育的研究

玉米的叶脉在单子叶植物中有一定的代表性,叶脉由四级组成,粗细不同的一、二、三级叶脉均从叶基向叶尖方向延伸,属叶片的纵向叶脉,四级脉横向与一、二、三级叶脉连接,是横向的叶脉,各级叶脉有各自的形成方式,由于它们有规律的分布,从而构成了叶片的输导网络,各级叶脉的发生和发育与叫片的生长有直接的关系。     

Water Deficit and Inflorescence Development in Zea mays L.

A water deficit imposed during the period of terminal male inflorescenceinitiation and early development reduced both the growth rateand the mature size of that organ in Zea mays (cv. Iochief).Growth and development of the axillary shoots, the potentialfemale inflorescences, was inhibited during the episode of waterdeficit but promoted thereafter. As a result, plants which hadbeen subjected to a water deficit at that period produced 2–3mature cobs and relatively large axillary shoots at the lowernodes, whereas plants supplied with water throughout produceda single mature cob and relatively small axillary shoots. A water deficit imposed during other growth phases did not producethis response and, moreover, a further period of deficit imposedlater in development, following a deficit at the sensitive stage,inhibited the enlargement of the axillary shoots invoked bythe earlier deficit. It did not, however, inhibit the enhancedfloral development of those axillary shoots nor reverse theinhibition of tassel growth. The data are discussed in relation to correlative inhibitionin Zea mays.     

The purification and physicochemical characterization of maize (Zea mays L.) isocitrate lyase.

A purification scheme is described for the glyoxylate cycle enzyme isocitrate lyase from maize scutella. Purification involves an acetone precipitation and a heat denaturation step, followed by ammonium sulfate precipitation and chromatography on DEAE-cellulose and on blue-Sepharose. The latter step results in the removal of the remaining malate dehydrogenase activity, and of a high molecular mass (62 kDa) but inactive degradation product of isocitrate lyase. Catalase can be completely removed by performing the DEAE-cellulose chromatography in the presence of Triton X-100. Pure isocitrate lyase can be stored without appreciable loss of activity at -70 degrees C in 5 mM triethanolamine buffer containing 6 mM MgCl2, 7 mM 2-mercaptoethanol, and 50% (v/v) glycerol, pH 7.6. Maize isocitrate lyase is a tetrameric protein with a subunit molecular mass of 64 kDa. Purity of the enzyme preparation was demonstrated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, in acid (pH 3.2) urea and by isoelectric focusing (pI = 5.1). Maize isocitrate lyase is devoid of covalently linked sugar residues. From circular dichroism measurements we estimate that its structure comprises 30% alpha-helical and 15% beta-pleated sheet segments. The enzyme requires Mg2+ ions for activity, and only Mn2+ apparently is able to replace this cation to a certain extent. The kinetics of the isocitrate lyase-catalyzed cleavage reaction were investigated, and the amino acid composition of the maize enzyme was determined. Finally the occurrence of an association between maize isocitrate lyase and catalase was observed. Such a multienzyme complex may be postulated to play a protective role in vivo.     

The phytochrome system in light-grown Zea mays L.

The phytochrome system is analyzed in light-grown maize (Zea mays L.) plants, which were prevented from greening by application of the herbicide SAN 9789. The dark kinetics of phytochrome are not different in the first, second or third leaf. It is concluded that in light-grown maize plants phytochrome levels are regulated by P_r formation and P_fr and P_r destruction, rather than by P_frP_r dark reversion. P_r undergoes destruction after it has been cycled through P_fr. The consequences of this P_r destruction on the phytochrome system are discussed.Abbreviations SAN 9789 4-chloro-5-(methylamino)-2-(,,-trifluoro-m-tolyl)-3(2H) pyridazinone - P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot P_fr+P_r